Next Article in Journal
Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling
Next Article in Special Issue
Penetration of the SARS-CoV-2 Spike Protein across the Blood–Brain Barrier, as Revealed by a Combination of a Human Cell Culture Model System and Optical Biosensing
Previous Article in Journal
Vaccinia Virus: From Crude Smallpox Vaccines to Elaborate Viral Vector Vaccine Design
Previous Article in Special Issue
Miniaturized Electrochemical Sensors to Monitor Fetal Hypoxia and Acidosis in a Pregnant Sheep Model
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation

Nanobiosensorics Group, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary
Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest, Hungary
Department of Pharmacognosy, Semmelweis University, Üllői út 26, 1085 Budapest, Hungary
Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary
Department of Immunology, Eötvös Loránd University, 1117 Budapest, Hungary
MTA-ELTE Immunology Research Group, Eötvös Loránd Research Network (ELKH), Eötvös Loránd University, 1117 Budapest, Hungary
Clore Laboratory, University of Buckingham, Buckingham MK18 1EG, UK
MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary
National Public Health Center, Albert Flórián út 2-6, 1097 Budapest, Hungary
Author to whom correspondence should be addressed.
Biomedicines 2021, 9(12), 1781;
Submission received: 25 October 2021 / Revised: 19 November 2021 / Accepted: 22 November 2021 / Published: 26 November 2021
(This article belongs to the Special Issue Biosensors at the Aid of Medicine)


Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.

1. Introduction

Natural medicines, extracted from herbs and other living sources such as serpent venoms, have been used by humans since the earliest times. In contrast, the use of mineral substances as medicines was an innovation of Paracelsus introduced as recently as the 16th century AD. Today, there is renewed interest in natural substances for curing illnesses, both directly and as inspirations for manufactured pharmaceuticals (for example, an anti-malaria agent extracted from the sweet wormwood plant [1]). Many people prefer to take mixtures of herbs as alternatives to industrial medicine, which often causes deleterious side effects [2]. Plants (and fungi) synthesize a large number of specific compounds called secondary metabolites (SMs). The function of SMs in plants and fungi is not fully understood; however, many of these compounds can be used for medicinal purposes. They have special significance in cancer therapy: among compounds introduced since the 1940s, almost 50% were isolated, purified SMs or their semi-synthetic derivatives [3].
The study of the mode of action of natural substances at the cellular and molecular levels only began a few years ago with the advent of modern techniques. Compared to manufactured pharmaceuticals systematic scientific evidence for the efficacy and safety of natural substances is generally still lacking [2]. When studying SMs, the major challenge is to elucidate their effective targets, which are responsible for the medicinal effects of these natural compounds. In contrast with synthetic drugs the target is usually preselected, and the challenge is to find a molecule that binds to the target and not the others.
We suggest that these substances influence primarily at the cellular level. The general approach has been to apply labeling techniques to investigate effects on cell adhesion, migration, motility etc. in vitro [1]. We shall review new, label-free techniques for monitoring cell adhesion and spreading directly, without label-induced perturbation.
Cell adhesion is crucial for the assembly of individual cells into tissues [4]. It is responsible for the overall architecture of the tissue [4]. Monitoring cell adhesion and spreading is important because these processes maintain the multicellular tissue structure, and individual cell migration, survival, proliferation, differentiation, gene expression, cell-cell communication and immunity, and cancer metastasis [1]. Thus, studying the adhesion and spreading of the treated cells by natural compounds helps to understand their effects on metabolism, development and physiology.
In this review we summarize the effects of natural compounds from plants, fungi and snake venom on cellular adhesion and movement, and the methods applied to reveal these effects. Finally, we mention some possible ways to prevent or reduce symptoms of COVID-19 by applying natural compounds according to the recent literature in this topic.

2. Relationship between Adhesion, Movement and Inflammation

Inflammation is typified by the accumulation of leukocytes and other mesenchymal cells in response to attractant molecules at sites of injury or infection [5,6,7]. Leukocytes become activated after being exposed to chemoattractants and are capable of adhering tightly to the endothelium [7]. Cytokines and endotoxins stimulate the endothelium to become more adhesive for leukocytes [5,7,8]. The general classes of cell adhesion molecules (CAM) are integrins, selectins, the immunoglobulin superfamily of cell adhesion molecules and cadherins [5,7,9]. Integrins and selectins on circulating leukocytes mediate their adhesion to the endothelium, whereas selectins and members of the immunoglobulin superfamily on the endothelium mediate their affinity for leukocytes [7,9]. CAMs are known to play a critical role in the recruitment of cells into various tissues and in the maintenance and regulation of the integrity of the tissues [5].
CAM expression is tightly regulated in normal tissue environment; however, inappropriate expression of CAMs disrupts normal cell-cell and cell-matrix interactions, facilitating tumour formation.
Integrins are prime regulators of communication between cells and their microenvironment. These evolutionarily old cellular adhesion receptors play an important role in physiological and pathological processes. These large molecules are responsible for the attachment of cells to the extracellular matrix (ECM) components and cell-to-cell interactions. Integrin heterodimers are composed of noncovalently bound α and β subunits.
In vertebrates the integrin family is composed of 18 α subunits and 8 β subunits that form 24 different heterodimeric complexes [10] (Figure 1). The integrins can be grouped into subgroups based on their subunit composition and ligand-binding specificity.
A subgroup of integrins (8 out of 24) recognize proteins (such as fibronectin and vitronectin) that contain the Arg-Gly-Asp (RGD) sequence. The collagen binding integrins α1β1, α2β1, α10β1, and α11β1 are able to recognize the triple-helical GFOGER collagen sequence. The laminin receptors α3β1, α6β1 and α7β1 mediate adhesion to basement membrane laminins. Vertebrates also have leukocyte-specific integrins that mediate cell-cell adhesion. The β2 integrins are present on the cell surface in a normally inactive state, in which they do not bind ligands. An activation signal like an inflammatory stimulus results in a conformational change of the integrin, increasing its ability to bind the ligand. Activated β2 integrins play an important role in cellular adhesion, migration and phagocytosis.
The lack of the β subunit can block preimplantation development (β1) and perinatal lethality (β8) and is involved in various defects of leukocyte function (β2, β7), inflammation (β6), hemostasis and angiogenesis (β3). Integrins frequently intercommunicate; that is, they are able to activate or inhibit each other’s function [10].
The CD11/CD18 β2 integrins are specifically expressed on circulating leukocytes, and play a significant role in fast adhesion to endothelial cells [7,9]. The intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) are well-characterized endothelial cell ligands for CD11/CD18 [7,11]. The endothelial cell surface quantity of molecules like ICAM-1 is increased with the release of some proinflammatory cytokines from lymphocytes; an environment is thereby generated in which the leukocytes are more chemoreceptive to arterial walls during inflammatory processes [5]. Recruitment of arterial leukocytes is a significant step in the progression of different inflammatory diseases, such as rheumatism [12,13], liver inflammation [13,14], atherosclerosis [13,14], and inflammatory bowel disease [13]. Medical plant extracts may alter pathological mechanisms via the modulation of adhesion molecules [5]; this general observation will be analyzed and systematized below.
All morphogenetic processes are affected by cell migration, which contributes to many illnesses, including cardiovascular disease and cancer [15]. In general, cell movement begins with extension of the membrane followed by the formation of new adhesive protrusions at the front, which link the actin cytoskeleton to the substratum, generating traction forces that move the cell forwards; adhesive protrusions at the rear are simultaneously dismantled [15]. The cycle of forming and dismantling of adhesive structures drives migration [15]. Rho GTPases have an important role in this process; they regulate actin polymerization and myosin II activity and, therefore, adhesion dynamics [15].
The adhesion molecules driving migration are the same as those involved in the inflammatory response, but their expression and ligand-binding capacity depend on the stimuli.
Figure 1. Integrin family members and their ligands. Integrins are transmembrane heterodimer molecules containing α and β subunits. The figure illustrates the association of these subunits occurring in mammalian cells. As shown, 18 α and 8 β subunits form 24 different, distinct integrins. These can be grouped in subfamilies based on evolutionary relationships (different colors of α subunits), ligand specificity and, in the case of β2 and β7 integrins, restricted expression on white blood cells. Integrins can be grouped into two larger classes that bind to cell surface cell adhesion molecules (CAMs) and ECM ligands. They can be further classified as collagen-binding integrins (α1β1, α2β1, α10β1, and α11β1), RGD-recognizing integrins (α5β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3), laminin-binding integrins (α3β1, α6β1, α7β1, and α6β4), and leukocyte integrins (αLβ2, αMβ2, αXβ2, and αDβ2). The β2 integrin subunit (CD18) can pair with one of the four α subunits (αL-CD11a, αM-CD11b, αX-CD11c, and αD-CD11d) [16]. The α4β1 and α9β1 integrins recognize fibronectin and VCAM-1. The β2 and β7 integrins are restrictedly expressed by leukocytes). Asterisks show the alternatively spliced cytoplasmic domains. This figure is based on the study of Hynes [10] and Yue et al. [17]. (RGD, Arg-Gly-Asp sequence; VCAM-1, a vascular cellular adhesion molecule-11).
Figure 1. Integrin family members and their ligands. Integrins are transmembrane heterodimer molecules containing α and β subunits. The figure illustrates the association of these subunits occurring in mammalian cells. As shown, 18 α and 8 β subunits form 24 different, distinct integrins. These can be grouped in subfamilies based on evolutionary relationships (different colors of α subunits), ligand specificity and, in the case of β2 and β7 integrins, restricted expression on white blood cells. Integrins can be grouped into two larger classes that bind to cell surface cell adhesion molecules (CAMs) and ECM ligands. They can be further classified as collagen-binding integrins (α1β1, α2β1, α10β1, and α11β1), RGD-recognizing integrins (α5β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3), laminin-binding integrins (α3β1, α6β1, α7β1, and α6β4), and leukocyte integrins (αLβ2, αMβ2, αXβ2, and αDβ2). The β2 integrin subunit (CD18) can pair with one of the four α subunits (αL-CD11a, αM-CD11b, αX-CD11c, and αD-CD11d) [16]. The α4β1 and α9β1 integrins recognize fibronectin and VCAM-1. The β2 and β7 integrins are restrictedly expressed by leukocytes). Asterisks show the alternatively spliced cytoplasmic domains. This figure is based on the study of Hynes [10] and Yue et al. [17]. (RGD, Arg-Gly-Asp sequence; VCAM-1, a vascular cellular adhesion molecule-11).
Biomedicines 09 01781 g001

3. Mechanisms of Action of Natural Compounds

3.1. Prestimulation with Cytokines

To demonstrate the inhibitory effect of a natural compound on cellular adhesion, cells, typically from the human umbilical vein endothelial cell line (HUVEC), are usually first treated with certain cytokines to stimulate the expression of CAM (Figure 2). In vivo, lipopolysaccharide (LPS, from Gram-negative bacteria) stimulates the immune response by interacting with its leukocyte membrane receptor, the Pattern Recognition Receptor (PRR) CD14 (with TLR4-MD2), to induce the generation of cytokines such as tumour necrosis factor α (TNF-α), interleukin-1 and -6 (IL-1, IL-6) (Figure 3). TNF-α is also involved in systemic inflammation [2,18]. It is primarily produced by activated monocytes or macrophages [19]. Note, cytokine generation, increase of expression of cytokines can also be stimulated by certain plant extracts (for example, among others, garlic (Allium sativum) decreases the level of IL-1α, IL-2, IL-6, IL-12, TNF- α and IFN-γ, however, increases IL-10), as summarized by Spelman, et al. [20]. After stimulation of the endothelial cells, the plant (or venom) extract is then added to them. The natural compound may downregulate expression of the adhesion molecules, resulting in the diminution of cell adhesion, and the compound has an antiinflammatory effect in consequence. The ICAMs and VCAMs are the most researched adhesion molecules [5]. We elaborate on this in the next section.

3.2. Inhibition of CAMs by Suppression of Their Expression (Downregulation)

The phytochemicals of olive oil (from Olea europaea) and red wine (from Vitis vinifera), oleuropein (monoterpene, seco-iridoid glucoside), hydroxytyrosol (phenylethanoid), tyrosol (phenylethanoid), elenolic acid (monoterpene, seco-iridoid) and resveratrol (stilbene) at nutritionally relevant concentrations have been shown to inhibit endothelial adhesion molecule expression (Figure 2). This provides a strongly suggestive basis for the atheroprotective property of the so-called “Mediterranean diet” [21].
Walnut (Juglans regia) extract and its principal active component ellagic acid decreased the stimulated endothelial expression of ICAM-1 and VCAM-1, indicating a mechanism for the known antiatherogenic and osteoblastic activity of the substance [18]. A walnut-enriched diet may therefore indeed be cardioprotective and inhibit osteoporosis [18].
Curcumin (diphenylheptanoid) from the Curcuma longa rhizome also downregulated the expression of adhesion molecules and, hence, monocyte adhesion [13]. Saponin (triterpene) astagaloside IV from Mongolian milkvetch (Astagalus membranaceus) decreased the LPS-induced expression of VCAM-1 and E-selectin on the surface of HUVEC, hence this Chinese traditional medicinal herb is predicted to have anti-inflammatory efficacy; however, ICAM-1 was not affected [22].
Tripertygium wilfordii is a vine-like plant that grows in south China, and in the Chinese pharmacopoeia the extract from its root is prescribed for treating long-term rheumatoid arthritis and systemic lupus erytnematosus [7,8]. Chang et al., applied IL-1α to stimulate HUVEC cells; treatment with a high concentration (50 ng/mL) of the herb extract (containing wilforonide, alkaloids, diterpenes, triterpenes, b-sitosterol, daucosterol, dulcitol and glycosides [7,23]) had a significant inhibitory effect on both the expression and secretion of the cellular adhesion molecules, and thus may be a potential therapeutic agent for the treatment of inflammatory diseases [7].

3.3. Mechanism of Downregulation

The downregulation of CAMs by natural products is achieved by inhibiting their gene expression.
The molecular details involve the uptake of the natural product by the cytoplasm followed by interaction between the compound and transcription factors for adhesion molecule genes. Activation of the transcription factor nuclear factor κB (NF-κB), is mediated by the proinflammatory cytokines mentioned above, for instance TNF-α, and triggers gene expression of adhesion molecules (Figure 4). NF-κB binding sites are found in the promoter region of E-selectin, ICAM-1 and VCAM-1 [13,24,25,26,27]. NF-κB is in the cytoplasm in inactive form, complexed to IκB “nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor” [13]. When cells are stimulated with TNF-α, the IκB is phosphorylated, ubiquitinated, and degraded. The thereby activated NF-κB translocates to the nucleus and transcriptionally up-regulates cytokine receptors as well as the adhesion molecules [13,26,28,29].
The expression and function of integrins on various immune cells are summarized in Table 1.
Kawasaki and co-workers demonstrated that hot-water extract of Curcuma longa also suppressed the phosphorylation and degradation of IκBα in endothelial cells [13]. Another extract, the triterpene saponin astragaloside IV (3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol) from the Chinese herb Astragalus membranaceus, shown to have anti-inflammatory effects in vivo, completely annulled LPS- and TNF-α-triggered nuclear translocation of NF-κB and NF-κB DNA-binding activity in endothelial cells [22] (Figure 4), furthermore, it has shown that it has antioxidative stress, antiapoptosis, and antifibrosis activities, both in vitro and in vivo [34,35].
Recent studies have shown that astragaloside IV (AS-IV) administration ameliorates diabetic neuropathy in streptozotocin (STZ)-induced diabetic rats via an anti-inflammatory mechanism [36], inhibits endoplasmic reticulum stress [37], and protects podocytes [34,38]. However, the effect and mechanism of AS-IV on diabetic neuropathy induced by type 2 diabetes remain unknown [34]. Flavonoid quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2–ERK1/2 and PKCδ–JNK1/2–c-Jun or NF-κB pathways [39]. Flavonoid apigenin significantly suppressed the TNF-α-stimulated upregulation of VCAM-1-, ICAM-1-, and E-selectin-mRNA to the basal levels [40]. Simple phenolic compound salicin inhibits IL-1β-induced production of pro-inflammatory cytokines such as TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecules such as (ICAM-1 and VCAM-1, and high-mobility group protein 1 (HMGB-1)) [41].
Lignan-type active compounds manassantin A and B, dineolignan compounds, inhibited the PMA-induced ICAM-1/LFA-1-mediated homotypic aggregation of the HL-60 cells without cytotoxicity, with MIC values of 1.0 and 5.5 nM, respectively. Even though these compounds did not affect the adhesion of ICAM-1 to LFA-1, they inhibited PMA-induced ICAM-1 expression in HL-60 cells in a dose-dependent fashion. These results suggest that manassantin A and B inhibit cell aggregation through downregulation of ICAM-1 expression [42].
HUVECs treated with sesquiterpene α-iso-cubebene showed markedly suppressed TNF-α-induced mRNA expression of VCAM-1 and E-selectin, but little alteration in ICAM-1 mRNA expression. α-iso-cubebene treatment also significantly decreased the TNF-α-induced cell surface and total protein expression of VCAM-1 and E-selectin without affecting ICAM-1 expression [43].
Diterpene andrographolide significantly reduced E-selectin expression of activated endothelial cells, and inhibited E-selectin expression at the mRNA level [44].
In vitro, triterpene saponine dioscin decreased monocyte adhesion to TNF-α-treated HUVECs by reducingvascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression and inhibiting endothelial lipase (EL) expression in TNF-α-treated HUVECs and macrophages by blocking the NF-kB pathway [45].
Ethyl 3′,4′,5′-trimethoxycinnamate and piperine are the two active principles of Piper longum. Using primary human umbilical vein endothelial cells, Kumar et al. [46] evaluated the activities of ethyl 3′,4′,5′-trimethoxycinnamate on TNF-α-induced expression of cell adhesion molecules, ICAM-1, VCAM-1 and E-selectin, which play key roles in controlling various inflammatory diseases. Both compounds inhibited the TNF-α-induced expression of ICAM-1 [46].
Ganoderma lucidum, a medicinal mushroom, has been used in traditional Chinese medicine to prevent and treat various diseases, for example cancer [47]. A polysaccharide derived from the fungus interacted with cell surface proteins and β1-integrin expression was diminished, while β-actin expression was not affected [47].
We summarize the different modes of pathway intervention involving gene expression in Table 2.

3.4. Intervention at the ECM

Flavonoid baicalein, derived from the root of Scutelaria baicalensis, a widely used Chinese herbal medicine that has been used in anti-cancer and anti-inflammatory therapy [48], has an inhibitory effect on the expression of matrix metalloproteinases (MMPs) [48], which are involved in the degradation of the extracellular matrix. Destruction of basement membranes and stromal extracellular matrix is critical for favoring metastasis and invasion of malignant cells [48]. The MMPs have therefore a role in promoting tumour growth, invasion and metastasis. Treatment of human breast carcinoma (MDA-MB-231) cells with baicalein inhibited the expression of MMP-2/9, which is a result of the mitogen-activated protein kinase (MAPK) signaling pathway [48] (Figure 5).
Flavonoid (chalcone) butein (3,3,2′,4′-tetrahydroxychalcone) is an active substance found in several plants, such as Semecarpus anacardium, Dalbergia odorifera, Caragana jubata and Rhus verniciflua [49]. It has been demonstrated that it decreased leukocyte adhesion to A549 cells through the inhibition of TNF-α-induced ICAM-1 and VCAM-1 expression by inhibiting the NF-κB/MAPK/Akt signaling pathway. Butein also inhibits ROS generation [49], and may prevent TNF-α-induced airway inflammation [49] (Figure 4).

3.5. Inhibition of CAM Binding by Blocking Specific Cell–Surface Receptor Sites

We can distinguish three types of mechanisms inhibiting cell adhesion by blocking (B) specific receptor sites (Table 3).
In contrast to intervention at the level of protein expression, natural compounds can also specifically block cell recognition motifs, such as the amino acid triplet RGD. For example, the polyphenol EGCG from green tea has been shown to block RGD motifs (B) (Arg-Gly-Asp) and, hence, inhibit adhesion [50].
We recall that integrins are transmembrane heterodimers, a family of plasma membrane receptors that mediate adhesion of leukocytes to ECM [51]. Integrins are also involved in pathophysiological processes as well, such as genetic and autoimmune diseases, metastasis and thrombosis and, thus, integrins are important therapeutic target structures [51,52]. Blocking or disruption of binding to integrin receptors is therefore an important topic in industrial drug discovery [51,53]. Serpent venom disintegrins, a family of low molecular weight proteins, typically contain the RGD motif, and are known to block integrin activities by binding with high affinity to the integrins [51,54]. For example, rhodostomin, a snake venom from Calloselasma rhodostoma, blocked the integrin ανβ3 and also affected pp125 FAK phosphorylation and the actin cytoskeleton [55]. Rhodostomin contains an RGD motif that specifically inhibits the integrin-binding function [56]. It can be produced in Pichia pastoris (methylotrophic yeast) as well and it inhibits platelet aggregation with a K(I) of 78 nM as potent as native rhodostomin [56]. However, its D51E mutant blocks platelet aggregation with a K(I) of 49 mM [56]. Structural analysis of rhodostomin and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets [56]. Two minor differences between them were inferred from their backbone dynamics and 3D structures [56]. The docking of rhodostomin into integrin αIIbβ3 showed that between the backbone amide and carbonyl groups of the D51 residue were formed hydrogen bonds with the integrin residues R216 and R214, respectively [56]. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex [56].
Another serpent venom, echistatin from Echis carinatus, inhibited integrin-mediated cell adhesion via selective recognition by αIIbβ3, α5β1, ανβ3 integrins [51], preventing their adhesion to the ECM.
Not only snake venom affects integrin-mediated adhesion, but herbs as well. Epigallocatechin-gallate (EGCG) (flavonoid ester) is the main polyphenol of green tea (Camellia sinensis). Many studies have shown its beneficial effect on human health [1]. The majority of them demonstrated direct effects on cell adhesion and movement. In a previous study we showed that EGCG indirectly affects HeLa cell adhesion: the cells cannot adhere onto EGCG-pre-treated model ECM coatings [50]. The polyphenol formed multilayers in poly-L-lysine polyethylene-glycol-RGD (PLL-g-PEG-RGD) chains, and blocked the RGD motifs [50]. EGCG alters the properties of mucin as well; EGCG-mucin mixtures showed that discrete particles are formed and their size increases with the ratio of EGCG to mucin [57]. Another natural compound, cistifolin (benzofuran derivative) from the rhizome of the gravel root (Eupatorium purpureum), known as an anti-rheumatic herbal drug, was identified as a potent inhibitor of β1 and β2 integrin-mediated cell adhesion and, thus, has therapeutic potential for diseases where integrin adhesion molecules play a significant role [58].

4. Measurement Techniques for Monitoring Cellular Functions, Adhesion and Viability

4.1. Classical Techniques for Measuring Cell Viability

Experimental natural compounds are added to stimulated cell cultures to induce a CAM response, which is commonly measured by labeling methods, mainly the enzyme-linked immunosorbent assay (ELISA), Western blot, and flow cytometry. ELISA uses antibodies linked to enzymes that create a color change (e.g., by altering a dye) to identify the examined substances [13]. The Western blot is a widely used technique in biology to detect specific proteins in a sample by letting animal-derived or synthetic antibodies bind to them [13]. ELISA and Western blot techniques cannot be used for measuring cell viability directly; however, for example, Western blot can be applied as a complementary method to study the mechanism of cell death (apoptosis, autophagy markers, etc.), thus it provides a lot of information about the mechanism. Flow cytometry is usually a laser-based technique for cell sorting and counting. A wide range of fluorophores can be used in flow cytometry measurements. Fluorophores are fluorescent labels that can attach to the antibody that recognizes the target molecule of the cell [2,59]. However, impedance-based flow cytometers also exist, known as Coulter counters, which are well established label-free methods for sizing and counting cells and particles [60].
Some workers have used staining to reveal the effect of natural compounds on cell viability, mainly the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and the trypan blue exclusion test. However, Wang et al. in 2010 showed that the MTT- and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-based assays (Figure 6) underestimate the antiproliferative effect of EGCG [1,61].
The terms “cell viability” or “compound cytotoxicity” have broad meanings in drug discovery. For in vitro monolayer cell cultures, a compound is considered to be cytotoxic if the compound interferes with cellular attachment, or significantly alters cellular morphology, cell growth and cell viability. A variety of assay methods can be used to estimate the number of viable eukaryotic cells after exposure of the investigated compounds. These cell-based assays are often used for screening collections of compounds to determine if the compounds have effects on cell proliferation or show cytotoxic and cytostatic effects. Cell-based assays are also widely used for monitoring organelle function. These screening methods have been devised to examine a broad variety of parameters associated with biochemical events necessary for sustaining viability, especially as evinced by membrane integrity. The quantities emerging from metabolism (especially ATP-based viability) assays are proportional to viable cell number. Cytotoxicity assays determine parameters proportional to the degree of cell death. The fundamental difference between the approaches depends on the length of exposure to the compound (short-term exposures (4 h or less) may adversely affect metabolism markers or ATP content before measurable membrane integrity changes, and long-term exposures (24 h or more), particularly after early primary necrosis, may lead to underestimation of cytotoxicity owing to degradation of marker enzyme activity after its release into the extracellular environment [62,63,64,65].
Most cell viability and cytotoxicity assays can be divided into three categories: those that (i) exploit the loss of membrane integrity; (ii) directly measure metabolic markers or ATP content; and (iii) assess metabolic activity. Other forms of detection exist. Crystal violet staining can reveal the adherence of cells and thus be used to measure the viability of adherent cells [66,67,68]. Determination of the loss of membrane integrity: these assays rely on the breakdown/disintegration of the cell membrane to allow different molecules to enter the cell, or allow intracellular compounds to be secreted to the extracellular area.
Metabolic assays primarily focus on measuring ATP levels, or the reduction of tetrazolium salts, or resazurin dyes inside living cells.
Cellular proliferation causes a change in the ratios of certain metabolites e.g., NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD. These metabolic intermediates are then capable of reducing tetrazolium salts to formazan product, which can be detected. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a non-fluorescent redox dye, which when reduced to resorufin becomes a red compound, so the color change can be detected.
The most common method for assaying live cell proliferation is measuring the amount of DNA synthesis, which is done by adding a labeled DNA analog called BrdU (5-bromo-2′-deoxyuridine (BrdU), which is incorporated into the DNA instead of thymidine. To assess the incorporation of BrdU into the DNA colorimetric ELISA or immunohistochemical staining methods are used. A newer approach is to detect the incorporation of the alkyne containing thymidine analog EdU. The incorporation can be detected by a copper catalyzed azide-alkyne cycloaddition.

4.2. Limitations and Considerations When Using Membrane Integrity or Metabolic Assays

The listed substrates (Table 4, Table 5, Table 6 and Table 7) all have distinct advantages and disadvantages when compared to each other. Assay sensitivity, noise-to-signal ratio, ease of use, and also reagent stability are all factors that have to be considered. Metabolic assays also need to consider that the reduction of said substrates are impacted by other intracellular metabolic activity, and have no direct effect on the cells viability or cytotoxicity of a studied compound.
In fixed samples such as animal tissues and cell population, proliferation can still be measured, but only by immunostaining for specific proliferative markers. Ki-67 is a nuclear protein that is associated with cell proliferation and ribosomal RNA transcription. Traditional antibodies for Ki-67 can only be used to stain frozen (not paraffin embedded) samples. MIB-1 antibodies however target a different epitope of Ki-67, and thus can be used to stain formalin and paraffin fixed samples, making the use of Ki-67 as a proliferative marker easier. Another commonly used marker is the proliferating cell nuclear antigen. This protein expedites DNA synthesis by holding the polymerase to the DNA, so it is expressed widely in the nucleus during DNA synthesis, making it an effective marker of cell proliferation. Other markers that can be used include MCM2 also. It has to be noted that all such assays measuring DNA synthesis directly or indirectly are sensitive to the stage of the cell in the cell cycle at the time the measurement is carried out.
The most frequently used techniques in cellular adhesion experiments with natural compounds are based on dyes (absorbing or fluorescing) as labels. These methods have several drawbacks; however, (i) in general they are not high-throughput techniques, since the procedures are time-consuming and complicated, (ii) many dyes are cytotoxic and therefore disturb the normal physiological activity of the cells; furthermore their structures are often quite comparable to those of the natural compounds under investigation, leading to interference, (iii) typically only end-point data is gathered; i.e., no information on the kinetics of the processes is obtained, (iv) many natural compounds investigated for possible therapeutic benefits have low molecular weight, and frequently are environmentally sensitive (e.g., to oxidation) and thermally or photolytically unstable.
A good example of this instability is the green tea polyphenol EGCG [1,50]. Investigating the physiological effects of such compounds by labeling techniques exacerbates the difficulties.
Recent developments, such as the use of quantum dots (semiconductor nanoparticles) and gold nanoparticles instead of organic dyes as labels do little to alleviate these disadvantages.

4.3. Measurement Techniques for Monitoring Cellular Movement and Adhesion

Cell migration takes place inside or on the extracellular matrix surface [157]. The lamellae of the cell have to adhere to the matrix components to generate traction forces for cell movement [157]. The actin filaments of the cytoskeleton are linked through the cell membrane to the substrate at focal adhesion points, which are connected by actin filaments such as to promote the direction of movement and are located under the entire body of the cell [157,158]. Hence, adhesion and migration are in close relation to each other; adhesion is essential to achieve cellular movement. Thus, measuring cell migration with migration assays provides information about cell adhesion as well [157]. In Table 8, we summarize the advantages and disadvantages of migration assays. Spreading and adhesion play crucial roles in development and maintenance of cell homeostasis and in many further complex functional processes [159,160]. Many types of assays have been developed to measure the adhesion strengths (forces) and can be categorized into single cell and population cell studies [158,161,162]. Cell adhesion detachment events for single cell studies can be initiated via the breakage of molecular bonds (e.g., optical tweezer techniques and micropipette aspiration), cell population studies via static adhesion (centrifugation technique), and cell population studies via dynamic adhesion (e.g., microfluidic techniques, spinning disk, and flow chamber) [158,161,162]. The measurement of migration and adhesion under experimental in vitro conditions can provide important information about natural compounds, yielding quantitative data about cellular movement and adhesion albeit in a drastically simplified environment compared to the in vivo situation [50].

4.3.1. Label-Free Biosensors

The concept of labeling is rooted in the poor sensitivity of many traditional biological molecular detection techniques. Biomolecules are mostly composed of light elements with small masses and optical polarizabilities and are only rarely fluorescent. The techniques based on electrons, X-rays, visible light and mass spectrometry, which have been so successful in physics and chemistry, become much less potent when moved to the world of biology. By artificially coupling a biomolecule to an artificial entity with an electron density, optical polarizability or mass that may be orders of magnitude greater than that of the biomolecule, the latter suddenly becomes “visible” to the technique being employed to detect it. The euphoria that followed this sudden visibility tended to completely overwhelm continuing appraisal of whether the labeling itself might mask the biological effect that was the primary object of investigation [189].
The advent of new technologies sufficiently sensitive to remove the need to label the biomolecules to make them visible opened up a new world of label-free detection [190,191,192]. Natural compounds usually have small molecular weight where labeling can be problematic or even impossible, especially if their binding pocket is small or embedded. Label-free biosensors are emerging tools to investigate the mode of action of small molecules as well. They eliminate all of the disadvantages of the classical techniques listed above, because these methods do not require labels or dyes, which may disturb the samples. Furthermore, in general, the measurement procedures are not difficult, relatively cheap, and not time-consuming. In the field of natural compound research (for example active compounds in traditional Chinese medicines), novel methods, for example, resonant waveguide grating (RWG) [50,193,194] and holographic microscopy can be applied without using any dyes or other labels [195].
These techniques, particularly the highly sensitive waveguide-based methods [57,194,196,197,198,199,200,201,202], constitute highly promising novel phenotypic assays for drug discovery [203] owing to their capacity to reveal the complexity of drug actions and interactions and provide a holistic view of receptor-ligand interactions in living cells. Waveguide sensors are based on the phenomenon of the evanescent field generated by waveguided (usually visible) light being modulated by the presence of drugs, proteins and other biomolecules and living cells [204,205]. An adlayer, which may be constituted from a phospholipid membrane, extracellular matrix proteins or tissue is firstly placed onto the planar waveguide, where it is in contact with physiological medium containing the analyte of interest, such as the natural compound [204,205]. Molecular processes taking place in or on this adlayer modify the parameters of light propagating in the waveguide, any of which can serve as signal transducers [205,206].
Label-free techniques are especially useful for kinetic monitoring of biological events [207,208,209], even at single-cell level [210]. So they have become more and more popular in the field of drug discovery and in other areas focusing on the biological roles of small molecules [1,203]. Furthermore, the same techniques can be readily adapted to measure the dynamics of cell shape changes [211]. Cell adhesion assays and cell migration tests can be achieved in a completely label-free way, with in situ exposure to the natural compounds and, indeed, any drug. For example, a recent study of Peter et al. applied holographic microscopy to quantitatively show that EGCG reduced the motility speed and migration of HeLa cells [195]. Holographic microscopy creates 3D images of cells and the changes in cell morphology due to EGCG could be observed in real time [195].
These techniques are more and more important to systematically study the effects of active substances on cell behavior, especially cell adhesion, and to investigate different biological materials (such as membranes and matrices) and living cells in a high-throughput format [50]. In a recent experiment, Peter et al. showed that EGCG can block the RGD (Arg-Gly-Asp) motif, an important cell adhesion ligand and directly affect matrix properties by hydrogen bonding (Figure 7) [50]. The other novelty of this study is that the polymer coating fabrication, its treatment with the small EGCG molecule (a green tea polyphenol), and the observation of cell adhesion could be all studied online using a high-throughput RWG biosensor, with different EGCG and oxidized EGCG concentrations [50].
This and similar methodologies should be applicable to other extracellular matrix interactions with small natural compounds (Figure 7).
Recently, it was concluded that the shape of the kinetic curves obtainable with the label-free biosensor can be used to quantify in vitro cell viability in a fast and highly sensitive manner [59].
Natural compounds are generally small molecules that can self-associate into colloidal aggregates in aqueous buffer [212]. In the early stage of drug discovery, this phenomena is the principal cause of false results [212]. Wang et al. reported RWG-based assays to identify natural compound aggregation and characterize its influence on membrane receptors in living cells [212]. They showed that the colloidal aggregates may cause false activity in DMR desensitization assays [212]. A series of RWG-based assays for colloidal aggregate detection and characterization of their promiscuity were developed [212]. RWG-based assays can be applied as practical tools to distinguish between real and false responses, providing useful reliable results in the early stages of drug discovery [212].

4.3.2. Living Cell Movements—Holographic Microscopy

The realization of in situ monitoring of living cell movements (i.e., crawling along the endothelium) is a very important advance [195,213]. There are anyway few techniques to study cell movements; classical ones are mostly directed at migration studies and they also have their drawbacks [195]. For instance, filter assays (Zigmond and Dunn chambers, Boyden chamber) measure cell migration over a membrane in response to chemoattractant compounds [195]. These assays are very specialized, requiring cells to migrate through both a matrix and the pores of a filter [213]; however, very few cell lines can migrate through both of them [195]. Single cell movements can be investigated by using time-lapse imaging, usually requiring fluorescent labels [195,213]. As discussed above, fluorescent imaging may disturb the cells and the imaging time is limited by bleaching of the fluorescent marker [195,213]. In contrast to fluorescent imaging, holographic microscopy is a label-free technique [195,214]. It is usually critical to observe and quantitatively record live cell behavior, especially directional movement (migration) and random movement (motility), and also the shape changes to understand the behavior of the cells in such environments and to be able to make inferences regarding further therapeutic possibilities [195]. Although traditional herbal extracts have become more and more popular for treating illnesses, the literature of systematic quantitative studies is still limited [1].

5. Preparation of Natural Compounds

Natural products with their broad chemical diversity and bioactivity spectrum are sought after by the pharmaceutical industry and they continue to provide new structures with promising effects and to offer templates for the development of scaffolds of novel drug candidates. However, natural product discovery programs have been abandoned over the past 30 years by many pharmaceutical companies [215,216,217].
Natural products present their own challenges for drug discovery, including screening, isolation, characterization and optimization, which doubtless contributed to the decline in their pursuit by the pharmaceutical industry. In recent years, several technological and scientific developments—such as improved analytical tools, genome mining, engineering strategies, and microbial culturing—are addressing these challenges, preparing the way for new opportunities and interest in natural compounds [218,219].
Here, we briefly summarize recent technological developments that are facilitating natural product-based drug discovery, highlight selected applications and discuss key opportunities. Innovative compound identification from natural products requires a multidisciplinary approach utilizing numerous technologies [220].
Isolation and purification of natural compounds are the most important and difficult steps for molecular structure identification, in vitro testing, quantity control and further industrial production. The natural compounds are usually in complex matrix material and the active moieties are present in low concentration [220] which are often accompanied by co-occurring irrelevant common metabolites accumulated in high amounts. Selection of appropriate techniques and strategies is essential for getting the target compounds at high yield. Over the past decades, many types of new extraction, isolation and purification techniques (classical solvent extraction procedures, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), extraction with ionic liquids, accelerated (pressurized) solvent extraction (ASE), supercritical fluid extraction (SFE), extraction on solid phases, distillation methods, membrane filtration, preparative high-performance liquid chromatography (HPLC), counter-current chromatography (CCC), supercritical fluid chromatography (SFC), etc.) have been applied. With developments in separation science and microplate-based in vitro high-throughput screening (HTS) assays, natural compound research gained remarkable momentum recently. Nevertheless, despite these developments of extraction, separation, analytical and structural elucidation techniques, isolation of natural compounds from plants, marine organisms or microorganisms is challenging, with problems including post-harvest changes in material quality [221,222]. Extraction is the first step to separate most natural products from the raw materials. The method progresses through the following stages: (i) solvent penetration into the matrix; (ii) the components dissolve in the solvent; (iii) the components diffuse out of the matrix. A brief but excellent summary of the various extraction methods used for natural products is presented by Zhang and co-workers [223]. A more detailed description is presented in a book chapter by Cavalcanti et al. [224]. The analyses of extracts, fractions, isolates, compounds from different natural matrices, studies on chemotaxonomy, chemical fingerprinting, metabolome elucidation, and structural evaluation have become easier because of the availability of a number of instrumental analytical techniques, e.g., GC-MS, LC-PDA, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, and CE-MS, NMR, NIR, FT-IR.
Atanasov and co-workers [219] have detailed the traditional bioactivity-guided isolation process and its limitations, and also ideas on addressing them. The general process begins with the extraction steps and the method applied determines which type of compounds will be present in the particular extract (solvents with hydrophilic chemical character will result in a higher abundance of hydrophilic compounds). Following the identification of an extract with promising bioactivity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the individual active compound or compounds are isolated. To isolate such new antiproliferative/antimicrobial compounds from a series of extracts, highthroughput label-free activity screening by label-free biosensors could be applied. The identities of the pure compounds responsible for an extract’s activity can be determined by bioactivity-guided fractionation using novel label-free biosensor systems [1,204,205,206,207,208,209,210,211,212,213,214].
A systems biology-guided approach coupled with application of commensurate technologies (genomics, proteomics, metabolomics, in silico modeling, etc.) should provide new opportunities for identifying new and better drug candidates. Molecular libraries of lead compounds from natural products will serve as sources of lead extracts/compounds for innovative drugs (Figure 8).

6. Natural Compounds of Plant, Animal and Fungal Origin

We summarize and categorize those natural compounds found in the literature that affect cellular adhesion and migration. The sources of the active compounds are divided into three groups; plants (Table 9), animals (Table 10), or fungi (Table 11). The compounds produced by these organisms include primary and secondary metabolites. They show structural and functional diversities, which are the result of biosynthetic processes modulated by natural selection. Defense molecules—antimicrobial peptides (AMPs)—are commonly produced to resist stresses. These oligo- and polypeptides are synthesized by ribosomes or via non-ribosomal peptide synthetases. Many AMPs exhibit broad-spectrum antimicrobial and anticancer activity. Plants are a remarkably promising source of these molecules and they are also considered to affect plant growth and development [225]. AMPs can be classified into cationic or anionic compounds according to their net charge. Their function, isolation and other aspects have been reviewed [225,226,227,228].
In the cases of plant or fungal origin, it has been well known for centuries that active substances can be extracted from individual plant organs (i.e., leaves, flowers, rhizomes, fruits, seeds, roots, stems, bark and xylem). These parts can be consumed as hot water infusions (e.g., green tea, herbal infusions), as spices (e.g., pepper, clove), as raw fruits or extruded/fermented products (e.g., red wine, olive oil). Such natural compounds are consumed day-by-day as food, but their ability to cure diverse diseases has been known for thousands of years (e.g., in Chinese traditional medicine).
In our tables, if the literature information is too scanty to allow us to be more specific, we simply write P (pathway intervention involving gene expression) or B (cell adhesion inhibition by blocking specific receptor sites) to indicate the mechanism.
In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238,239,240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion (Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides produced by several fungal strains, for example Microsphaeropsis sp. [241]. These compounds and metabolites from the peribysin group are also detailed in Table 11.
Table 11. Effects of fungi-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Table 11. Effects of fungi-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Active SubstanceIUPACName (CAS Number)SourceCellular EffectEffect TypeMolecular MechanismsTested Cell Line 1, AnimalMethodRef.
Ganoderma lucidum
Ganoderma lucidumInhibition of tumour cell adhesionPAteration in β1-integrin expressionMT-1Coomassie blue staining,
Western blot
Wu et al. 2006 [47]
Biomedicines 09 01781 i052
Pichia pastorisInhibition of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet by ARGDWN mutantsBPrevention of integrin αIIbβ3 interactionCHO
Mass spectrometry,
Fibrinogen binding assay,
Flow cytometry,
Platelet aggregation assay,
Nuclear magnetic resonance spectroscopy,
Molecular docking
Chang et al. 2017 [56]
Cyclopeptolide HUN-7293
Biomedicines 09 01781 i053
Bartalinia robillardoidesInhibition of VCAM-1 and ICAM-1PInhibition of the expression of VCAM-1 and ICAM-1HMEC-1ELISASchreiner et al. 2004 [242]
Biomedicines 09 01781 i054
Mycotypha sp. UMF-006 (soil)Inhibition of cell aggregation and adhesionNo dataNo dataHL-60
Cell aggregation assay,
Adhesion assay (CFSE-labelling
Takamatsu et al.n 2002 [243]
Biomedicines 09 01781 i055
Cordiceps militarisInhibition of motility, invasion and migrationPInhibition of PI3K/Akt pathway and expression of claudin family proteins. Downregulation of MMP activityLNCaPWound healing migration assay,
MTT assay,
Boyden chamber,
Matrigel invasion assay,
Measurement of TER (EVOM voltohmmeter)
Jeong et al. 2012 [244]
Fusarisetin A
Biomedicines 09 01781 i056
Fusarium sp. FN080326Inhibition of migrationPPathway inhibitionMDA-MB-231Scratch wound assay,
Boyden chamber transwell assay
Xu et al. 2012 [245]
Biomedicines 09 01781 i057
Aspergillus terreus CRI301Inhibition of adhesion and migrationPDownregulation of MMP-2 and MMP-9 transcription. Reduction of protein levels for the Rho GTPasesMDA-MB-231
Cell adhesion assay (crystal violet staining),
Wound healing assay,
Matrigel cel migration assay
Kasorn et al. 2018 [246]
Ophiobolin A
Biomedicines 09 01781 i058
Drechslera giganteaDecreased the 2D-migration potentialNo dataNo dataU373 MG
Glioblastoma multiforme (GBM)
MTT assay,
Quantitative video microscopy,
Immunofluorescence/pseudo-confocal microscopy, Flow cytometry
Bury et al. 2013 [247]
Fusicoccin A
Biomedicines 09 01781 i059
(2S)-2-((1S,4R,5S,6S,9S,10aR,E)-4-(((2S,3R,4S,5R,6R)-4-acetoxy-3,5-dihydroxy-6-(((2-methylbut-3-en-2-yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-6-formyl-1,5-dihydroxy-9-(methoxymethyl)-6,10a-dimethyl-1,2,4,5,6,6a,7,8,9,10a-decahydrodicyclopenta[a,d][8]annulen-3-yl)propyl acetate
Fusicoccum amygdaliDecreased the 2D and 3D-migration potential.
Decreased adhesion
No dataNo dataU373-MG
Glioblastoma multiforme (GBM)
Quantitative video microscopy,
Boyden chamber assay,
In vitro adhesion assay (hematoxylin staining)
Bury et al. 2013 [248]
Altersolanol A
Biomedicines 09 01781 i060
Stemphylium globuliferumInhibition of migrationPInhibition of TNFα-activated NF-κB signalling pathway. Inhibition of NF-κB-mediated MMP expressionA549Incucyte Live-Cell imaging system,
Videomicroscopy analysis,
Scratch wound assay, flow cytometry, Western blot
Teiten et al. 2013 [249]
Sphaeropsidin A
Biomedicines 09 01781 i061
Smardea sp. AZ0432Inhibition of migrationNo dataNo dataMDA-MB-231Wound healing assayWang et al. 2011 [250]
Biomedicines 09 01781 i062
Unidentified fungal strains Y90086/Y80805Inhibition of migrationNo dataNo dataHUVECWound migration assayLee et al. 2001 [251]
Biomedicines 09 01781 i063
Cytochalasin D
Biomedicines 09 01781 i064
(3S,3aR,4S,6S,6aR,7E,10S,12R,13E,15R,15aS)-3-benzyl-6,12-dihydroxy-4,10,12-trimethyl-5-methylene-1,11-dioxo-2,3,3a,4,5,6,6a,9,10,11,12,15-dodecahydro-1H-cycloundeca[d]isoindol-15-yl acetate
Not mentioned (but many fungi produce, e.g., Zygosporium mansonii)Inhibition of actin polymerization, decreased cell motility and colonization. Inhibition of actin polymerizationNo dataNo dataMFC-7
Quantitative videomicroscopy,
Scratch wound assay
Hayot et al. 2006 [252]
FTY720 (fingolimod)
Biomedicines 09 01781 i065
Isaria sinclairiiInhibition of cell migration, motility and invasionPInhibition of RhoA-GTPase expressionDU145
Wound closure assay,
3D collagen colony forming assay
Zhou et al. 2006 [253]
Biomedicines 09 01781 i066
* (Fluvastatin (syn))
Biomedicines 09 01781 i067
(1S,3R,7S,8S,8aR)-8-(2-((2S,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (S)-2-methylbutanoate
Pleurotus ostreatus **Inhibition of EGF-induced migration and invasivenessPInhibition of translocation of RhoAPANC-1Matrigel invasion assayKusama et al. 2001
(3S,5R,E)-7-(3-(4-fluorophenyl)-1-isopropyl-1H-indol-2-yl)-3,5-dihydroxyhept-6-enoic acid
Inhibition of tumor cell attachement and migrationNo dataNo dataF3IIAdhesion assay,
Migration assay (in vitro wound assay)
Alonso et al., 1998 [255]
Suppressed the EGF-induced cell adhesion, actin filament reorganization and transmigrationPSuppression of Rho/ROCK and FAK/paxillin signalingAROMatrigel invasion assay,
Cell adhesion assay
Zhong et al., 2005 [256]
Inhibition of adhesion. Reduced tumor cell migration, attachement and motility. Changed the tumor cell shapePReduced membrane localization of Rho proteinF3IIAdhesion assay,
Migration assay (in vitro wound assay)
Farina et al., 2002 [257]
Reduced migration and invasionPDiminished ERK signaling. Impaired the regulation of the mevalonate- and the Ras-Raf-MEK-ERK pathway. Affected the post-translational modification of H-Ras and Rac1U87
Migration assay,
Matrigel invasion assay
Afshordel et al., 2014
Biomedicines 09 01781 i068
(1S,7S,8S,8aR)-8-(2-((2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (S)-2-methylbutanoate
Pleurotus ostreatus **Inhibition of invasion and migrationPAlteration in expression of matrix-metalloproteasesM14Matrigel invasion assay,
Integrin-mediated binding assays
Glynn et al., 2008 [259]
Biomedicines 09 01781 i069
(1S,7S,8S,8aR)-8-(2-((2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (S)-2-methylbutanoate
Penicillium citrinum **HT144
Simvastain (syn)
Biomedicines 09 01781 i070
(1S,3R,7S,8S,8aR)-8-(2-((2S,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate
Aspergillus terreusSK-MEL-28
Macrosphelides (MSs)
Biomedicines 09 01781 i071
Microsphaeropsis sp. FO-5050 (soil)Inhibition of HL-60 cell adhesion to HUVECBBlocking the binding of SLex to ELAM-1HL-60Cell adhesion assayHayashi et al., 1995 [260]
Biomedicines 09 01781 i072
Biomedicines 09 01781 i073
Microsphaeropsis sp. FO-5050 (soil)Inhibition of adhesionNo dataNot discussedHL-60Cell adhesion assayTakamatsu et al., 1997 [261]
Biomedicines 09 01781 i074
Macrosphelide J
Biomedicines 09 01781 i075
Microsphaeropsis sp. FO-5050 (soil)No effectNo effectNo effectHL-60
Cell adhesion assayFukami et al., 1999 [262]
Biomedicines 09 01781 i076
Biomedicines 09 01781 i077
Macroshelide A
Biomedicines 09 01781 i078
and structurally related compounds of Macroshelide A (Macroshelide C, E, F, G, H, I, L and M)
Periconia byssoides OUPS-N133Inhibition of HL-60 cell adhesion to HUVECNo dataNo dataHL-60
Cell adhesion assay with MTT staining (“Miki’s method” [263])Yamada et al., 2001, 2002, 2007 [264,265,266]
Macroshelide B
Biomedicines 09 01781 i079
Microsphaeropsis sp. FO-5050 (soil)Inhibition of cell binding to HUVECB, PBlocking the binding of sLex to E-selectin.
Suppressed the expression of adhesion molecules.
Adhesion assay (CFSE-labelling),
flow cytometry analysis
Fukami et al., 2002
Peribysin A
Biomedicines 09 01781 i080
and structurally related compounds of Peribysin A (Peribysin B, C, D, E, F, G, H, I and J )
(1aR,2S,3aR,7S,7aR,7bS)-1a-(3-hydroxyprop-1-en-2-yl)-7,7a-dimethyldecahydronaphtho[1,2-b]oxiren-2-olPericonia byssoides OUPS-N133Inhibition of HL-60 cell adhesion to HUVECNo dataNo dataHL-60
Cell adhesion assay with MTT staining (“Miki’s method” [263])Yamada et al., 2004,
1 MT-1: human breast malignant carcinoma, CHO: Chinese hamster ovary cells, K562: human immortalized myelogenous leukemia cells, HMEC-1:human dermal microvascular endothelial cells, HL-60: human leukemia cells, LNCaP: androgen-sensitive human prostate adenocarcinoma, MDA-MB-231: human breast adenocarcinoma, MCF-7: human breast adenocarcinoma, U373-MG: human glioblastoma astrocytoma, A549: human lung epithelial cells, HUVEC: human umbilical vein endothelial cells, DU145:human prostate cancer cells, PC3: human prostate cancer cells, PANC-1: human pancreatic cancer cells, F3II: sarcomatoid mammary carcinoma, ARO: anaplastic thyroid cancer cells, U87: human primary glioblastoma cells, U343: glioblastoma cells, M14: human melanoma cells, HT144: human melanoma cells, SK-MEL-28:human melanoma cells, B16/BL6: mouse melanoma cells, L5178Y-ML: mouse lymphoma cells. * Fluvastatin is a synthetic derivative of lovastatin. Experimental results of fluvastatin are only related to the work of Kusama et al. [254] ** In general, the cited publications do not detail the source of statins. In this table, the typical natural sources of these compounds are given; Pleurotus ostreatus and Penicillium citrinum. In the case of lovastatin, Aspergillus terreus is a main source as well.

7. SARS-CoV-2 and Possible Treatments with Herbal Extracts

A novel coronavirus (nCoV) started infecting humans since late 2019. The pathogen is called “Severe Acute Respiratory Syndrome-related Coronavirus 2” (SARS-CoV-2). It can cause a fatal respiratory disease, called Coronavirus disease 2019 (COVID-19), and acute respiratory distress syndrome (ARDS) as well [271]. The COVID-19 pandemic spread quickly inducing a worldwide problem, because the virus is highly contagious; transmission occurs presumably mainly via airborne droplets [271]. SARS-CoV-2 belongs to the genus Betacoronavirus of the large family of Coronaviridae [271]. Externally, the virus displays a corona-shaped layer of spikes that play a significant role in the infection process [272]. The virions are able to recover from the drastic mechanical perturbation imposed by atomic force microscopy [272]. The global structure is temperature-resistant, but the virion surface is denuded of spikes if heated [272]. These properties obviously influence the infectivity of the virus. The spike protein is a large glycoprotein trimer, which contributes to host receptor binding, cell tropism and pathogenesis [271]. A recent study showed that the surface of the native virion displays a dynamic brush owing to the rapid motions and flexibility of the spikes [272]. By binding host receptors, the virus genome penetrates into the cytoplasm of the host cell [271]. The angiotensin-converting enzyme II (ACE2) is a receptor for SARS-CoV-2, but it is suggested that the virus may use integrins too as receptors, binding to them through the conserved RGD motif [50,51,273]) present on the spike protein [271]. Members of the integrin family are commonly used as receptors by many other human viruses as well, and RGD is the minimal sequence required for binding [271]. Integrins have high expression in lungs and other vital organs whereas ACE2 is found to have negligible occurrence in the lungs [274]. Expression of integrins is high in lung cells (especially αVβ6, αVβ8, α5β1) and the ICAM-1. Thus, the high infectivity of the virus may be at least partly due to the RGD-integrin-mediated cell-adhesive property [274]. Phosphorylation sites on the spike protein induce Tyr, PKC and cAMP signaling pathways, which activate calcium ion channels or get activated by calcium [274]. Thus the RGD-integrin interaction clearly occurs in a calcium-dependent manner [274]. This interaction may then unleash a “cytokine storm” due to TNFα and IL-6 activation [274]. The lowering of divalent ion concentrations in the lungs by pulmonary EDTA chelation therapy may inhibit virus-host attachment [274]. Glycans may have multiple roles during viral entry [275]. Inhibition of N-glycan biosynthesis was shown to enhance spike protein proteolysis, leading to a decrease of receptor-binding domain presentation on the virus [275]. Thus, another idea for treatment is to administer chemical inhibitors of glycosylation [275]. Hence, although vaccines have already been developed and authorized for emergency use, extensive research work and brainstorming continue for the development of future drugs against SARS-CoV-2 (Figure 9).
Already-existing synthetic drugs (favipiravir, ivermectin, remdesivir, among others) have been shown to be effective for treatment, but the application of these drugs may have direct or indirect side effects (for example, pain, liver problems, allergy, etc.) [276]. Plants are an abundant source of natural antiviral compounds that may be an effective option, because most of them are safer compared to synthetic drugs, although there are exceptions [276]. Some of these metabolites have protective effects against different microbes; however, the role of most metabolites still remains unknown for us. Bhuiyan et al. created a large collection of antiviral compounds from 219 medicinal plants [276], from which, it can be inferred that polyphenols work against coronaviruses by actuating or inhibiting cellular signaling pathways or inhibiting 3-chymotripsin-like protease (3CLpro) and papain-like protease (PLpro) [276]. Polyphenol compounds from Broussonetia papyrifera, Sambucus and Pelargonium, and flavonoid-type compounds (quercetin, apigenin) showed activity against human coronavirus [276,277]. Different types of alkaloids (for example tylophorine, 7-methoxy cryptopleurine, etc.) have anti-SARS activity by inhibiting protease and RNA and protein synthesis, and chloroquine has been reported to have anti-SARS-CoV-2 activity [276]. Saponins (amphipatic triterpenes) showed antiviral effects against a lot of viruses (Influenza virus, Dengue virus, rotaviruses, among others) [276,278]. Among triterpenes, ginkgolide A can strongly inhibit the SARS-CoV-2 protease [276], and glycyrrhizin was found to be effective against influenza [278]. In the case of SARS-CoV-2, glycyrrhizin was found to have the highest binding affinity with the viral S protein [276,279], but curcumin, apigenin and chrisophanol also bind to this part of the virus according to in silico molecular docking [279]. In a recent study, EGCG from green tea beverage was shown to inhibit infection of live virus and its variants by inhibiting spike binding to ACE2 receptor [280].
It should be noted that the risk factors for a severe course of COVID-19 in intensive care unit patients are chronic obstructive pulmonary disease, renal dysfunction, hypertension, diabetes mellitus and coronary heart disease. Elderly adults and patients with chronic illnesses and obesity are vulnerable [278]. There is preliminary evidence that nutrient-related disorders are associated with greater susceptibility to infection [278].
Herbal remedies have a potentially preventive effect, mainly acting through supporting the immune system, for example Astragalus membranaceus or Echinacea purpurea [277]. Clinical studies showed that extracts from Pelargonium sidoides, Sambucus nigra and Cistus incanus are effective treatments of infectious respiratory illnesses [277,278]. Kalus et al., a decade before the COVID-19 pandemic, showed that Cistus incanus extract significantly decreased the symptoms of 160 patients with infections of the upper respiratory tract (caused by bacteria, influenza, and other viruses) and the level of C-reactive protein inflammatory marker was also decreased [281]. EGCG green tea polyphenol was shown to bind to the hemagglutinin of influenza virus [281] as well, implying that regular consumption of green tea should decrease the influenza infection rate, too [278]. In summary, consuming extracts of herbs, vegetables and fruits improve overall health due to their phytochemicals and nutrients, and these compounds may prevent or attenuate the symptoms of COVID-19.

8. Conclusions

Most investigations into the effect of natural products on cellular adhesion are focused on the adhesion molecules belonging to the immunoglobulin superfamily of CAMs (ICAM-1 and VCAM-1), and selectins and integrins [5]. These molecules are implicated in several widespread diseases, such as various types of inflammation, rheumatism, atherosclerosis and cancer. Metabolites obtained from plants, fungi and venoms may offer therapeutic potential by regulating adhesion molecules, generally down-regulation [5]. We summarized natural metabolites having diverse structures, which influence cellular adhesion and migration by modulation of adhesion molecules. The reported effects of natural products, either presented to model cell systems as complex extracts or as purified active principles, generally manifest themselves as: (i) inhibition of cell-cell adhesion (i.e., circulating to endothelial cells); and (ii) a general anti-inflammatory effect. The mechanism of (i) is either the blocking of specific adhesion sites (such as the RGD motif and/or its binding complement) on the cell surface (the group of mechanisms labeled B—blocking adhesion receptors on the surface of immune or tissue cells, or motifs in the ECM), or the downregulation of the cell adhesion molecules (the group of mechanisms labeled P—inhibiting the dephosphorylation, translocation or binding of certain factors). The latter group is often effected by inhibition of signaling pathways involving NF-κB. The mechanism of (ii) is by suppressing ROS, achieved by the sacrificial oxidation of the natural product. To map the properties and effects of natural compounds, “classical” labeling techniques have been applied to monitor cellular adhesion and movement. New label-free methods can provide more information (especially kinetic information) about the effects of extracts more sensitively and conveniently without using any cell physiology-altering dyes or other labels. In this review, the preparation method of natural compounds was summarized and the natural product banks were mentioned as well. We systematized recent studies about natural compounds referring to their effects on cell adhesion and movement; the active substances were categorized based on their origin (floral, faunal or fungal). From these collected results it can be clearly seen that applying natural compounds can be a cure against tumors and inflammatory diseases, but more clinical tests would be desirable. Synthetically modified versions of the substances can be also used to cure illness. Finding new, still unknown natural compounds and to map the exact effects of already known extracts from traditional medicine are both important to create new, better, and more effective therapeutic drugs. Some plant extracts showed antiviral activity on SARS-CoV-2 as well, thus natural compounds may be used to attenuate or even prevent the symptoms of COVID-19 as well.
Our review aimed at collecting evidence in order to answer a specific research question [282], namely the role of natural compounds in cellular adhesion and migration, including the characterization methods of these bioactive compounds. We have elucidated the main points of interest (definition of natural compounds and their isolation, adhesion and migration process, effects on cell viability, invasive and non-invasive assays, label free techniques) with inclusion and exclusion criteria. We have made a careful and systematic search of the literature, using the following keywords: natural compound of plant, fungi and other origin, cell adhesion, migration, motility, movement, CAM, integrin, cancer cell, stimulation, inflammation, viability, cytotoxicity, flow cytometry, dyes, label-free, biosensors, preparation, isolation, intracellular pathogens SARS-CoV-2. Approximately 280 relevant paper wereselected. Very recent (2021) and old references (articles from the 1970s and even from 1962) were evaluated. Our criteria were that the compound must be a natural one (i.e., natural origin), with effect on cell adhesion and/or migration.

Author Contributions

Conceptualization: B.P., S.B., G.M.K., I.B. and R.H. Preparation of tables: B.P., I.S. Literature searching and writing: all authors. Visualization and figures: B.P., S.B. and I.S. All authors have read and agreed to the published version of the manuscript.


This research was funded by the Hungarian Academy of Sciences [Lendület (Momentum) Program], the National Research, Development and Innovation Office (NKFIH) [ERC_HU, PD 131543 and KKP_19 Programs] and grant OTKA (number 135712). S.B., I.B. and G.M.K. acknowledge the support of the ELTE Thematic Excellence Programme supported by the Hungarian Ministry for Innovation and Technology and ELTE Thematic Excellence Programme 2020 supported by the National Research, Development and Innovation Office (TKP2020-IKA-05). I.S., S.B, I.B. and G.M.K. thank for the support of grant EFOP-1.8.0-VEKOP-17-2017-00001.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All relevant data are available in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Peter, B.; Bosze, S.; Horvath, R. Biophysical characteristics of living cells exposed to the green tea polyphenol epigallocatecin-3-gallate (EGCg): Review of recent advances from molecular mechanisms to clinical trials. Eur. Biophys. J. 2016, 46, 1–35. [Google Scholar] [CrossRef]
  2. Ling, S.; Nheu, L.; Dai, A.; Guo, Z.; Komesaroff, P. Effects of four medicinal herbs on human vascular endothelial cells in culture. Int. J. Cardiol. 2008, 128, 350–358. [Google Scholar] [CrossRef] [PubMed]
  3. Kinghorn, A.D.; De Blanco, E.J.C.; Chai, H.; Orjala, J.; Farnsworth, N.R.; Soejarto, D.D.; Oberlies, N.H.; Wani, M.C.; Kroll, D.J.; Pearce, C.J.; et al. Discovery of anticancer agents of diverse natural origin. Pure Appl. Chem. 2009, 81, 1051–1063. [Google Scholar] [CrossRef] [Green Version]
  4. Gumbiner, B.M. Cell adhesion: The molecular basis of tissue architecture and morphogenesis. Cell 1996, 84, 345–357. [Google Scholar] [CrossRef] [Green Version]
  5. Spelman, K.; Aldag, R.; Hamman, A.; Kwasnik, E.M.; Mahendra, M.A.; Obasi, T.M.; Morse, J.; Williams, E.J. Traditional herbal remedies that influence cell adhesion molecule activity. Phytother. Res. 2011, 25, 473–483. [Google Scholar] [CrossRef]
  6. Goldstein, I.M.; Malmsten, C.L.; Samuelsson, B.; Weissmann, G. Prostaglandins, thromboxanes, and polymorphonuclear leukocytes. Inflammation 1977, 2, 309–317. [Google Scholar] [CrossRef] [PubMed]
  7. Chang, D.-M.; Kuo, S.-Y.; Lai, J.-H.; Chang, M.-L. Effects of anti-rheumatic herbal medicines on cellular adhesion molecules. Ann. Rheum. Dis. 1999, 58, 366–371. [Google Scholar] [CrossRef] [PubMed]
  8. Bevilacqua, M.P.; Pober, J.S.; Wheeler, M.E.; Cotran, R.S.; Gimbrone, M.A. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J. Clin. Investig. 1985, 76, 2003–2011. [Google Scholar] [CrossRef]
  9. Cronstein, B.N.; Weissmann, G. The adhesion molecules of inflammation. Arthritis Rheum. 1993, 36, 147–157. [Google Scholar] [CrossRef]
  10. Hynes, R.O. Integrins: Bidirectional, Allosteric Signaling Machines. Cell 2002, 110, 673–687. [Google Scholar] [CrossRef] [Green Version]
  11. Bevilacqua, M.P. Endothelial-leukocyte cellular adhesion molecules. Annu. Rev. Immunol. 1993, 11, 767–804. [Google Scholar] [CrossRef]
  12. Grober, J.S.; Bowen, B.L.; Ebling, H.; Athey, B.; Thompson, C.B.; Fox, D.A.; Stoolman, L.M. Monocyte-endothelial adhesion in chronic rheumatoid arthritis: In situ detection of selectin and integrin-dependent interactions. J. Clin. Investig. 1993, 91, 2609–2619. [Google Scholar] [CrossRef]
  13. Kawasaki, K.; Muroyama, K.; Yamamoto, N.; Murosaki, S. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-alpha-stimulated human endothelial cells. Biosci. Biotechnol. Biochem. 2015, 79, 1654–1659. [Google Scholar] [CrossRef]
  14. Ramadori, G.; Moriconi, F.; Malik, I.; Dudas, J. Physiology and pathophysiology of liver inflammation, damage and repair. J. Physiol. Pharmacol. 2008, 59, 107–117. [Google Scholar]
  15. Parsons, J.T.; Horwitz, A.R.; Schwartz, M.A. Cell adhesion: Integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 2010, 11, 633–643. [Google Scholar] [CrossRef]
  16. Mezu-Ndubuisi, O.J.; Maheshwari, A. The role of integrins in inflammation and angiogenesis. Pediatr. Res. 2020, 89, 1619–1626. [Google Scholar] [CrossRef]
  17. Yue, J.; Zhang, K.; Chen, J.F. Role of integrins in regulating proteases to mediate extracellular matrix remodeling. Cancer Microenviron. 2012, 5, 275–283. [Google Scholar] [CrossRef] [Green Version]
  18. Papoutsi, Z.; Kassi, E.; Chinou, I.; Halabalaki, M.; Skaltsounis, L.A.; Moutsatsou, P. Walnut extract (Juglans regia L.) and its component ellagic acid exhibit anti-inflammatory activity in human aorta endothelial cells and osteoblastic activity in the cell line KS483. Br. J. Nutr. 2008, 99, 715–722. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Tang, P.; Hung, M.C.; Klostergaard, J. Human pro-tumor necrosis factor is a homotrimer. Biochemistry 1996, 35, 8216–8225. [Google Scholar] [CrossRef]
  20. Spelman, K.; Burns, J.J.; Nichols, D.; Winters, N.; Ottersberg, S.; Tenborg, M. Modulation of Cytokine Expression by Traditional Medicines: A Review of Herbal Immunomodulators. Altern. Med. Rev. 2006, 11, 128–150. [Google Scholar]
  21. Carluccio, M.A.; Siculella, L.; Ancora, M.A.; Massaro, M.; Scoditti, E.; Storelli, C.; Visioli, F.; Distante, A.; De Caterina, R. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: Antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler. Thromb. Vasc. Biol. 2003, 23, 622–629. [Google Scholar] [CrossRef] [Green Version]
  22. Zhang, W.-J.; Hufnag, P.; Binder, B.R.; Wojta, J. Antiinflammatory activity of astragaloside IV is mediated by inhibition of NF-κB activation and adhesion molecule expression. Thromb. Haemost. 2003, 90, 904–914. [Google Scholar] [CrossRef]
  23. Tao, X.; Lipsky, P.E. The Chinese anti-inflammatory and immunosuppressive herbal remedy Tripterygium Wilfordii Hook F. Rheum. Dis. Clin. N. Am. 2000, 26, 29–50. [Google Scholar] [CrossRef]
  24. Iademarco, M.F.; McQuillan, J.J.; Rosen, G.D.; Dean, D.C. Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1). J. Biol. Chem. 1992, 267, 16323–16329. [Google Scholar] [CrossRef]
  25. Schindler, U.; Baichwal, V.R. Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol. Cell. Biol. 1994, 14, 5820–5831. [Google Scholar] [CrossRef] [Green Version]
  26. Collins, T.; Read, M.A.; Neish, A.S.; Whitley, M.Z.; Thanos, D.; Maniatis, T. Transcriptional regulation of endothelial cell adhe- sion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J. 1995, 10, 899–909. [Google Scholar] [CrossRef] [Green Version]
  27. Voraberger, G.; Schäfer, R.; Stratowa, C. Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5′- regulatory region. Induction by cytokines and phorbol ester. J. Immunol. 1991, 147, 2777–2786. [Google Scholar]
  28. Tak, P.P.; Firestein, G.S. NF-κB: A key role in inflammatory diseases. J. Clin. Investig. 2001, 107, 7–11. [Google Scholar] [CrossRef] [PubMed]
  29. Baldwin, A.S., Jr. The NF-κB and I kappa B proteins: New discoveries and insights. Annu. Rev. Immunol. 1996, 14, 649–683. [Google Scholar] [CrossRef] [Green Version]
  30. Kurucz, I.; Peter, B.; Prosz, A.; Szekacs, I.; Erdei, A.; Horvath, R. Label-free optical biosensor for on-line monitoring the integrated response of human B cells upon the engagement of stimulatory and inhibitory immune receptors. Sens. Actuators B 2017, 240, 528–535. [Google Scholar] [CrossRef]
  31. Erdei, A.; Sándor, N.; Szilvia, B.M.; Kremlitzka, M.; Bajtay, Z. The versatile functions of complement C3-derived ligands. Immunol. Rev. 2016, 274, 127–140. [Google Scholar] [CrossRef] [Green Version]
  32. Gorfu, G.; Rivera-Nieves, J.; Ley, K. Role of β7 integrins in intestinal lymphocyte homing and retention. Curr. Mol. Med. 2009, 9, 836–850. [Google Scholar] [CrossRef]
  33. Caswell, P.T.; Vadrevu, S.; Norman, J.C. Integrins: Masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 2009, 10, 843–853. [Google Scholar] [CrossRef]
  34. Liu, X.; Wang, W.; Song, G.; Wei, X.; Zeng, Y.; Han, P.; Wang, D.; Shao, M.; Wu, J.; Sun, H.; et al. Astragaloside IV ameliorates diabetic nephropathy by modulating the mitochondrial quality control network. PLoS ONE 2017, 12, e0182558. [Google Scholar] [CrossRef] [Green Version]
  35. Date, R. Research review on the pharmacological effects of Astragaloside IV. Fundam. Clin. Pharmacol. 2017, 31, 17–36. [Google Scholar] [CrossRef]
  36. Gui, D.; Huang, J.; Guo, Y.; Chen, J.; Chen, Y.; Xiao, W.; Liu, X.; Wang, N. Cytokine Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF- j B-mediated inflammatory genes expression. Cytokine 2013, 61, 970–977. [Google Scholar] [CrossRef]
  37. Wang, Z.S.; Xiong, F.; Xie, X.H.; Chen, D.; Pan, J.H.; Cheng, L. Astragaloside IV attenuates proteinuria in streptozotocin-induced diabetic nephropathy via the inhibition of endoplasmic reticulum stress. Nephrology 2015, 16, 44. [Google Scholar] [CrossRef] [Green Version]
  38. Chen, J.; Chen, Y.; Luo, Y.; Gui, D.; Huang, J.; He, D. Astragaloside IV ameliorates diabetic nephropathy involving protection of podocytes in streptozotocin induced diabetic rats. Eur. J. Pharmacol. 2014, 736, 86–94. [Google Scholar] [CrossRef]
  39. Cheng, S.; Wu, Y.; Huang, W.; Pang, J.S. Cytokine Anti-in fl ammatory property of quercetin through downregulation of ICAM-1 and MMP-9 in TNF- α -activated retinal pigment epithelial cells. Cytokine 2019, 116, 48–60. [Google Scholar] [CrossRef]
  40. Lee, J.; Zhou, H.Y.; Cho, S.Y.; Kim, Y.S.; Lee, Y.S.; Jeong, C.S. Anti-inflammatory Mechanisms of Apigenin: Inhibition of Cyclooxygenase-2 Expression, Adhesion of Monocytes to Human Umbilical Vein Endothelial Cells, and Expression of Cellular Adhesion Molecules. Arch. Pharm. Res. 2007, 30, 1318–1327. [Google Scholar] [CrossRef] [PubMed]
  41. Journal, A.I.; Song, Y.; Tian, X.; Wang, X.; Feng, H. Vascular protection of salicin on IL-1 β -induced endothelial inflammatory response and damages in retinal endothelial cells. Artif. Cells Nanomed. Biotechnol. 2019, 47, 1995–2002. [Google Scholar] [CrossRef] [Green Version]
  42. Rho, M.; Kwon, O.E.; Kim, K.; Lee, S.W.; Chung, M.Y.; Kim, Y.H.; Hayashi, M.; Lee, H.S.; Kim, Y. Inhibitory Effects of Manassantin A and B Isolated from the Roots of Saururus chinensis on PMA-Induced ICAM-1 Expression. Planta Med. 2003, 69, 1147–1149. [Google Scholar]
  43. Choi, Y.; Jin, H.; Park, S.; Chung, J.; Lee, H.; Oh, S.; Kim, B.; Kim, J.; Chung, H.; Yu, B.; et al. Inhibition of endothelial cell adhesion by the new anti-inflammatory agent α -iso-cubebene. Vasc. Pharmacol. 2009, 51, 215–224. [Google Scholar] [CrossRef]
  44. Jiang, C.; Li, J.; Liu, F.; Wu, T.A.O.; Yu, M.; Xu, H. Andrographolide Inhibits the Adhesion of Gastric Cancer Cells to Endothelial Cells by Blocking E-selectin Expression. Anticancer Res. 2007, 2448, 2439–2447. [Google Scholar]
  45. Wu, S.; Xu, H.; Peng, J.; Wang, C.; Jin, Y.; Liu, K. Biochimie Potent anti-inflammatory effect of dioscin mediated by suppression of TNF- a -induced VCAM-1, ICAM-1and EL expression via the NF- k B pathway. Biochimie 2015, 110, 62–72. [Google Scholar] [CrossRef]
  46. Kumar, S.; Arya, P.; Mukherjee, C.; Singh, B.K.; Singh, N.; Parmar, V.S.; Prasad, A.K.; Ghosh, B. Novel aromatic ester from Piper longum and its analogues inhibit expression of cell adhesion molecules on endothelial cells. Biochemistry 2005, 44, 15944–15952. [Google Scholar] [CrossRef]
  47. Wu, Q.P.; Xie, Y.Z.; Li, S.Z.; La Pierre, D.P.; Deng, Z.; Chen, Q.; Li, C.; Zhang, Z.; Guo, J.; Wong, C.K.A.; et al. Tumour cell adhesion and integrin expression affected by Ganoderma lucidum. Enzyme Microb. Technol. 2006, 40, 32–41. [Google Scholar] [CrossRef]
  48. Wang, L.; Ling, Y.; Chen, Y.; Li, C.L.; Feng, F.; You, Q.D.; Lu, N.; Guo, Q.L. Flavonoid baicalein suppresses adhesion, migration and invasion of MDA-MB-231 human breast cancer cells. Cancer Lett. 2010, 297, 42–48. [Google Scholar] [CrossRef]
  49. Jang, J.H.; Yang, E.S.; Min, K.-J.; Kwon, T.K. Inhibitory effect of butein on tumor necrosis factor-α-induced expression of cell adhesion molecules in human lung epithelial cells via inhibition of reactive oxygen species generation, NF-κB activation and Akt phosphorylation. Int. J. Mol. Med. 2012, 30, 1357–1364. [Google Scholar] [CrossRef] [Green Version]
  50. Peter, B.; Farkas, E.; Forgacs, E.; Saftics, A.; Kovacs, B.; Kurunczi, S.; Szekacs, I.; Csampai, A.; Bosze, S.; Horvath, R. Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: Multicomponent models monitored optically. Nat. Publ. Gr. 2017, 7, 42220. [Google Scholar] [CrossRef] [Green Version]
  51. Szekacs, I.; Orgovan, N.; Peter, B.; Kovacs, B.; Horvath, R. Receptor specific adhesion assay for the quantification of integrin–ligand interactions in intact cells using a microplate based, label-free optical biosensor. Sens. Actuators B Chem. 2018, 256, 729–734. [Google Scholar] [CrossRef]
  52. Cox, D.; Brennan, M.; Moran, N. Integrins as therapeutic targets: Lessons and opportunities. Nat. Rev. Drug Discov. 2010, 9, 804–820. [Google Scholar] [CrossRef] [PubMed]
  53. Ley, K.; Rivera-Nieves, J.; Sandborn, W.J.; Shattil, S. Integrin-based therapeutics: Biological basis, clinical use and new drugs. Nat. Rev. Drug Discov. 2016, 15, 173–183. [Google Scholar] [CrossRef] [Green Version]
  54. McLane, M.A.; Sanchez, E.E.; Wong, A.; Paquette-Straub, C.; Perez, J.C. Disintegrins. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2004, 4, 327–355. [Google Scholar] [CrossRef] [PubMed]
  55. Wu, W.-B.; Peng, H.-C.; Huang, T.-F. Disintegrin causes proteolysis of β-catenin and apoptosis of endothelial cells: Involvement of cell—cell and cell—ECM interactions in regulating cell viability. Exp. Cell Res. 2003, 286, 115–127. [Google Scholar] [CrossRef]
  56. Chang, Y.; Shiu, J.; Huang, C.; Chen, Y.; Chen, C.; Chang, Y.; Chuang, W. Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin α IIb β 3 recognition. PLoS ONE 2017, 12, e0175321. [Google Scholar] [CrossRef] [Green Version]
  57. McColl, J.; Horvath, R.; Yakubov, G.E.; Ramsden, J.J. Surface rearrangement of adsorbed EGCG–mucin complexes on hydrophilic surfaces. Int. J. Biol. Macromol. 2017, 95, 704–712. [Google Scholar] [CrossRef] [Green Version]
  58. Habtemariam, S. Cistifolin, an Integrin-Dependent Cell Adhesion Blocker from the Anti- Rheumatic Herbal Drug, Gravel Root (Rhizome of Eupatorium purpureum). Planta Med. 1998, 64, 683–685. [Google Scholar] [CrossRef]
  59. Peter, B.; Ungai-Salanki, R.; Szabó, B.; Nagy, A.G.; Szekacs, I.; Bösze, S.; Horvath, R. High-Resolution Adhesion Kinetics of EGCG-Exposed Tumor Cells on Biomimetic Interfaces: Comparative Monitoring of Cell Viability Using Label-Free Biosensor and Classic End-Point Assays. ACS Omega 2018, 3, 3882–3891. [Google Scholar] [CrossRef]
  60. Cheung, K.C.; Di Berardino, M.; Schade-Kampmann, G.; Hebeisen, M.; Pierzchalski, A.; Bocsi, J.; Mittag, A.; Tárnok, A. Microfluidic impedance-based flow cytometry. Cytom. Part A 2010, 77, 648–666. [Google Scholar] [CrossRef]
  61. Wang, P.; Henning, S.M.; Heber, D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS ONE 2010, 5, e10202. [Google Scholar] [CrossRef] [PubMed]
  62. Niles, A.L.; Moravec, R.A.; Riss, T.L. In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening. Curr. Chem. Genom. 2009, 3, 33–41. [Google Scholar] [CrossRef]
  63. Mahto, S.K.; Chandra, P.; Rhee, S.W. In vitro models, endpoints and assessment methods for the measurement of cytotoxicity. Toxicol. Environ. Health Sci. 2010, 2, 87–93. [Google Scholar] [CrossRef]
  64. Menyhárt, O.; Harami-Papp, H.; Sukumar, S.; Schäfer, R.; Magnani, L.; de Barrios, O.; Győrffy, B. Guidelines for the selection of functional assays to evaluate the hallmarks of cancer. Biochim. Biophys. Acta Rev. Cancer 2016, 1866, 300–319. [Google Scholar] [CrossRef] [Green Version]
  65. Markossian, S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C.P.; Baell, J.; Chung, T.D.Y.; Coussens, N.P.; Dahlin, J.L.; et al. (Eds.) Assay Guidance Manual; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, MD, USA, 2004; p. 540958. Available online: (accessed on 23 November 2021).
  66. Feoktistova, M.; Geserick, P.; Leverkus, M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc. 2016, 2016, 343–346. [Google Scholar] [CrossRef]
  67. Lee, Y.; Chen, M.; Lee, J.D.; Zhang, J.; Lin, S.; Fu, M.; Chen, H.; Ishikawa, T.; Chiang, S.; Katon, J.; et al. HHS Public Access through inhibition of a MYC-WWP1 inhibitory pathway. Science 2020, 364, 1–40. [Google Scholar] [CrossRef]
  68. Vasan, N.; Razavi, P.; Johnson, J.L.; Shao, H.; Shah, H.; Antoine, A.; Ladewig, E.; Gorelick, A.; Lin, T.Y.; Toska, E.; et al. Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Ka inhibitors. Science 2019, 366, 714–723. [Google Scholar] [CrossRef] [PubMed]
  69. Johnston, G. Automated handheld instrument improves counting precision across multiple cell lines. Biotechniques 2010, 48, 325–327. [Google Scholar] [CrossRef]
  70. Strober, W. Trypan Blue Exclusion Test of Cell Viability. Curr. Protoc. Immunol. 2001, 21, A-3B. [Google Scholar] [CrossRef]
  71. Aslantürk, S.; Çelik, T.A. Antioxidant activity and anticancer effect of Vitex agnus-castus L. (Verbenaceae) seed extracts on MCF–7 breast cancer cells. Caryologia 2013, 66, 257–267. [Google Scholar] [CrossRef] [Green Version]
  72. Stone, V.; Johnston, H.; Schins, R.P.F. Development ofin vitrosystems for nanotoxicology: Methodological considerations. Crit. Rev. Toxicol. 2009, 39, 613–626. [Google Scholar] [CrossRef]
  73. Yip, D.K.; Auersperg, N. The dye-exclusion test for cell viability: Persistence of differential staining following fixation. In Vitro J. Tissue Cult. Assoc. 1972, 7, 323–329. [Google Scholar] [CrossRef]
  74. Ruben, R.L. Cell culture for testing anticancer compounds. In Advances in Cell Culture; Marqmarosh, K., Sato, G.H., Eds.; Academic Press: San Diego, CA, USA, 1988; Volume 6, pp. 161–188. [Google Scholar]
  75. Kim, S.I.; Kim, H.J.; Lee, H.J.; Lee, K.; Hong, D.; Lim, H.; Cho, K.; Jung, N.; Yi, Y.W. Application of a non-hazardous vital dye for cell counting with automated cell counters. Anal. Biochem. 2016, 492, 8–12. [Google Scholar] [CrossRef] [Green Version]
  76. Marmion, D.M. Handbook of U.S. Colortants for Foods, Drugs, and Cosmetics; Wiley Interscience: New York, NY, USA, 1979. [Google Scholar]
  77. Aslantürk, Ö.; Çelik, T.; Karabey, B.; Karabey, F. Active Phytochemical Detecting, Antioxidant, Cytotoxic, Apoptotic Activities of Ethyl Acetate and Methanol Extracts of Galium aparine L. Br. J. Pharm. Res. 2017, 15, 1–16. [Google Scholar] [CrossRef]
  78. Bopp, S.K.; Lettieri, T. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol. 2008, 8, 8. [Google Scholar] [CrossRef] [PubMed]
  79. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  80. Berg, K.; Zhai, L.; Chen, M.; Kharazmi, A.; Owen, T.C. The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitol. Res. 1994, 80, 235–239. [Google Scholar] [CrossRef]
  81. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 1999, 36, 47–50. [Google Scholar] [CrossRef]
  82. Rotter, B.A.; Thompson, B.K.; Clarkin, S.; Owen, T.C. Rapid colorimetric bioassay for screening of fusarium mycotoxins. Nat. Toxins 1993, 1, 303–307. [Google Scholar] [CrossRef] [PubMed]
  83. Buttke, T.M.; McCubrey, J.A.; Owen, T.C. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. J. Immunol. Methods 1993, 157, 233–240. [Google Scholar] [CrossRef]
  84. Promega Corparation Technical Bulletin. CellTitre 96 AQueous Non-Radioactive Cell Proliferation Assay. 2006. Available online: (accessed on 23 November 2021).
  85. Cory, A.H.; Owen, T.C.; Barltrop, J.A.; Cory, J.G. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Comm. 1991, 3, 207–212. [Google Scholar] [CrossRef]
  86. Riss, T.L.; Moravec, R.A. Comparison of MTT, XTT, and a novel tetrazolium compound MTS for in vitro proliferation and chemosensitivity assays. Mol. Biol. Cell 1992, 3, A184. [Google Scholar]
  87. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.; Boyd, M.R. Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines. Cancer Res. 1988, 48, 4827–4833. [Google Scholar] [PubMed]
  88. Ishiyama, M.; Shiga, M.; Sasamoto, K.; Mizoguchi, M.; He, P. A new sulfonated tetrazolium salt that produces a high water-soluble formazan dye. Chem. Pharm. Bull. 1993, 41, 1118–1122. [Google Scholar] [CrossRef] [Green Version]
  89. Decker, T.; Lohmann-Matthes, M.L. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods 1988, 115, 61–69. [Google Scholar] [CrossRef]
  90. Schins, R.P.F.; Duffin, R.; Höhr, D.; Knaapen, A.M.; Shi, T.; Weishaupt, C.; Stone, V.; Donaldson, K.; Borm, P.J.A. Surface modification of quartz inhibits toxicity, particle uptake, and oxidative DNA damage in human lung epithelial cells. Chem. Res. Toxicol. 2002, 15, 1166–1173. [Google Scholar] [CrossRef]
  91. Fotakis, G.; Timbrell, J.A. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol. Lett. 2006, 160, 171–177. [Google Scholar] [CrossRef] [PubMed]
  92. Lappalanien, K.; Jaaskelainen, L.; Syrjanen, K.; Urtti, A.; Syrjanen, S. Comparison of cell proliferation and toxicity assays using two catronic liposomes. Pharm. Res. 1994, 11, 1127–1131. [Google Scholar] [CrossRef]
  93. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mcmahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82, 1107–1112. [Google Scholar] [CrossRef]
  94. Borenfreund, E.; Puerner, J.A. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Methods 1985, 9, 7–9. [Google Scholar] [CrossRef]
  95. Repetto, G.; del Peso, A.; Zurita, J.L. Neutral red uptake assay for the estimation of cell viability/ cytotoxicity. Nat. Protoc. 2008, 3, 1125–1131. [Google Scholar] [CrossRef]
  96. Ringwood, A.H.; Conners, D.E.; Hoguet, J. Effects of natural and anthropogenic stressors on lysosomal destabilization in oysters Crassostrea virginica. Mar. Ecol. Prog. Ser. 1998, 166, 163–171. [Google Scholar] [CrossRef] [Green Version]
  97. Geserick, P.; Hupe, M.; Moulin, M.; Wong, W.W.L.; Feoktistova, M.; Kellert, B.; Gollnick, H.; Silke, J.; Leverkus, M. Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment. J. Cell Biol. 2009, 187, 1037–1054. [Google Scholar] [CrossRef] [Green Version]
  98. Degterev, A.; Hitomi, J.; Germscheid, M.; Ch’en, I.L.; Korkina, O.; Teng, X.; Abbott, D.; Cuny, G.D.; Yuan, C.; Wagner, G.; et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 2008, 4, 313–321. [Google Scholar] [CrossRef] [Green Version]
  99. Sun, L.; Wang, H.; Wang, Z.; He, S.; Chen, S.; Liao, D.; Wang, L.; Yan, J.; Liu, W.; Lei, X.; et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012, 148, 213–227. [Google Scholar] [CrossRef] [Green Version]
  100. Feoktistova, M.; Geserick, P.; Kellert, B.; Dimitrova, D.P.; Langlais, C.; Hupe, M.; Cain, K.; MacFarlane, M.; Häcker, G.; Leverkus, M. CIAPs Block Ripoptosome Formation, a RIP1/Caspase-8 Containing Intracellular Cell Death Complex Differentially Regulated by cFLIP Isoforms. Mol. Cell 2011, 43, 449–463. [Google Scholar] [CrossRef] [Green Version]
  101. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421–5426. [Google Scholar] [CrossRef]
  102. Ansar Ahmed, S.; Gogal, R.M.; Walsh, J.E. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorporation assay. J. Immunol. Methods 1994, 170, 211–224. [Google Scholar] [CrossRef]
  103. Page, B.; Page, M.; Noel, C. A new fluorometric assay for cytotoxicity measurements in vitro. Int. J. Oncol. 1993, 3, 473–476. [Google Scholar] [CrossRef]
  104. Pace, R.T.; Burg, K.J.L. Toxic effects of resazurin on cell cultures. Cytotechnology 2015, 67, 13–17. [Google Scholar] [CrossRef] [Green Version]
  105. Schreer, A.; Tinson, C.; Sherry, J.P.; Schirmer, K. Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout. Anal. Biochem. 2005, 344, 76–85. [Google Scholar] [CrossRef]
  106. Ganassi, R.C.; Schirmer, K.; Bols, N.C. Cell and tissue culture. In The Laboratory Fish; Ostrander, G.K., Ed.; Academic Press: San Diego, CA, USA, 2000; pp. 631–651. [Google Scholar]
  107. Sidman, R.L.; Miale, I.L.; Feder, N. Cell proliferation and migration in the primitive ependymal zone; An autoradiographic study of histogenesis in the nervous system. Exp. Neurol. 1959, 1, 322–333. [Google Scholar] [CrossRef]
  108. Miller, M.W.; Nowakowski, R.S. Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res. 1988, 457, 44–52. [Google Scholar] [CrossRef]
  109. Salic, A.; Mitchison, T.J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. USA 2008, 105, 2415–2420. [Google Scholar] [CrossRef] [Green Version]
  110. Nowakowski, R.S.; Lewin, S.B.; Miller, M.W. Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population. J. Neurocytol. 1989, 18, 311–318. [Google Scholar] [CrossRef]
  111. Taupin, P. BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation. Brain Res. Rev. 2007, 53, 198–214. [Google Scholar] [CrossRef]
  112. Duque, A.; Rakic, P. Different effects of BrdU and (3)H-thymidine incorporation into DNA on cell proliferation, position and fate. J. Neurosci. Off. J. Soc. Neurosci. 2001, 31, 15205–15217. [Google Scholar] [CrossRef]
  113. Maehara, Y.; Anai, H.; Tamada, R.; Sugimachi, K. The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer Clin. Oncol. 1987, 23, 273–276. [Google Scholar] [CrossRef]
  114. García, O.; Massieu, L. Glutamate Uptake Inhibitor L-Trans-Pyrrolidine 2,4-Dicarboxylate Becomes Neurotoxic in the Presence of Subthreshold Concentrations of Mitochondrial Toxin 3-Nitropropionate: Involvement of Mitochondrial Reducing Activity and ATP Production. J. Neurosci. Res. 2003, 74, 956–966. [Google Scholar] [CrossRef]
  115. Andreotti, P.E.; Hartmann, D.M.; Linder, D.; Harel, G.; Gleiberman, I.; Caruso, P.A.; Ricks, S.H.; Cree, I.A.; Kurbacher, C.M.; Untch, M.; et al. Chemosensitivity Testing of Human Tumors Using a Microplate Adenosine Triphosphate Luminescence Assay: Clinical Correlation for Cisplatin Resistance of Ovarian Carcinoma. Cancer Res. 1995, 55, 5276–5282. [Google Scholar]
  116. Duellman, S.J.; Zhou, W.; Meisenheimer, P.; Vidugiris, G.; Cali, J.J.; Gautam, P.; Wennerberg, K.; Vidugiriene, J. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening. Assay Drug Dev. Technol. 2015, 13, 456–465. [Google Scholar] [CrossRef] [Green Version]
  117. Suzuki, T.; Fujikura, K.; Higashiyama, T.; Takata, K. DNA staining for fluorescence and laser confocal microscopy. J. Histochem. Cytochem. 1997, 45, 49–53. [Google Scholar] [CrossRef] [Green Version]
  118. Liu, X.; Chen, H.; Patel, D.J. Solution structure of actinomycin-DNA complexes: Drug intercalation at isolated G-C sites. J. Biomol. NMR 1991, 1, 323–347. [Google Scholar] [CrossRef]
  119. Latt, S.A. Fluorescent probes of chromosome structure and replication. Can. J. Genet. Cytol. 1977, 19, 603–623. [Google Scholar] [CrossRef]
  120. Liu, W.; Liang, Y.; Chan, Q.; Jiang, L.; Dong, J. CX3CL1 promotes lung cancer cell migration and invasion via the Src/focal adhesion kinase signaling pathway. Oncol. Rep. 2019, 41, 1911–1917. [Google Scholar] [CrossRef]
  121. Réu, P.; Svedberg, G.; Hässler, L.; Möller, B.; Svahn, H.A.; Gantelius, J. A 61% lighter cell culture dish to reduce plastic waste. PLoS ONE 2019, 14, e0216251. [Google Scholar] [CrossRef]
  122. Avlasevich, S.L.; Bryce, S.M.; Cairns, S.E.; Dertinger, S.D. In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability. Environ. Mol. Mutagen. 2006, 47, 56–66. [Google Scholar] [CrossRef]
  123. Yan, X.; Habbersett, R.C.; Cordek, J.M.; Nolan, J.P.; Yoshida, T.M.; Jett, J.H.; Marrone, B.L. Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry. Anal. Biochem. 2000, 286, 138–148. [Google Scholar] [CrossRef]
  124. Bryce, S.M.; Bemis, J.C.; Avlasevich, S.L.; Dertinger, S.D. In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat. Res. 2007, 630, 78–91. [Google Scholar] [CrossRef] [Green Version]
  125. Mukhopadhyay, P.; Rajesh, M.; Haskó, G.; Hawkins, B.J.; Madesh, M.; Pacher, P. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat. Protoc. 2007, 2, 2295–2301. [Google Scholar] [CrossRef]
  126. Kerscher, B.; Barlow, J.L.; Rana, B.M.; Jolin, H.E.; Gogoi, M.; Bartholomew, M.A.; Jhamb, D.; Pandey, A.; Tough, D.F.; Van Oosterhout, A.J.M.; et al. BET bromodomain inhibitor IBET151 impedes human ILC2 activation and prevents experimental allergic lung inflammation. Front. Immunol. 2019, 10, 678. [Google Scholar] [CrossRef]
  127. Paivandy, A.; Eriksson, J.; Melo, F.R.; Sellin, M.E.; Pejler, G. Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production. Cell Death Discov. 2019, 5, 95. [Google Scholar] [CrossRef]
  128. Vig, S.; Buitinga, M.; Rondas, D.; Crèvecoeur, I.; van Zandvoort, M.; Waelkens, E.; Eizirik, D.L.; Gysemans, C.; Baatsen, P.; Mathieu, C.; et al. Cytokine-induced translocation of GRP78 to the plasma membrane triggers a pro-apoptotic feedback loop in pancreatic beta cells. Cell Death Dis. 2019, 10, 309. [Google Scholar] [CrossRef]
  129. Akagi, J.; Kordon, M.; Zhao, H.; Matuszek, A.; Dobrucki, J.; Errington, R.; Smith, P.J.; Takeda, K.; Darzynkiewicz, Z.; Wlodkowic, D. Real-time cell viability assays using a new anthracycline derivative DRAQ7®. Cytom. Part A 2013, 83 A, 227–234. [Google Scholar] [CrossRef] [Green Version]
  130. Severini, A.; Richard Morgan, A. An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence. Anal. Biochem. 1991, 193, 83–89. [Google Scholar] [CrossRef]
  131. Zipper, H.; Brunner, H.; Bernhagen, J.; Vitzthum, F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res. 2004, 32, e103. [Google Scholar] [CrossRef]
  132. Singer, V.L.; Lawlor, T.E.; Yue, S. Comparison of SYBR® Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test). Mutat. Res. Genet. Toxicol. Environ. Mutagen. 1999, 439, 37–47. [Google Scholar] [CrossRef]
  133. Mirrett, S. Acridin orange stain. Infect. Control. 1982, 3, 250–252. [Google Scholar] [CrossRef] [Green Version]
  134. Kumar, R.; Kaur, M.; Kumari, M. Acridine: A versatile heterocyclic nucleus. Acta Pol. Pharm. Drug Res. 2012, 69, 3–9. [Google Scholar]
  135. Darzynkiewicz, Z.; Juan, G.; Srour, E.F. Differential staining of DNA and RNA. Curr. Protoc. Cytom. 2004, 30, 7.3.1–7.3.16. [Google Scholar] [CrossRef]
  136. Lekishvili, T.; Campbell, J.J. Rapid comparative immunophenotyping of human mesenchymal stromal cells by a modified fluorescent cell barcoding flow cytometric assay. Cytom. Part A 2018, 93, 905–915. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  137. BD Biosciences. BD Biosciences Brochure. Available online: (accessed on 23 November 2021).
  138. Prado-Garcia, H.; Romero-Garcia, S.; Rumbo-Nava, U.; Lopez-Gonzalez, J.S. Predominance of Th17 over regulatory T-cells in pleural effusions of patients with lung cancer implicates a proinflammatory profile. Anticancer Res. 2015, 35, 1529–1536. [Google Scholar]
  139. McMaster, S.R.; Gabbard, J.D.; Koutsonanos, D.G.; Compans, R.W.; Tripp, R.A.; Tompkins, S.M.; Kohlmeier, J.E. Memory T cells generated by prior exposure to influenza cross react with the novel H7N9 influenza virus and confer protective heterosubtypic immunity. PLoS ONE 2015, 10, e0115725. [Google Scholar] [CrossRef] [PubMed]
  140. Rodríguez-Rodríguez, N.; Apostolidis, S.A.; Penaloza-MacMaster, P.; Martín Villa, J.M.; Barouch, D.H.; Tsokos, G.C.; Crispín, J.C. Programmed Cell Death 1 and Helios Distinguish TCR-αβ + Double-Negative (CD4 − CD8 −) T Cells That Derive from Self-Reactive CD8 T Cells. J. Immunol. 2015, 194, 4207–4214. [Google Scholar] [CrossRef] [Green Version]
  141. Flies, D.B.; Higuchi, T.; Chen, L. Mechanistic Assessment of PD-1H Coinhibitory Receptor–Induced T Cell Tolerance to Allogeneic Antigens. J. Immunol. 2015, 194, 5294–5304. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  142. Akabane, S.; Matsuzaki, K.; Yamashita, S.I.; Arai, K.; Okatsu, K.; Kanki, T.; Matsuda, N.; Oka, T. Constitutive activation of PINK1 protein leads to proteasome-mediated and non-apoptotic cell death independently of mitochondrial autophagy. J. Biol. Chem. 2016, 291, 16162–16174. [Google Scholar] [CrossRef] [Green Version]
  143. Iraolagoitia, X.L.R.; Spallanzani, R.G.; Torres, N.I.; Araya, R.E.; Ziblat, A.; Domaica, C.I.; Sierra, J.M.; Nuñez, S.Y.; Secchiari, F.; Gajewski, T.F.; et al. NK Cells Restrain Spontaneous Antitumor CD8 + T Cell Priming through PD-1/PD-L1 Interactions with Dendritic Cells. J. Immunol. 2016, 197, 953–961. [Google Scholar] [CrossRef] [Green Version]
  144. Mercer, F.; Diala, F.G.I.; Chen, Y.P.; Molgora, B.M.; Ng, S.H.; Johnson, P.J. Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis. PLoS Negl. Trop. Dis. 2016, 10, e0004913. [Google Scholar] [CrossRef]
  145. Souza-Fonseca-Guimaraes, F.; Young, A.; Mittal, D.; Martinet, L.; Bruedigam, C.; Takeda, K.; Andoniou, C.E.; Degli-Esposti, M.A.; Hill, G.R.; Smyth, M.J. NK cells require IL-28R for optimal in vivo activity. Proc. Natl. Acad. Sci. USA 2015, 112, E2376–E2384. [Google Scholar] [CrossRef] [Green Version]
  146. Matsui, K.; Adelsberger, J.W.; Kemp, T.J.; Baseler, M.W.; Ledgerwood, J.E.; Pinto, L.A. Circulating CXCR5+CD4+ T follicular-like helper cell and memory B cell responses to human papillomavirus vaccines. PLoS ONE 2015, 10, e0137195. [Google Scholar] [CrossRef] [Green Version]
  147. Jones, A.; Opejin, A.; Henderson, J.G.; Gross, C.; Jain, R.; Epstein, J.A.; Flavell, R.A.; Hawiger, D. Peripherally Induced Tolerance Depends on Peripheral Regulatory T Cells That Require Hopx to Inhibit Intrinsic IL-2 Expression. J. Immunol. 2015, 195, 1489–1497. [Google Scholar] [CrossRef] [Green Version]
  148. Nath, A.; Li, I.; Roberts, L.R.; Chan, C. Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma. Sci. Rep. 2015, 5, 14752. [Google Scholar] [CrossRef] [Green Version]
  149. Kanemaru, K.; Noguchi, E.; Tokunaga, T.; Nagai, K.; Hiroyama, T.; Nakamura, Y.; Tahara-Hanaoka, S.; Shibuya, A. Tie2 Signaling Enhances Mast Cell Progenitor Adhesion to Vascular Cell Adhesion Molecule-1 (VCAM-1) through α4β1 Integrin. PLoS ONE 2015, 10, e0144436. [Google Scholar] [CrossRef] [Green Version]
  150. Talabot-Ayer, D.; Martin, P.; Vesin, C.; Seemayer, C.A.; Vigne, S.; Gabay, C.; Palmer, G. Severe Neutrophil-Dominated Inflammation and Enhanced Myelopoiesis in IL-33–Overexpressing CMV/IL33 Mice. J. Immunol. 2015, 194, 750–760. [Google Scholar] [CrossRef] [Green Version]
  151. Keppel, M.P.; Saucier, N.; Mah, A.Y.; Vogel, T.P.; Cooper, M.A. Activation-Specific Metabolic Requirements for NK Cell IFN-γ Production. J. Immunol. 2015, 194, 1954–1962. [Google Scholar] [CrossRef]
  152. Shade, K.T.C.; Platzer, B.; Washburn, N.; Mani, V.; Bartsch, Y.C.; Conroy, M.; Pagan, J.D.; Bosques, C.; Mempel, T.R.; Fiebiger, E.; et al. A single glycan on IgE is indispensable for initiation of anaphylaxis. J. Exp. Med. 2015, 212, 457–467. [Google Scholar] [CrossRef] [Green Version]
  153. Weiser, J.N.; Roche, A.M.; Hergott, C.B.; LaRose, M.I.; Connolly, T.; Jorgensen, W.L.; Leng, L.; Bucala, R.; Das, R. Macrophage Migration Inhibitory Factor Is Detrimental in Pneumococcal Pneumonia and a Target for Therapeutic Immunomodulation. J. Infect. Dis. 2015, 212, 1677–1682. [Google Scholar] [CrossRef] [Green Version]
  154. Allen, T.M.; Cleland, L.G. Serum-induced leakage of liposome contents. BBA Biomembr. 1980, 597, 418–426. [Google Scholar] [CrossRef]
  155. Patel, H.; Tscheka, C.; Heerklotz, H. Characterizing vesicle leakage by fluorescence lifetime measurements. Soft Matter 2009, 5, 2849–2851. [Google Scholar] [CrossRef]
  156. Szakács, G.; Váradi, A.; Özvegy-Laczka, C.; Sarkadi, B. The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Drug Discov. Today 2008, 13, 379–393. [Google Scholar] [CrossRef]
  157. Tóvári, J.; Futosi, K.; Bartal, A.; Tátrai, E.; Gacs, A.; Kenessey, I.; Paku, S. Boyden chamber-based method for characterizing the distribution of adhesions and cytoskeletal structure in HT1080 fibrosarcoma cells. Cell Adhes. Migr. 2014, 8, 509–516. [Google Scholar] [CrossRef] [Green Version]
  158. Khalili, A.A.; Ahmad, M.R. A Review of cell adhesion studies for biomedical and biological applications. Int. J. Mol. Sci. 2015, 16, 18149–18184. [Google Scholar] [CrossRef] [Green Version]
  159. Zepeda-Moreno, A.; Taubert, I.; Hellwig, I.; Hoang, V.; Pietsch, L.; Lakshmanan, V.K.; Wagner, W.; Ho, A.D. Innovative method for quantification of cell-cell adhesion in 96-well plates. Cell Adhes. Migr. 2011, 5, 215–219. [Google Scholar] [CrossRef] [Green Version]
  160. Kashef, J.; Franz, C.M. Quantitative methods for analyzing cell-cell adhesion in development. Dev. Biol. 2015, 401, 165–174. [Google Scholar] [CrossRef] [Green Version]
  161. Zhou, D.W.; García, A.J. Measurement Systems for Cell Adhesive Forces. J. Biomech. Eng. 2015, 137, 020908. [Google Scholar] [CrossRef] [Green Version]
  162. Ungai-Salánki, R.; Peter, B.; Gerecsei, T.; Orgovan, N.; Horvath, R.; Szabó, B. A practical review on the measurement tools for cellular adhesion force. Adv. Colloid Interface Sci. 2019, 269, 309–333. [Google Scholar] [CrossRef] [Green Version]
  163. Kramer, N.; Walzl, A.; Unger, C.; Rosner, M.; Krupitza, G.; Hengstschläger, M.; Dolznig, H. In vitro cell migration and invasion assays. Mutat. Res. Rev. Mutat. Res. 2013, 752, 10–24. [Google Scholar] [CrossRef]
  164. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 1962, 115, 453–466. [Google Scholar] [CrossRef] [Green Version]
  165. Restouin, A.; Aresta, S.; Prébet, T.; Borg, J.P.; Badache, A.; Collette, Y. A simplified, 96-well-adapted, ATP luminescence-based motility assay. Biotechniques 2009, 47, 871–875. [Google Scholar] [CrossRef]
  166. Marshall, J. Transwell((R)) invasion assays. Methods Mol. Biol. 2011, 769, 97–110. [Google Scholar]
  167. Liang, C.C.; Park, A.Y.; Guan, J.L. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007, 2, 329–333. [Google Scholar] [CrossRef] [Green Version]
  168. Gyorffy, B.; Stelniec-Klotz, I.; Sigler, C.; Kasack, K.; Redmer, T.; Qian, Y.; Schäfer, R. Effects of RAL signal transduction in KRAS-and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer. Oncotarget 2015, 6, 13334–13346. [Google Scholar] [CrossRef] [Green Version]
  169. Gorshkova, I.; He, D.; Berdyshev, E.; Usatuyk, P.; Burns, M.; Kalari, S.; Zhao, Y.; Pendyala, S.; Garcia, J.G.N.; Pyne, N.J.; et al. Protein kinase C-ε regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-ζ, and Rac1. J. Biol. Chem. 2008, 283, 11794–11806. [Google Scholar] [CrossRef] [Green Version]
  170. Lo, C.M.; Keese, C.R.; Giaever, I. Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing. Biophys. J. 1995, 69, 2800–2807. [Google Scholar] [CrossRef] [Green Version]
  171. Tamada, M.; Perez, T.D.; Nelson, W.J.; Sheetz, M.P. Two distinct modes of myosin assembly and dynamics during epithelial wound closure. J. Cell Biol. 2007, 176, 27–33. [Google Scholar] [CrossRef]
  172. Zordan, M.D.; Mill, C.P.; Riese, D.J.; Leary, J.F. A high throughput, interactive imaging, bright-field wound healing assay. Cytom. Part A 2011, 79 A, 227–232. [Google Scholar] [CrossRef] [Green Version]
  173. Poujade, M.; Grasland-Mongrain, E.; Hertzog, A.; Jouanneau, J.; Chavrier, P.; Ladoux, B.; Buguin, A.; Silberzan, P. Collective migration of an epithelial monolayer in response to a model wound. Proc. Natl. Acad. Sci. USA 2007, 104, 15988–15993. [Google Scholar] [CrossRef] [Green Version]
  174. Simpson, K.J.; Selfors, L.M.; Bui, J.; Reynolds, A.; Leake, D.; Khvorova, A.; Brugge, J.S. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat. Cell Biol. 2008, 10, 1027–1038. [Google Scholar] [CrossRef]
  175. Gough, W.; Hulkower, K.I.; Lynch, R.; McGlynn, P.; Uhlik, M.; Yan, L.; Lee, J.A. A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay. J. Biomol. Screen. 2011, 16, 155–163. [Google Scholar] [CrossRef] [Green Version]
  176. Leto, T.L.; Pratt, B.M.; Madri, J.A. Mechanisms of cytoskeletal regulation: Modulation of aortic endothelial cell protein band 4.1 by the extracellular matrix. J. Cell. Physiol. 1986, 127, 423–431. [Google Scholar] [CrossRef]
  177. Varani, J.; Orr, W.; Ward, P.A. A comparison of the migration patterns of normal and malignant cells in two assay systems. Am. J. Pathol. 1978, 90, 159–172. [Google Scholar]
  178. Kroening, S.; Goppelt-Struebe, M. Analysis of matrix-dependent cell migration with a barrier migration assay. Sci. Signal. 2010, 3, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  179. Rosen, E.M.; Meromsky, L.; Setter, E.; Vinter, D.W.; Goldberg, I.D. Quantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads. Exp. Cell Res. 1990, 186, 22–31. [Google Scholar] [CrossRef]
  180. Konduri, S.D.; Tasiou, A.; Chandrasekar, N.; Rao, J.S. Overexpression of tissue factor pathway inhibitor-2 (TFPI-2), decreases the invasiveness of prostate cancer cells in vitro. Int. J. Oncol. 2001, 18, 127–131. [Google Scholar] [CrossRef] [PubMed]
  181. Chaubey, S.; Ridley, A.J.; Wells, C.M. Using the Dunn Chemotaxis Chamber to Analyze Primary Cell Migration in Real Time, in Cell Migration: Developmental Methods and Protocols. Methods Mol. Biol. 2011, 769, 41–51. [Google Scholar] [CrossRef]
  182. Echeverria, V.; Meyvantsson, I.; Skoien, A.; Worzella, T.; Lamers, C.; Hayes, S. An automated high-content assay for tumor cell migration through 3-Dimensional matrices. J. Biomol. Screen. 2010, 15, 1144–1151. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  183. Miura, K. Tracking movement in cell biology. Adv. Biochem. Eng. Biotechnol. 2005, 95, 267–295. [Google Scholar] [CrossRef] [PubMed]
  184. Gu, W.; Pellegrino, T.; Parak, W.J.; Boudreau, R.; Le Gros, M.A.; Alivisatos, A.P.; Larabell, C.A. Measuring cell motility using quantum dot probes. Methods Mol. Biol. 2007, 374, 125–131. [Google Scholar] [CrossRef]
  185. Lin, M.; DiVito, M.M.; Merajver, S.D.; Boyanapalli, M.; van Golen, K.L. Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and caveolin-1. Mol. Cancer 2005, 4, 21. [Google Scholar] [CrossRef] [Green Version]
  186. Niinaka, Y.; Haga, A.; Raz, A. Quantification of cell motility. Metastasis Res. Protoc. 2001, 58, 55–60. [Google Scholar]
  187. Swaminathan, V.; Mythreye, K.; Tim O’Brien, E.; Berchuck, A.; Blobe, G.C.; Superfine, R. Mechanical Stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Res. 2011, 71, 5075–5080. [Google Scholar] [CrossRef] [Green Version]
  188. Cribb, J.A.; Osborne, L.D.; Beicker, K.; Psioda, M.; Chen, J.; O’Brien, E.T.; Taylor, R.M.; Vicci, L.; Hsiao, J.P.L.; Shao, C.; et al. An Automated High-throughput Array Microscope for Cancer Cell Mechanics. Sci. Rep. 2016, 6, 27371. [Google Scholar] [CrossRef] [Green Version]
  189. Hillman, H. Procedures used in modern cell biology ignore natural laws. J. Biol. Phys. Chem. 2009, 9, 130–135. [Google Scholar] [CrossRef]
  190. Ramsden, J.J. Towards zero-perturbation methods for investigating biomolecular interactions. Colloids Surf. A Physicochem. Eng. Asp. 1998, 141, 287–294. [Google Scholar] [CrossRef]
  191. Ramsden, J.J. Experimental methods for investigating protein adsorption kinetics at surfaces. Q. Rev. Biophys. 1993, 27, 41–105. [Google Scholar] [CrossRef] [Green Version]
  192. Ramsden, J.J. Optical biosensors. J. Mol. Recog. 1997, 10, 109–120. [Google Scholar] [CrossRef]
  193. Song, H.P.; Wang, H.; Zhao, X.; He, L.; Zhong, H.; Wu, S.Q.; Li, P.; Yang, H. Label-free pharmacological profiling based on dynamic mass redistribution for characterization and authentication of hazardous natural products. J. Hazard. Mater. 2017, 333, 265–274. [Google Scholar] [CrossRef]
  194. Zhang, X.; Deng, H.; Xiao, Y.; Xue, X.; Ferrie, A.M.; Tran, E.; Liang, X.; Fang, Y. Label-free cell phenotypic profiling identifies pharmacologically active compounds in two traditional Chinese medicinal plants. RSC Adv. 2014, 4, 26368–26377. [Google Scholar] [CrossRef]
  195. Peter, B.; Nador, J.; Juhasz, K.; Dobos, A.; Korosi, L.; Székács, I.; Patko, D.; Horvath, R. Incubator proof miniaturized Holomonitor to in situ monitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: Validity of the measured parameters and their corrections. J. Biomed. Opt. 2015, 20, 067002. [Google Scholar] [CrossRef]
  196. McColl, J.; Horvath, R.; Aref, A.; Larcombe, L.; Chianella, I.; Morgan, S.; Yakubov, G.E.; Ramsden, J.J. Polyphenol control of cell spreading on glycoprotein substrata. J. Biomater. Sci. Polym. Ed. 2009, 20, 841–851. [Google Scholar] [CrossRef]
  197. Orgovan, N.; Peter, B.; Bősze, S.; Ramsden, J.J.; Szabó, B.; Horvath, R. Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor. Sci. Rep. 2014, 4, 4034. [Google Scholar] [CrossRef] [Green Version]
  198. Aref, A.; Horvath, R.; Ramsden, J.J. Spreading kinetics for quantifying cell state during stem cell differentiation. J. Biol. Phys. Chem. 2010, 10, 145–151. [Google Scholar] [CrossRef]
  199. Ramsden, J.J. Biospecific interaction analysis using integrated optics techniques. In Quantitative Analysis of Biospecific Interactions; Lundahl, P., Lundqvist, A., Greijer, E., Eds.; Harwood: Amsterdam, The Netherlands, 1998; pp. 149–162. [Google Scholar]
  200. Michielin, O.; Vergères, G.; Ramsden, J.J. Myristoylation-induced compaction of a membrane-binding protein. J. Am. Chem. Soc. 1999, 121, 6523–6526. [Google Scholar] [CrossRef]
  201. Shanshiashvili, L.V.; Suknidze, N.C.; Machaidze, G.G.; Mikeladze, D.G.; Ramsden, J.J. Adhesion and clustering of charge isomers of myelin basic protein at model myelin membranes. Arch. Biochem. Biophys. 2003, 419, 170–177. [Google Scholar] [CrossRef] [PubMed]
  202. Vergères, G. and Ramsden, J.J. Regulation of the binding of MARCKS-related protein to lipid bilayer membranes by calmodulin. Arch. Biochem. Biophys. 2000, 378, 45–50. [Google Scholar] [CrossRef]
  203. Ramsden, J.J. OWLS–A versatile technique for drug discovery. Front. Drug Des. Discov. 2006, 2, 211–223. [Google Scholar]
  204. Nikolelis, D.P.; Nikoleli, G.P. Biosensors for Security and Bioterrorism Applications; Springer International Publishing: Cham, Switzerland, 2016. [Google Scholar] [CrossRef]
  205. Ramsden, J.J. From Kinetics to Structure: High resolution molecular microscopy. In Proteins at Solid-Liquid Interfaces; Dejardin, P., Ed.; Springer: Berlin/Heidelberg, Germany, 2006; pp. 23–49. [Google Scholar]
  206. Horvath, R.; McColl, J.; Yakubov, G.E.; Ramsden, J.J. Structural hysteresis and hierarchy in adsorbed glycoproteins. J. Chem. Phys. 2008, 129, 08B401. [Google Scholar] [CrossRef] [Green Version]
  207. Saftics, A.; Kurunczi, S.; Peter, B.; Szekacs, I.; Ramsden, J.J.; Horvath, R. Data evaluation for surface-sensitive label-free methods to obtain real-time kinetic and structural information of thin films: A practical review with related software packages. Adv. Colloid Interface Sci. 2021, 294, 102431. [Google Scholar] [CrossRef] [PubMed]
  208. Jankovics, H.; Kovacs, B.; Saftics, A.; Gerecsei, T.; Tóth, É.; Szekacs, I.; Vonderviszt, F.; Horvath, R. Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers. Sci. Rep. 2020, 10, 22253. [Google Scholar] [CrossRef]
  209. Kliment, K.; Szekacs, I.; Peter, B.; Erdei, A.; Kurucz, I.; Horvath, R. Biosensors and Bioelectronics Label-free real-time monitoring of the BCR-triggered activation of primary human B cells modulated by the simultaneous engagement of inhibitory receptors. Biosens. Bioelectron. 2021, 191, 113469. [Google Scholar] [CrossRef]
  210. Sztilkovics, M.; Gerecsei, T.; Peter, B.; Saftics, A.; Kurunczi, S.; Szekacs, I.; Szabo, B.; Horvath, R. Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy. Sci. Rep. 2020, 10, 61. [Google Scholar] [CrossRef] [Green Version]
  211. Ramsden, J.J.; Li, S.-Y.; Heinzle, E.; Prenosil, J.E. Optical method for measurement of number and shape of attached cells in real time. Cytometry 1995, 1, 97–102. [Google Scholar] [CrossRef]
  212. Wang, R.; Wang, J.; Liu, Y.; Zhang, X.; Liang, X. Resonant waveguide grating based assays for colloidal aggregate detection and promiscuity characterization in natural products. RSC Adv. 2019, 9, 38055–38064. [Google Scholar] [CrossRef]
  213. Persson, J.; Mölder, A.; Pettersson, S.; Alm, K. Cell motility studies using digital holographic microscopy. Education 2010, 1063–1072. [Google Scholar]
  214. El-Schish, Z.; Mölder, A.; Sebesta, M.; Gisselsson, L.; Alm, K.; Gjörloff, A.; Wingren, A. Digital holographic microscopy–Innovative and non-destructive analysis of living cells. Microsc. Sci. Technol. Appl. Educ. 2010, 4, 1055–1062. [Google Scholar]
  215. Murray, E.M.; Allen, C.F.; Handy, T.E.; Hu, C.A.; Craig, W.R.; Seaton, S.C.; Wolfe, A.L. Development of a Robust and Quantitative High-Throughput Screening Method for Antibiotic Production in Bacterial Libraries. ACS Omega 2019, 4, 15414–15420. [Google Scholar] [CrossRef]
  216. Cordell, G.A.; Shin, Y.G. Finding the needle in the haystack. The dereplication of natural product extracts. Pure Appl. Chem. 1999, 71, 1089–1094. [Google Scholar] [CrossRef] [Green Version]
  217. Dias, D.A.; Urban, S.; Roessner, U. A Historical Overview of Natural Products in Drug Discovery. Metabolites 2012, 303–336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  218. Newman, D.J.; Cragg, G.M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J. Nat. Prod. 2020, 83, 770–803. [Google Scholar] [CrossRef] [PubMed]
  219. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Orhan, I.E.; Banach, M.; Rollinger, J.M.; Barreca, D.; Weckwerth, W.; Bauer, R.; Bayer, E.A.; et al. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [Google Scholar] [CrossRef]
  220. Thomford, N.E.; Senthebane, D.A.; Rowe, A.; Munro, D.; Seele, P.; Maroyi, A.; Dzobo, K. Natural products for drug discovery in the 21st century: Innovations for novel drug discovery. Int. J. Mol. Sci. 2018, 19, 1578. [Google Scholar] [CrossRef] [Green Version]
  221. Walker, J.M. Natural products isolation. In Methods in Biotechnology; Sarker, S.D., Latif, Z., Gray, A.I., Eds.; Humana Press Inc: Totowa, NJ, USA, 2005. [Google Scholar]
  222. Sarker, S.D.; Nahar, L. An Introduction to Natural Products Isolation. Methods Mol. Biol. 2012, 864, 1–25. [Google Scholar] [CrossRef] [PubMed]
  223. Zhang, Q.W.; Lin, L.G.; Ye, W.C. Techniques for extraction and isolation of natural products: A comprehensive review. Chin. Med. 2018, 13, 20. [Google Scholar] [CrossRef] [Green Version]
  224. Cavalcanti, R.N. Uses and Applications of Extracts from Natural Sources. In RSC Green Chemistry No. 21 Natural Product Extraction: Principles and Applications; Rostagno, M.A., Prado, J.M., Eds.; The Royal Society of Chemistry: London, UK, 2013. [Google Scholar]
  225. Li, J.; Hu, S.; Jian, W.; Xie, C.; Yang, X. Plant antimicrobial peptides: Structures, functions, and applications. Bot. Stud. 2021, 62, 5. [Google Scholar] [CrossRef] [PubMed]
  226. Dos Santos-Silva, C.A.; Zupin, L.; Oliveira-Lima, M.; Vilela, L.M.B.; Bezerra-Neto, J.P.; Ferreira-Neto, J.R.; Ferreira, J.D.C.; de Oliveira-Silva, R.L.; de J. Pires, C.; Aburjaile, F.F.; et al. Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era. Bioinform. Biol. Insights 2020, 14, 1177932220952739. [Google Scholar] [CrossRef]
  227. Hegedüs, N.; Marx, F. Antifungal proteins: More than antimicrobials? Fungal Biol. Rev. 2013, 26, 132–145. [Google Scholar] [CrossRef]
  228. Shwaiki, L.N.; Lynch, K.M.; Arendt, E.K. Future of antimicrobial peptides derived from plants in food application–A focus on synthetic peptides. Trends Food Sci. Technol. 2021, 112, 312–324. [Google Scholar] [CrossRef]
  229. Xiang, L.; Chi, T.; Tang, Q.; Yang, X.; Ou, M.; Chen, X.; Yu, X.; Chen, J.; Ho, R.J.Y.; Shao, J.; et al. A pentacyclic triterpene natural product, ursolic acid and its prodrug US597 inhibit targets within cell adhesion pathway and prevent cancer metastasis. Oncotarget 2015, 6, 9295–9312. [Google Scholar] [CrossRef] [Green Version]
  230. Rahimi-Esboei, B.; Ebrahimzadeh, M.A.; Gholami, S.H.; Falah-Omrani, V. Anti-giardial activity of Sambucus ebulus. Eur. Rev. Med. Pharmacol. Sci. 2013, 17, 2047–2050. [Google Scholar] [CrossRef] [PubMed]
  231. Suzuki, Y.; Isemura, M. Inhibitory effect of epigallocatechin gallate on adhesion of murine melanoma cells to laminin. Cancer Lett. 2001, 173, 15–20. [Google Scholar] [CrossRef]
  232. De Campos, P.S.; Matte, B.F.; Diel, L.F.; Jesus, L.H.; Bernardi, L.; Alves, A.M.; Rados, P.V.; Lamers, M.L. Low Doses of Curcuma longa Modulates Cell Migration and Cell-Cell Adhesion. Phyther. Res. 2017, 31, 1433–1440. [Google Scholar] [CrossRef] [PubMed]
  233. Kim, J.D.; Liu, L.; Guo, W.; Meydani, M. Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. J. Nutr. Biochem. 2006, 17, 165–176. [Google Scholar] [CrossRef] [PubMed]
  234. Liu, J.M.; Bignon, J.; Haroun-Bouhedja, F.; Bittoun, P.; Vassy, J.; Fermandjian, S.; Wdzieczak-Bakala, J.; Boisson-Vidal, C. Inhibitory effect of fucoidan on the adhesion of adenocarcinoma cells to fibronectin. Anticancer Res. 2005, 25, 2129–2133. [Google Scholar]
  235. Di Mattia, C.; Battista, N.; Sacchetti, G.; Serafini, M. Antioxidant Activities in vitro of Water and Liposoluble Extracts Obtained by Different Species of Edible Insects and Invertebrates. Front. Nutr. 2019, 6, 106. [Google Scholar] [CrossRef] [Green Version]
  236. Arancibia-avila, P.; Fuentes, O.; Alarco, J.; Zamorano-ponce, E.; Herna, M. Production and Purification of Statins from Pleurotus ostreatus (Basidiomycetes ) Strains. Z. Naturforsch. C J. Biosci. 2003, 58, 62–64. [Google Scholar]
  237. Tamura, C.; Terahara, A. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinium. J. Antibiot. 1976, 29, 1346–1348. [Google Scholar]
  238. Altwairgi, A.K. Statins are potential anticancerous agents. Oncol. Rep. 2015, 33, 1019–1039. [Google Scholar] [CrossRef] [Green Version]
  239. Gazzerro, P.; Proto, M.C.; Gangemi, G.; Malfitano, A.M.; Ciaglia, E.; Pisanti, S.; Santoro, A. Pharmacological Actions of Statins: A Critical Appraisal in the Management of Cancer. Pharmacol. Rev. 2012, 64, 102–146. [Google Scholar] [CrossRef]
  240. Iannelli, F.; Lombardi, R.; Milone, M.R.; Pucci, B.; Rienzo, S. De Targeting Mevalonate Pathway in Cancer Treatment: Repurposing of Statins. Recent Pat. Anticancer Drug Discov. 2018, 13, 184–200. [Google Scholar] [CrossRef]
  241. Takamatsu, S. Naturally occurring cell adhesion inhibitors. J. Nat. Med. 2018, 72, 817–835. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  242. Schreiner, E.P.; Kern, M.; Steck, A.; Foster, C.A. Synthesis of ether analogues derived from HUN-7293 and evaluation as inhibitors of VCAM-1 expression. Bioorgan. Med. Chem. Lett. 2004, 14, 5003–5006. [Google Scholar] [CrossRef]
  243. Takamatsu, S.; Zhang, Q.; Schrader, K.K.; ElSohly, H.N.; Walker, L.A. Characterization of Mycotypha metabolites found ro be inhibitors of cell adhesion molecules. J. Antibiot. 2002, 55, 585–592. [Google Scholar] [CrossRef] [Green Version]
  244. Jeong, J.; Jin, C.; Park, C.; Han, M.I.N.H.O.; Kim, G.; Moon, S.; Kim, C.G.I.L.; Jeong, Y.K.E.E.; Kim, W.; Lee, J.; et al. Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt. Int. J. Oncol. 2012, 40, 1697–1704. [Google Scholar] [CrossRef] [PubMed]
  245. Xu, J.; Caro-diaz, E.J.E.; Lacoske, M.H.; Hung, C.; Theodorakis, E.A. EDGE ARTICLE Fusarisetin A: Scalable total synthesis and related studies. Chem. Sci. 2012, 12, 3378–3386. [Google Scholar] [CrossRef] [Green Version]
  246. Kasorn, A.; Loison, F.; Kangsamaksin, T. Terrein inhibits migration of human breast cancer cells via inhibition of the Rho and Rac signaling pathways. Oncol. Rep. 2018, 39, 1378–1386. [Google Scholar] [CrossRef] [Green Version]
  247. Mathieu, V.; Berger, W.; Evidente, A.; Kornienko, A.; Gailly, P.; Vandier, C. Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity. Cell Death Dis. 2013, 4, e561. [Google Scholar] [CrossRef] [Green Version]
  248. Bury, M.; Andolfi, A.; Rogister, B.; Cimmino, A.; Mégalizzi, V.; Mathieu, V.; Feron, O.; Evidente, A.; Kiss, R. Fusicoccin A, a phytotoxic carbotricyclic diterpene glucoside of fungal Origin, reduces proliferation and invasion of glioblastoma cells by targeting multiple tyrosine kinases. Transl. Oncol. 2013, 6, 112–123. [Google Scholar] [CrossRef] [Green Version]
  249. Teiten, M.; Mack, F.; Debbab, A.; Aly, A.H.; Dicato, M.; Proksch, P.; Diederich, M. Bioorganic & Medicinal Chemistry Anticancer effect of altersolanol A, a metabolite produced by the endophytic fungus Stemphylium globuliferum, mediated by its pro-apoptotic and anti-invasive potential via the inhibition of NF- j B activity. Bioorgan. Med. Chem. 2013, 21, 3850–3858. [Google Scholar] [CrossRef]
  250. Wang, X.; Bashyal, B.P.; Wijeratne, E.M.K.; Ren, J.M.U.; Liu, M.X.; Gunatilaka, M.K.; Arnold, A.E.; Gunatilaka, A.A.L. Smardaesidins A–G, Isopimarane and 20- nor -Isopimarane Diterpenoids from Smardaea sp., a Fungal Endophyte of the Moss Ceratodon purpureus 1. J. Nat. Prod. 2011, 74, 2052–2061. [Google Scholar] [CrossRef] [Green Version]
  251. Lee, H.J.; Lee, J.H.; Hwang, B.Y.; Kim, H.S.; Lee, J.J. Anti-Angiogenic Activities of Gliotoxin and Its Methylthio- Derivative, Fungal Metabolites. Arch. Pharm. Res. 2001, 24, 397–401. [Google Scholar] [CrossRef]
  252. Hayot, C.; Debeir, O.; Van Ham, P.; Van Damme, M.; Kiss, R.; Decaestecker, C. Characterization of the activities of actin-affecting drugs on tumor cell migration. Toxicol. Appl. Pharmacol. 2006, 211, 30–40. [Google Scholar] [CrossRef] [PubMed]
  253. Zhou, C.; Ling, M.; Lee, T.K.; Man, K.; Wang, X.; Wong, Y. FTY720, a fungus metabolite, inhibits invasion ability of androgen-independent prostate cancer cells through. Cancer Lett. 2006, 233, 36–47. [Google Scholar] [CrossRef] [PubMed]
  254. Kusama, T.; Mukai, M.; Iwasaki, T.; Tatsuta, M.; Matsumoto, Y.; Akedo, H. Inhibition of Epidermal Growth Factor-induced RhoA Translocation and Invasion of Human Pancreatic Cancer Cells by 3-Hydroxy-3-methylglutaryl-coenzyme A. Cancer Res. 2001, 61, 4885–4891. [Google Scholar]
  255. Alonso, D.F.; Farina, H.G.; Skilton, G.; Gabri, M.R.; de Lorenzo, M.S.; Gomez, D.E. Reduction of mouse mammary tumor formation and metastasis by lovastatin, an inhibitor of the mevalonate pathway ofcholesterol synthesis. Breast Cancer Res. Treat. 1998, 50, 83–93. [Google Scholar] [CrossRef]
  256. Zhong, W.; Liang, Y.; Wang, C.; Chang, T.; Lee, W. Lovastatin suppresses invasiveness of anaplastic thyroid cancer cells by inhibiting Rho geranylgeranylation and RhoA / ROCK signaling. Endocr. Relat. Cancer 2005, 3, 615–630. [Google Scholar] [CrossRef]
  257. Bublik, R.; Alonso, D.F.; Gomez, D.E. Lovastatin alters cytoskeleton organization and inhibits experimental metastasis of mammary carcinoma cells. Clin. Exp. Metastasis 2002, 19, 551–559. [Google Scholar]
  258. Afshordel, S.; Kern, B.; Clasohm, J.; König, H.; Priester, M.; Weissenberger, J.; Kögel, D.; Eckert, G.P. Lovastatin and perillyl alcohol inhibit glioma cell invasion, migration, and proliferation–Impact of Ras-/Rho-prenylation. Pharmacol. Res. 2015, 91, 69–77. [Google Scholar] [CrossRef]
  259. Glynn, S.A.; Sullivan, D.O.; Eustace, A.J.; Clynes, M.; Donovan, N.O. The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, invasion of melanoma cells. BMC Cancer 2008, 8, 9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  260. Hayashi, M.; Kim, Y.P.; Hiraoka, H.; Natori, M.; Takamatsu, S.; Kawakubo, T.; Masuma, R.; Komiyama, K.; Somura, S. Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 1995, 48, 1435–1439. [Google Scholar] [CrossRef] [Green Version]
  261. Takamatsu, S.; Hiraoka, H.; Kim, Y.; Hayashi, M.; Natori, M.; Komiyama, K.; Omura, S. Macrosphelides C and D, Novel Inhibitors of Cell Adhesion. J. Antibiot. 1997, 50, 878–880. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  262. Fukami, A.; Taniguchi, Y.; Nakamura, T.; Rho, M.; Kawaguchi, K.; Hayashi, M.; Komiyama, K.; Omura, S. New Membersof the Macrosphelides from Microsphaeropsis sp. FO-5050 IV. J. Antibiot. 1999, 52, 501–504. [Google Scholar] [CrossRef] [Green Version]
  263. Miki, I.; Ishihara, N.; Otoshi, M.; Kase, H. Simple colorimetric cell-cell adhesion assay using MTT-stained leukemia cells. J. Immunol. Methods 1993, 164, 255–261. [Google Scholar] [CrossRef]
  264. Yamada, T.; Iritani, M.; Doi, M.; Minoura, K.; Ito, T.; Numata, A. Absolute stereostructures of cell-adhesion inhibitors, macrosphelides C, E–G and I, produced by a sea hare-derived Periconia sp. J. Antibiot. 2001, 3046–3053. [Google Scholar] [CrossRef]
  265. Yamada, T.; Iritani, M.; Minoura, K.; Numata, A. Absolute Stereostructures of Cell Adhesion Inhibitors, Macrosphelides H and L, from Periconia byssoides OUPS-N133. J. Antibiot. 2002, 55, 147–154. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  266. Yamada, T.; Minoura, K.; Tanaka, R.; Numata, A. Cell-adhesion Inhibitors Produced by a Sea Hare-derived Periconia sp. J. Antibiot. 2007, 60, 370–375. [Google Scholar] [CrossRef]
  267. Fukami, A.; Iijima, K.; Hayashi, M.; Komiyama, K.; Omura, S. Macrosphelide B Suppressed Metastasis through Inhibition of Adhesion of sLe x / E-Selectin Molecules. Biochem. Biophys. Res. Commun. 2002, 1070, 1065–1070. [Google Scholar] [CrossRef]
  268. Yamada, T.; Iritani, M.; Minoura, K.; Kawai, K.; Numata, A. Peribysins A–D, potent cell-adhesion inhibitors from a sea hare-derived culture of Periconia species. Org. Biomol. Chem. 2004, 2, 2131–2135. [Google Scholar] [CrossRef]
  269. Yamada, T.; Doi, M.; Miura, A.; Harada, W.; Hiramura, M.; Minoura, K.; Tanaka, R.; Numata, A. Absolute Stereostructures of Cell-adhesion Inhibitors, Peribysins A, E, F and G, Produced by a Sea Hare-derived Periconia sp. J. Antibiot. 2005, 58, 185–191. [Google Scholar] [CrossRef]
  270. Yamada, T.; Minoura, K.; Tanaka, R.; Numata, A. Cell-adhesion Inhibitors Produced by a Sea Hare-derived Periconia sp. II. J. Antibiot. 2006, 59, 345–350. [Google Scholar] [CrossRef]
  271. Sigrist, C.J.A.; Bridge, A.; Le Mercier, P. A potential role for integrins in host cell entry by SARS-CoV-2. Antiviral Res. 2020, 177, 104759. [Google Scholar] [CrossRef] [PubMed]
  272. Kiss, B.; Kis, Z.; Pályi, B.; Kellermayer, M.S.Z. Topography, Spike Dynamics, and Nanomechanics of Individual Native SARS-CoV-2 Virions. Nano Lett. 2021, 21, 2675–2680. [Google Scholar] [CrossRef] [PubMed]
  273. Kanyo, N.; Kovacs, K.D.; Saftics, A.; Szekacs, I.; Peter, B.; Santa-Maria, A.R.; Walter, F.R.; Dér, A.; Deli, M.A.; Horvath, R. Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data. Sci. Rep. 2020, 10, 22422. [Google Scholar] [CrossRef]
  274. Dakal, T.C. SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19. Immunobiology 2021, 226, 152021. [Google Scholar] [CrossRef] [PubMed]
  275. Yang, Q.; Hughes, T.A.; Kelkar, A.; Yu, X.; Cheng, K.; Park, S.J.; Huang, W.C.; Lovell, J.F.; Neelamegham, S. Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration. bioRxiv 2020, 1–19. [Google Scholar] [CrossRef]
  276. Bhuiyan, F.R.; Howlader, S.; Raihan, T.; Hasan, M. Plants Metabolites: Possibility of Natural Therapeutics Against the COVID-19 Pandemic. Front. Med. 2020, 7, 444. [Google Scholar] [CrossRef]
  277. Antonelli, M.; Donelli, D.; Maggini, V.; Firenzuoli, F. Phytotherapic compounds against coronaviruses: Possible streams for future research. Phyther. Res. 2020, 34, 1469–1470. [Google Scholar] [CrossRef]
  278. Seifert, G.; Jeitler, M.; Stange, R.; Michalsen, A.; Cramer, H.; Brinkhaus, B.; Esch, T.; Kerckhoff, A.; Paul, A.; Teut, M.; et al. The Relevance of Complementary and Integrative Medicine in the COVID-19 Pandemic: A Qualitative Review of the Literature. Front. Med. 2020, 7, 946. [Google Scholar] [CrossRef]
  279. Subbaiyan, A.; Ravichandran, K.; Singh, S.V.; Sankar, M.; Thomas, P.; Dhama, K.; Malik, Y.S.; Singh, R.K.; Chaudhuri, P. In silico molecular docking analysis targeting SARS-CoV-2 Spike protein and selected herbal constituents. J. Pure Appl. Microbiol. 2020, 14, 989–998. [Google Scholar] [CrossRef]
  280. Liu, J.; Bodnar, B.H.; Meng, F.; Khan, A.I.; Wang, X.; Saribas, S.; Wang, T.; Lohani, S.C.; Wang, P.; Wei, Z.; et al. Epigallocatechin gallate from green tea effectively blocks infection of SARS-CoV-2 and new variants by inhibiting spike binding to ACE2 receptor. Cell Biosci. 2021, 11, 1–15. [Google Scholar] [CrossRef]
  281. Kalus, U.; Grigorov, A.; Kadecki, O.; Jansen, J.P.; Kiesewetter, H.; Radtke, H. Cistus incanus (CYSTUS052) for treating patients with infection of the upper respiratory tract. A prospective, randomised, placebo-controlled clinical study. Antiviral Res. 2009, 84, 267–271. [Google Scholar] [CrossRef] [PubMed]
  282. Bramer, W.M.; De Jonge, G.B.; Rethlefsen, M.L.; Mast, F.; Kleijnen, J. A systematic approach to searching: An efficient and complete method to develop literature searches. J. Med. Libr. Assoc. 2018, 106, 531–541. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Figure 2. Adhesion molecule stimulation and mechanisms in endothelial cells. Inhibitory effect of a natural compound on cellular adhesion, typically of the human umbilical vein endothelial cell line (HUVEC), which are usually first treated with certain cytokines to stimulate the expression of CAMs.
Figure 2. Adhesion molecule stimulation and mechanisms in endothelial cells. Inhibitory effect of a natural compound on cellular adhesion, typically of the human umbilical vein endothelial cell line (HUVEC), which are usually first treated with certain cytokines to stimulate the expression of CAMs.
Biomedicines 09 01781 g002
Figure 3. Stimulation mechanism for cytokine modulation in lymphocytes. In vivo, lipopolysaccharide (LPS, from Gram-negative bacteria) stimulates the immune response by interacting with its leukocyte membrane receptor, CD14 (with TLR4-MD2), to induce the generation of cytokines such as TNF- α, IL-1, IL-1 α, IL-6.
Figure 3. Stimulation mechanism for cytokine modulation in lymphocytes. In vivo, lipopolysaccharide (LPS, from Gram-negative bacteria) stimulates the immune response by interacting with its leukocyte membrane receptor, CD14 (with TLR4-MD2), to induce the generation of cytokines such as TNF- α, IL-1, IL-1 α, IL-6.
Biomedicines 09 01781 g003
Figure 4. The NF-κB pathway in endothelial cells. The compounds shown to have anti-inflammatory effects in vivo completely annulled LPS- and TNF-α-triggered nuclear translocation of NF-κB and NF-κB DNA-binding activity in endothelial cells, thus the production of cytokine receptors and ICAM, VCAM is inhibited.
Figure 4. The NF-κB pathway in endothelial cells. The compounds shown to have anti-inflammatory effects in vivo completely annulled LPS- and TNF-α-triggered nuclear translocation of NF-κB and NF-κB DNA-binding activity in endothelial cells, thus the production of cytokine receptors and ICAM, VCAM is inhibited.
Biomedicines 09 01781 g004
Figure 5. The interaction between the ECM, MMPs and natural compounds in breast carcinoma. Baicalein blocked the expression of MMP-2/9, thus the degradation of the extracellular matrix is also inhibited.
Figure 5. The interaction between the ECM, MMPs and natural compounds in breast carcinoma. Baicalein blocked the expression of MMP-2/9, thus the degradation of the extracellular matrix is also inhibited.
Biomedicines 09 01781 g005
Figure 6. Schematic illustration of the measurement steps of flow cytometry (A) and tetrazolium-based colorimetric MTT viability tests (B). (A) Propidium iodide is a popular red-fluorescent counterstain. Living cells are not permeable to it, but dead cells are (hence acquire dark coloring) and can be detected in the population after exposure treatment of the cells. (B) The basis of the MTT assay is that the yellow, water-soluble tetrazole becomes purple, insoluble formazan by the action of mitochondrial dehydrogenase of living cells. Cytotoxic and cytostatic activities can be determined from the optical density of the control and treated cells (ICM: incomplete medium, CM: complete medium). The formazan can be conveniently extracted by DMSO for colorimetric measurement.
Figure 6. Schematic illustration of the measurement steps of flow cytometry (A) and tetrazolium-based colorimetric MTT viability tests (B). (A) Propidium iodide is a popular red-fluorescent counterstain. Living cells are not permeable to it, but dead cells are (hence acquire dark coloring) and can be detected in the population after exposure treatment of the cells. (B) The basis of the MTT assay is that the yellow, water-soluble tetrazole becomes purple, insoluble formazan by the action of mitochondrial dehydrogenase of living cells. Cytotoxic and cytostatic activities can be determined from the optical density of the control and treated cells (ICM: incomplete medium, CM: complete medium). The formazan can be conveniently extracted by DMSO for colorimetric measurement.
Biomedicines 09 01781 g006
Figure 7. Multicomponent study measuring cancer cell adhesion on a natural compound-treated coating [50]. A 384-well biosensor plate was used in the experiment (left) comprising the manipulation steps in a typical well (right). Representative kinetic curves are also shown (bottom).
Figure 7. Multicomponent study measuring cancer cell adhesion on a natural compound-treated coating [50]. A 384-well biosensor plate was used in the experiment (left) comprising the manipulation steps in a typical well (right). Representative kinetic curves are also shown (bottom).
Biomedicines 09 01781 g007
Figure 8. Bioactivity-guided isolation of natural compounds.
Figure 8. Bioactivity-guided isolation of natural compounds.
Biomedicines 09 01781 g008
Figure 9. Schematic illustration of SARS-CoV-2 attachment and two possible examples of ways to inhibit this process. (A) Virus can attach to ACE2 receptor or integrins of the host cell. RGD-integrin interaction occurs in calcium-dependent manner [274]. As the result of the process, the virus penetrates into the cell and starts to copy itself. (B) Several compounds bind to the spike protein or even may alter it [275] and prevent virus-receptor attachment. (C) Lowering divalent ion concentrations in the lungs with pulmonary EDTA chelation therapy may inhibit virus-host interaction [274].
Figure 9. Schematic illustration of SARS-CoV-2 attachment and two possible examples of ways to inhibit this process. (A) Virus can attach to ACE2 receptor or integrins of the host cell. RGD-integrin interaction occurs in calcium-dependent manner [274]. As the result of the process, the virus penetrates into the cell and starts to copy itself. (B) Several compounds bind to the spike protein or even may alter it [275] and prevent virus-receptor attachment. (C) Lowering divalent ion concentrations in the lungs with pulmonary EDTA chelation therapy may inhibit virus-host interaction [274].
Biomedicines 09 01781 g009
Table 1. Ligands and functions of different integrins of human leukocytes (RGD, Arg-Gly-Asp sequence; VCAM-1, vascular cellular adhesion molecule-1; ICAM-1, Intercellular adhesion molecule-1) [10,30,31,32,33].
Table 1. Ligands and functions of different integrins of human leukocytes (RGD, Arg-Gly-Asp sequence; VCAM-1, vascular cellular adhesion molecule-1; ICAM-1, Intercellular adhesion molecule-1) [10,30,31,32,33].
β1RGD, VCAM-1, E-cadherinAdhesion
β2iC3b, fibrinogen, ICAM-1Adhesion, phagocytosis, apoptotic cell clearance
β6RGD, fibronectinAdhesion, endocytosis, inflammation
β7RGD, VCAM-1Adhesion, inflammation
Table 2. Different modes of pathway intervention involving gene expression by natural compounds.
Table 2. Different modes of pathway intervention involving gene expression by natural compounds.
P1Inhibiting the dephosphorylation of I κB and, hence, the activation of NF-κB
P2Inhibiting translocation of activated NF-κB into the nucleus
P3Inhibition of binding of activated NF-κB to promoter sites for CAM expression
Table 3. Three types of mechanisms inhibiting cell adhesion by blocking specific receptor sites.
Table 3. Three types of mechanisms inhibiting cell adhesion by blocking specific receptor sites.
B1Blocking adhesion receptors on the surface of mobile cells (e.g., leukocytes)
B2Blocking adhesion receptors on the surface of tissue cells (e.g., of the endothelium)
B3Blocking adhesion motifs in the extracellular matrix (ECM)
Table 4. Absorbance-based and colorimetric cell viability and cytotoxicity assays 1.
Table 4. Absorbance-based and colorimetric cell viability and cytotoxicity assays 1.
Cell Viability and Cytotoxicity Assays
Absorbance-Based or Colorimetric
Dye exclusion assaysTrypan bluemembrane integritysimple, easy to use, economic, fasthemocytometer required, counting error, difficult to process large number of samples, cannot be distinguished healthy cells and cell function lost alive cells, toxic on mammalian cellsJonston 2010 [69],
Strober 2001 [70],
Aslantürk and Celik 2013 [71],
Stone et al., 2009 [72],
Yip and Auersperg 1972 [73],
Ruben 1988 [74]
(Erythrosin B)
membrane integrityeconomic, versatility, biosafetytime-consuming, labor-intensive, sensitive for the contamination, filling rate, and inter-user variationKim et al., 2016 [75],
Marmion 1979 [76]
Colorymetric assaysMTTenzyme (mitochondrial) activityeasy to use, safe, high reproductibility, widly usedorganic solvents required, significant well-to-well error, interaction with compounds results false positive, or false negative dataStone et al., 2009 [72],
Aslantürk et al. [77],
Bopp and Lettieri 2008 [78],
Mosmann 1983 [79]
MTSeasy to use, rapid, sensitive, economic, specific, inexpensivemeasured absorbance level is influenced by incubation time, cell type, and cell number; optimal incubation time: 1–3 hBerg et al., 1994 [80],
Tominaga et al., 1999 [81],
Rotter et al., 1993 [82],
Buttke et al., 1993 [83],
Promega Technical Bulletin [84],
Cory et al., 1991 [85],
Riss et al., 1992 [86]
XTTresults water soluble crystals, quick, sensitive, easy-to-use, safe; highly sensitive and accuratestrongly depends on reductive capacity of viable cells due to pH, cellular ion concentration, cell cycle variation, environmental factorsScudiero et al.1988 [87]
WST-1easy to use, high reproducibility, wildly used, no interference with phenol red, water soluble dye, no additional incubation timerelatively long incubation time (2 h)Ishiyama et al., 1993 [88]
WST-8not cell permeable; cells can be used after the assay; water soluble formazanintracellular metabolic activity can influence the reduction of WST-8Tominaga et al., 1999 [81],
Strober 2001 [70]
LDH (lactate dehydrogenase)enzyme (lactate dehydrogenase) activityreliability; quick, simple evaluation, indicator of cell deathserum incompetenceDecker and Lohmann-Matthes 1988 [89],
Schins et al., 2002 [90],
Fotakis et al., 2006 [91],
Lappalanien et al., 1994 [92]
SRB (sulforhodamine B)SRB-protein interactionsimple, fast, sensitive, good linearity with cell number, metabolism independent, high reproducibilityhomogenous cell suspension is required; cellular clumps and aggregates interfere with SRBSkehan et al., 1990 [93]
NRU (neutral red uptake)neutral red uptake and lysosomal accumulationgood marker for lysosomal damage, fast and simple evaluationinfluenced by pollutantsBorenfreund and Puerner 1984 [94],
Repetto et al., 2008 [95],
Ringwood et al.1998 [96]
Crystal violetbinding for proteins and DNA of viable cellsquick; chemical inhibtors can be incorporated into the assaymetabolism affected compounds can not be tested, not able to measure cell proliferation rateFeoktisova et al., 2016 [66],
Geserick et al., 2009 [97],
Degterev et al., 2008 [98],
Sun et al., 2012 [99],
Feoktisova et al., 2011 [100]
1 Trypan Blue: Staining with trypan blue is one of the oldest viability assays. In a viable cell, the intact membrane will prevent trypan blue from entering cells. In dead or dying cells, trypan blue will enter the cell, staining it blue. This method was traditionally quantified manually using microscopes and hemocytometers, making it very labor-intensive. However, the recent availability of affordable automated cell counters makes this assay less time consuming and more accurate. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) is a tetrazolium salt that gets reduced by both mitochondrial and extra-mitochondrial dehydrogenases to form insoluble blue formazan crystals, meaning a solubilization step is required before the assay can be read. MTS/XTT: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) substrates are similar to MTT. However, one advantage is that the reactions are carried out intracellularly in the presence of the intermediate electron acceptor phenazine methosulfate (PMS), which enhances their sensitivity. In addition, the reduced formazan product is soluble and gets released to the culture media, removing the need for the extra solubility step that is required with MTT. However, phenol red in cell culture media, fatty acids, and serum albumin have all been reported to distort data obtained from MTS, XTT, and WST assays over prolonged incubation periods. WST: Water-soluble tetrazolium salts (WSTs) are cell-impermeable tetrazolium dyes that get reduced extracellularly via plasma membrane electron transport, and combined with the electron acceptor PMS to generate water-soluble formazan dyes. LDH assay: Lactate dehydrogenase is a ubiquitous, stable cytoplasmic enzyme that converts lactate to pyruvate. If the cell membrane has been damaged, LDH, and therefore, its enzymatic activity is released from cells and can be detected in cell culture media.
Table 5. Fluorometric cell viability and cytotoxicity assays 1.
Table 5. Fluorometric cell viability and cytotoxicity assays 1.
Fluorometric Assay
AlamarBlue (resazurin reduction assay)enzyme (mitochondrial and other (e.g., Diaphorases)) activityrelatively inexpensive, sensitive, multiplexed with other methods (e.g., Caspase activity)fluorescence interference with tested compound; direct cytotoxic effect can be occured (depending on incubation time)O’Brien et al., 2000 [101],
Ahmed et al., 1994 [102],
Page et al., 1993 [103],
Markossian et al., 2004 [65],
Pace et al., 2013 [104]
CFDA-AM (5-carboxyfluorescein diacetate, acetoxymethyl ester)plasma membrane integritycan be used parallel with alamarBlue on the same set of cellsfluorescence interference with tested compoundBopp et al., 2006 [103],
Schreer et al., 2005 [105],
Ganassi 2000 [106]
protease viability marker assay;
GF-AFC assay (glycylphenyl-alaninyl)-aminofluoro-cumarine)
enzyme (aminopeptidase) activityrelatively nontoxic; multiplex with other assays, short incubation time (30–60 min)fluorescence interference with tested compoundNiles et al., 2009 [62]
(bromoeoxyuridin) and EdU (5-ethynyl-2 deoxyuridine)
DNA synthesisrapid, highly selective, results in the most reliable and direct index of proliferation, in contrast to 3H-thymidine incorporation assay, which requires a scintillation beta-counter, BrdU and EdU can be detected by antibodies, allowing analysis by flow cytometry or by immuno-histochemistryBrdU is toxic and mutagenic, alters cell cycle, required DNA denaturationSidman et al., 1959 [107]
Miller and Nowakowski 1988 [108]
Salic and Mitchison 2008 [109]
Nowakowski et al. 1989 [110]
Taupin 2007 [111]
Duque and Rakic 2011 [112]
1 Alamar Blue: is a resazurin compound that gets reduced to resorufin and dihydroresorufin in viable cells. It can enter live cells so does not require cell lysis, and is stable in culture media. This assay has the added advantage that it can be measured in both fluorimetric and colorimetric plate readers. Pace et al., reported incubation time dependent cytotoxic effect [104]. Calcein-AM: Calcein-acetoxymethylester is a non-fluorescent dye that is used in both cell viability and apoptosis assays and its lipophilic, allowing easy passage through the cell membrane. Once inside the viable cell, intracellular esterases cleave the ester bonds of the acetomethoxy group, resulting in the formation of a fluorescent anionic and hydrophilic calcein dye. Non-viable cells do not contain active esterases. Also need to consider that: Cu2+, Co2+, Fe3+, Mn2+, and Ni2+ quench the fluorescent signal from calcein at physiological pH, which means care must be taken to select the appropriate cell culture media.
Table 6. Luminometric cell viability and cell cytotoxicity assays 1.
Table 6. Luminometric cell viability and cell cytotoxicity assays 1.
Luminometric Assay
ATP assaymembrane integritythe fastest and the most sensitive assay to use; no artefacts; no plate handling stepsensitivity depends on reproducibility of pipettingMaehara et al., 1987 [113],
García et al., 2003 [114],
Andreotti et al., [115],
Markossian et al., 2004 [65]
Real-time viability assaymetabolic activityreal-time measurement; multiplexincubation time is cell type and seeding density dependentDuellman et al., 2015 [116],
Markossian et al., 2004 [65]
1 ATP content: there are numerously available assays that measure ATP levels as an output, when cells begin to undergo cell death process (apoptosis) or lose membrane integrity, ATP stocks become depleted through the activity of ATPases that concurrently prevent any new ATP synthesis. This leads to a rapid depletion of intracellular ATP levels. Luminescent ATP assays function by lysing cells to release ATP stores, while concurrently inhibiting ATPases. Luciferase catalyzes the oxidation of luciferin to oxyluciferin in the presence of magnesium and ATP, resulting in a luminescent signal that directly correlates with the intracellular ATP concentration.
Table 7. Cell viability assays for flow cytometry and microscopic imaging 1.
Table 7. Cell viability assays for flow cytometry and microscopic imaging 1.
Cell Viability Assays for Flow Cytometry and Microscopic Imaging
Nucleic acid diesPropidium iodide488 and 561 nmcan be added directly to the samplesnot membrane permeable to live cells, not possible to use on fixed cells, can also bind to RNA not only to DNA, toxigenic and mutagenicSuzuki et al., 1997 [117]
7-amino-actinomycin-D (7-AAD)488 and 561 nmcan be added directly to the samples, can be used in combination with formaldehyde fixationnot membrane permeable to live cells, potential carcinogenLiu et al., 1991 [118]
Latt 1977 [119]
Hoechst 33342350 and 454 nm not membrane permeable to live cellsLiu et al., 2019 [120],
Réu et al., 2019 [121]
Helix NP™ Green, NIR and Blue
and SYTOX® based dyes
wide range from 444–640 nmit can also be used for viability in microscopy on live cells or as a nuclear counterstain on fixed and permeabilized cells and tissue sensitive nucleic acid stain, in combination with resuzarine can be used, works with mammalian and Gram-positive and Gram-negative bacteria, can be incorporated SYTOX® stains into a number of assays for apoptosis, cell viability, and metabolism, easy-to-use.non cell-permeable to live cellsAvlasevich et al., 2006 [122],
Yan et al., 2000 [123],
Bryce et al., 2007 [124],
Mukhopadhyay et al., 2007 [125]
far-red fluorescent DNA dye
633 nmcan be combined with FITC, PE, and other UV or violet excitable dyes for multicolor analysis, non toxic, can be used for siRNA studies and other dynamic viablity assaysnot membrane-permeable to live cellsKerscher et al., 2019 [126]
Paivandy et al., 2019 [127]
Vig et al., 2019 [128]
Akagi et al., 2013 [129]
Ethidium bromide518 nmcan be used on fixed cells, economicnot membrane-permeable, intercalates double-stranded DNA and RNA, mutagen, carcinogen,Severini and Morgan 1991 [130]
SYBR® Green494 nmhighly selective for DNA, can be used in combination with propidium iodidecarcinogen, less mutagenic than ethidium bromideZipper et al., 2004 [131],
Singer et al., 1999 [132]
Acridine orange500 nmcell permeable, low-cost, sensitive, rapid, intercalate to DNA, and electrostatically interact with RNA, sensitive for pH, acidic organells can be also detected, compatible for ethidium bromide and propidium iodidenonfixableMirrett 1982 [133],
Kumar et al., 2012 [134],
Darzynkiewicz et al., 2004 [135]
Protein binding dieseFluor fixable dyeswide range from 401–645 nmtraditional organic fluorescence dies, multiple application, fully compatible with most convential dies Lekishvili et al., 2018 [136]
BD Horizon Brilliant dyes polymer dyes with brighter fluorescence signal BD Biosciences products [137]
Biolegend Zombie dyeswide range from 360–633 nm Pardo-Garcia et al., 2015 [138],
McMaster et al., 2015 [139],
Rodríguez- Rodríguez et al., 2015 [140],
Files et al., 2015 [141],
Akabane et al., 2016 [142],
Iraolagoitia et al., 2016 [143],
Mercer et al., 2016 [144],
Souza-Fonseca-Guimaraes et al., 2015 [145],
Matsui et al., 2015 [146],
Jones et al., 2015 [147],
Nath et al., 2015 [148],
Kanemaru et al., 2015 [149],
Tabalot-Ayer et al., 2015 [150],
Keppel et al., 2015 [151],
Shade et al., 2015 [152],
Weiser et al., 2015 [153]
Calcein AM496cell permeant, indicator of lipid vesicle leakage, neutral substrate for MDR efflux transporters, selective for live cells; suitable for proliferating and non-proliferating cells; ideal for suspension and adherent cells, rapid, ideal for high-throughput assays; commonly used for cell tracing and in studies of endocytosis, cell migration, and gap junctions; adaptable to a wide variety of techniques, including: microplate assays, immunocytochemistry, flow cytometry, and in vivo cell tracking Allen and Cleland 1980 [154],
Patel et al., 2009 [155],
Glavinas et al., 2004 [156]
1 Calcein-AM: Calcein-acetoxymethylester is a non-fluorescent dye that is used in both cell viability and apoptosis assays and is lipophilic, allowing easy passage through the cell membrane. Once inside the viable cell, intracellular esterases cleave the ester bonds of the acetomethoxy group, resulting in the formation of a fluorescent anionic and hydrophilic calcein dye. Non-viable cells do not contain active esterases. Also need to consider that: Cu2+, Co2+, Fe3+, Mn2+, and Ni2+ quench the fluorescent signal from calcein at physiological pH, which means care must be taken to select the appropriate cell culture media. Propidium Iodide/7-AAD: These intercalating agents are frequently used to study the cell cycle and they are membrane-impermeable, they are excluded from viable cells. This means that the fluorescence signal emitted by PI or 7-AAD in non-viable cells can be measured either by fluorescence microscopy or FACS analysis. Cell-impermeable DNA-binding dyes such (DRAQ7 from Abcam, Cambridge, UK or SYTOX from Thermo Fisher, Waltham, MA, USA) enter cells through compromised cell membranes and display strong fluorescence upon binding with DNA.
Table 8. Conventional assay systems to quantify cell migration.
Table 8. Conventional assay systems to quantify cell migration.
Migration Assays
Transwell migration and invasion assay (Boyden chamber)chemotaxis, migration, invasioneasy setup, most frequently used method, can be visualized by cytological dyes,
stained fluorescent, or lysed and assessed by a plate reader; invasive index can be calculated
endpoint measurement, only adherent cells can be used, only vertical movement can be detected, must be optimized for each cell typeMenyhárt et al., 2016 [64],
Kramer et al., 2013 [163],
Boyden 1962 [164],
Restouin et al., 2009 [165],
Marshall 2011 [166]
Scratch (wound healing) assaytwo dimensional (2D) cell migration in confluent, monolayer cell culturessimple, easy setup, low cost, kinetic measurement, can be combined with other techniques (e.g., gene transfections), allows high -throughput screening (HTS)only adherent cells can be used, only horizontal movement can be detected, require relatively large cell and reagent quantities, is not ideal for chemotaxis studiesMenyhárt et al., 2016 [64],
Liang et al., 2007 [167],
Győrffy et al., 2015 [168],
Gorshkova et al., 2008 [169],
Lo et al., 1995 [170],
Tamada 2007 [171],
Zordan et al., 2011 [172],
Poujade et al., 2007 [173],
Simpson et al., 2008 [174]
Cell exclusion zone assaysmigrationSeveral kinds of barrier can be used (e.g., glass, silicone, metal, Teflon, microfabricated soft and elastic “stencils” or agarose gels), cells are visualized several manner (e.g., photomicrography or labeled with fluorescence and measured with a microplate reader), migratory capacity and interaction between two different populations can be compared, kinetic measurement, this is currently the only method that allows investigation of the effects of ECM proteins on cell motility, allows high -throughput screening (HTS)only adherent cells can be used, only horizontal movement can be detectedMenyhárt et al., 2016 [64],
Poujade et al., 2007 [173],
Gough et al., 2011 [175],
Pratt et al., 1984 [176]
Varani et al., 1978 [177]
Kroening 2010 [178]
Microcarrier bead and spheroid migration assaysmigrationadherent cells and spheroids can be used, vertical and horizontal movement, plastic surface and beads can be used, microscope, time-lapse microscope and fluorescent staining can be used to count the cells, endpoint and kinetic measurement can be performed Menyhárt et al., 2016 [64],
Rosen et al., 1990 [179],
Konduri et al., 2001 [180]
The capillary chamber migration assaymigration and morphologyadherent and suspension cells can be used, migratory behavior and morphological responses can be visualized in real time by time-lapse microscopy, endpoint and kinetic measurement can be performed, suitable for rare cell types and expensive compounds due to its small cell and volume require, liquid handling and image processing can be fully automated, allows high- throughput imaging system (HTIS)only horizontal movement can be detectedMenyhárt et al., 2016 [64],
Chaubey et al., 2011 [181],
Echeverria et al., 2010 [182]
Motility of individual cellsmigrationadherent and suspension cells can be used, time-lapse video microscopy, 3D tracking is also possible, analysis of invasive properties of single cells as well as of populations can be performed, kinetic measurementonly horizontal movement can be detected, requires specialized microscopes and image analyzing softwareMenyhárt et al., 2016 [64],
Miura 2005 [183],
Gu et al., 2007 [184],
Lin et al., 2005 [185],
Niinaka et al., 2001 [186]
Mechanical propertiesmigration and invasion in 3D structures3D force microscope and coated magnetic beads can be also used, high-speed video camera, video spot tracker can be appliedonly adherent cells can be tested, only horizontal movement can be detected, endpoint assayMenyhárt el al. 2016 [64],
Swaminathan et al., 2011 [187],
Cribb et al., 2016 [188]
Table 9. Effects of plant-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Table 9. Effects of plant-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Active SubstanceIUPAC Name (CAS Number)SourceCellular EffectEffect TypeMolecular MechanismsTested Cell Line 1, AnimalMethodRef.
Biomedicines 09 01781 i001
5-acetyl-6-hydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-3-yl (E)-2-methylbut-2-enoate
Eupatorium purpureum (rhizome)
Biomedicines 09 01781 i002
Inhibition of cell adhesionNo dataModulation of integrin-dependent cell-cell and cell-protein interactionU937
Homotypic cell aggregation assay,
Cell attachment assay,
Carrageenan oedema test
Habtemariam. 1998
Ursolic acid (UA)
Biomedicines 09 01781 i003
(3β)-3-Hydroxyurs-12-en-28-oic acid
Berries and wax-like coatings of apples, pears, etc.
Biomedicines 09 01781 i004
Inhibition of cancer cell adhesion, invasion and migration. Preventing cancer metastasis. Inhibition on proliferation. Inhibition of cell invasionPICAM-1 downregulationMHCC-97H
Sprague Dawley rats (males)
MTT cell viability assay,
Adhesion assay (fluorescence microscope photographed method),
Invasion assay (matrigel coated transwell inserts),
Wound healing assay,
Flow cytometry,
Western blot,
Microarray assay,
Real-time PCR,
In vivo tumor growth and metastasis assay,
ICAM-1 immunohistochemistry
Xiang et al., 2015
Ellagic acid
Biomedicines 09 01781 i005
Juglans regia
Biomedicines 09 01781 i006
Antiinflammatory activity and osteoblastic activityPInhibition of the effect on TNF-α-induced VCAM-1 and ICAM-1 expressionHAEC
Cell-enzyme-linked immunosorbent assay (cell-ELISA),
MTT assay
Papoutsi et al., 2007
Biomedicines 09 01781 i007
Syzygium aromaticum
Biomedicines 09 01781 i008
Inhibition of trophozoite adhesion. Anti-Giardia activityNo dataNo dataGardia lamblia
(WB strain [ATCC 30957])
Light microscope,
Neubauer cell-counter chamber,
Morphological assay,
Transmission and scanning electron microscopy,
MTT assay
Machado et al., 2011
Elenolic acid,
Biomedicines 09 01781 i009
2-[(2S,3S,4S)-3-formyl-5-methoxycarbonyl-2-methyl-3,4-dihydro-2H-pyran-4-yl]acetic acid (34422-12-3)Olea europaea and Vitis vinifera
Biomedicines 09 01781 i014
Decreased monocytoid cell adhesion to HUVECs. Anti-inflammatory activityPVCAM-1 down-expressionHUEVEC
Enzyme immunoassay (EIA),
Cell number and viability assessment (Trypan blue,)
Adhesion assay,
Northern analysis,
Transfection assays (calcium phosphate precipitation method),
Electrophoretic mobility shift assay (EMSA)
Carluccio et al., 2003 [21]
Biomedicines 09 01781 i010
Biomedicines 09 01781 i011
(2S,3E,4S)-3-ethylidene-2-(β-D-glucopyranosyloxy)-3,4-dihydro-5-(methoxycarbonyl)-2H-pyran-4-acetic acid, 2-(3,4-dihydroxyphenyl)ethyl ester
Biomedicines 09 01781 i012
Biomedicines 09 01781 i013
Epigallocatechin-gallate (EGCG)
Biomedicines 09 01781 i015
Camellia sinensis
Biomedicines 09 01781 i016
Inhibition of cancer cell adhesionBEGCG forms multilayers in the PLL-g-PEG–RGD chains, bocking the RGD motifsHeLaEpic BT (RWG label-free biosensor)Peter et al., 2017 [50]
(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl 3,4,5-trihydroxybenzoate
Inhibition of cancer cell movement (motility, average motility speed and migration)No dataNo dataHeLaHoloMonitor M4Peter et al., 2015 [195]
Inhibition of cancer cell adhesion to lamininBBinding of EGCG to lamininB16Trypan blue dye exclusion assay
Affinity chromatography
Suzuki and Isemura, 2001 [231]
Biomedicines 09 01781 i017
Curcuma longa
Biomedicines 09 01781 i018
Suppressed monocyte and HUVEC adhesionPDown-expression of adhesion molecules. Blocks the NF- κB signaling pathwayHUVEC
WST-1 colorimetry assay,
Western Blot
Kawasaki et al., 2015
Dose dependent decrease of cell-cell adhesion, especially on tumor-derived spheroids. Decreased cell proliferation in cell lines with mesenchymal characteristics. Decreased the migration speed of highly migratory cells. Decreased tumor growth and aggressiveness.No dataNo dataHACAT
HCPA.BALB/c (nude mice)
CyQUANT NF cell proliferation assay kit,
Spheroid assay,
Time-lapse analysis (phase microscopy, inverted microscope),
Flow cytometry,
Xenograft model
Santos de Campos et al., 2017
Ethyl 3’,4’,5’-trimethoxycinamate (1),
Biomedicines 09 01781 i019
(1) ethyl 3,4,5-trimethoxybenzoate
Piper longum
Biomedicines 09 01781 i021
Anti-inflammatory effect.
(1) Significantly but reversibly blocked the adhesion of neutrophils to endothelium
PDown-expression of ICAM-1Primary endothelial cells
from human umblical cord
Trypan blue exclusion test,
MTT assay,
Flow cytometry,
Kumar et al., 2005
piperine (2)
Biomedicines 09 01781 i020
(2) (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)penta-2,4-dien-1-one
Neutrophil cells from peripheral blood of healthy individuals
Saponin astagaloside IV (AS-IV)
Biomedicines 09 01781 i022
(3β,6α,9β,16β,20R,24S)-16,25-dihydroxy-3-(β-D-xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl β-D-glucopyranoside
Astagalus membranaceus
Biomedicines 09 01781 i023
Reduced adhesion of LPS-stimulated HUVECs for PMNs and THP-1 cellsPDecreased the LPS-induced expression of E-selectin and VCAM-1 on the surface of HUVECs. Decreased the LPS- and TNFα-induced specific mRNA levels for E-selectin and VCAM-1. Abolished LPS- and TNFα-induced nuclear translocation of NF- κB and NF- κB DNA binding activity in endothelial cellsHUVEC
Northern blot,
Electrophoretic mobility shift assay (EMSA),
Immunofluorescence staining
Zhang et al., 2003
Galangin (G)
Biomedicines 09 01781 i024
Alpinia officinarium (G)
Biomedicines 09 01781 i028
Dose-dependent suppression of U937 cell adhesion to HUVEC
PM and Q reduced the IL-1β stimulated expression of VCAM-1, ICAM-1 and E-selectinHUVEC
Trypan blue exclusion test,
Ferric-reducing ability assay of plasma method (FRAP),
Cytofluor fluorescence multiwell plate reader (dichlorofluorescein),
Fluoresent labeling (BCECF-AM),
Kim et al., 2006
Kaempferol (K)
Biomedicines 09 01781 i025
Gingko biloba, fruits and vegetables (K)
Biomedicines 09 01781 i029
Myricetin (M)
Biomedicines 09 01781 i026
Berries, Camellia sinensis, etc. (M)
Biomedicines 09 01781 i030
Quercetin (Q)
Biomedicines 09 01781 i027
Vitis vinifera, Camellia sinensis,fruits and vegetables (Q)
Biomedicines 09 01781 i031
Biomedicines 09 01781 i032
Scutelaria baicalensis
Biomedicines 09 01781 i033
Suppressed MDA-MB-231 cell adhesion to fibronectin-coated substratePDown-regulated the expression of MMP-2/9 involved MAPK signaling pathwayMDA-MB-231MTT assay,
Wound healing assay,
Invasion assay,
Gelatin zymography,
Western blot
Wang et al., 2010
Biomedicines 09 01781 i034
(2S,3S,4S,5S,6R)-4,5-dihydroxy-2,6-dimethyltetrahydro-2H-pyran-3-yl hydrogen sulfate
Ascophyllum nodosum
Biomedicines 09 01781 i035
Inhibits cell adhesion to fibronectinBBinds to fibronectin with high affinity. Binds to MDA-MB 231 cell membrane and is internalized. Compromises α5 subunit distribution on MDA-MB-231 cell membraneMDA-MB-231Western blot,
Flow cytometer,
Scatchard plot analysis,
Fluorescence microscopy,
Immunofluorescence and confocal laser imaging,
Lab-tek chamber
Liu et al., 2005
Biomedicines 09 01781 i036
Semecarpus anacardium,
Biomedicines 09 01781 i037
Dalbergia odorifera,
Biomedicines 09 01781 i038
Caragana jubata,
Biomedicines 09 01781 i039
Rhus verniciflua
Biomedicines 09 01781 i040
Significantly decreased TNF-α -induced U937 cell adhesion to lung epithelial cells in a dose dependent manner. Anti-inflammatoryPInhibition of TNF-α-induced ICAM-1 and VCAM-1 expression by inhibiting the NF- κB/ MAPK/Akt signaling pathway. Inhibited expression of ICAM-1 and VCAM-1A549
Western blot,
Fluorescent labeling (BCECF-AM),
Luciferase reporter gene assay,
Fluorescence microscopy,
Fluocytometer (H2DCFDA)
Jang et al., 2012
Tripterygium wilfordii extract (TWH-f), and tetrandrine
Biomedicines 09 01781 i041
Tripterygium wilfordii
Biomedicines 09 01781 i042
Affects the secretion and expression of adhesion molecules on cellsPTWH-f at high concentration has significant inhibitory effect on expression and secretion of E-selectin, ICAM-1, VCAM-1. Tetrandrine did not demonstrate the same effects.Neutrophils
Flow cytometry,
Chang et al., 1999
„Danshen” (DS) Salvia miltiorrhiza (DS)
Biomedicines 09 01781 i043
SZ and HSW significantly inhibited apoptosis in HUVEC undergoing serum deprivation and TNF-α stimulationPDown-regulation of caspase-3-gene expression. DS and SQ significantly attenuated TNF-α-induced expression of VCAM-1 and ICAM-1HUVECAnnexin-V staining,
Western blot,
Flow cytometry,
[3H]-thymidine incorporation assay
Ling et al., 2007
„Sanchi” (SQ)Panax notoginseng (SQ)
Biomedicines 09 01781 i044
„Shanzai” (SZ)Crataegus (SZ)
Biomedicines 09 01781 i045
„Heshouwu”(HSW)Polygonum multiflorum (HSW)
Biomedicines 09 01781 i046
1 U937: human monocyte leukemia cells, EA.hy926: human somatic cell hybrid endothelial cells, MHCC-97H: human hepatocellular carcinoma, MHCC-97L: human hepatocellular carcinoma, HepG2: human hepatocellular carcinoma, M619: human uveal melanoma, MDA-MB-231: human breast adenocarcinoma, MCF-7: human breast adenocarcinoma, HT29: human colorectal adenocarcinoma, SW620: human colorectal adenocarcinoma, B16-F10: mouse melanoma cells, HAEC: human aorta endothelial cells, KS483: murine preosteoblastic cells, HUVEC: human umbilical vein endothelial cells, BAEC: bovine aortic endothelial cells, U937: human monocyte leukemia cells, HeLa: human cervix epithelial adenocarcinoma, THP-1: human monocytic cells, PMN: polymorph-nuclear leukocytes, A549: human lung epithelial cells, HACAT: human spontaneously transformed aneuploid immortal keratinocyte cell line, NIH-3T3: mouse fibroblast, SCC25: human oral squamous cell carcinoma, CAL27: oral adenosquamous carcinoma cell line, HCPA.BALB/c (nude mice), HFB: human synovial fibroblast, B16: mouse melanoma cell line.
Table 10. Effects of animal-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Table 10. Effects of animal-derived natural compounds on cell adhesion and movement. The table summarizes the applied methods and techniques as well.
Active SubstanceSourceCellular EffectEffect TypeMolecular MechanismsTested Cell Line 1, AnimalMethodRef.
Biomedicines 09 01781 i047
Biomedicines 09 01781 i048
Calloselasma rhodostoma (venom)
Biomedicines 09 01781 i049
Cell detachment.
Actin cytoskeleton perturbed
B, PPrevention of integrin αvβ3 interaction.
Decreased pp125FAK phosphorylation
HUVECCell detachment assay (Trypan blue),
MTT assay,
Flow cytometry,
DNA fragmentation assay,
Precipitation of cell extracts and immunoprecipitation,
Western blot,
Immunofluorescence microscopy
Wu et al., 2002 [55]
Biomedicines 09 01781 i050
Echis carinatus
Biomedicines 09 01781 i051
Decreased cell adhesionBPrevention of αIIbβ3, αvβ3 and α5β1 integrin interactionsHeLaEpic BT (RWG label-free biosensor)Szekacs et al., 2018 [51]
1 HUVEC: human umbilical vein endothelial cells, HeLa: human cervix epithelial adenocarcinoma.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Péter, B.; Boldizsár, I.; Kovács, G.M.; Erdei, A.; Bajtay, Z.; Vörös, A.; Ramsden, J.J.; Szabó, I.; Bősze, S.; Horvath, R. Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation. Biomedicines 2021, 9, 1781.

AMA Style

Péter B, Boldizsár I, Kovács GM, Erdei A, Bajtay Z, Vörös A, Ramsden JJ, Szabó I, Bősze S, Horvath R. Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation. Biomedicines. 2021; 9(12):1781.

Chicago/Turabian Style

Péter, Beatrix, Imre Boldizsár, Gábor M. Kovács, Anna Erdei, Zsuzsa Bajtay, Alexandra Vörös, Jeremy J. Ramsden, Ildikó Szabó, Szilvia Bősze, and Robert Horvath. 2021. "Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation" Biomedicines 9, no. 12: 1781.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop