Modulation of Cytoskeleton, Protein Trafficking, and Signaling Pathways by Metabolites from Cucurbitaceae, Ericaceae, and Rosaceae Plant Families

One promising frontier within the field of Medical Botany is the study of the bioactivity of plant metabolites on human health. Although plant metabolites are metabolic byproducts that commonly regulate ecological interactions and biochemical processes in plant species, such metabolites also elicit profound effects on the cellular processes of human and other mammalian cells. In this regard, due to their potential as therapeutic agents for a variety of human diseases and induction of toxic cellular responses, further research advances are direly needed to fully understand the molecular mechanisms induced by these agents. Herein, we focus our investigation on metabolites from the Cucurbitaceae, Ericaceae, and Rosaceae plant families, for which several plant species are found within the state of Florida in Hillsborough County. Specifically, we compare the molecular mechanisms by which metabolites and/or plant extracts from these plant families modulate the cytoskeleton, protein trafficking, and cell signaling to mediate functional outcomes, as well as a discussion of current gaps in knowledge. Our efforts to lay the molecular groundwork in this broad manner hold promise in supporting future research efforts in pharmacology and drug discovery.


Objective of Review
For approximately 60,000 years, plants have been utilized across the world for treating a diverse array of health conditions and diseases [1]. Indeed, plants are used not only as a medicinal source, but their metabolites also provide the foundation for developing new chemotherapies [1]. For example, a metabolite from the Apocynaceae plant family (i.e., vinblastine, a monoterpenoid indole alkaloid from Catharanthus roseus) elicits antineoplastic properties and is an FDA-approved drug for use in patients afflicted by various cancers such as non-Hodgkin lymphomas [2]. Along with the goal of identifying bioactive metabolites from plants, their subsequent synthetic/semi-synthetic development would ideally minimize toxicity and improve efficacy for use in patients [1]. However, to generate sufficient industrial-level quantities of vinblastine, for example, necessary for clinical use, its production has been based on extracting low yields followed by purification of precursors, chemical coupling, and reduction reactions [3]. Since up to 2 tons of leaves are needed to obtain 1 g of the final product, vinblastine has unfortunately been listed as a drug with supply chain issues by the FDA in recent years [3]. In spite of the numerous challenges [1], this prior work sets a strong framework for future synthesis platforms (i.e.,

Methods-Search Terms and Pubmed Results
We exclusively utilized PubMed to identify primary research articles for the critical evaluation of the cytoskeleton, protein trafficking, and signaling pathways. From the search results, the abstracts were first reviewed, and those that were irrelevant were eliminated. It was further noted that there were multiple articles that overlapped across the various search terms utilized. The full primary research articles were accessed through our institution and reviewed; those that were irrelevant were also discarded upon review. We utilized similar search terms for each plant family; the number of identified articles is indicated in Supplementary Table S1 (Cucurbitaceae), Supplementary Table S2 (Ericaceae),  and Supplementary Table S3 (Rosaceae). Out of all these search results, >200 primary research articles were reviewed comprehensively.
We also direct the reader to additional, comprehensive review articles for these plant families or specific species that may present other valuable information at a broader scope. The highly medicinally rich flowering plant family, Rosaceae, comprises over 100 genera [16], of which several are present within Hillsborough County, Florida (Table 3) [9]. Similar to the Ericaceae family, it is also economically important as valuable crop for fruit consumption as well as the production of perfume and cosmetics. The Rosaceae family contains a variety of phytochemicals; within the red raspberry from Rubus idaeus L, the bioactive compounds are classified based on their structural similarities, including phenolic acids and complex polyphenols, amongst others [17]. With respect to polyphenols, the major compound groups include anthocyanins (9 components in red raspberries) and ellagitannins (i.e., sanguiin H-6 in red raspberries), as well as other phenolics such as hydroxycinnamic acids (i.e., caffeic, p-coumaric, and ferulic acids), hydroxybenzoic acids (i.e., ellagic and p-hydroxybenzoic acids), flavonols (i.e., quercetin and kaempferol), and tannins [17]. Many of these phytochemicals, such as the Ericaceae family, are also subjected to their bioavailability due to their degradation in vivo [17]. The extracts and/or purified phytochemicals from species from Rosaceae induce anti-neoplastic, anti-inflammatory, and anti-diabetic responses in addition to protection against obesity and neurodegenerative diseases [17]. Based on our search terms, in the majority of the research literature reviewed for Cucurbitaceae, highly purified agents purchased from companies were utilized in the studies described herein. This is in contrast to the Ericaceae and Rosaceae families, in which the majority of studies utilized plant extracts and/or fractions, with only a subset utilizing purified compounds. Supplementary Files S1-S3 (Cucurbitaceae, Ericaceae, and Rosaceae, respectively) summarize the use of these agents as either purified, mixed, or fractionated components together with originating plant details if provided.
Furthermore, based on our focused search term analyses, we present similarities and differences amongst these three plant families in terms of research area foci to specific human diseases; the results show higher diversity in human diseases within the Rosaceae family in contrast to the Cucurbitaceae family, which appears to be mostly focused on anti-neoplastic responses. These results are presented in Supplementary File S4 and schematically represented as a Venn diagram in Figure 2.

Importance of the Cytoskeleton
Multiple cellular events (i.e., angiogenesis, migration, invasion, cell division, intracellular protein trafficking) require dynamic cytoskeletal rearrangements invol changes in microtubules, actin, and intermediate filaments, amongst other regula components [18]. Hallmarks of cancer include dissemination from the primary tumo followed by re-establishment at a secondary site and increased cellular prolifer capacity. These events are essential to support tumor growth, angiogenesis, metastasis, for which the latter involves cell invasion and migration [18,19]. Drugs disrupt the cytoskeleton (i.e., vinblastine) are utilized in the clinic as a treatment stra for cancer. With an improved understanding of the mechanisms underlying cytoske regulation, improved targeting agents could be designed to improve patient survival Please see Table 4 for a summary of plant components investigated with respect to h relevance or associated disease models.

Importance of the Cytoskeleton
Multiple cellular events (i.e., angiogenesis, migration, invasion, cell division, and intracellular protein trafficking) require dynamic cytoskeletal rearrangements involving changes in microtubules, actin, and intermediate filaments, amongst other regulatory components [18]. Hallmarks of cancer include dissemination from the primary tumor site followed by re-establishment at a secondary site and increased cellular proliferative capacity. These events are essential to support tumor growth, angiogenesis, and metastasis, for which the latter involves cell invasion and migration [18,19]. Drugs that disrupt the cytoskeleton (i.e., vinblastine) are utilized in the clinic as a treatment strategy for cancer. With an improved understanding of the mechanisms underlying cytoskeletal regulation, improved targeting agents could be designed to improve patient survival [18]. Please see Table 4 for a summary of plant components investigated with respect to health relevance or associated disease models.

Effect of Cucurbitaceae Phytochemicals on the Cytoskeleton
For the Cucurbitaceae family, although it is recognized that species contain multiple phytochemicals, including carotenoids and saponins (amongst other constituents), we focused exclusively on the effect of cucurbitacins (which are highly abundant in various genera of this family) on cytoskeletal alterations. Using our above-described search terms, the research findings that were uncovered primarily revolved around the contribution of cucurbitacin B, E, I, and some derivatives. There were also a few studies identifying key regulatory molecules involved in the cytoskeletal organization (i.e., paxillin, cofilin, Arp2/3, LIMK, and VASP), whose expression and/or activities were modulated by cucurbitacins. The effects of these phytochemicals on actin/microtubule organization and expression of intermediate filaments (i.e., vimentin) were also noted to be altered. The details of these studies are described below in Sections 3.2.1-3.2.4, of which the majority were conducted in cancer cell lines associated with in vitro and/or in vivo anti-neoplastic responses (i.e., reduction in cellular viability, cell cycle arrest, induction of apoptosis, reduced migration/invasion, reduced adhesion, and diminished tumor growth in murine xenograft models). A schematic of the key findings is displayed in Figure 3.
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 11 o were conducted in cancer cell lines associated with in vitro and/or in vivo anti-neopla responses (i.e., reduction in cellular viability, cell cycle arrest, induction of apopto reduced migration/invasion, reduced adhesion, and diminished tumor growth in mur xenograft models). A schematic of the key findings is displayed in Figure 3.

Cucurbitacin B (CuB)
Blood Cancers: CuB-induced alterations in actin filament organization in cells fr different blood cancer types within minutes to a few hours following exposu Specifically, in an acute promyelocytic leukemia cell line (HL60), CuB (0.5 µM) elicite disorganized accumulation of F-actin into aggregates, as observed via rhodami phalloidin immunofluorescence staining [20]. In support, in the Jurkat T lymphocytic line, CuB (1 µM) diminished the pool of monomeric G-actin with corresponding increa in F-actin [21]. The changes in actin organization occurred within 30 min and coincid with cofilin dephosphorylation (an actin-interacting molecule that regenerates ac filaments) [21]. Similar cytoskeletal alterations were noted in primary effusion lympho (PEL) cell lines (i.e., BCBL-1) in which CuB (30 nM) reduced G-actin levels leading to ac aggregation and decreased p-cofilin levels within 1-6 h of treatment [22].
Solid Tumors: Alterations in a cytoskeletal organization upon exposure to CuB w also reported in cells derived from solid tumors, including breast, lung, brain, a melanoma. In breast cancer cell lines, CuB-induced alterations in all three ma components of the cytoskeleton: actin, microtubules, and intermediate filaments. In MCF-7 breast cancer cell line, aggregation of F-actin filaments and microtubules occur upon exposure to CuB (0.5 µM) within 20 min [23]. In an independent report using same breast cancer cell lines (MDA-MB-231 and MCF-7), CuB (2.5-10 µM) inhibited assembly of microtubules within 15 min, as noted via immunofluorescence staining o tubulin [24]. In MDA-MB-231 and SKBR-3 breast cancer cell lines, CuB (30 nM) altered cytoskeletal organization with reduced vimentin along with increased F-actin aggrega in the perinuclear area [25]. In one report studying H1299 lung cancer cells, cytoplasm aggregates of F-actin were observed following 2 h of CuB (0.1-0.35 µM) exposure, wh partially required activation of p38 MAPK, as shown through the use of the SB203 inhibitor [26]. In human T98G and U87 glioblastoma multiforme (GBM) cell lines, wit 30 min exposure to CuB (0.1 µM), F-actin formed aggregates, and the microtub network became disrupted [27]. In human A375 and murine B16F10 melanoma cell lin CuB (0.1 µM) induced F-actin aggregation within 30 min [28], an event that w dependent on VASP (barbed-end F-actin binding protein) clustering and co-localizati

Cucurbitacin B (CuB)
Blood Cancers: CuB-induced alterations in actin filament organization in cells from different blood cancer types within minutes to a few hours following exposure. Specifically, in an acute promyelocytic leukemia cell line (HL60), CuB (0.5 µM) elicited a disorganized accumulation of F-actin into aggregates, as observed via rhodamine-phalloidin immunofluorescence staining [20]. In support, in the Jurkat T lymphocytic cell line, CuB (1 µM) diminished the pool of monomeric G-actin with corresponding increases in F-actin [21]. The changes in actin organization occurred within 30 min and coincided with cofilin dephosphorylation (an actin-interacting molecule that regenerates actin filaments) [21]. Similar cytoskeletal alterations were noted in primary effusion lymphoma (PEL) cell lines (i.e., BCBL-1) in which CuB (30 nM) reduced G-actin levels leading to actin aggregation and decreased p-cofilin levels within 1-6 h of treatment [22].
Solid Tumors: Alterations in a cytoskeletal organization upon exposure to CuB were also reported in cells derived from solid tumors, including breast, lung, brain, and melanoma. In breast cancer cell lines, CuB-induced alterations in all three major components of the cytoskeleton: actin, microtubules, and intermediate filaments. In the MCF-7 breast cancer cell line, aggregation of F-actin filaments and microtubules occurred upon exposure to CuB (0.5 µM) within 20 min [23]. In an independent report using the same breast cancer cell lines (MDA-MB-231 and MCF-7), CuB (2.5-10 µM) inhibited the assembly of microtubules within 15 min, as noted via immunofluorescence staining of α-tubulin [24]. In MDA-MB-231 and SKBR-3 breast cancer cell lines, CuB (30 nM) altered the cytoskeletal organization with reduced vimentin along with increased F-actin aggregates in the perinuclear area [25]. In one report studying H1299 lung cancer cells, cytoplasmic aggregates of F-actin were observed following 2 h of CuB (0.1-0.35 µM) exposure, which partially required activation of p38 MAPK, as shown through the use of the SB203580 inhibitor [26]. In human T98G and U87 glioblastoma multiforme (GBM) cell lines, within 30 min exposure to CuB (0.1 µM), F-actin formed aggregates, and the microtubule network became disrupted [27]. In human A375 and murine B16F10 melanoma cell lines, CuB (0.1 µM) induced F-actin aggregation within 30 min [28], an event that was dependent on VASP (barbed-end F-actin binding protein) clustering and co-localization. CuB also induced VASP phosphorylation, mediated by Protein Kinase A (PKA) in a cAMP-independent manner [28]. Furthermore, Gα13 and RhoA were shown to contribute to PKA activation, as demonstrated through siRNA targeting these upstream molecules [28].

Cucurbitacin E (CuE)
Blood Cancers: Reports of CuE-induced alterations in cytoskeletal organization in blood cancer cells is minimal, with only one report in a leukemia cell line. In human U937 leukemia cells, CuE (1-100 nM, 24 h) reduced p-cofilin levels in the absence of a change in p-LIMK1/2, which is the kinase involved in regulating cofilin phosphorylation [29]. Interestingly, using biotin-linked CuE in an affinity binding assay in these cells, a 20kDa cofilin protein was identified as an interacting partner [29]. Further studies involving molecular docking simulation may be needed to determine whether this is a direct and high-affinity interaction.
Solid Tumors: Alterations in a cytoskeletal organization upon exposure to CuE are comparatively more extensive in cells derived from solid tumors, including prostate, breast, lung, and intestinal epithelial cells. In the prostatic PC-3 cell line, CuE (50 nM, 24 h) altered the F-actin intracellular arrangement to an aggregated network in the cytoplasm with a loss of G-actin [30]. CuE treatment, however, did not disrupt the microtubular network (using staining of β-tubulin) [30]. Although the intermediate filament cytokeratin was not altered, CuE disrupted the vimentin distribution to cell-surface membrane blebs [30]. In the MDA-MB-231 breast cancer cell line, CuE (0.2 µM, 1-6 h) reduced focal adhesions (i.e., through paxillin staining) and polymerized actin (i.e., through phalloidin staining) [31]. Using an in vitro assay, CuE inhibited the activity of Arp2/3, a critical mediator of lamellipodia formation [31], and reduced its protein levels in cells [31]; furthermore, when Arp2/3 was overexpressed in the breast cancer cells, the cellular response to CuE was diminished in terms of modulating the actin network [31]. In lung 95D cancer cells, CuE (50-1000 nM, 24 h) induced aggregation of F-actin with no marked alteration on the intracellular distribution of β-tubulin [32]. In human Caco-2 intestinal epithelial cells, CuE (0.1 µM, 6-24 h) induced the aggregation of F-actin filaments as well as reduced p-cofilin and p-LIMK protein expression [33].
Other: In an in vitro study, CuE (0.5 µM) inhibited rabbit skeletal muscle actin depolymerization by binding to F-actin through a covalent bond at Cys257, simulating the behavior of a small molecule modulator [34].

Cucurbitacin I (CuI)
Solid Tumors: Alterations in the cytoskeletal organization were reported with CuI in cells derived from cervical cancers. Using the cervical HeLa cell line, CuI (10-100 nM, 2 h) caused aggregation of actin in a disulfide bridge-independent manner (at C272, C374, and C257) [35]. Furthermore, CuI reduced p-cofilin levels by interacting directly with LIMK1 [35]; through molecular docking simulations and taking advantage of the atomic structure of LIMK1-staurosporine complex, CuI was identified to dock into the nucleotide pocket of LIMK1 [35].

Ericaceae Phytochemicals on the Cytoskeleton
For the Ericaceae family, using our above-described search terms, the research findings that were uncovered utilized extracts as well as purified phytochemicals. Amongst these studies, the majority were descriptive of the visual alteration in the cytoskeletal network and thus limited with respect to the underlying mechanism of action. These are described below in Sections 3.3.1 and 3.3.2, primarily conducted in cancer cell lines associated with in vitro and in vivo anti-neoplastic responses as described for Cucurbitaceae. A schematic of the key findings is displayed in Figure 4. LIMK1/2 levels [38]. IsoCuD also resulted in a reduced F-actin/G-actin ratio elucidated b examining the polymerization rate for 24 h [38].

Ericaceae Phytochemicals on the Cytoskeleton
For the Ericaceae family, using our above-described search terms, the researc findings that were uncovered utilized extracts as well as purified phytochemica Amongst these studies, the majority were descriptive of the visual alteration in th cytoskeletal network and thus limited with respect to the underlying mechanism of actio These are described below in Sections 3.3.1 and 3.3.2, primarily conducted in cancer ce lines associated with in vitro and in vivo anti-neoplastic responses as described f Cucurbitaceae. A schematic of the key findings is displayed in Figure 4.

Extracts
Solid Tumors: Using the cervical HeLa cancer cell line, cranberry proanthocyanid extract (CPAC, from Vaccinium macrocarpon, up to 100 µg/mL, 4 h) disrupted act polymerization along with delocalization of VASP to focal adhesions and α actinin/paxillin [39]. CPAC also altered the phagocytic response in a J774 murin macrophage cell line, which was independent of any alteration in host cellular viabili [39]. In another study, using an extract derived from a capsule-form of bilberr deterioration of the actin filament network (F-actin aggregation in cytoplasm, 0.25 mg/m 24 h) with changes in the microtubule network (aggregation at specific areas in th cytoplasm, 0.5 mg/mL, 24 h) were noted in MCF-7 breast cancer cells overexpressing GF tubulin [40].

Purified Components
Solid Tumors: Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, a phytochemic component of berries, 2.5-10 µM) was tested in MCF-7 and MDA-MB-231 breast canc cell lines and found to inhibit the production of cancer stem-like cells (CSCs characterize by CD44+/CD24-expression) through an NFκB pathway [41]. Specifically, this age inhibited tumor-associated macrophage (i.e., M2-polarized THP-1) induced CSCs fro enriching along with reduced migratory and invasive potential [41]. These changes we accompanied by reduced NFkB expression as well as mediators involved in epithelia mesenchymal transition (EMT); one such mediator is vimentin, which was reduce (coinciding with increased E-cadherin) [41]. These effects were supported in an in vi

Extracts
Solid Tumors: Using the cervical HeLa cancer cell line, cranberry proanthocyanidin extract (CPAC, from Vaccinium macrocarpon, up to 100 µg/mL, 4 h) disrupted actin polymerization along with delocalization of VASP to focal adhesions and α-actinin/paxillin [39]. CPAC also altered the phagocytic response in a J774 murine macrophage cell line, which was independent of any alteration in host cellular viability [39]. In another study, using an extract derived from a capsule-form of bilberry, deterioration of the actin filament network (F-actin aggregation in cytoplasm, 0.25 mg/mL, 24 h) with changes in the microtubule network (aggregation at specific areas in the cytoplasm, 0.5 mg/mL, 24 h) were noted in MCF-7 breast cancer cells overexpressing GFP-tubulin [40].

Purified Components
Solid Tumors: Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, a phytochemical component of berries, 2.5-10 µM) was tested in MCF-7 and MDA-MB-231 breast cancer cell lines and found to inhibit the production of cancer stem-like cells (CSCs characterized by CD44+/CD24-expression) through an NFκB pathway [41]. Specifically, this agent inhibited tumor-associated macrophage (i.e., M2-polarized THP-1) induced CSCs from enriching along with reduced migratory and invasive potential [41]. These changes were accompanied by reduced NFkB expression as well as mediators involved in epithelial-mesenchymal transition (EMT); one such mediator is vimentin, which was reduced (coinciding with increased E-cadherin) [41]. These effects were supported in an in vivo mouse model xenograft model with pterostilbene (5 mg/kg) with reduced NFκB and vimentin protein [41].

Rosaceae Phytochemicals on the Cytoskeleton
Similar to the Ericaceae family, the research findings that were uncovered for the Rosaceae family utilized both extracts and purified phytochemicals. Amongst these studies, the majority were descriptive of the visual alteration in the cytoskeletal network and thus limited to the underlying mechanism of action. These are described below in Sections 3.4.1 and 3.4.2, conducted across a diverse range of cell lines and some conditions outside the realm of tumorigenesis. A schematic of the key findings is displayed in Figure 5.
Similar to the Ericaceae family, the research findings that were uncovered for the Rosaceae family utilized both extracts and purified phytochemicals. Amongst these studies, the majority were descriptive of the visual alteration in the cytoskeletal network and thus limited to the underlying mechanism of action. These are described below in Sections 3.4.1-3.4.2, conducted across a diverse range of cell lines and some conditions outside the realm of tumorigenesis. A schematic of the key findings is displayed in Figure  5.

Extracts
Endothelial Permeability and Neovascularization: WS1442, an herbal extract derived from Crataegus spp hawthorn (100 µg/mL), hindered thrombin-induced adherens junction dysfunction and stress fiber formation (i.e., F-actin stress fibers and the contractile network involving myosin) in human umbilical vein endothelial cells (HUVEC) [42]. These alterations were accompanied by reduced intracellular calcium levels, PKC/RhoA inactivation, as well as elevated cAMP and PKA activation leading to VASP phosphorylation [42]. In another study, a red raspberry phenolic extract (50 µg/mL) also induced changes in F-actin, leading to its disorganization in human microvascular endothelial cells (HMVECs) [43].
Hepatic Fibrosis: A red raspberry extract (RBE, 250 µg/mL) mediated protection against fibrotic response in hepatic stellate cells (HSCs); interestingly, a proteomicbiological network analysis identified changes in pathways involved in cell adhesion and cytoskeletal remodeling [44]. Further, in an in vivo model using dimethylnitrosamine-(DMN) treated rats with RBE (25-50 mg/kg), diminished levels of alpha-smooth muscle actin, an indicator of liver fibrogenesis, were noted [44].
Solid Tumors: Reports of Rosaceae extract-induced alterations in cytoskeletal organization encompass multiple tumor types, including liver, brain, prostate, lung, ovarian, and colorectal. In the human HepG3 hepatoma cell lines, stem and cortex extracts from Sorbus commixta Hedl (SC, 250 µg/mL, 24 h) inhibited actin filament organization

Extracts
Endothelial Permeability and Neovascularization: WS1442, an herbal extract derived from Crataegus spp hawthorn (100 µg/mL), hindered thrombin-induced adherens junction dysfunction and stress fiber formation (i.e., F-actin stress fibers and the contractile network involving myosin) in human umbilical vein endothelial cells (HUVEC) [42]. These alterations were accompanied by reduced intracellular calcium levels, PKC/RhoA inactivation, as well as elevated cAMP and PKA activation leading to VASP phosphorylation [42]. In another study, a red raspberry phenolic extract (50 µg/mL) also induced changes in F-actin, leading to its disorganization in human microvascular endothelial cells (HMVECs) [43].
Hepatic Fibrosis: A red raspberry extract (RBE, 250 µg/mL) mediated protection against fibrotic response in hepatic stellate cells (HSCs); interestingly, a proteomic-biological network analysis identified changes in pathways involved in cell adhesion and cytoskeletal remodeling [44]. Further, in an in vivo model using dimethylnitrosamine-(DMN) treated rats with RBE (25-50 mg/kg), diminished levels of alpha-smooth muscle actin, an indicator of liver fibrogenesis, were noted [44].
Solid Tumors: Reports of Rosaceae extract-induced alterations in cytoskeletal organization encompass multiple tumor types, including liver, brain, prostate, lung, ovarian, and colorectal. In the human HepG3 hepatoma cell lines, stem and cortex extracts from Sorbus commixta Hedl (SC, 250 µg/mL, 24 h) inhibited actin filament organization [45]. Likewise, extract preparations from Crataegus berries, leaves, and flowers from 6 species reduced cell polarity, coinciding with increased actin aggregates and actin stress fibers in the highly malignant U87MG human glioblastoma cell line [46]. In prostate stromal cancer (PSC) cells, an extract from Pygeum africanum (PA, 7.35 µg/mL, 24 h) reduced levels of vimentin and alpha-smooth muscle actin proteins [47]. A raspberry extract from Rubus idaeus L (RIE, 100 µg/mL) reduced p-FAK, p-paxillin, vimentin, and N-cadherin protein levels in A549 lung cancer cells; furthermore, using an in vivo mouse xenograft model, RIE (500 mg/kg) not only reduced tumor volume but also diminished vimentin protein expression, as noted via immunohistochemical analyses [48]. Leaf extracts from Duchesnea indica (DIE, 100 µg/mL, 24 h) also increased E-cadherin protein while decreasing N-cadherin, vimentin, as well as p-paxillin levels in A549 cells [49]. Selenium-enriched polysaccharides from Pyracantha fortuneana (Se-PFPs, 200-400 µg/mL, 48 h) increased E-cadherin while reducing N-cadherin and vimentin protein expression in the ovarian cancer HEY and SKOV3 cell lines [50]. Along with reduced tumor volume, diminished expression of vimentin and E- cadherin were also noted in an in vivo xenograft model using HEY cells following treatment with Se-PFPs (400 mg/kg) [50]. In colorectal RKO and HCT15 cancer cell lines, extracts from the roots from Sanguisorba officinalis L (DY, 100 µg/mL, 24 h) increased E-cadherin levels while reducing N-cadherin and vimentin protein levels [51].

Purified Components
Blood Cancers: Aruncin B (a monoterpenoid, 30 µg/mL, 36 h) derived from a methylene chloride extract from the aerial parts of the goats' beard, Aruncus dioicus var. kamtschaticus, was observed (via α-tubulin staining) to induce damage on the microtubule network in Jurkat T cells [52].
Solid Tumors: In an in vivo xenograft model using breast cancer cells, phloretin (Ph, 100-150 mg/kg), a polyphenol from apple, reduced tumor weight as well as N-cadherin and vimentin expression [53]. Moreover, Ph (10-150 µM, 24 h) reduced mediators involved in the cytoskeletal organization, including p-FAK, paxillin, and alpha-smooth muscle actin in the breast cancer MDA-MB-231 cell line [53]. In addition to a black raspberry extract (BRB), some purified components, namely ellagic acid (EA), urolithin A (UA), and protocatechuic acid (PCA), were tested in combination with docetaxel-and cabazitaxel in castration-resistant prostatic cancer (CRPC) cell lines [54]. Differing effects were noted between the extract (1 mg/mL) and the purified components; specifically, EA (as well as PCA and UA, 10-30 µM) increased microtubule assembly, whereas BRB extract inhibited assembly [54]. Together with cabazitaxel (a chemotherapeutic agent), all the compounds and the extract decreased tubulin polymerization [54]. The roots of Sanguisorba officinalis L have been used traditionally for loss of pigment; in this context, kaempferol (15 µM) markedly increased melanin content while also increasing dendrite length and cytoskeletal F-actin filaments in the malignant SK-MEL-28 melanoma cell line [55]. Kaempferol also elevated RhoA and CDC42 expression, which are critical in mediating actin-dependent melanosome transport [55].

Importance of Protein Trafficking Dynamics
In order to support a variety of cellular functional outcomes (i.e., growth, migration, invasion, etc.), numerous molecules must traffic from various originating locations to a specific destination to mediate a specific molecular function [56]. This may include movement from the endoplasmic reticulum (ER) along the secretory route to the cell surface or plasma membrane, where they could potentially function as mediators in signal transduction [57]. Along this route, these proteins pass through a series of compartments, including various stacks of the golgi apparatus and transport vesicles, that eventually fuse with the cell surface in an exocytic event. Proteins are also internalized from the cell surface, moving into endosomal compartments, which are then distributed to secondary locations that may include the lysosome [57]. Other molecules may be retrograde transported to the trans-golgi network or directly to the endoplasmic reticulum. Some proteins may be translocated to other organelles, including the nuclear or mitochondrial compartments using unique transport mechanisms. Cytoskeletal involvement, including actin, microtubules, and intermediate filaments, in addition to motor proteins (i.e., dynein and kinesin) and targeting molecules (i.e., SNAREs and Rabs), are all critically involved in protein trafficking events. These need to be critically regulated to support cellular homeostasis and, when dysregulated, can result in cellular abnormalities contributing to the pathogenesis of specific diseases [56]. The endoplasmic reticulum is also involved in quality control, and when this is dysregulated, accumulated misfolded proteins may lead to ER stress response [57]. Drugs that target these events to restore cellular homeostasis would be of clinical benefit in various diseases [56]. We urge the reader to review the literature such as [57] for a broader background in this research field. Herein, we focus on organellar dynamics, trafficking of proteins along the secretory route, as well as translocation of key mediators involved in signaling events, including cytoplasmic to nuclear movements. Please see Table 5 for a summary of plant components investigated with respect to health relevance or associated disease models.

Cucurbitaceae Phytochemicals in Protein Trafficking Dynamics
The literature describes cucurbitacins' cellular response primarily in altering the nuclear translocation of key signaling molecules such as β-catenin and the p65 subunit of NFκB. Only CuB and CuE were thus far reported to modulate such protein trafficking events. A schematic of the key findings is displayed in Figure 6. Solid Tumors: β-catenin is a critical Wnt signaling mediator that normally translocates from the cytoplasmic compartment to the nucleus; it is frequently deregulated in breast

Cucurbitacin B (CuB)
Solid Tumors: β-catenin is a critical Wnt signaling mediator that normally translocates from the cytoplasmic compartment to the nucleus; it is frequently deregulated in breast tumors leading to increased gene expression of cyclin D1 and c-Myc. In a series of breast cancer cell lines (MCF-7, SKBR-3, and T47D), CuB (25 µg/mL, 24 h) not only reduced β-catenin protein expression but it hindered its ability to translocate into the nuclear compartment [58].

Cucurbitacin E (CuE)
Rheumatoid Arthritis: This disease is characterized by inflammation, damage to bone or cartilage, and deformed joints. In the synoviocyte MH7A cell line stimulated with TNFα, CuE treatment (10 nM) reduced the gene expression of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and IL-8) together with a reduction in p-NFκB levels [59]. The nuclear translocation of p65, a subunit of NFκB, was also markedly reduced upon CuE treatment [59].

Ericaceae Phytochemicals in Protein Trafficking Dynamics
The phytochemical literature for the Ericaceae plant family for protein trafficking primarily focused on the dysregulated nuclear translocation of the p65 subunit of NFκB in addition to descriptive analyses of ER stress markers in cell model systems of cancers, inflammation, and neurodegenerative diseases. A schematic of the key findings is displayed in Figure 7.

Extracts
Solid Tumors: Blueberry powder was utilized to supplement a basal diet in a 7,12dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch cancer model [60]. The blueberry supplementation mediated a protective response (100-200 mg/kg) that entailed reduced tumor burden, expression of NFκB, and nuclear translocation of the p65 subunit [60]. In the cervical HeLa cancer cell line, the Rhododendron luteum extract (RLE, 40 µg/mL, 72 h) increased the RNA expression of CHOP, an ER stress marker [61]. Since the ER is a critical regulator of apoptotic response, increased CHOP may thus contribute to the cell death response.
Neurodegenerative Disease: Neurodegenerative diseases such as Parkinson's (PD) are characterized by misfolding and aggregation of alpha-synuclein (aSyn) as well as by ER stress and induction of autophagy [62]. The (poly)phenol-digested metabolites from the leaves of Arbutus unedo (LPDMs, 2 µg/mL) antagonized aSyn aggregation in the human neuroglioma H4 cell line while decreasing RNA levels of ER stress markers (LPDMs, 62 µg/mL) in yeast cells [62].

Purified Components
Inflammation: In the endothelial HUVEC cell line treated with TNFα, combinatorial

Extracts
Solid Tumors: Blueberry powder was utilized to supplement a basal diet in a 7,12dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch cancer model [60]. The blueberry supplementation mediated a protective response (100-200 mg/kg) that entailed reduced tumor burden, expression of NFκB, and nuclear translocation of the p65 subunit [60]. In the cervical HeLa cancer cell line, the Rhododendron luteum extract (RLE, 40 µg/mL, 72 h) increased the RNA expression of CHOP, an ER stress marker [61]. Since the ER is a critical regulator of apoptotic response, increased CHOP may thus contribute to the cell death response.
Neurodegenerative Disease: Neurodegenerative diseases such as Parkinson's (PD) are characterized by misfolding and aggregation of alpha-synuclein (aSyn) as well as by ER stress and induction of autophagy [62]. The (poly)phenol-digested metabolites from the leaves of Arbutus unedo (LPDMs, 2 µg/mL) antagonized aSyn aggregation in the human neuroglioma H4 cell line while decreasing RNA levels of ER stress markers (LPDMs, 62 µg/mL) in yeast cells [62].

Rosaceae Phytochemicals in Protein Trafficking Dynamics
The phytochemical literature for the Rosaceae plant family in protein trafficking primarily focused on expression patterns of ER stress markers in multiple in vitro and/or in vivo model systems of obesity, cancer, inflammation, heart and liver diseases, as well as heavy metal toxicity. Interestingly, one study featured the trafficking of a key enzyme from the ER to the cell surface in cells relevant to intestinal bowel disease (IBD). A schematic of the key findings is displayed in Figure 8.

Extracts
Obesity: Raspberry seed powder (RSF) supplementation was investigated on metabolic outcomes of a high-sucrose diet in mice [64]. In this in vivo murine model, RSF recovered glucose metabolism and reduced triglycerides to normal levels, but it also reduced liver ER stress (assessed via western analyses of the stress markers p-JNK, p-p38, and p-eIF2α) [64]. In an ovariectomy-induced obese rat model, raspberry ketone (RK, 160 mg/kg) reduced body weight and protein expression of ER stress markers (i.e., reduced BIP and IRE1α) [65]. In a high-fat, high-sucrose (HFHS) fed mouse model, the Saskatoon berry powder (SBp, 5%) and one of its major components cyanidin-3-glucoside (C3B, 5%) reduced glucose, triacylglycerides, as well as ER stress markers (i.e., CHOP) [66].
Solid Tumors: Increased levels of ER stress markers (i.e., ATF-6 and XBP-1) were reported alongside a series of apoptotic markers in colorectal HCT-116 cancer cells that were treated with strawberry tree honey from Arubutus unedo L. (STH), which was more potent in combination with 5-fluorouracil (5-FU) [67].
Inflammation: One potential key therapeutic target in intestinal bowel diseases (IBD) is intestinal sucrase-isomaltase (SI), responsible for digesting disaccharides [68]. Reduced levels of SI at brush border membrane (BBM) lipid rafts may give rise to symptoms such as diarrhea and abdominal pain [68]. In this context, the response of a methanol extract from Rosa canina (RCME) was investigated on dextran sodium sulfate-(DSS) induced dysregulation of ER homeostasis and protein trafficking using Caco-2 intestinal cancer cells [68]. RCME (50-1000 µg/mL) reduced the expression of ER sensors (including CHOP,

Extracts
Obesity: Raspberry seed powder (RSF) supplementation was investigated on metabolic outcomes of a high-sucrose diet in mice [64]. In this in vivo murine model, RSF recovered glucose metabolism and reduced triglycerides to normal levels, but it also reduced liver ER stress (assessed via western analyses of the stress markers p-JNK, p-p38, and p-eIF2α) [64]. In an ovariectomy-induced obese rat model, raspberry ketone (RK, 160 mg/kg) reduced body weight and protein expression of ER stress markers (i.e., reduced BIP and IRE1α) [65]. In a high-fat, high-sucrose (HFHS) fed mouse model, the Saskatoon berry powder (SBp, 5%) and one of its major components cyanidin-3-glucoside (C3B, 5%) reduced glucose, triacylglycerides, as well as ER stress markers (i.e., CHOP) [66].
Solid Tumors: Increased levels of ER stress markers (i.e., ATF-6 and XBP-1) were reported alongside a series of apoptotic markers in colorectal HCT-116 cancer cells that were treated with strawberry tree honey from Arubutus unedo L. (STH), which was more potent in combination with 5-fluorouracil (5-FU) [67].
Inflammation: One potential key therapeutic target in intestinal bowel diseases (IBD) is intestinal sucrase-isomaltase (SI), responsible for digesting disaccharides [68]. Reduced levels of SI at brush border membrane (BBM) lipid rafts may give rise to symptoms such as diarrhea and abdominal pain [68]. In this context, the response of a methanol extract from Rosa canina (RCME) was investigated on dextran sodium sulfate-(DSS) induced dysregulation of ER homeostasis and protein trafficking using Caco-2 intestinal cancer cells [68]. RCME (50-1000 µg/mL) reduced the expression of ER sensors (including CHOP, ATG4, BiP, GRP94, and XBP1) and restored the trafficking of SI to cell surface lipid rafts [68].
Heavy Metal Toxicity: Lead exposure typically targets the liver and kidney, leading to oxidative stress within these tissues and possibly ER stress [69]. A polyphenol extract from the pulp of Malus micromalus Makino (MMPE, 100 mg/kg) was tested in lead-treated mice [69]. Along with recovered body weight, reduced erythrocyte lysis, and improved liver and kidney functions, MMPE reduced calreticulin (CRT) protein expression, which is associated with the ER stress pathway [69].
Liver Disease: In a rat model of high-fat diet-induced non-alcoholic fatty liver disease (NAFLD), a compound present in Potentilla chinensis, namely Asiatic acid (AAPC, at 4 and 8 mg/kg), was found to improve liver injury and reduce ER stress markers (i.e., GRP78, PERK, eIF2α, and CHOP) [71].

Importance of Signaling Events
Cytoskeletal alterations and protein trafficking dynamics are intertwined into signaling events. Indeed, these cellular activities are modulated by stimulation of a signaling cascade mediated by receptor activation. The contributors to these signaling pathways are numerous; their complexity is further increased through the large array of intracellular binding partners for each of these mediators [72]. Another level of complexity arises from post-translational modifications on each of these signaling mediators [72]. In sum, these all contribute to the challenges in defining the underlying aberration in a pathological disease. Drugs that target signaling events can include those that (1) elicit structural change, (2) inhibit enzyme activities through binding to catalytic sites, (3) inhibit enzymes by binding to an allosteric site, (4) are agonists or antagonists to ligand-receptor interactions, or (5) deregulate expression of a signaling molecule [72]. In each of our selected plant families, the major pathways that we assessed herein are the MAPK, PI3K/AKT/mTOR, and JAK/STAT pathways. Please see Table 6 for a summary of plant components investigated with respect to health relevance or associated disease models.

Cucurbitaceae Phytochemicals in Signaling
A schematic of the key findings is displayed in Figure 9.

Cucurbitacin A (CuA) PI3K/AKT/mTOR Signaling
Solid Tumors: In lung A549 adenocarcinoma cells, CuA (200 µM) reduced cell viability and clonogenic potential by inducing apoptosis and G2/M phase arrest [73]. These cellular alterations were accompanied by reduced activation of signaling mediators, including p-AKT, p-mTOR, and p-PI3K, although the total proteins for these latter two were also reduced [73]. In ovarian SKOV3 cancer cells, CuA (300 µM) similarly reduced cellular viability and colony formation ability, possibly by induction of G2/M arrest and apopto-sis [74]. Similar to A549 cells, the cellular alterations in SKOV3 cells were accompanied by reduced p-AKT, p-mTOR, and p-PI3K, although the total proteins of the latter two were also reduced [74].
sum, these all contribute to the challenges in defining the underlying aberration in a pathological disease. Drugs that target signaling events can include those that (1) elicit structural change, (2) inhibit enzyme activities through binding to catalytic sites, (3) inhibit enzymes by binding to an allosteric site, (4) are agonists or antagonists to ligandreceptor interactions, or (5) deregulate expression of a signaling molecule [72]. In each of our selected plant families, the major pathways that we assessed herein are the MAPK, PI3K/AKT/mTOR, and JAK/STAT pathways. Please see Table 6 for a summary of plant components investigated with respect to health relevance or associated disease models.

Cucurbitaceae Phytochemicals in Signaling
A schematic of the key findings is displayed in Figure 9.  [73]. These cellular alterations were accompanied by reduced activation of signaling mediators, including p-
Solid Tumors: In a neuroblastoma cell line (SHSY5Y), CuB (5 µM)-mediated reduction in cellular viability and the proliferative index was accompanied by a reduction in p-AKT, which coincided with increased PTEN [76]. Knockdown of PTEN resulted in the recovery of cell survival and proliferation upon CuB treatment, implicating the involvement of PTEN in CuB-induced cellular responses [76]. In a series of human astrocytoma cell lines, CuB (IC 50 of 0.93-0.49 µM) inhibited cell viability, clonogenic potential, invasion, and migration with an increased apoptotic response [77]. These functional outcomes were also accompanied by reduced p-AKT protein [77]. Similarly, in breast cancer cell lines, CuB (75 nM) inhibited cell survival and induced apoptosis. These cellular outcomes were accompanied by reduced cell surface tyrosine kinase receptor expression (i.e., HER2/neu and EGFR), suppression of integrins (another cell surface receptor), and reduction in p-AKT as well as p-ERK [78]. However, in an independent study, CuB (1.6 µM) reduced cellular viability and increased apoptosis, but reductions in p-AKT were not aligned with alterations in MAPK signaling [79]. In BEL-7402 hepatocellular carcinoma cells, CuB (100 nM) promoted apoptosis with increased DNA damage response [80]. Together with these alterations, CuB reduced p-mTOR, p-AKT, and p62 with increased levels of p-ULK1 and LC3B-II (which are autophagy markers) as well as elevated p-PTEN [80]. In the KKU-100 cholangiocarcinoma cell line, CuB reduced cell viability and increased apoptotic response (20 µM) [81]. These functional changes were accompanied by changes in the expression of p-PI3K and p-AKT [81]. In a series of colorectal cell lines, CuB (0.742 µM) hindered the interaction between laminin and integrin, which reduces tumor budding [82]. Via microarray profiling, it was demonstrated that gene expression of mediators within the PI3K/AKT and focal adhesion signaling events were altered by CuB [82].  These alterations were accompanied by reduced p-STAT3, p-STAT5, p-JAK2, and increased p-MAPK [83]. Changes in JAK-STAT signaling (i.e., reduced p-STAT3) were also noted in an independent study using Panc-1 pancreatic cancer cell line, though at higher doses of CuB (up to 3 µM) that coincided with reduced cell viability, G2/M phase arrest, and increased apoptosis [84]. In yet another Panc-1 study in which CuB (at 0.5-1.0 mg/kg) reduced tumor volume when cells in Matrigel were grafted into nude mice, alterations in JAK-STAT signaling (i.e., reduced levels of p-STAT3 and p-JAK2) were also noted [85]. In the human U2OS osteosarcoma cell line, CuB reduced cell viability (100 µM) and migration with increased apoptosis [86]. These alterations were accompanied by reduced p-JNK, p-ERK, p-p38, p-JAK2, and p-STAT3; however, all of their total proteins were also reduced [86]. In human HCT116 colorectal cancer cells, CuB reduced clonogenic potential and cellular viability (800 nM) along with an apoptotic response [87]. These cellular outcomes were associated with reduced p-JAK2 and p-STAT3 proteins [87]. In the lung A549 adenocarcinoma cell line, CuB hindered cell viability (0.9 µM), increased apoptotic response, and reduced clonogenic potential [88]. These changes were also associated with reduced p-STAT3 levels [88].

Cucurbitacin C (CuC) JAK/STAT Signaling
Solid Tumors: Across an array of cancer cell lines, CuC reduced their viability (IC 50 of 10-100 nM) along with reduced clonogenicity, migration, G2/M or G1 phase arrest, and elevated apoptotic response [92]. These events were accompanied by reduced p-AKT, although the GO/KEGG pathway analyses also identified the JAK/STAT pathway as the most altered in the hepatoma HepG2 cell line [92].

Cucurbitacin D (CuD) PI3K/AKT/mTOR Signaling
Cancers: In a series of human gastric cancer cell lines, including AGS cells, CuD (2 µM) induced apoptosis with increased reactive oxygen species (ROS) generation; these functional alterations were accompanied by reduced p-AKT and p-mTOR levels [93].

MAPK Signaling
Cancers: In MT-4 adult T cell leukemia cells, CuD (1 µM) promoted cell death and was associated with reduced p-MAPK, p-p38, and p-JNK [94]. In a series of pancreatic cancer cell lines including Capan-1, CuD (0.8 µM) reduced cellular viability with a G2/M phase arrest, increased apoptotic response and elevated ROS levels, which coincided with increased p-p38 levels, in the absence of change in p-JNK [95].

MAPK Signaling
Solid Tumors: In A549 cells, CuE (2.5 µM) induced apoptosis along with reduced p-STAT3 and p-MEK1/2 levels, whereas p-EGFR and p-ERK were elevated [100]. Interestingly, molecular docking simulation of the CuE-EGFR complex identified CuE interaction with the ATP binding site of the EGFR kinase domain; this interaction was stabilized by Hbonds with Leu694, Met769, Arg817, and Asp831 [100]. Across a series of cancer cell lines, including MDA-MB-468, CuE (100-200 nM) induced G2/M phase arrest and apoptosis; these cellular outcomes were associated with reduced p-STAT3, p-AKT, and p-ERK with increased levels of p-JNK [101].

JAK/STAT Signaling
Blood Cancers: Along with CuI-mediated apoptotic response in CD4+T cells from patients with Sezary syndrome, an aggressive type of lymphoma, CuI (30 µM) also induced a reduction in p-STAT3 (as well as total STAT3 levels) [103].
Solid Tumors: CuI (10 µM) treatment in NIH 3T3 cells led to a reduction in p-STAT3 and p-JAK2, which was confirmed in an array of tumor cell lines [104]. CuI was also shown to mediate this inhibition by disrupting the DNA binding activity of STAT3 and, henceforth, its subsequent effect on gene expression [104]. In an independent study, inhibition of CuI on p-STAT3 was confirmed across an array of cancer cells [105]. In cancer-associated fibroblasts (CAFs), CuI (50 nM) promoted apoptosis with inhibition of p-STAT3 [106]. In human malignant glioma cell lines, CuI (up to 400 nM) reduced cellular viability with an apoptotic response and induction of G2/M phase arrest [107]. These cellular outcomes were accompanied by reduced p-STAT3 [107]. CuI (IC 50 of 170-245 nM) response in glioblastoma cell lines resulted in increased p-AMPK with decreased p-p70S6K, p-mTOR, p-JAK, and p-STAT3 [108]. These changes coincided with cell death accompanied by the increased autophagic response (i.e., LC3B-II punctae and autophagosomes identified via transmission electron microscopy) [108]. In DU145 prostate cancer cells treated with EGF or IL-6, CuI (50 nM) was noted to reduce STAT3 activity and STAT3 nuclear localization [109]. In nasopharyngeal cell lines, CuI (1 µM) reduced cellular viability, and clonogenicity, while simultaneously inducing apoptosis [110]. These cellular outcomes were associated with reduced p-STAT3 [110]. In MDA-MB-468 breast cancer cells, CuI (1 µM) reduced p-STAT3 levels, which accompanied reduction in cellular viability, adhesion, migration, and tube formation [111].

MAPK Signaling
Blood Cancers: CuI (1 µM, non-toxic doses) was found to induce p-JNK in the BJAB Burkitt lymphoma cell line and the pre-acute NALM-6 lymphocytic leukemia cell line; CuI treatment, however, increased VEGF levels [112].
Solid Tumors: In colon cancer cell lines, CuI (10 nM-1 µM) was most effective in reducing cellular viability and inducing apoptosis in cells lacking a K-RAS activating mutation [113]. In A549 cells, CuI (400 nM) reduced cellular viability and clonogenicity with increased apoptosis [114]. These changes were accompanied by elevated LC3B-II (autophagy marker) with reduced AKT, PI3K, p-PI3K, p-mTOR, p-ERK, and p-STAT3 [114]. When autophagy was inhibited using an inhibitor (3-methyladenine, 3-MA), the effect of CuI on ERK/mTOR/STAT3 signaling was reversed, suggesting that autophagic induction is responsible for the CuI-mediated alterations through this signaling pathway [114]. In gastric cancer cell lines, CuI reduced cellular viability (IC 50 of 97.4 to 123 nM) with G2/M phase arrest, induction of apoptosis, and DNA damage [115]. These changes, however, were not accompanied by changes in p-STAT3 but only elevated p-p38 and p-JNK proteins [115]. Furthermore, pretreatment with a JNK inhibitor (SP600125) antagonized the effect of CuI on cellular viability and apoptosis, implicating the JNK pathway in mediating these observed cellular responses of CuI [115].

Cucurbitacin Q (CuQ) JAK/STAT Signaling
Solid Tumors: Although CuQ (0.5-1 mg/kg) inhibited tumor growth most effectively using A549 cells in a mouse xenograft model along with reduced p-STAT3 levels in a series of cancer cell lines, CuQ (10 µM) did not elicit a change in p-JAK2 (with contrasts with responses to CuA, CuB, CuE, and CuI) [117].

Cucurbitacin Derivatives JAK/STAT and AKT Signaling
Solid Tumors: An extract from the leaves of C. colocynthis (L.) Shrad (containing cucurbitacinglycosides) was tested in breast cancer cell lines. The cucurbitacin treatment surprisingly increased p-STAT3 levels, which was associated with G2/M phase arrest and apoptosis; however, p-AKT and cell survival response was reduced [118].

Ericaceae Phytochemicals in Signaling
A schematic of the key findings is displayed in Figure 10. that the long side chain of CuII sits into the binding pocket by two H-bonds with Met769 while making additional H-bonds at the active site with Arg817, Thr830, and Asp831 [120]. In A549 cells, CuIIb (80 µM) reduced cellular viability and increased apoptosis along with G2/M phase arrest [121]. These cellular outcomes were accompanied by reduced p-STAT3, p-EGFR, p-MEK1/2, and p-ERK1/2 [121]. With the finding that EGFR kinase activity was reduced, molecular docking was performed; this analysis investigated the interaction between CuIIb and EGFR (using the X-ray crystal structure of the kinase domain of EGFR) [121]. CuIIb was identified to fit into the hydrophobic cleft of the ATPbinding site with multiple hydrophobic interactions arising from Leu694, Phe699, Val702, Ala719, Met742, Leu768, Met769, Phe771, and Leu829 in addition to H-bonds at Leu694, Met769, Arg817, and Asp831 [121].

Ericaceae Phytochemicals in Signaling
A schematic of the key findings is displayed in Figure 10.

PI3K/AKT/mTOR Signaling
Blood Cancers: Antho 50 (25 µg/mL, a bilberry extract from Vaccinium myrtillus L.) induced apoptosis in chronic lymphocytic leukemia (CLL) cells; this cellular outcome was associated with reduced pro-survival signaling, including p-AKT and p-BAD levels [122]. Studies have also been performed using whole cranberry extracts from berry juice (CB, 25-50 µg/mL) in the human leukemia HL-60 cell line leading to apoptosis, which was associated with increased dephosphorylation of BAD along with reduced p-AKT [123].
Solid Tumors: In the neuroblastoma SMS-KCNR cells, an enriched fraction of cranberry oligomeric proanthocyanidins (PAC-1A, 25 µg/mL) induced a cytotoxic response along with G2/M phase arrest and increased apoptotic response [124]. These cellular outcomes were associated with reduced pro-survival (i.e., p-AKT, p-PI3K, and p-mTOR) and increased pro-death (i.e., increased p-JNK) signaling events [124]. Another cranberry proanthocyanidin extract (PAC-1, 50 µg/mL) was tested on SKOV3 ovarian cancer cells, which reduced cellular viability along with G2/M phase arrest and increased apoptosis; these outcomes were similarly associated with reduced p-AKT levels [125]. In addition, proanthocyanidin-enriched extracts from cranberries (CPAC, 25 µg/mL) reduced cellular viability in DU145 prostate cancer cells that were associated with alterations in signaling pathways, including reduced p-AKT (and total AKT, however) with increased p-p38 and p-ERK1/2 [126]. In an in vivo study, whole cranberry powder

Ericaceae Extracts PI3K/AKT/mTOR Signaling
Blood Cancers: Antho 50 (25 µg/mL, a bilberry extract from Vaccinium myrtillus L.) induced apoptosis in chronic lymphocytic leukemia (CLL) cells; this cellular outcome was associated with reduced pro-survival signaling, including p-AKT and p-BAD levels [122]. Studies have also been performed using whole cranberry extracts from berry juice (CB, 25-50 µg/mL) in the human leukemia HL-60 cell line leading to apoptosis, which was associated with increased dephosphorylation of BAD along with reduced p-AKT [123].
In colorectal HCT-116 cancer cells, a blueberry extract (BE, IC 50 of 1.26 mg/mL) reduced cellular viability along with an apoptotic response and G0/G1 cell cycle arrest [128]. Associated with these functional changes was reduced expression of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNFα) in addition to reduced levels of p-AKT protein [128]. In breast MDA-MB-231 cancer cells, a whole blueberry extract from Vaccinium angustifolium (30 µL/mL) hindered hepatocyte growth factor (HGF)-induced migration; this cellular outcome was associated with reduced p-AKT levels [129]. In another study with relevance to chronic use of Snus (smokeless tobacco containing N-nitrosamines), blueberry supplementation (Vaccinium myrtillus, 0.5 g/kg) in a rat model (administered snus intragastrically) promoted the health of epithelium in the forestomach, which was associated with reduced expression of p-AKT [130]. Phytochemicals may also offer protection against dysregulated angiogenic events [131]. In this regard, blueberry components (anthocyanins (ACN, 60 µg/mL) and phenolic acids (PA, 60 µg/mL) from whole blueberries) were tested in the HUVEC endothelial cell system [131]. Although ACN reduced tube formation with reduced AKT1 protein in an in vitro angiogenesis assay, PA increased angiogenesis with elevated AKT1 protein [131]. These results implicate different cellular outcomes to specific phytochemical fractions within a specific extract.
Cardiovascular Disease: One characteristic associated with cardiovascular disease is elevated blood levels of microvesicles (MVs) [132]. The effect of a bilberry extract (BE) was investigated on MVs using patient samples wherein they analyzed baseline and 8 weeks post-treatment levels [132]. The findings show that BE reduced blood-derived MVs, in the absence of BE-mediated toxicity (up to 1000 µg/mL) on HUVECs, and reduced p-AKT with no change in p-p38 expression [132].
Perimenopause: Towards a search for alternative treatments to hormone replacement therapy, bilberry anthocyanins (BA, similar in structure to phytoestrogens, at 25, 70, and 140 mg/kg) was examined in a premenopausal rat model [133]. In addition to reduced levels of cholesterol and triglycerides along with improved ovarian morphology and function, elevated levels of GPR30, AKT, and ERK2 mRNA were noted [133], suggesting that activation of AKT signaling may contribute to some health benefits to alleviate perimenopausal symptoms.
Metabolic Disorders: An anthocyanin-enriched blueberry extract (BAE, 200 mg/kg) was tested in a high-fat diet (HFD) mouse model and found to reduce body weight, the weight of liver/adipose tissues, and function/histology of liver/adipose tissues [134]. BAE also reduced triacylglycerides and ceramide synthesis, which were associated with reduced PKC-zeta expression, which is proposed to alter AKT signaling [134]. In human retinal capillary endothelial cells (HRCECs), a blueberry anthocyanin extract (BAE, from Vaccinium ashei) not only antagonized the reduction in cellular viability induced by high glucose (HG) but also reduced AKT and VEGF protein levels [135].
Cognitive Functions: Spatial working memory may also be affected by the consumption of components from blueberries [137]. Compared to aged rats on an unsupplemented diet, aged rats on a diet supplemented with blueberries (BB) performed better in spatial working memory tasks [137]. These changes were correlated with the activation of CREB (cAMP response element binding protein) along with increased BDNF (brain-derived neurotrophic factor) and p-ERK1/2, p-AKT, and p-mTOR in the hippocampal region [137].
Solid Tumors: In a dimethylbenzanthracene (DMBA)-induced hamster model of oral cancer, blueberry supplementation (200 mg/kg) reduced tumor burden; this outcome was associated with reduced JAK2 and STAT3 RNA expression and reduced p-JAK2 and p-STAT3 protein (along with reducing its nuclear translocation) [139]. Although the blueberry supplement failed to alter cellular viability in SCC131 oral cancer cells, the purified malvidin component induced a marked growth reduction (IC 50 of 62 µM) associated with reduced p-JAK2 and nuclear p-STAT3 levels [139].

Ericaceae Purified Phytochemicals PI3K/AKT/mTOR Signaling
Solid Tumors: Estrogen receptors (ER) may contribute to the pathogenesis of colorectal carcinoma (CRC) with support of ER pathway targeting by pterostilbene (Pter, a component in blueberries and analog of resveratrol); via molecular docking simulations, Pter was identified to dock into the ER-β active site with high affinity, similar to 17-β-estradiol [153]. Using Caco-2 and HCT-116 colorectal cancer cells, Pter (IC 50 from 2.44 to 1.07 µM) not only reduced cellular viability mediated by 5-fluorouracil (5-FU) along with reducing ER-β levels (9%), but it also reduced p-AKT and p-ERK levels [153]. In human myeloma cell lines, Pter (IC 50 of 24-22.8 µM) not only reduced cellular viability, but it also increased apoptosis; these cellular outcomes were associated with reduced PI3K and p-AKT along with increased p-p38 but no change in p-JNK [154]. In A549 and H460 non-small cell lung cancer cell lines, Cinnamtannin D1 (CNT D1, A-type procyanidin trimer, a component isolated from Rhododendron formosanum leaf extracts, 50-200 µM) reduced cellular viability along with G1 phase arrest; while there was an absence of apoptosis, autophagic induction was identified through vacuole formation and an LC3B-II punctate pattern [155]. Together with these cellular features, CNT D1 also reduced p-mTOR and p-AKT levels while also increasing p-ERK1/2 levels [155].
Inflammation: The cellular effect of rhodomeroterpene (RMT, 30 mg/kg, a meroterpenoid from Rhododendron) was tested in a murine ischemia-reperfusion (I/R)-induced AKI mouse model and found to improve the inflammatory response and kidney health as assessed via kidney injury markers and histological analyses [156]. In support of these in vivo findings, when macrophages were co-treated with LPS/IFNγ, RMT (40 µM) also reduced the expression of pro-inflammatory mediators (i.e., IL-1β, IL-6, and TNFα) as well as p-PI3K and p-AKT [156]. In HUVEC endothelial cells, hyperoside (purified component from the leaves of Rhododendron brachycarpum) protected against the pro-inflammatory response by diminishing high mobility group box 1 (HMGB1)-induced activation of p-AKT and p-ERK1/2 [157].

Rosaceae Phytochemicals in Signaling
A schematic of the key findings is displayed in Figure 11.

PI3K/AKT/mTOR Signaling
Blood Cancers: Using adult MOLT-4 lymphoblastic leukemia cells, extracts from dried fruits of Rosa cymosa (RCE, 60 µg/g) reduced tumor volume in an in vivo mouse xenograft model [164]. In in vitro assays, RCE reduced cellular viability (IC50 of 88.8-114.8 µg/mL) with induction of apoptosis [164]. These changes were associated with changes in ER stress markers as well as elevated PTEN, p-PTEN, and p-c-Raf with decreased p-AKT and p-STAT3 proteins [164].

Rosaceae Extracts PI3K/AKT/mTOR Signaling
Blood Cancers: Using adult MOLT-4 lymphoblastic leukemia cells, extracts from dried fruits of Rosa cymosa (RCE, 60 µg/g) reduced tumor volume in an in vivo mouse xenograft model [164]. In in vitro assays, RCE reduced cellular viability (IC 50 of 88.8-114.8 µg/mL) with induction of apoptosis [164]. These changes were associated with changes in ER stress markers as well as elevated PTEN, p-PTEN, and p-c-Raf with decreased p-AKT and p-STAT3 proteins [164].
In lung A549 cells, an extract from the petals of Rosa gallica (RPE, 100-400 µg/mL) reduced cellular viability as well as inhibited migration and invasion; these changes were accompanied by reduced expression of p-EGFR, p-c-Raf, p-MEK1/2, p-mTOR, and p-AKT proteins [169]. In this same lung cancer cell line, a polyphenol-enriched plum pulp extract (PPP, 160 µg/mL) from Wushancuili elicited a marked reduction in cellular viability, which coincided with reduced p-PI3K and p-AKT levels [170]. Red-flesh (AFP) or peel (APP, phenolic content is higher) component of apples (Meihong variety) was tested on breast cancer cell lines (250-1000 µg/mL); it was identified that the APP was more potent in mediating reduction in cellular viability along with G0/G1 phase arrest and apoptotic response; these functional changes were associated with reduced p-AKT and p-BAD [171]. In human prostate cancer cells, four extracts from the fruits of Rubus coreanus Miquel (RCM, 100 µg/mL) (i.e., 50% ethanol extract of unripe RCM (UE), aqueous extract of unripe RCM (UH), 50% ethanol extract of ripe RCM (RE), and aqueous extract of ripe RCM (RH) did not mediate any alteration in cellular viability [172]. However, most of the extracts (except for RE) inhibited cellular migration and invasion, which was accompanied by reduced p-PI3K and p-AKT levels following UA treatment [172]. In HeLa and SiHa cervical cancer cell lines, a polysaccharide extract from Rosa rugosa petals (RRP, 800 µg/mL) reduced cellular viability along with induction of apoptosis and autophagy (i.e., increased LC3B-II and reduced p62 levels) [173]. The autophagic-induced cell death response by RRP involved reducing p-AKT and p-mTOR levels [173].
Tissue Regeneration: Tissue regeneration using stem cells is a valuable option for therapeutic purposes, such as in the process of aging in which stem cell quality and quantity are reduced [174]. Ethanol extracts from the apple, Malus pumila Mill, were tested on adult stem cells from adipose tissue (ADSCs) and cord-blood mesenchyme (CB-MSCs) [174]. Using these cells, the apple extract (0.5-1%) supported stem cell proliferation and cytokine production (i.e., VEGF and IL-6), which were accompanied by activation of p-p70S6K, p-S6RP, p-eIF4E, and p-Raptor [174].
Inflammation: Intestinal bowel diseases (IBD) involves the stimulation of microvascular endothelial cells to which circulating immune cells adhere [175]. The effect of a black raspberry extract (BRE, 100 µg/mL) on primary human esophageal microvascular endothelial cells (HEMEC, stimulated with TNFα/IL-1β) reduced gene expression of cell adhesion molecules (i.e., ICAM-1 and VCAM-1), which likely caused diminished adhesion of U937 monocytes [175]. Along with these cellular changes, BRE diminished gene expression of pro-inflammatory mediators (i.e., COX-2 and PGE2 activity) and reductions in VEGFinduced p-AKT in addition to p-ERK1/2 and p-JNK [175]. In another study, total flavonoids (TFs, 50-100 mg/kg, intragastric administration) from the leaves of Eriobotrya japonica were tested on mice exposed to cigarette smoke (CS) to examine protective effects and underlying mechanism of action [176]. The treatment with TF reversed the loss of body weight and pulmonary edema; these changes were associated with improved lung histology and lung health as well as reduced levels of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNFα in serum) with increased p-AKT and reduced p-JNK levels [176]. The anti-inflammatory activity of a water extract using the dried inner bark of the stems of Sorbus commixta (Sc-WE, 100 mg/kg) was tested in an in vivo mouse ear edema model; in this system, Sc-WE reduced edema without any cytotoxic response [177]. In the in vitro model using macrophages stimulated with LPS, Sc-WE (300 µg/mL) hindered the activation of p-AKT, p-PI3K, and p-PDK1 [177]. Another bark extract from Prunus jamasakura (and the purified component, sakuranetin at 60 µM) was tested in rat hepatocytes stimulated with IL-1β and found to reduce pro-inflammatory nitric oxide (NO) production along with diminished levels of p-AKT and IL-1 receptor [178].
Muscle Aging: During the process of aging, loss of muscle mass is one characteristic leading to diminished quality of life in the elderly population [179]. A leaf extract from Eriobotrya japonica (LE, 50 mg·kg −1 ·day −1 ) was tested on aged rat muscles, and although there was no alteration in body weight, forelimb grip strength was increased together with increased muscle mass [179]. In C2C12 murine myoblasts, LE (0.25-2.5 µg/mL) supported their differentiation without a change in cell survival; these cellular outcomes were accompanied by elevated p-AKT (as well as total AKT) and p-4E-BP1 [179]. Since the quality of life is promoted by maintaining the appropriate mass of skeletal muscles, compounds with a high ursolic acid content, such as that present in an extract from Aronia melanocarpa (AME), were tested in rats [180]. Although AME (2.9 g/kg) did not alter muscle weight or muscle protein synthesis after resistance exercise, there were elevated p-AKT levels along with elevated p-ERK1/2, p-mTOR, and p70S6K [180].
Neurodegeneration Diseases: The nervous system is the primary accumulation point for heavy metal cadmium toxicity, potentially leading to neurogenerative diseases [181]. The protective action of a polysaccharide ethanol extract (PAP) derived from the roots of Potentilla anserine L. was noted in a mouse model, which led to reduced cadmium-induced reductions in food consumption, diarrhea and convulsions, amongst other symptoms of cadmium-induced toxicity [181]. Likewise, in human and murine neuroblastoma cell lines, although PAP did not alter cellular viability (up to 400 g/L) alone, it did promote viability (25 mg/L) against the detrimental effects of cadmium along with a reduction in apoptotic response and reduced ROS levels [181]. Furthermore, PAP reduced cadmium-induced activation of p-AKT and p-mTOR [181].
Metabolic Diseases: Extracts prepared from the fruit of the chokeberry (CBE), Aronia melanocarpa, were tested in vivo (at 100-200 mg·kg −1 ·day −1 ) to determine its efficacy in improving the metabolic condition of rats fed with a high-fructose diet [182]. Together with reduced gain in body weight and adipose tissue, the fasting blood glucose, insulin levels in plasma, triacylglycerides, and total cholesterols were reduced by CBE treatment [182]. In addition, there were diminished levels of the pro-inflammatory cytokines (i.e., IL-6 and TNFα) with altered gene expression of molecules involved in insulin signaling (i.e., elevated PI3KR1 and reduced PTEN RNA) in the epididymal adipose tissues [182]. The anti-diabetic activity of root, fruit, and leaf extracts from Sarcopoterium spinosum sp. were tested for their effects on 3T3-L1 pre-adipocytes [183]. Using non-toxic doses of these plant extracts (<1 mg/mL), it mediated glucose uptake (more potent with root and leaf than fruit extracts) as well as inhibition of the PI3K/AKT pathway [183]. In diabetic rats, extracts from fruits (without seeds) of Crataegus pinnatifida Bge (HPE, 300 mg/kg) reduced body weights, fasting blood glucose, cholesterol, triacyglyceride, and insulin levels along with improved tissue histology [184]. HPE also reduced the expression of pro-inflammatory mediators (i.e., IL-6 and TNFα) and recovered p-AKT and p-PI3K levels [184]. A methanol extract derived from the aerial components of Alchemilla monticola (ALM, 5-25 µg/mL), in which the major components are kaempferol-3-O-glucoside (AST, 5-25 µM) and quercetin-3-O-rhamnoside (QUE, 5-25 µM), were all tested for anti-adipogenic effects in human adipocytes [185]. In a molecular docking simulation, it was noted that AST and QUE had the lowest binding constants with PI3K and PPARγ, which may provide a mechanism of action for these specific metabolites [185]. The ALM extract was reported to diminish the abundance of lipid droplets (LDs) while reducing gene expression of adipogenic genes (i.e., CEBPA and PPARG) [185]. The ALM extract was also the most potent in reducing the protein levels of AKT and PI3K [185].

JAK/STAT Signaling
Inflammation: In LPS-activated macrophages, a water extract from the unripe fruits of Rubus coreanus Miquel (RF) reduced p-p38 levels and NO generation [186]. Associated with this response, RF diminished the gene expression of the ER stress marker, CHOP, as well as STAT1, STAT3, and JAK2, in the absence of cytotoxicity (25-200 µg/mL) [186]. In another study using HaCaT cells, a water extract from Sanguisorbae Radix (WSR) did not mediate any change in cellular viability (up to 50 µg/mL) with only a subtle effect at a higher dose (100 µg/mL) [187]. In response to TNFα/IFNγ stimulation, WSR (low doses) inhibited pro-inflammatory chemokine expression (i.e., TARC, RANTES, MDC, and IL-8) and diminished expression of p-JAK2 and p-STAT1 [187]. An ethanol extract from black raspberry powder (BRB-E, containing diverse phytochemical constituents, 200 µg/mL) from Rubus occidentalis was tested on CD4+ and CD8+ T cells; in response to CD3/CD28 activation, BRB-E reduced cellular viability, as well as IL-6, mediated p-STAT3 and IL-2 mediated p-STAT5 levels [188].

MAPK Signaling
Solid Tumors: Pre-treatment of human lung cancer cells (A549) with strawberry extracts (Fragaria x ananassas cv. Earliglow, diluted at 1:250-1:500) resulted in a reduction in cellular viability as well as antagonism towards ultraviolet irradiation (UVB)-induced activation of p-ERK and p-JNK with no change in p-p38 [189]. In B16 murine melanoma cells, a methylene chloride fraction (1mg/mL, from Whole Geum japonicum Thunberg plant powder) inhibited cell attachment and migration; this fraction also inhibited angiogenesis in HUVEC endothelial cells [190]. Together with these cellular outcomes, this fraction reduced gene expression of CD44 (involved in tumorigenic response) and TIMP2 (involved in activation of MMP-2) along with increased p-JNK and p-p38 but no change in p-ERK [190]. In HepG2 cells, three different extracts from different plum (Prunus salicina Lindl cv Soldam) stages of maturity (immature extract (IPE), mid mature extract (MMPE), and mature extract (MPE)) were tested [191]. Phorbol ester (PMA) induction of cellular migration, as well as MMP-9 (expression and activity), were markedly reduced by IPE (12.5-100 µg/mL) [191]. In addition, other alterations by IPE in PMA-treated HepG2 cells included reduced p-p38, p-JNK, and p-ERK levels [191]. The effect of total phenolics from dark sweet cherries (WE) along with enriched fractions of anthocyanins (ACN) and proanthocyanidins (PCA) were tested towards their anti-neoplastic effects using breast cancer MDA-MB-453 cells in an in vivo mouse xenograft model [192]. In this regard, treatments with these agents (150 mg/kg) reduced tumor growth along with elevated levels of p-ERK1/2 [192]. With ACN, there were enhanced anti-neoplastic effects via downregulation in the expression of signaling proteins (i.e., AKT, STAT3, p38, and JNK) [192]. By using these same breast cancer cells, the dark sweet cherry anthocyanin-enriched phenolics (WE, 83 µg/mL) along with ACN (anthocyanin-rich, 19 µg/mL) and PCA (proanthocyanidin-rich, 22.5 µg/mL) from Prunus avium were found to reduce p-AKT while increasing p-p38, p-JNK, and p-ERK1/2 [193]. In the HT1080 fibrosarcoma cell line, a methanolic extract from Agrimona Pilosa Ledeb (APLME, 20-80 µg/mL) reduced cellular viability and invasion upon VEGF stimulation; these cellular outcomes also reduced MMP-2 and MMP-9 levels [194]. Furthermore, the levels of p-ERK and p-JNK were reduced following stimulation with PMA, as a tumor promoting agent [194]. In colorectal SW-480 and HT-29 cancer cells, a leaf extract derived from Chaenomeles japonica L. (PRE) inhibited migration and invasion by reducing the activities of MMP-2 and MMP-9 [195]. These changes were accompanied by diminished levels of p-ERK and p-AKT in SW-480 but increased p-ERK with reduced p-AKT in HT-29 cells [195].
Inflammation: With attempts to uncover dietary considerations for intestinal bowel diseases (IBD), apple powder (from the skin and pulp) from two varieties of apples (Marie Menard and Golden Delicious) were tested in a mouse model of colitis (HLA-B27 transgenic rats, characterized by intestinal inflammation with histological alterations similar to human IBD) [197]. After a 12-week treatment period, symptoms of IBD were reduced (i.e., diarrhea) with extensive changes in gene expression patterns (i.e., reduction in MAPK pathway, TNFα-NFkB), which were more marked with the Marie Menard variety [197]. An apple polyphenol extract (APP) was tested to identify whether it could mediate protection against cigarette-smoke-induced lung injury and inflammation using mice exposed to cigarette smoke [198]. APP treatment (up to 300 mg/kg) via intragastric administration diminished the inflammatory cell infiltration in the lung tissue with improved lung tissue histology [198]. At a cellular level, APP treatment reduced gene expression of the pro-inflammatory cytokines (i.e., TNFα and IL-1β) and reduced p-p38, as noted in histochemical sections [198]. In LPS-stimulated mice, extracts from the bark of Prunus yedoensis (PYE, (10, 50, 250 mg/kg) markedly reduced pro-inflammatory cytokines in serum (i.e., TNFα and IL-6) [199]. This supports findings from in vitro macrophage studies wherein PYE (up to 200 µg/mL) also reduced the gene expression of these cytokines induced by LPS, which were accompanied by reduced levels of p-p38, p-JNK, and p-ERK1/2 [199].
The anti-inflammatory potential of extracts from the berries of Crataegus laevigata (CLE) was tested on HaCaT cells [200]. In the absence of any changes in cytotoxicity from CLE (10-100 µg/mL) with LPS-stimulated HaCaT cells, CLE reduced the expression of pro-inflammatory cytokines (i.e., IL-8 and TARC) as well as p-p38, p-ERK, and p-JNK [200]. HaCaT cells treated with TNFα/IFNγ were also utilized to test anti-inflammatory responses with extracts from the leaves of the Rosa davurica Pall plant (RDL) [201]. RDL (100 µg/mL) did not alter cellular viability in cells, but it did suppress TARC and IL-6 secretion along with p-p38, p-JNK, and p-ERK [201]. The anti-inflammatory action of the peel extract from Cydonia oblonga miller was tested in THP-1 human myelomonocytic cells [202]. Although the extract (20 µg/mL) did not alter cellular viability, it diminished LPS-stimulated secretion of TNFα (partly via IL-6), IL-10, and IL-8 [202]. The signaling cascade mediated through this extract was the inhibition of p-p38 and p-AKT [202]. The skin moisturizing and antiinflammatory responses of an extract from the whole plant of Filipendula palmata (FPE) were tested on HaCaT cells [203]. Although FPE (10-200 µg/mL) did not alter cellular viability, it reduced ROS generation when cells were stimulated with TNFα/IFNγ [203]. Furthermore, FPE inhibited the gene expression of pro-inflammatory cytokines (IL-8 and TARC) as well as promoted elevated levels of hyaluronic acid (HA) [203]. FPE also inhibited the p-ERK1/2, p-JNK, and p-p38 levels [203].
Allergic Responses: In addition to unripe fruit extracts of the black raspberry plant, namely Rubus coreanus Miquel, that antagonize allergic responses, ripe fruit extracts from this plant (RFRC) were also tested in an animal model [204]. In the in vivo mouse model, mortality was reduced when the mast cell degranulation compound 48/80 was intraperitoneally injected following anal administration of RFRC (10-1000 mg/kg) [204]. Likewise, in human mast cell lines, RFRC (100 µg/mL) inhibited the release of histamine, intracellular calcium, and pro-inflammatory cytokine expression and secretion (i.e., TNFα, IL-1β, IL-6) [204]. Furthermore, there was a notable reduction in p-p38, p-JNK, and p-ERK upon mast cell activation [204].
Neurodegenerative Disease: A total flavonol extract (TFs) from Rosa laevigata Michx was tested for its ability to mediate protection against neurodegenerative diseases [205]. Specifically, TFs were examined with respect to protection against oxidative injury in which hydrogen peroxide can be detrimental to neuronal health by inducing cell death [205]. In this regard, H 2 O 2 -stimulated pheochromocytoma PC-12 cells treated with TFs (100-500 µg/mL) recovered cell health in addition to the reduction in pro-inflammatory cytokine production (i.e., IL-1, IL-6, and TNFα) and reduction in p-ERK, p-JNK, and p-p38 levels [205].
In an animal model subjected to BCCAo (bilateral common carotid artery occlusion) in which there is reduced cerebral blood flow leading to neurological complications, an extract from unripe fruit of Prunus mume (200 mg·kg −1 ·day −1 ) was tested [206]. The extract decreased both microglial and astrocytic activation with a reduction in pro-inflammatory mediators (i.e., COX-2, IL-1β, and IL-6) protein expression and a reduction in p-p38 activation in the hippocampus [206].
Liver Injury: Carbon tetrachloride (CCl 4 )-induced liver injury is associated with the induction of oxidative stress, and thus, antioxidants arising from phytochemicals may pro-vide some health benefits to hinder the detrimental effects associated with this injury [207]. In this regard, extracts from the fruits of Chaenomeles thibetica (CTE, 40-100 mg/kg) were tested in a rat model of CCl 4 -induced liver injury [207]. CTE elevated glutathione (GSH) levels, as well as decreased liver enzymes (ALP) and total bilirubin, as a marker of liver function, with decreased histological evidence of liver damage [207]. Along with these outcomes, CTE was noted to recover cellular viability (up to 250 µg/mL) as well as elevate p-JNK, p-ERK, and p-p38 levels in HepG2 cells treated with CCl 4 [207].
Skin Aging: Photoaging of the skin, a critical barrier between organs and environment, can be induced by ultraviolet B irradiation (UVB) [208]. Ethanol extracts from the twigs of Sorbus commixta (STE) were found to mediate protection from UVB-stimulated human dermal fibroblast cells (NHDF); STE (200 µg/mL) treatment increased cellular viability, reduced ROS generation, and protein expression of secreted MMP-1, MMP-2, and MMP-3 [208]. These changes were accompanied by reduced p-p38, p-ERK, and p-JNK (and total JNK) [208]. Extracts from cherry blossoms of Prubus yeonesis (CBE) were also tested on UVB-irradiated NHDFs [209]. In the absence of any alteration in cellular viability, CBE (100 µg/mL) reduced UVB-induced gene expression of MMP-1 and MMP-3 along with increased type I pro-collagen RNA levels [209]. These changes were accompanied by reduced p-ERK, p-JNK, and p-p38 [209]. Furthermore, an extract from the dried fruit of Rubus idaeus L (RI, 1-100 µg/mL) also increased cellular viability, reduced ROS production, and levels of secreted MMP-1 and IL-6 along with increased pro-collagen type I RNA [210]. These changes were accompanied by reduced p-ERK, p-JNK, and p-p38 under these conditions [210].
Polyphenol extracts from the fruits of Crataegus pinnatifida (HPE) were also tested for their protective benefit towards UVB-induced photoaging on dermal fibroblasts and keratinocytes (HDF and HaCaT cells) [211]. HPE was noted to recover UVB-induced reduction in cellular viability (5-10 µg/mL) along with reducing ROS production and MMP protein production [211]. Pro-collagen type I protein expression was also elevated with HPE treatments [211]. In an in vivo mouse study wherein mice were orally exposed to HPE (100 or 300 mg·kg −1 ·day −1 ) along with UVB irradiation [211], the extract reduced MMP-1, MMP-3, and MMP-9 with improvements in histology (i.e., reversal of elastic fiber thickening, disorganization, and hyperplasia along with improved skin moisture) [211]; moreover, HPE reduced UVB-induced p-p38, p-ERK, and p-JNK levels [211]. Extracts prepared from the leaves of Pourthiaea villosa (PVDE, 50-100 µg/mL) were tested on HDFs and found to mediate protection against H 2 O 2 -induced cell death [212]. PVDE also reduced ROS production, which occurred along with the reduction in MMP-2, MMP-3, and MMP-9 activities as well as p-p38 and p-JNK (although p-ERK was elevated) [212]. Ethanol extracts from Potentilla glabra (Pg-EE) were also tested in HaCaT cells in response to UVB-induced photoaging [213]. With only a subtle growth-promoting response on cellular viability of Pg-EE (200 µg/mL), there was the diminished expression of pro-inflammatory mediators (i.e., IL-1β and IL-6), elevated RNA gene expression of the skin barrier and hydration factors, as well as reduced levels of p-ERK1/2 and p-p38 levels [213].
Hair Growth: An extract prepared from Crataegus pinnatifia fruits was tested on human dermal papilla cells (hDPCs) to investigate its effects on modulating hair growth, which may offer some benefit to improving blood circulation to the hair follicle to stimulate its growth [214]. While the extract increased the proliferative capacity (1 µg/mL, 40%), it also elevated p-p38, p-ERK, and p-JNK, as well as p-AKT [214].

Rosaceae Purified Phytochemicals PI3K/AKT/mTOR Signaling
Solid Tumors: In an array of cancer cell lines, a compound isolated from Potentilla discolor Bunge, namely PDB-1 (C-27-carboxylated-lupane-triterpenoid derivative), was found to reduce cellular viability (most potent at IC 50 of 7.8 µM in A549) along with induction of G2/M phase arrest; these cellular outcomes were associated with reduced p-PI3K, p-mTOR, and p-AKT levels [215]. A compound (ellagic acid) isolated from an ethanol extract from the root of Sanguisorba officinalis was tested on murine melanoma cells (B16F10) to uncover its mechanism of action [216]. Along with its growth-reducing potential (100-300 µg/mL), it induced apoptosis and G1 arrest, which was associated with reduced levels of p-AKT, p-p70S6K, and p-ERK1/2 along with increased PTEN activity [216].
Cardiovascular Diseases: Pentacyclic triterpenoids, euscaphic acid (EA), and tormentic acid (TA) were isolated from roots from Potentilla anserine L and tested in EA.hy926 cells, a human umbilical vein endothelial cell line [217]. While both agents protected the cells against hypoxia-induced cellular damage, they mediated differential responses in signaling events wherein EA induced p-ERK1/2 (with reduced p-AKT) and TA activated both p-ERK1/2 and p-AKT [217].
Inflammation: The critical cellular component of articular cartilage, namely chondrocytes, contributes to the pathogenesis of osteoarthritis (OA) in which IL-1β is a critical contributor to OA development; antagonizing IL-1β activities may be of benefit to these patients [219]. One compound from Rosa agrestis, namely astragalin, did not affect cellular viability (20-80 µg/mL) in these cells, but it did reduce IL-1β-induced responses, including expression of pro-inflammatory mediators (i.e., NO and PGE2) as well as p-ERK1/2, p-JNK, and p-p38 levels [219]. In A549 cells, the apple tree flavonoid phloretin was also tested for its effects on eliciting anti-inflammatory responses [220]. In response to IL-1β stimulation, phloretin (3-100 µM) reduced the expression of pro-inflammatory cytokines (i.e., PGE2, COX-2, IL-8, and IL-6) [220]. Furthermore, these changes were accompanied by reduced levels of p-AKT, p-ERK, p-JNK, and p-p38 [220]. Since inappropriate activation of human neutrophils can lead to damage to tissues along with the pathogenesis of the disease, finding therapies to hinder their aberrant activation would be of high clinical benefit [221]. In in vitro studies, N-formylated peptides (fMLP, which activates GPCRs) were utilized to simulate neutrophil activation and examine protection mediated by 2 -3-dihydroxy-5methoxybiphenyl (RIR-2), which is obtained from a methanol extract derived from the roots of a variety of Rhaphiolepsis indica [221]. In normal human neutrophils, RIR-2 (IC 50 of 2.57 µM) diminished fMLP-stimulated migration with no change in cellular viability [221]. Interestingly, although RIR-2 did not alter ligand-binding to the receptor, it did hinder the interaction between G i β and p-Src and with PLC, and moreover, RIR-2 diminished fMLP-induced activation of p-p38, p-AKT, and p-ERK along with reduced p-PLC and p-PKC [221].
Skeletal Maintenance: Maintenance of the skeleton is critical in post-menopausal women, who commonly undergo hormone therapy to oppose osteoporosis [222]. However, this treatment regimen is associated with an increased risk of myocardial infarctions, strokes, and cancer [222]. In this regard, using calvarial osteoblasts isolated from rats, prunetin (from Prunus avium) increased cellular proliferative indices (0.1 nM-1 µM) and osteoblast differentiation along with induction of genes involved in osteogenesis (i.e., RUNX2, OCN, Col-1). These alterations were dependent on cAMP production [222], while the increase in RUNX2 was dependent on activation of the MAPK signaling cascade (pMEK1/2 and p-ERK1/2) through adenylyl cyclase (AC) and increased G-protein coupled receptor (GPR30) expression [222].

Cytoskeletal Alterations-Future Perspectives and Gaps in Knowledge
In all of the above-described CuB studies, the cytoskeletal alterations were mostly focused on changes in actin filaments. Further efforts are needed to investigate CuB's effects on microtubules and intermediate filaments, including the detailed mechanism of action. These cytoskeletal events occurred within a short time period (minutes to a few hours), which is in contrast to the slow responses that are induced to mediate functional responses such as cellular growth, migration, invasion, and tumor growth (24 h to a few days). Further evidence using targeting strategies (i.e., siRNA) against proteins involved in regulating the cytoskeletal network would provide evidence of their contribution to the observed cellular outcomes (cell death, tumor reduction, migration, invasion, etc.) in response to the cucurbitacins. Apart from evidence associating CuB-induced changes in cytoskeletal alterations across all the above-described cancer cell types, there is limited data on the mechanism of these changes, with the exception of one report presenting evidence of the involvement of a PKA-dependent pathway leading to VASP activation required for F-actin aggregate formation [28]. Since PKA is downstream of G-protein coupled receptors (GPCRs), a current gap in knowledge is the identity of such cell-surface GPCRs that may become activated in response to CuB exposure. As reviewed in [18], there exist numerous regulatory proteins which participate in cytoskeletal organization, and further research could be focused on how CuB exposure might regulate their activities.
The majority of the studies with CuE appear to be conducted at longer time periods (i.e., 24 h), which contrasts with the shorter time course performed with CuB. Along with limited data in blood cancers, there is yet again little to no examination of the detailed mechanism of action of CuE on modulating cytoskeletal elements. Therefore, further efforts could focus on comparing the efficacies and detailed mechanistic contribution to these descriptive intracellular cytoskeletal outcomes. Although it was demonstrated that human glioma tumor (GBM) specimens are characterized by an upregulated cofilin pathway including elevated LIMK1/2 (relative to the normal brain) [224], and CuI-treated GBM cell lines led to multiple altered cellular outcomes (i.e., cytotoxicity, adhesion, migration, and invasion), no further mechanistic insights into cytoskeletal alterations induced by cucurbitacins were established, which would be a noteworthy future direction. Furthermore, such as CuE, there is limited data for CuI in both blood and solid cancers, along with mechanisms of action on actin filaments. Further efforts can investigate alterations in microtubules and intermediate filaments in response to CuI as well. There is also limited data in both blood and solid cancers with respect to cucurbitacin derivatives, along with their mechanisms of action on the cytoskeletal network. Further efforts can investigate such alterations in actin, microtubules, and intermediate filaments in response to these cucurbitacin derivatives as well.
Farrerol, a flavanone in the Ericaceae family from Rhododendron dauricum L., elicits functional outcomes by modulating numerous signaling cascades, including MAPK and AKT, amongst others [225]. However, to the best of our knowledge, there are no published reports of its effects on the cytoskeleton. Little to no mechanistic insights into cytoskeletal alterations induced by phytochemicals from the Ericaceae family were noted, which is a noteworthy research direction. There is also limited data across different cell model systems with respect to mechanisms of action on the cytoskeletal network. It is also unknown which phytochemical(s) are responsible for mediating alterations in the cytoskeleton in the CPAC and blueberry capsule extracts. Similar to Ericaceae, there are little to no mechanistic insights into cytoskeletal alterations induced by either purified phytochemicals or mixtures from the Rosaceae family, which should be considered a future research direction. Similar to the Ericaceae family, it is unknown which phytochemical(s) are responsible for mediating alterations in the cytoskeleton in the WS1442, RBE, SC, PA, DIE, RIE, PY, and Se-PFPs mixtures, although prepared from a rich diversity of plant species from this family.

Protein Trafficking Dynamics-Future Perspectives and Gaps in Knowledge
Regardless of the agent under investigation, with the exception of one study investigating the trafficking of sucrase-isomaltase to the BBM [68], the majority of the studies focused on expression profiling of key molecules in the ER stress pathway or in signaling events that were involved in modulating translocation of a cytoplasmically localized protein to the nuclear compartment. Therefore, this is a research area that could benefit from a broader focus using high-throughput screening strategies such as using tag-based fluorescence methods. This would enable an unbiased investigation of the detailed underlying mechanism of action of the phytochemical(s) in the relevant cell model system, specifically with efforts to study their effects on the alteration of organelle and trafficking dynamics.
As discussed earlier, while the role of cytoskeletal dynamics is primarily understood as being related to cellular structure, shape, and motility, these elements also play an intimate role in protein trafficking dynamics. For example, it is well known that actin assembly contributes to the process of clathrin-mediated endocytosis, which consequently affects signaling events [226]. Furthermore, the process underlying clathrin-mediated endocytosis has also been linked to a range of human diseases, including cancers, neurogenerative conditions, and even infectious diseases in which pathogens harness this mechanism to enter cells [227]. Such evidence corroborates the need to further explore these roles for these above-described plant metabolites, for which there is evidence of their ability to influence cytoskeletal integrity.

Signaling-Future Perspectives and Gaps in Knowledge
To a large extent, across the three plant families (Cucurbitaceae, Ericaceae, and Rosaceae), the effects of the phytochemicals (in extract or purified form) on signaling events were highly descriptive via assessment of changes in expression. However, there were a few studies that progressed to the next step in assessing whether the observed expression changes in the signaling molecule contributed to the functional outcomes (i.e., reduced cellular viability, reduced migration/invasion, decreased angiogenesis, and apoptosis). We propose that specific drugs targeting these pathways (i.e., PI3K, MAPK, EGFR, etc.), knockdown strategies (i.e., siRNA, shRNA, etc.), or overexpression approaches can be performed in combination with these plant phytochemicals. Likewise, only a few reports described efforts at molecular docking simulations to predict the affinity of their interac-tions with specific signaling mediators, such as EGFR. In some studies, changes in both phosphorylated and total signaling proteins were noted, and thus the dose or time of the agent utilized may be correlated with an apoptotic outcome, which includes dismantling and subsequent breakdown of the intracellular components; thus, interpretation of these findings of how the altered signaling mediator contributes to the mechanism by which the phytochemical alters the signaling event remains challenging.
Furthermore, although there were findings demonstrating the effect of various phytochemicals on EGFR kinase activity, future research directions could be focused on GPCRs, for which reports in this area are limiting. With respect to Rosaceae and Ericaceae, most of the studies utilized whole plant extracts or enriched fractions; in comparison, the use of purified phytochemicals was fewer, with, at times, opposing responses being elicited for more than one purified phytochemical from one plant species. Thus, further investigation into the effect and dosages of purified phytochemicals on signaling events in vitro and in vivo model systems, as well as it is in vivo bioactivity, is warranted. Finally, based on our search terms used in the initial literature screening for cucurbitacins from the Cucurbitaceae family, we uncovered findings focused predominantly on the JAK/STAT signaling cascade. This contrasts with the Ericaceae and Rosaceae families in which MAPK and PI3K/AKT/mTOR signaling events were predominantly uncovered. Therefore, towards the goal of uncovering whether the phytochemicals from these three different plant families uniquely target one or more of these pathways, one could apply an unbiased proteomic profiling approach such as Reverse Phase Protein Array (RPPA) or Mass-Spectrometric based approaches.

Limitations of This Study
Since our PubMed searches utilized specific terms, including the plant family, human, and a subset of specific molecules, this process of identifying articles may have eliminated other articles relevant to this area of research. For example, there may be specific species of plants within each family for which there may be information that was not identified in our broad search efforts. Furthermore, since there exists a large array of cytoskeletal regulatory factors and our searches did not focus on each one of these but only the major cytoskeletal elements, this would have also limited the articles identified. The use of the term "human" in all of our searches (to eliminate plant-based focused research) may also limit our findings by eliminating articles describing pre-clinical models. We also focused on specific signaling pathway elements (PI3K/AKT, JAK/STAT, and MAPK), although there is much crosstalk amongst these and with other transduction events for which we did not focus on. In addition, there were articles that intertwined cytoskeletal alterations, changes in translocation of key molecules, and signaling changes; however, we did not list these articles across multiple categories but focused on the major pathway of interest presented in the article.
With respect to the Cucurbitaceae family of plant metabolites, we focused on the cucurbitacins due to their high enrichment in this family. However, there are other relevant plant metabolites in the Cucurbitaceae family for which we did not pursue searches. Therefore, in this regard, this is another limitation of our study.
The methods utilized for the preparation of plant extracts and fractions may also be diverse across the various papers we reviewed, and this may thus affect the final biological outcomes. Future studies could focus on relating these specific methods (including doses used in cell systems and handling methods) to the biological outcomes, which may uncover differences due to differences in composition or loss in metabolite bioactivities. We also did not describe the side effects observed, if any, in any of the model organism studies. These would be highly relevant to translating such pre-clinical findings to the clinic.

Overall Future Perspectives
As discussed earlier, towards our review objective and for comparative purposes, the three plant families that were selected had at least 10 species in Hillsborough County, with two families associated with a high medicinal value (Ericaceae and Rosaceae) and one associated with a lesser medicinal value (Cucurbitaceae) in North America. Out of the plant genera in the Cucurbitaceae family that were uncovered in our searches, only two were described, namely Cucumis and Thricosanthes. With respect to the Ericaceae plant family, the Rhododendron and Vaccinium genera were reported in the analyzed studies. Within the Rosaceae family, the Agrimonia, Aphanes, Aronia, Crataegus, Eriobotrya, Fragaria, Malus, Potentilla, Prunus, Pyracantha, Rosa, and Rubus genera were analyzed within the framework of our search methods. Therefore, we propose that future research efforts could focus on the species from these specific genera and species (summarized in Tables 1-3) within our region of Hillsborough County to assess their medicinal value and underlying mechanisms of action on cytoskeletal organization, protein trafficking dynamics, and signaling events. With respect to our data mining of the Florida Plant Atlas, in which we identify the high abundance species across a large array of plant genera in Hillsborough county (Figure 1), we do not know of their relative abundance for each, which could be a future research direction.
Challenges have been encountered regarding the isolation of specific phytochemicals, including those from cranberry [160], and as such, current research has been restricted to the use of crude extracts with varying phytochemical contents, which may lead to variability in research findings. Wang and colleagues isolated specific flavonoids from cranberry extracts to the highest purity achieved thus far using high-performance column chromatography and subsequent characterization methods (i.e., HPLC and MS-approaches) [160]. Similar experimental approaches can be applied to the isolation of specific components from other plant extracts. However, there is still a need to critically evaluate published research studies and direct future research based on the feasibility of isolating metabolites from such plant species and/or applying them to synthesis platforms [3].
Overall, the majority of the studies analyzed herein were descriptive in nature within the cytoskeletal organization, protein trafficking dynamics, and signaling research topics. Further insight into uncovering the underlying mechanism of action can be pursued by using a combination of genetic approaches. Moreover, efforts to move forward using molecular docking simulations to investigate drug-protein interactions together with in silico experimental approaches would be valuable as a critical step in drug discovery [228,229].  were project leaders (along with the next three authors in the list, as subleaders) who were involved in supervising, directing members in each subgroup, and leading the research efforts in their subgroups. The corresponding author supervised all the contributors as well as contributing substantially to both the intellectual and writing components (including data analyses and figure preparation).