An In Vitro Evaluation of the Molecular Mechanisms of Action of Medical Plants from the Lamiaceae Family as Effective Sources of Active Compounds against Human Cancer Cell Lines

Simple Summary Plants have been used in folk medicine for thousands of years. The Lamiaceae family is one of the largest families of flowering plants and includes a wide variety of species with biological and medical uses. They are mainly herbs and shrubs with an aromatic scent and rich in valuable compounds of great value in medicine. The article focuses on the assessment of the anticancer properties of extracts, essential oils, and pure compounds derived from various species of the Lamiaceae family and their potential molecular mechanisms of action in in vitro studies against the four most common types of cancer in women and men: breast, lung, prostate, and colon. Abstract It is predicted that 1.8 million new cancer cases will be diagnosed worldwide in 2020; of these, the incidence of lung, colon, breast, and prostate cancers will be 22%, 9%, 7%, and 5%, respectively according to the National Cancer Institute. As the global medical cost of cancer in 2020 will exceed about $150 billion, new approaches and novel alternative chemoprevention molecules are needed. Research indicates that the plants of the Lamiaceae family may offer such potential. The present study reviews selected species from the Lamiaceae and their active compounds that may have the potential to inhibit the growth of lung, breast, prostate, and colon cancer cells; it examines the effects of whole extracts, individual compounds, and essential oils, and it discusses their underlying molecular mechanisms of action. The studied members of the Lamiaceae are sources of crucial phytochemicals that may be important modulators of cancer-related molecular targets and can be used as effective factors to support anti-tumor treatment.


Introduction
Cancer is an important health problem and leading cause of death globally. The development of cancer is a multistage process that begins with genetic alteration and is followed by abnormal cell proliferation. Carcinogenesis is strictly related to the activation of oncogenes (induction of cell

The Lamiaceae Family Plants
The Lamiaceae family is one of the largest families of flowering plants and includes a wide variety of species with biological and medical uses. They are mainly herbs and shrubs with an aromatic scent and rich in valuable compounds of great value in natural medicine. Plants of this family are characterized by square stems and opposite leaves [72,73]. The most famous representatives are thyme, mint, oregano, basil, sage, savory, rosemary, hyssop, and lemon balm, which are used as aromatic spices, and some others with more limited uses [74]. Historically, species in the Lamiaceae family have enjoyed a long history of use in flavoring, preserving food, and for medicinal purposes. This family includes about 250 genera and about 7000 species, with the largest genera being Salvia, Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium, Vitex, Thymus, or Nepeta ( Figure 1). It is one of the most economically important families with great diversity and cosmopolitan distribution due to the aromatic properties of most of its members [75]. It is well known that each species produces a wide variety of secondary metabolites with strong antibacterial, antioxidant, anti-inflammatory, antiviral or anticancer properties; the oils comprise a complex mixture of bioactive compounds, in which each ingredient contributes to its overall bioactivity [56].
Cancers 2020, 12, x 5 of 47 genera being Salvia, Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium, Vitex, Thymus, or Nepeta ( Figure  1). It is one of the most economically important families with great diversity and cosmopolitan distribution due to the aromatic properties of most of its members [75]. It is well known that each species produces a wide variety of secondary metabolites with strong antibacterial, antioxidant, antiinflammatory, antiviral or anticancer properties; the oils comprise a complex mixture of bioactive compounds, in which each ingredient contributes to its overall bioactivity [56].

The Lamiaceae Family as a Source of Valuable Secondary Metabolites With Anti-Cancer Potential
The first and largest group of secondary metabolites occurring in the Lamiaceae is polyphenols, which are characterized by at least one aromatic ring having hydroxyl groups. Based on their numbers of phenolic groups and structural elements, polyphenols can be divided into phenolic acids, flavonoids, stilbenes, lignans, lignins, coumarins, anthraquinones, and xanthones [76][77][78]. In the human body, polyphenols exhibit antioxidant properties, which protect against chronic diseases caused by free radicals damaging of tissues and organs [72], as well as various anticancer [79] antidiabetic [80], neuroprotective [81], anti-inflammatory [82], antiviral [83], antifungal [84] or antibacterial properties [85]. Polyphenols are believed to cause cancer cell death by apoptosis through several mechanisms, such as DNA fragmentation, alteration of the level of apoptotic proteins, and mitochondrial membrane potential and cell cycle arrest [86,87].
Another potent group of secondary compounds is the terpenes, in particular oxygenated terpenoids [88]. In turn, terpenoids are categorized into monoterpenes, sesquiterpenes, diterpenes, sesterpenes, and triterpenes depending on the number of their isoprene units. Diterpenes constitute a diverse class of plant metabolites with more than 10,000 different structures, isolated in the

The Lamiaceae Family as a Source of Valuable Secondary Metabolites with Anti-Cancer Potential
The first and largest group of secondary metabolites occurring in the Lamiaceae is polyphenols, which are characterized by at least one aromatic ring having hydroxyl groups. Based on their numbers of phenolic groups and structural elements, polyphenols can be divided into phenolic acids, flavonoids, stilbenes, lignans, lignins, coumarins, anthraquinones, and xanthones [76][77][78]. In the human body, polyphenols exhibit antioxidant properties, which protect against chronic diseases caused by free radicals damaging of tissues and organs [72], as well as various anticancer [79] anti-diabetic [80], neuroprotective [81], anti-inflammatory [82], antiviral [83], antifungal [84] or antibacterial properties [85]. Polyphenols are believed to cause cancer cell death by apoptosis through several mechanisms, such as DNA fragmentation, alteration of the level of apoptotic proteins, and mitochondrial membrane potential and cell cycle arrest [86,87].
Another potent group of secondary compounds is the terpenes, in particular oxygenated terpenoids [88]. In turn, terpenoids are categorized into monoterpenes, sesquiterpenes, diterpenes, sesterpenes, and triterpenes depending on the number of their isoprene units. Diterpenes constitute a diverse class of plant metabolites with more than 10,000 different structures, isolated in the Lamiaceae present about 50 different skeletons [89,90]. Most diterpenes play a critical role in ecological interactions of plants and show interesting biological activities both in vitro and in vivo [91,92], such as antibacterial, antifungal, antiprotozoal, enzyme-inducing, anti-inflammatory, and the modulation of immune cell function and anticancer properties [92,93]. Studies have shown that some diterpenes have significant cytotoxic and cytostatic effects on various cell lines of human origin, interfere with the biochemical pathways of apoptosis and the cell cycle phase, and can influence the expression of proto-oncogenes such as avian myelocytomatosis viral oncogene homolog (c-Myc) and B-cell lymphoma 2 (Bcl-2) [93,94].
Many members of the Lamiaceae also produce alkaloids, which is an extremely diverse chemical group based on a ring structure including a nitrogen atom. Many alkaloids are toxic and are used by plants to protect themselves against aggression from other organisms [95,96]. Many such compounds have a strong cytotoxic effect and induce apoptosis through various pathways in different cell lines [97,98].
Essential oils (EO) are volatile and complex mixtures of diverse compounds, typically with a strong odor, synthesized as secondary metabolites by aromatic plants, mainly from the Lamiaceae family (132). Although EO are mainly contained in the leaves, they can be found in all the above-ground parts of plants. They are widely used in cosmetics, flavors, fragrances, perfumes, pesticides, and the pharmaceutical industry [99]. These phytocomplexes can be obtained by hydro or dry distillation. Their ingredients include sesquiterpenes, oxygenated sesquiterpenes, monoterpenes, oxygenated monoterpenes, and phenols, among others [100,101]. Many preclinical studies have found some anticancer, antibacterial, antioxidant, or anti-inflammatory effects in a range of cellular and animal models [102], and they have examined their mechanism of action and pharmacological targets [101].
The particular signaling pathways and related factors that constitute a target for natural anticancer modulators from Lamiaceae discussed in this paper are given in Figure 2.

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of Cell Cycle
The cell cycle events controlling cell duplication and arrest are highly dysregulated in cancer cells. Cell cycle arrest is closely related to the G1/S, G2/M and M phases' checkpoints perturbations. Progression is mediated by the activation and deactivation of cyclin-dependent kinases (CDKs), while activation depends on the presence of activated subunits named cyclins. The levels of cyclins and CDKs are changed in human cancers; for example, the levels of CDK1 and cyclin B1 are reduced in human breast adenocarcinoma (MCF-7) and human colorectal cancer (HCT-116) cell lines following treatment with Micromeria fruticosa aerial part extract, resulting in G2/M arrest [143,144]. In addition, treatment with Melissa officinalis leaf extract blocked the expression of CDKs 2, 4, and 6 and cyclin D3 in the human colon carcinoma (HT-29 and T84) cell lines, and specifically activated an important CDK inhibitor named p18 [145]; in addition, treatment with Vitex rotundifolia fruit extract downregulated cyclin D1 and CDK 4 levels in HCT-116 and human colon adenocarcinoma (SW480) [146]. Manipulation of the cell cycle may induce an apoptotic response [147]. Ocimum basilicum leaf extract induced cell cycle arrest in MCF-7 cells [148], and Salvia miltiorrhiza root extract induced a G2/M phase arrest in human lung adenocarcinoma (Glc-82) cells [149], as did Melissa officinalis leaf extract in HT-29 and T84 cells and Prunella vulgaris root extract in human breast carcinoma MCF-5 cells [150]. Satureja khuzistanica extract enriched with rosmarinic acid [151] was found to increase the size of the apoptotic sub-G0/G1 population in MCF-7 cells.

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of Apoptosis Signaling
Apoptosis or programmed cell death is a crucial mechanism for maintaining cell homeostasis. The process can be triggered by various conditions, including immune reaction or reactive oxygen species (ROS). The study of Ocimum sanctum roots extract suggested that it may increase ROS production in the HCI-H460 lung carcinoma cell line and may decrease viability via apoptosis. The same phenomenon is observed for Melissa officinalis leaf extracts against HT-29 and T84 cell lines [145]. That strategy is confirmed by the occurrence of excessive numbers of apoptotic cells [152]. It was concluded that also stress originating from the endoplasmic reticulum may play an important role in triggering apoptosis; for example, Scutellaria barbata extract induced human lung cancer (CL1-5) cell death [153].
The signaling mechanisms of apoptotic cell death are divided into two pathways: an intrinsic pathway mediated by mitochondria and an extrinsic one mediated by death receptors. Cell size reduction, membrane blebbing, and apoptotic bodies were observed in HT-29 cells after Stachys pilifera leaf extract treatment; these were all signs of apoptosis [154]. MCF-7 cells showed a modified nucleus following incubation with Caryopteris x clandonensis stem extract [155], and changes in cell rounding, shrinkage, and detachment from other cells after treatment with Teucrium mascatense whole plant extract [156]. Another important future of apoptosis is the translocation of phosphatidylserine phospholipid from the inner to the outer plasma membrane, resulting inter alia in recognition by phagocytes [157]; this was observed against HT-29 and T84 cell lines after the administration of Teucrium mascatense extract [156] and Melissa officinalis leaf extract [145]. Teucrium flavum whole plant extracts were found to induce DNA fragmentation in breast carcinoma cells (MDA-MB-231) [158], as well as Vitex rotundifolia leaf extract in human breast cancer T-47D cells [159] and Ocimum sanctum leaf extract in human prostate adenocarcinoma LNCaP cells [160]; such DNA cleavage is a hallmark of apoptosis. Perovskia abrotanoides flower extract has also demonstrated proapoptotic effects against MCF-7 cells [161] and Salvia chorassanica root extract against MCF-7 and prostate cancer cells (DU-145) [162].
The treatment of human non-small cell lung carcinoma cells (NCI-H460) with Ocimum sanctum root extract [152] and MCF-7 cells with Teucrium sandrasicum leaf extract [163] resulted in increased mitochondrial membrane permeability, which is characteristic of the intrinsic pathway. The Bcl-2 protein family also induces a loss of mitochondrial membrane potential. This family is separated into two groups: one, including Bak and Bax, which possesses proapoptotic potential, and another, including Bcl-2 and Bcl-xL, which has antiapoptotic activities. The balance between the two groups influences the progression of apoptosis. The Bcl-2 protein family was found to be modulated by the Origanum compactum aerial part extract in A549 cells [164]. Changes in the level of Bcl-2 family members may result in the release of numerous pro-apoptotic molecules. The expression of Bcl-2 protein family members in different lines of human lung cancer cells became unregulated following the administration of extracts from Salvia milithoriza roots (Glc-82 cells) [149], Scutellaria baicalensis root (H358 and H2087 cells) [165], and Melissa officinalis leaves (A549) [166]. A similar result was observed for several human colon cancer cell lines treated with Coleus amboinicus leaves (WiDr cells) [167] and Vitex rotundifolia fruit (HCT-116, SW480, LoVo and HT-29 cells) extracts [146]. In addition, apoptotic signals have been triggered in breast cancer cell lines after incubation with extracts of Plectranthus amboinicus leaves (MCF-7 cells) [120], Orthosiphon stamineus leaves (MCF-7 cells) [168], Melissa officinalis leaves (MCF-7 cells) [166], Vitex rotundifolia leaves (T-47D cells) [159], and Prunella vulgaris root (MCF-5 cells) [150]. Changes in the expression level of Bcl-2 family protein are also induced in prostate cancer cell lines by extracts of Ocimum sanctum leaves (LNCaP cells) [160], Dracocephalum palmatum (PC-3 cells) [169], and Melissa officinalis leaves (PC-3) [166].
The insertion of Bax/Bak into the mitochondrial membrane results in the formation of a pore complex and release of cytochrome c into the cytosol from the intermembrane space. In contrast, Bcl-2 and Bcl-xl prevent the release of cytochrome c. Excessive levels of cytochrome c were observed in MCF-7 cells after Orthosiphon stamineus leaf extract treatment [168]. Cytochrome c binds to apoptotic protease-activating factor 1 (Apaf-1) to create an apoptosome that activates caspase-9. Caspase-9 was found to be upregulated following treatment with Coleus amboinicus leaf extract in WiDr cells [167], Teucrium sandrasicum leaf extract in MCF-7 cells [163], Vitex rotundifolia leaf extract in T-47D cells [159], Micromeria fruticose aerial part extract in MCF-7 cells [144], Stachys pilifera leaf extract in HT-29 cell line [154], and Teucrium chamaedrys aerial and flowering parts in SW480 cells [170].
In apoptosis, the detection of poly-ADP-ribose-polymerase (PARP) is an important diagnostic method because it produces specific patterns of proteolytic cleavage fragments [173]. Increased expression of PARP1, a hallmark of apoptotic death, was observed in Glc-82 cells treated with Salvia miltiorrhiza roots extract [149], LNCaP cells incubated with Ocimum sanctum leaf extract [160], and PC-3 cells interacted with Nepeta cataria aerial part extract [172]. PARP activation was also demonstrated after the treatment of Teucrium mascatense whole plant extract in MCF-7 cells [156], Rosmarinus officinalis leaf extract in A549 cells [174], and Scutellaria baicalensis root extract in H358 and H2087 cells [165], suggesting the involvement of mitochondria in the apoptotic signals.
The extrinsic pathway is initiated by the TNF (tumor necrosis factor), TRAIL (TNF-related apoptosis-inducing ligand), and Fas ligands binding to the extracellular domain of death receptors, such as type 1 TNF receptor (TNFR1), TRAIL, and Fas receptors. The Teucrium chamaedrys aerial flowering part extract initiated an excessive expression of Fas in SW480 cells [170]. The Fas ligand-receptor junction is inhibited by nucleolin, which is a protein that protects against apoptotic death [175]. Orthosiphon stamineus leaf extract treatment significantly decreased the nucleolin level in MCF-7 cells [168]. The attachment of a Fas to a specific receptor triggers the formation of specific death-inducing signaling complex (DISC) possessing a Fas-associated death domain (FADD); this complex can recruit an adaptor protein and then activate initiator caspase-8 and caspase-10. Caspase-8 induction was found to induce apoptosis in MCF-7 and HCT-116 cells following Micromeria fruticose aerial part extract administration [144], as well as treatment with Vitex rotundifolia leaf extract in T-47D cells [159], Stachys pilifera leaf extract in the HT-29 cell line [154], and for Teucrium chamaedrys aerial flowering part extract in SW480 cells [170]. In addition, it was speculated that Satureja khuzistanica extract enriched in rosmarinic acid can induce apoptosis through activation of the extrinsic pathway in MCF-7 cells [151]. Then, the activation of caspase-8 and caspase-10 is followed by the activation effector caspases 3/7, enzymatic cleavage of numerous downstream targets, and cell death [176,177]. The Fas-mediated apoptosis pathway is believed to play a role in Scutellaria barbata extract-induced CL1-5 cell cytotoxicity [153].
An important inhibitor of apoptosis is called survivin. This protein is under-expressed in cancer cells and is related to poor clinical outcome through the blockage of apoptosis by caspase inhibition [178]. Significant reductions of survivin levels were observed for Micromeria fruticosa aerial part extract in MCF-7 and HCT-116 cell lines [143,144], Origanum majorana leaf extract in HT-29 cells [171], and for Scutellaria barbata whole plant extract in HT-29 cells [179]. Another key molecule that plays a crucial role in the negative regulation of apoptosis is Her2, belonging to the human epidermal growth factor receptor family. Excessive Her2 expression is related to antiapoptotic signals via the activation of survivin and Bcl2 protein. Melissa officinalis extract shows significant potential to reduce Her2 levels in PC-3 cells [166].
Another regulator of apoptosis and autophagy is mammalian target of rapamycin kinase (mTOR), which also demonstrates pleiotropic features [180]. The activation of the mTOR pathway was promoted by Ocimum basilicum leaf extract in MCF-7 cells with p70S6K, which is a downstream target kinase [181]. Similar features were shown for Scutellaria baicalensis root extract in H358 and H2087 cell lines [165].

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of p53 Signaling
DNA damage occurs due to metabolic processes and environmental factors including ROS and results in an increase in the levels of tumor suppressor protein p53. The protein serves as a key regulator of the cellular response and triggers target genes including CDK and inhibitor p21, taking part in cell cycle arrest, and the proapoptotic protein Bax [182]. p53 expression was dysregulated in WiDr cells by Coleus amboinicus leaf extract [167], and the levels of both p53 and p21 increase in Glc-82 after treatment with Salvia miltiorrhiza root extract [149] and in MCF-7 cells after treatment with Plectranthus amboinicus leaf extract [120]. The level of p21 was induced in cell lines HT-29 and T84 after incubation with Melissa officinalis leaf extract [145]. p53 may be regulated by Sirtuin 1 (SIRT1), which is a member of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases. The inhibition of SIRT1 was found to enhance apoptosis in CL1-5 cells treated with Scutellaria barbata extract [153]. One of the outcomes of p53 activation is cell cycle arrest, and these mechanisms have potent antitumor effects.

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of PI3K/AKT Signaling
Apoptosis and cell cycle progression are mediated by phosphatidylinositol 3-kinase (PI3K) signal transduction. Activated PI3K is responsible for the conversion of phosphatidylinositol (4,5)-phosphate into phosphatidylinositol (3,4,5)-phosphate [183]. PI3K levels were significantly reduced in A549 cells after the administration of Nepeta cataria whole plant extract [184]. That PI3K signaling cascade induces protein kinase B, which is also known as PKB or Akt. Akt is a pro-oncogene and enables tumor proliferation. Akt has numerous downstream effects and controls several biological responses, including the phosphorylation of apoptosis signal proteins Bcl-2 and Bcl-xL followed by the suppression of apoptosis and also caspase-9 and p53. Prunella vulgaris root extract was found to modulate the PI3K/Akt signaling pathway in MCF-5 cells [150]. Akt may inactivate mammalian target of rapamycin complex 1 (mTORC1), p70S6 kinase, and p21, which are known to stimulate cell growth and proliferation. The levels of Akt, mTOR, and p70S6K were reduced in A549 cells after treatment with Rosmarinus officinalis leaf extract [174].
An important suppressor of PI3K is called PTEN: phosphatase and tensin homolog deleted on chromosome 10 [183]. PTEN expression was detected in A549 cells after Nepeta cataria whole plant extract [184]. An increased expression of PTEN was also observed in Glc-82 cells after Salvia miltiorrhiza root extract treatment, which then resulted in a reduced level of Akt [149]. The levels of phosphorylated Akt were reduced in human prostate cancer PC-3 cells following treatment with Dracocephalum palmatum leaf extract [169].

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of NF-κB Signaling
In addition, the stimulation of nuclear factor kappa B (NF-κB) signaling cascade is mainly related to antiapoptotic properties. The NF-κB protein family has five members: p50, p52, p65, RelB, and c-Rel. NF-KB pathways are divided into the canonical and non-canonical. The canonical one is activated by a range of cell stressor molecules such as TNF-α that interact with tumor necrosis factor (TNF) receptors. A first step is the induction of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), which promote stimulation of the IkB kinase (IKK) complex composed of IKKγ, IKKα, and IKKβ that phosphorylates IκB, which results in degradation. The remaining NF-κB (p50 and p65) migrate to the nucleus, bind to the DNA, and activate the transcription of numerous genes such as Bcl-2 and Bcl-xL. [185]. Hence, the suppression of NF-κB by Stachys pilifera leaf extract is believed to be responsible for the induction of apoptosis in HT-29 cells [154]. The NF-κB signaling cascade also plays a pivotal role in the inflammatory response by activating the transcription of several pro-inflammatory genes such as interleukin (IL) 1β, IL-8, and cyclooxygenase-2 (COX-2) [186]. Commercial standardized Thymus vulgaris extract (thymol 0.3% w/w) was found to downregulate the activity of p65 and modulate the release of IL-1β and IL-8, which play roles part in the inflammatory mechanisms in the H460 human lung cancer cell line [187]. COX-2 plays a part in numerous malignancies, including several human cancers. COX-2 is associated with apoptosis suppression followed by uncontrolled proliferation, metastasis, and angiogenesis as a consequence of tumor growth [188]. COX-2 expression was dysregulated by high concentrations of Origanum majorana extract in A549 cells [189]. 5.2.6. The Activity of Plant Extracts from the Lamiaceae Family as Modulators of Wnt/β-catenin Signaling CDK/cyclin complexes control Wnt/β-catenin signaling [190]. Scutellaria barbata extract is related to decreased the expression of β-catenin in HT-29 cells [179], which is a component of the Wnt/β-catenin signaling pathway responsible for regulating cell growth. The signaling cascade is triggered by binding Wnt ligands to their receptors called Frizzled (Fz) and LDL receptor-related proteins 5 and 6 (LRP5 and LRP6). In the inactive state, a scaffolding protein Axin ensures β-catenin phosphorylation and promotes its degradation, but during the activation of the pathway, the Wnt ligand binds to Fz receptor, and it also allows β-catenin dephosphorylation and accumulation in the nucleus by Axin inhibition and the transcription of Wnt targeted genes such as c-Myc oncogene [191]. The suppression of Wnt signaling cascades induces apoptosis in SW480 and HCT-116 cells [192]. Additionally, Scutellaria barbata extract showed a potency to decrease the expression of the c-Myc in human colon adenocarcinoma HT-29 cells; excessive expression results in rapid cellular growth [193].

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of Autophagy Signaling
Another type of cell death connected with self-degradation is autophagy. That process may be caused by stress signals that originate from extracellular, intracellular, and endoplasmic reticulum such as growth factor deprivation, nutrient starvation, oxidative stress, protein aggregation, and pathogen infection. Upon autophagy, the organelles named autophagosomes capsules other organelles or a portion of cytosol and then fused them into lysosome and breakdown by lysosomal hydrolases. Nucleation of the phagophore is initiated by the activation of the Unc-51-like kinase 1 (ULK1) complex that triggered the phosphorylation the class IIIPI3K (PI3KC3) complex 1. This step is blocked by Bcl-2 proteins by direct association with becyclin-1, which is a component of that complex. The activated PI3KC complex mediates the production of phospatydyloinositol-3-posphate (PI3P). The first elongation step involves the enrollment of numerous autophagy-related (Atg) proteins by PI3P. The second phase of the elongation step is the formation of autophagosomal membrane-associated protein light chain 3 (LC3)-II from LC3-I via conjugation with phosphatidyl ethanolamine, which revealed the overexpression ratio of LC3-II/LC3-I in H358 and H2087 cells treated with Scutellaria baicalensis root extract [165]. Finally, autophagosomes fuse with lysosome and lysosomal enzymes with proteolytic activity degrade its cargo [194,195]. The autophagy is triggered in HT-29 cells by Origanum majorana leaf extract [171] and in CL1-5 cells by Scutellaria barbata extract [153].
The negative regulator of autophagy is mTOR. mTOR nucleates two distinct multi-protein complexes, mTORC1 and mTORC2. mTORC1 is composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with Sec13 protein 8 (mLST8), proline-rich AKT substrate 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting protein (Deptor). mTORC1 positively regulates proliferation and cell growth. One of the most important factors involved in the regulation of mTORC1 activity is the tuberous sclerosis complex (TSC1/2). TSC1/2 functions as an activating protein for Rheb (Ras homolog enriched in brain). In turn, Rheb is able to activate mTORC1 [196]. One of the components of the mTOR pathway, Ras small GTPases-Rags, interact with mTORC1 and promote their translocation to a lysosomal membrane where Rheb resides [197].

The Activity of Plant Extracts from the Lamiaceae Family as Modulators of Necrosis Signaling
Necrosis is referred to as accidental cell death. It is frequently detected following harmful physical and chemical conditions, adverse stimuli, or deleterious mutations. The process is mediated by an imbalance of calcium flux, oxidative stress, and PARP. The physiological role of PARP is participation in DNA repair signaling in response to cell injury [198]. Excessive ROS levels, increased intracellular calcium, and the inhibition of PARP result in damage to cell components, degradation of proteins, and DNA damage. Necrosis is characterized by damage of the cell membrane and the release of its components into the extracellular space, resulting in inflammation and damage to the surrounding tissues [199,200]. This pathway is observed to take place in colon adenocarcinoma HT-29 and SW480 cell lines after Rosmarinus officinalis extract treatment. Rosemary extract possesses a strong antiproliferative activity, and its mechanism of action suggests the role of excessive intracellular ROS formation [201].

Plant Extracts from the Lamiaceae family and their Impact on Angiogenesis
The critical moment is tumor-induced angiogenesis. Melissa officinalis leaf extract halted neovascularization in breast adenocarcinoma MDA-MB-231 cells [202]. Antiangiogenic effects were also exerted by Prunella vulgaris root extract in MCF-5 cells [150]. The formation of new blood vessels from pre-existing ones may be regulated by numerous factors. One of them is epidermal growth factor (EGF) and its receptors (EGFR). It was observed that tumor cells demonstrate abnormally high EGFR activity and enhanced sensitivity to their ligands and the progression of tumor [203]. In turn, EGF was found to target vascular endothelial growth factor (VEGF) and induce their activity, whereas VEGF may modulate the EGFR signaling pathway [204]. VEGF demonstrated an ability to increase the permeability of existing blood vessels. The levels of both EGF and VEGF factors were found to be lowered in PC-3 cells and VEGF in DU-145, after Salvia triloba extract incubation [205]. Nepeta cataria whole plant extract exhibits a preventive effect against A549 cell invasiveness by reducing the level of VEGF [184], whereas Melissa officinalis leaf extract acts against PC-3, MCF-7, and A549 cells spread [166]. VEGF may be activated by human telomerase reverse transcriptase (hTERT), specific oncogene, catalytic subunit of the enzyme telomerase essential for chromosome termini replication. hTERT downregulation was observed in PC-3, MCF-7, and A549 cancer cell lines after Melissa officinalis leaf extract usage [166].
Other proangiogenic factors are angiogenin (ANG), IL-8 [206], leptin [207], and RANTES (regulated upon activation, normal T-cell expressed and secreted) [208]. ANG is a protein belonging to the RNase A superfamily, whose activity is related to the formation of blood vessels and tumor growth [209], ENA-78 is a chemokine associated with the activation of granulocytic immune cells and vascularity of the tumors, whereas leptin is an endocrine hormone produced by adipocytes. ENA-78 is strongly related to prostate cancer progression [210]. RANTES is an anti-inflammatory chemokine that recruits inflammatory cells and controls the secretion of growth factors included in the angiogenic process [208]. Salvia triloba extract was found to have the opposite effect on angiogenesis in PC-3 and DU-145 cells by reducing the levels of VEGF, ANG, ENA-78, IL-8, leptin, and RANTES [205].

The Anticancer Activity of Phenolics Compounds from the Lamiaceae Family
Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; CAS 20283-92-5), an ester of caffeic acid, and 3,4-dihydroxyphenyllactic acid is commonly found in species of the Lamiaceae family. Rosmarinic acids and their derivatives possess a range of antioxidant, anti-inflammatory, antitumor, anti-angiogenic, and antimicrobial activities, among others [254]. Rosmarinic acid extracted from Salvia glabra demonstrates antitumor activity against breast cancer stem-like cells (BCSCs); this has been attributed to apoptosis induction by suppressing the expression of Bcl-2 and increasing that of Bax [255].
Wogonin (5,7-dihydroxy-8-methoxyflavone; CAS 632-85-9) is a flavone with numerous antitumor, anti-inflammatory, antiviral, and neuroprotective properties [256]. A natural source of wogonin is Scutellariae radix, which is the dried root of Scutellaria baicalensis. It has been shown to promote both autophagy and apoptosis processes in SW48 cells via the upregulation of autophagic factors including LC3II and Beclin 1 proteins and apoptotic factors such as caspase-3, caspase-8, caspase-9, and Bax proteins. The exposure to wogonin results in G2/M cell cycle arrest and the inhibition of PI3K/Akt signaling through attenuating PI3K protein expression [257]. Wogonin has been found to inhibit the invasiveness of MDA-MB-231 cells via suppressing the synthesis of two key factors related to new blood vessel formation: IL-8 and matrix metallopeptidase-9 (MMP-9) [258]. MMP-9 is critical for the progression of a pro-angiogenic outcome and the release of VEGF during carcinogenesis [259]. Scutellarein (5,6,7,4'-tetrahydroxyflavone), a flavone that is particularly present in the genus Scutellaria, has been found to be effective for the prevention and treatment of Helicobacter pylori infection, Alzheimer's disease, and vascular complications of diabetes; it has also been found to inhibit certain carcinomas [260]. It was found that the exposure of the HCT116 cells to the scutellarein extracted from Scutellaria barbata induces apoptosis via ROS-mediated mitochondrial membrane permeability and cytochrome c release, and by downregulating the expression of Bcl-2, increasing Bax and cleaved-caspase-3 activity [261].

The Anticancer Activity of Terpenoids Compounds from the Lamiaceae Family
Arguably, the most important class of chemicals produced by the Lamiaceae family is the terpenoids; of these, the most numerous subclass of compounds with confirmed anticancer properties is that of the diterpenoids.
An important subgroup of terpenoid compounds is tanshinone diterpenoids, which are produced by Salvia miltiorrhiza roots; these are also connected with the induction of apoptosis in cancer cells. Increases in apoptosis were observed in colon cancer cells (human colorectal cancer DLD-1, human colon carcinoma COLO 205, and human colorectal adenocarcinoma Caco-2) exposed to trijuganone C [271], prostate cancer cells (PC3 and LNCaP) treated with tanshinone analog 2-(Glycine [methyl ester]methyl)-naphtho [272], and lung (human non-small cell lung cancer PC9 and A549) and breast cancer (MCF-7) cells administered by tanshinone [273]; these changes were attributed to the upregulation of numerous apoptotic factors including cytochrome c, pro-and antiapoptotic proteins ratio, caspases, PARP, p53, and p38. In addition, MCF-7 cells treated with three tanshinones isolated from the roots of Perovskia abrotanoides (cryptotanshinone, tanshinone 2A, and hydroxycryptotanshinone) exhibit high amounts of PARP protein cleavage, which is a hallmark of apoptosis [274].
Another diterpenoid, 11α, 12α-epoxyleukamenin E isolated from whole plants of Salvia cavaleriei show anticancer potential against HCT116 and SW480 cell lines [275]; this activity was attributed to suppression of the crucial Wnt signaling pathway, which regulates cell fate and the activation of targeted genes, including c-Myc and survivin. The diterpene clerodermic acid found in aerial parts of four Salvia species including S. spinosa, S. santolinifolia, S. syriaca, and S. nemorosa was also shown to be effective in suppressing Hypoxia-Inducible Factor (HIF) 1 alpha in A549 cells [276], which correlates with tumor metastasis and angiogenesis. Hypoxia is a common microenvironment in many types of solid tumors, and the HIF-1α pathway is crucial for their survival; therefore, the reduction of its expression may serve as a potential cancer therapy target [277].

The Anticancer Activity of Polysaccharides Compounds from the Lamiaceae Family
Biologically active polysaccharides from natural plant sources also act as potent antitumor agents. They are also believed to be nontoxic and more preferred for living organisms [281]. SPS2p, a polysaccharide composed of carbohydrates, uronic acid, and proteins from Scutellaria barbata demonstrated pro-apoptotic activity against HT29 cells, probably by suppressing the PI3K/Akt pathway [282]. Whereas SBPW3, a polysaccharide containing rhamnose, arabinose, xylose, mannose, glucose, and galactose isolated from the same species also demonstrated anti-metastatic activity via the suppression of EMT in the same cell line [283].

The Anticancer Activity of Essential Oils from the Lamiaceae Family
The members of the Lamiaceae are sources of essential oils. Their cytotoxic potency against cancer cell lines are presented in Table 3. This section discusses their mechanisms of action against lung, colon, breast, and prostate cancer cell lines. Table 3. Cytotoxic properties of selected essential oils from the Lamiaceae family against lung, colon, breast, and prostate cancer cells.

Name of The Species
Part of the Plant Compounds Identified in Essential Oils Cancer Cell Lines Ref.    Nepeta rtanjensis essential oils rich in trans,cis-nepetalactone induced programmed cell death against A549 and MDA-MB-231 cells [319], as did Origanum onites essential oil, which is rich in carvacrol, against Ht-29 cells [320]. Ocimum viride essential oils, which have a high content of γ-terpinene, induced apoptosis as a consequence of DNA damage in HT-29 cells [321], while Thymus revolutus essential oil with a high content of γ-terpinene and p-cymene induced apoptosis in A549 cells [322]. Salvia aurea, S. judaica, and S. viscosa essential oils containing caryophyllene oxide as a main constituent induced apoptosis triggered by excessive ROS formation in DU-145 cells [323], as did Zataria multiflora essential oils in HCT-116 and SW48 cell lines [324].

Anisomeles indica
G2/M cell cycle delay and apoptosis were observed in PC-3 cells after exposure to Lavandula angustifolia essential oil, with linalool and linalyl acetate as major components [325]. Plectranthus amboinicus essential oil, containing high amounts of carvacrol, dysregulated levels of pro-and antiapoptotic factors and induced caspase-9 and caspase-3 in A549 cells [326].

Conclusions
Cancer causes the greatest economic burden of any of the top 15 causes of death worldwide, both for the patient and society in general. Therefore, there is a great need to identify new active molecules with anticancer activities. Medicinal plants and their bioactive compounds are widely used in the treatment of numerous diseases. The species of the Lamiaceae are considered important because of their use in traditional medicine throughout the world. The Lamiaceae include a range of secondary metabolites including polyphenols, terpenoids, alkaloids, or essential oils that demonstrate promising cytotoxic activity against lung, breast, prostate, and colorectal cancer cell lines mainly via the apoptosis pathway by the modulation of cell cycle progression, changes in genes expression, and impact on various signaling cascades. These biologically active compounds represent promising candidates for supportive use in anticancer therapy; however, further extensive scientific and clinical investigations are required. Funding: This review received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.