Phytomedicines Targeting Cancer Stem Cells: Therapeutic Opportunities and Prospects for Pharmaceutical Development

The presence of small subpopulations of cells within tumor cells are known as cancer stem cells (CSCs). These cells have been the reason for metastasis, resistance with chemotherapy or radiotherapy, and tumor relapse in several types of cancers. CSCs underwent to epithelial–mesenchymal transition (EMT) and resulted in the development of aggressive tumors. CSCs have potential to modulate numerous signaling pathways including Wnt, Hh, and Notch, therefore increasing the stem-like characteristics of cancer cells. The raised expression of drug efflux pump and suppression of apoptosis has shown increased resistance with anti-cancer drugs. Among many agents which were shown to modulate these, the plant-derived bioactive agents appear to modulate these key regulators and were shown to remove CSCs. This review aims to comprehensively scrutinize the preclinical and clinical studies demonstrating the effects of phytocompounds on CSCs isolated from various tumors. Based on the available convincing literature from preclinical studies, with some clinical data, it is apparent that selective targeting of CSCs with plants, plant preparations, and plant-derived bioactive compounds, termed phytochemicals, may be a promising strategy for the treatment of relapsed cancers.


Introduction
Cancer is one of the deadly diseases affecting the population worldwide, despite advancements in numerous therapeutic interventions. One of the major problems in cancer treatment is drug resistance, and cancer stem cells (CSCs) have been found as one of the important mediators to impart resistance [1,2]. CSCs are the drug-resistant cells that possess a unique ability for self-renewal, which makes them immortal [3]. There are numerous pluripotency-associated transcription factors, such as Oct4, Sox2, and Nanog, which play an essential role in maintaining the stemness of these CSCs [4]. Due to the stemness, CSCs lead to tumor heterogeneity and aggressiveness, which eventually results in metastasis [5]. CSCs also impart the dormancy of the tumors that causes treatment resistance and increases the chance of relapse [6]. CSCs are responsible for the initiation and progression of cancer as well as recurrence after treatment. Thus, CSCs have generated interest in understanding cancer treatment and prognosis in recent years [7,8]. CSCs account for EMT, which makes the cells more motile and invasive. Aberration or dysregulation of various molecular and cellular signaling pathways as well as altered metabolism of CSCs and dysregulated EMT further exacerbate the tumor heterogeneity. Altogether, the evidence points out that CSCs play a crucial role in cancer dissemination, from initiation to progression and relapse [9].
Since cancer is a fatal disease affecting millions of people worldwide, there is a great necessity of different treatment options to overcome the drug resistance or recurrence conditions in cancer patients. For the treatment of cancer, the conventional modalities are radiotherapy and chemotherapy. However, in recent years, accumulating experimental and epidemiologic studies have demonstrated that the plants and plant-derived bioactive agents, popularly known as phytochemicals, showed medicinal value, and appear beneficial as chemo-preventive and chemotherapeutic agents. Medicinal plants and their bioactive compounds have been a very good and easily accessible source for the development of novel therapeutics for various cancer diseases. Many of them have also been found to exert chemo-sensitizing effects and synergize the anticancer effects, and thus may be useful in drug-resistant cancer cells. Collectively, the plant-based formulations available either as a single herb formulation, or a mixture of many plant extracts or the plant-derived compounds, are called phytomedicines, and these have attracted interest to be future candidates in cancer therapy, owing to their selective cytotoxicity against cancer cells as well as fewer or negligible adverse effects [2]. Phytomedicines exist either in isolated or purified form or as a mixture of different secondary metabolites, and are used to prevent and cure different diseases [10]. Phytomedicines may also have vitamins and minerals which are believed to synergize preventive and therapeutic effects, and additionally be useful in treatment of drug-resistant cancers [10]. The plant extracts or plant-derived bioactive constituents have been tested for several years and showed anti-tumor activity by modulating the dysregulated signaling pathways, targeting efflux pump or transporters, and/or inducing apoptotic cell death and cell cycle arrest. Even though several purified bioactive phytocompounds and crude extracts of thousands of medicinal plants have been tested for their therapeutic effects during cancer disease treatment, very few of them have been studied on both in vitro and in vivo platforms, and only a few of them are under clinical trials.
In recent years, the utilization of many plant extracts and plant-derived agents has gained momentum for their activity against CSCs. The available studies are indicative of their anti-CSC properties mediating the modulation of numerous signaling pathways, which participate in the physiological and molecular regulation of CSCs. Many studies have shown that medicinal plants, plant-derived bioactive compounds, or the plant formulations commonly used in traditional Chinese medicines (TCM) reduce the stem-like characteristics of CSCs. They exhibit their activities by interfering with EMT genes, reducing invasiveness, and inhibiting migratory properties of CSCs [11]. In purview of the increasing understanding of the role of CSCs in many cancer types, in the present review, we comprehensively discussed the recent studies showing targeting of the CSCs by phytomedicines. The mechanisms and effects are presented in synoptic tables and schemes. The present review is suggestive of the therapeutic opportunities and prospects of phytomedicines and encouraging further studies for their pharmaceutical development.

Cancer Stem Cells and Their Markers
The process of tumorigenesis has been explained by two different models, namely the stochastic model and the hierarchical model, also known as the CSC model. According to the stochastic model, the transformation of somatic cells leads to the generation of tumors. In contrast, the hierarchical model states that CSCs are the mainstay of the tumor origin and growth [12,13]. In general, the CSCs are derived clonally by multiple symmetric or asymmetric cell divisions of cancer progenitor cells (CPCs) or transformed stem cells [8]. Further, the CSCs lead to the development of the aggressive or relapsed form of metastasizing tumors. CSCs are also known to express certain specific antigens which act as molecular biomarkers and help in their validation and identification, as illustrated in Figure 1. These overexpressed biomarkers are often employed to characterize and isolate different types of CSCs from drug-resistant cancer cell populations [14,15]. Further, these biomarkers of the CSCs are also utilized as a target to develop novel therapeutic targeted therapies, as summarized in Table 1.
Tumor cells are known to undergo phenotypic alteration as a consequence of EMT during cancer progression. In EMT, the epithelial cells develop the traits of mesenchymal cells, which are characterized by downregulation of E-cadherin and upregulation of Ncadherin mediated by numerous transcription factors such as Snail, Slug, and Twist [27,28]. Tumor cells under EMT undergo enhanced motility and migration properties [29]. Additionally, when tumor cells undergo metastasis, they possess through five orchestrated steps, such as invasion, intravasation, transport, extravasation, and colonization [30]. EMT is required for the intravasation and extravasation of tumor cells, but its loss is also eventually needed to achieve the proliferation of tumor cells. The reversal of the EMT process, termed mesenchymal-epithelial transition (MET), helps in tumor proliferation and growth [30,31]. Due to such transition process, tumor cells become more invasive, gain capability to metastasize, and impart resistance to the chemotherapy and radiotherapy in cancer treatment [32]. have shown that medicinal plants, plant-derived bioactive compounds, or the plant formulations commonly used in traditional Chinese medicines (TCM) reduce the stem-like characteristics of CSCs. They exhibit their activities by interfering with EMT genes, reducing invasiveness, and inhibiting migratory properties of CSCs [11]. In purview of the increasing understanding of the role of CSCs in many cancer types, in the present review, we comprehensively discussed the recent studies showing targeting of the CSCs by phytomedicines. The mechanisms and effects are presented in synoptic tables and schemes. The present review is suggestive of the therapeutic opportunities and prospects of phytomedicines and encouraging further studies for their pharmaceutical development.

Cancer Stem Cells and Their Markers
The process of tumorigenesis has been explained by two different models, namely the stochastic model and the hierarchical model, also known as the CSC model. According to the stochastic model, the transformation of somatic cells leads to the generation of tumors. In contrast, the hierarchical model states that CSCs are the mainstay of the tumor origin and growth [12,13]. In general, the CSCs are derived clonally by multiple symmetric or asymmetric cell divisions of cancer progenitor cells (CPCs) or transformed stem cells [8]. Further, the CSCs lead to the development of the aggressive or relapsed form of metastasizing tumors. CSCs are also known to express certain specific antigens which act as molecular biomarkers and help in their validation and identification, as illustrated in Figure 1. These overexpressed biomarkers are often employed to characterize and isolate different types of CSCs from drug-resistant cancer cell populations [14,15]. Further, these biomarkers of the CSCs are also utilized as a target to develop novel therapeutic targeted therapies, as summarized in Table 1.

JAK/STAT Pathway
The JAK/STAT pathway is one of the important pleiotropic signaling pathways which play a vital role in transmission of signals from cell-membrane receptors to the nucleus and contribute to the immune-inflammatory mechanisms mediating cytokines and growth factors. The ligands such as interleukins, growth factors, or hormones bind to the receptors and bring together two associated JAKs that facilitate the phosphorylation of each other on tyrosines, and become fully activated. Consequently, they phosphorylate the receptors and generate binding sites for STAT proteins. Further, the JAKs phosphorylate the STAT proteins, which dissociate from the receptor to form dimers and enter into the nucleus to regulate the gene expression. The overexpression of several genes such as IL-6 and CSF2 as well as highly activated STAT1 or STAT3 constitute a check on the aberration of this pathway in CSCs [34].

PI3K-Akt Pathway
The PI3K/Akt signaling pathway plays an important role in the regulation of physiological processes and controls cell survival and proliferation by checking cell cycle, growth, metabolism, proliferation, growth, and angiogenesis. Overactivation of this intracellular pathway has been demonstrated to play a crucial role in numerous cancer types. Mechanistically, when ligands bind to the receptor tyrosine kinases, then plasma membrane-bound enzyme PI3K is activated and converts phosphatidylinositol (3,4)-bis-phosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 acts as a docking site for protein kinase B (PKB), also called Akt. Further, PKB undergoes phosphorylation and is activated by mammalian target of rapamycin (mTOR) and the phosphoinositide-dependent kinase 1 (PDK1). The activated PKB inhibits the apoptosis by phosphorylating Bad. PTEN, a phosphatase, acts as a negative regulator of the process, causing the dephosphorylation of PIP3 to PIP2. Additionally, the constitutive activation of PKB or inactivation of PTEN has been observed as a reason for tumor generation in various cancers [35].

NF-κB Pathway
The NF-κB pathway, the most conserved and well-studied master regulator of innate immunity, exists in an inactive form in the cytoplasm. It is known to play complex roles in linking pathogenic signals and cellular danger signals and can be either canonical or non-canonical in nature. It has been shown to play an important role in regulating the transcription of DNA, cytokine production, and cell survival, and is crucial in mitochondrial function and dysfunction. In the canonical NF-κB pathway, the binding of ligands (IL-1β or the components derived from bacterial cell wall) to their respective receptors (IL-1 receptor or toll-like receptors) causes the recruitment of adaptor proteins, which in turn causes phosphorylation of IkB, making it available for the ubiquitination and proteasome degradation. As a result, NF-κB is released, which translocates to the nucleus and facilitates the gene transcription. Whereas, in the noncanonical NF-κB pathway, it involves activation by the receptor activator of NF-κB (RANK) and CD40. Thereafter, the kinases ensue the phosphorylation and process p100/RelB dimers into p52/RelB dimers. Consequently, NF-κB is released and translocates into the nucleus, where it facilitates the transcription [36].

Hedgehog Pathway
The Hh pathway is a relatively recent signaling cascade that has been identified to play an important role in many processes, including embryonic development and tissue homeostasis. The mammalian cells have three hedgehog homologues, including Sonic Hedgehog (SHh), Indian Hedgehog (IHh), and Desert Hedgehog (DHh). When these homologues interact with the target cells, they bind with Patched 1 (PTCH) cognate receptors involved in this pathway. When the receptors are unoccupied by the ligands, then it acts as a constitutive inhibitor of a transmembrane protein, i.e., Smoothened (Smo). Further, the transcription of the target gene is repressed by the Gli repressor. When ligands occupy the Patched 1 receptor, it causes the release of Smo and allows the Gli transcriptional activators to enhance the transcription of target genes [37].

Wnt/β-Catenin Pathway
The Wnt/β-catenin pathway comprises of a group of signal transduction pathways that start with proteins passing cellular signals, either in the closest cell-to-cell communication (paracrine) or communication within the same cell (autocrine). The Wnt pathway comprises of canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) signaling pathways. The canonical pathway is of paramount importance due to its role in survival of the CSCs. In the canonical pathway, β-catenin phosphorylates in the absence of Wnt ligands (Wnt3a and Wnt1) following its interaction with the destruction complex, which consists of the scaffold proteins, Axin, APC, GSK3β kinase, and casein kinase (CK1α). This phosphorylation brings the ubiquitination and degradation of β-catenin. On the other hand, when Wnt binds to the frizzled (Fzd) receptors and/or the low-density lipoproteinrelated protein (LRP) co-receptors, it results in activation of this pathway. Consequently, disheveled (Dvl) proteins are recruited that inactivate the destruction complex and result in stabilization and accumulation of β-catenin. This further translocates into the nucleus, and binds to the lymphoid enhancer factor (LEF)/T-cell factor (TCF) and facilitates the transcription of various target genes [38].

Notch Pathway
The Notch signaling pathway, a well-conserved cell signaling pathway, mediates juxtracrine cellular signaling by regulating cell fate decisions and tissue differentiation in neuronal, cardiac, immune, and endocrine tissues in embryonic development, and maintains homeostasis. The Notch pathway comprises of different types of Notch receptors, such as Notch1, Notch2, Notch3, and Notch4. The interaction and binding of Notch ligands (Delta-like-1, DLL3, DLL4, Jagged1, or JAG2) with NOTCH receptors (Notch1-4), ADAM/TACE, and γ-secretase in a sequential manner commence the proteolytic cleavage of the cytoplasmic domain of the receptor. This dual cleavage results in the release of Notch intracellular domain (NICD) into the cytoplasm. Then, it translocates into the nucleus and activates the transcription of target genes via the CBF1, suppressor of hairless (Su(H)), and LAG-1/recombining binding protein J-kappa (CSL/RBPJ) transcription factors [39].

Phytomedicines Targeting Key Regulators of Anti-Cancer Drug Resistance in CSCs
CSCs contribute to the anticancer drug resistance by numerous mechanisms, including EMT regulation, enhanced expression of ABC transporters, overexpression of aldehyde dehydrogenase (ALDH) enzyme, slow cycling of microRNAs, regulation of tumor microenvironment, as well as resistance to DNA damage and cell death [15]. Phytomedicines target any one of these key regulating machineries in the resistance of CSCs to the anticancer drugs ( Figure 2). The targeting of these mechanisms could be important in ceasing or eliminating CSCs and it may improve the outcome of anticancer drugs during chemotherapy of cancer. Some of these phytomedicines are discussed in this section and their related information and figures are provided in Figure 3 and summarized in Table 2. PienTze Huang    Curcumin, a polyphenol from rhizomes of turmeric (Curcuma longa), a popular dietary component, is one of the highly studied and regarded natural agents for numerous biological properties, including potent anticancer, chemo-preventive, and chemo-sensitizing activities, and benefits in synergizing anticancer activity and reducing dose-limiting organ toxicity. Recently, many reports revealed that curcumin targets CSCs in cancers of breast, thyroid, and brain. Curcumin was shown to act in many ways and appeared to be a polypharmacological agent in modulating numerous signaling pathways and transcription factors. All these mechanisms finally converge in reducing the tumor cells. In one study, curcumin downregulated EMT (vimentin, fibronectin, β-catenin) and stemness markers (Sox2, Nanog, Oct4) [47]. In another study, it reduced the overexpression of ABC transporters in breast CSCs [48]. In papillary thyroid CSCs, CUR dysregulated the JAK/STAT3 signaling pathway [49]. One of the major barriers in pharmaceutical development of curcumin is its bioavailability. Thus, in order to achieve the better stability, good aqueous solubility, and bioavailability of CUR, many novel drug delivery systems have been developed and are underway for further evaluation [50,59,60]. Correspondingly, the liposomal curcumin showed enhanced permeability and strong anticancer therapeutic efficacy for various drug-resistant cancers, including glioblastoma [50]. Next, Ovatodiolide (Ova) is a macrocyclic diterpenoid, isolated from Anisomeles indica. It exhibited potent anticancer actions on glioblastoma, nasopharyngeal carcinoma, and oral cancer cells [23,24,26]. Mechanistically, Ova reduced the stemness markers (CD44, CD133, Sox2, Klf4, Nanog, and Oct4) of CSCs and decreased the expression of EMT genes [23,24]. It also modulated the JAK2/STAT3 signaling pathway by inhibiting either JAK2 or STAT3 protein, and thus dysregulated the gene transcription [24]. In addition, Ova induced apoptotic cell death and exerted cytotoxicity of the cancer cells. The findings were further translated in an in vivo study, in an oral carcinoma (SAS cells) xenograft mice model, wherein Ova (3.6 mg/kg) reduced tumor growth, 2.2-folds less, compared to the untreated mice [24].     • Increased expression of cyclin D1 --Melanoma stem-like cells [25] --= NA.
Curcumin, a polyphenol from rhizomes of turmeric (Curcuma longa), a popular dietary component, is one of the highly studied and regarded natural agents for numerous biological properties, including potent anticancer, chemo-preventive, and chemo-sensitizing activities, and benefits in synergizing anticancer activity and reducing dose-limiting organ toxicity. Recently, many reports revealed that curcumin targets CSCs in cancers of breast, thyroid, and brain. Curcumin was shown to act in many ways and appeared to be a polypharmacological agent in modulating numerous signaling pathways and transcription factors. All these mechanisms finally converge in reducing the tumor cells. In one study, curcumin downregulated EMT (vimentin, fibronectin, β-catenin) and stemness markers (Sox2, Nanog, Oct4) [47]. In another study, it reduced the overexpression of ABC transporters in breast CSCs [48]. In papillary thyroid CSCs, CUR dysregulated the JAK/STAT3 signaling pathway [49]. One of the major barriers in pharmaceutical development of curcumin is its bioavailability. Thus, in order to achieve the better stability, good aqueous solubility, and bioavailability of CUR, many novel drug delivery systems have been developed and are underway for further evaluation [50,59,60]. Correspondingly, the liposomal curcumin showed enhanced permeability and strong anticancer therapeutic efficacy for various drug-resistant cancers, including glioblastoma [50]. Next, Ovatodiolide (Ova) is a macrocyclic diterpenoid, isolated from Anisomeles indica. It exhibited potent anticancer actions on glioblastoma, nasopharyngeal carcinoma, and oral cancer cells [23,24,26].
Mechanistically, Ova reduced the stemness markers (CD44, CD133, Sox2, Klf4, Nanog, and Oct4) of CSCs and decreased the expression of EMT genes [23,24]. It also modulated the JAK2/STAT3 signaling pathway by inhibiting either JAK2 or STAT3 protein, and thus dysregulated the gene transcription [24]. In addition, Ova induced apoptotic cell death and exerted cytotoxicity of the cancer cells. The findings were further translated in an in vivo study, in an oral carcinoma (SAS cells) xenograft mice model, wherein Ova (3.6 mg/kg) reduced tumor growth, 2.2-folds less, compared to the untreated mice [24].
Lusianthridin, a phenanthrene derivative and phenolic compound isolated from the stem of the plant Dendrobium venustum, was shown to downregulate the Src-STAT3-c-Myc signaling pathway and suppress CD133, ABCG2, and ALDH1A1 stemness markers, which induced the apoptosis in lung CSCs [22]. The root extract of Polygonum cuspidatum consisting of 2-ethoxystypandrone, i.e., a novel analogue of juglone, was shown to exhibit inhibition of the STAT3 signaling pathway in hepatocellular carcinoma cells (HCC cells). It ceased the growth and proliferation of HCC cells in a dose-dependent manner and induced the programmed cell death of CSCs in HCC [53].
Although a majority of the phytomedicines have been shown to target the cell death or apoptosis pathway, a few of them were also shown to target the cell cycle arrest, such as two monoterpenoid indole alkaloids, Scholarisine Q(1) and R(2), isolated from the fruit extract of Alstonia scholaris, which were found to induce apoptosis in glioma stem cells [40]. Another phytoconstituent, berberine, an isoquinoline alkaloid compound abundantly found in the plant Berberis vulgaris, was found to be effective in reducing the stemness, cell migration, and cell growth (via G0-G1 cell cycle arrest) of neuroblastoma and prostate CSCs, respectively [32,46]. Similarly, extract of Viola odorata rich in numerous bioactive components, such as saponin, salicylic acid derivatives, glycosides, alkaloids, anthocyanidins, and cyclotides, was shown to induce apoptosis and reduce the growth and migration of breast CSCs [52].
Carnosol, a polyphenolic diterpene abundantly found in Rosmarinus officinalis, showed modulation of the EMT genes and induced apoptosis in glioblastoma CSCs [57]. Phenethyl isothiocyanate (PEITC), a natural isothiocyanate predominantly present in the cruciferous vegetables such as broccoli and watercress, was shown to enhance oxidative stress in CSCs and downregulate the expression of stemness genes in various cancers, such as cervical and colon cancers [42]. In an in vivo study employing an ALDH + HeLa CSCs xenografted NOD-SCID mice model, PEITC at the concentration of 10 µM exhibited a reduction in the tumor volume when compared to the untreated mice serving as control mice [42]. In another study, PEITC at the dose of 20 mg/kg suppressed the growth of EpCAM + CSCs isolated from HCT116 cancer cells and also displayed a reduction in the tumor growth of a colon cancer xenograft mice model injected with EpCAM + CSCs [41]. Atractylenolide-1 (ATL-1) is a sesquiterpene isolated from the rhizome extract of Atractylodes macrocephala Koidz, a popular Chinese medicinal herb. ATL-1 (25-75 mg/kg) downregulated the Akt/mTOR pathway and altered the glucose metabolism and stem-like behavior in colon cancer cells. It also inhibited the colorectal tumor progression in xenografted nude mice [43]. The total polyphenolic fractions obtained from Fructus viticis were shown to modulate the Akt/mTOR pathway and repressed the stemness characteristics in lung CSCs [44].
Cinnamic acid, a monocarboxylic acid isolated from the bark of cinnamon (Cinnamomum zeylanicum), has been shown to decrease the stemness of colon CSCs [19]. Shikonin, a naphthoquinone derivative abundantly found in the roots of a Chinese medicinal herb, Lithospermum erythrorhizon, modulated the JNK/c-Jun pathway and augmented its cytotoxicity in glioblastoma stem cells (GSCs). The findings observed in the in vitro studies were confirmed in a GSCs-xenografted mice model [56]. Morusin, a flavonoid present in the roots of Morus australis, was found to attenuate NF-κB activity in cervical CSCs and induced apoptotic cell death in these CSCs by curbing their migration and growth. Furthermore, this compound exerted cytotoxicity of the cervical CSCs [55]. Glabridin, an isoflavane obtained from the roots of Glycyrrhiza glabra, modulated epigenetic regulation of miR-148a/SMAD2 signaling and inhibited stem cell-like properties of human breast cancer cells. Additionally, Glabridin (20 mg/kg) improved the survival of breast cancer mouse xenografts [54]. Similarly, Cajaninstilbene acid derivatives of pigeon pea modulated the cytotoxicity (pathway not deduced) in breast cancer stem-like cells [45].
It has been reported that ABC transporter genes such as ABCG2 and ABCB5 often become upregulated in cancers of pancreas, breast, lung, ovary, and skin, and can be the potential targets for therapy [15]. PienTze Huang (PZH), a traditional Chinese medicine (TCM) consisting of Moschus, Calculus Bovis, Snake Gall, and Radix Notoginseng, showed inhibition of the mRNA levels of ABCB1 and ABCG2 transporters in HT29 side population cells (HT29 CSCs) [58]. It also suppressed the growth of colorectal CSCs in a dose-dependent manner [58].
Similar to the plant-derived bioactive compounds, plant extracts have been tested in many studies. The crude extracts prepared from whole plant or certain plant parts showed potent anticancer effects in many cancer types by targeting drug-resistant CSCs in their cell populations. For example, the bark extract of Walsura pinnata Hassk and the rhizome extract of Costus speciosus induced the apoptotic cell death in the leukemic and prostate CSCs, respectively [16,51]. In the next sections, we comprehensively discuss targeting of several stemness markers, EMT genes, and cellular signaling pathways, which could be an important therapeutic approach for the elimination of CSCs from the drug-resistant cancer cell populations, as well as to keep a check on the cell growth and proliferation, therefore ceasing the tumor growth.

Phytomedicines Targeting Wnt/β-Catenin, Notch, and Hedgehog Signaling in CSCs
As we discussed earlier, the Wnt, Notch, and Hh signaling pathways are responsible for the stem-like characteristics of cancer cells and account for their self-renewal [11]. Therefore, we primarily focused on reviewing the phytomedicines which particularly target these pathways. Therapeutic targeting of these pathways by phytomedicines could pave the design and development of natural therapeutics. Phytomedicines targeting these pathways are schematically elucidated in Figure 4.

Phytomedicines Targeting Wnt/β-Catenin Signaling Pathway
Phytomedicines have been shown to eliminate CSCs by modulating the Wnt/βcatenin signaling pathway. In a study, diallyl trisulfide, an organosulfur compound predominantly present in garlic, was found to enhance the expression of GSK3-β and reduce the β-catenin, signifying the suppression of the Wnt/β-catenin pathway in colorectal CSCs [61]. Likewise, Koenimbin, an alkaloidal compound extracted from the leaves of Murraya koenigii (L) Spreng, a plant popularly known as curry tree used in dietary preparations across Indian subcontinent and reputed for its medicinal properties, has been found to reduce the expression level of β-catenin and cyclin D1 in MCF7 CSCs. It results in suppressed formation of mammospheres and diminishes the ADH + MCF7 CSC population, mediating downregulation of the Wnt/β-catenin pathway [21]. In another study, ginsenoside-Rb1 (Rb1), a natural triterpenoid saponin abundantly found in the rhizome of Panax quinquefolius plant and Notoginseng (a Chinese herbal medicine), was shown to target ovarian CSCs, mediating inhibition of the Wnt/β-catenin signaling pathway along with a reversal of EMT. A metabolite of Ginsenoside-Rb1, called compound K, in combination with Rb1 inhibited the self-renewal capacity of ovarian CSCs (isolated from patients) in a xenograft tumor mice model and sensitized these CSCs for their cytotoxic actions by chemotherapeutic agents, such as cisplatin and paclitaxel [62].

Phytomedicines Targeting Wnt/β-Catenin, Notch, and Hedgehog Signaling in CSCs
As we discussed earlier, the Wnt, Notch, and Hh signaling pathways are responsible for the stem-like characteristics of cancer cells and account for their self-renewal [11]. Therefore, we primarily focused on reviewing the phytomedicines which particularly target these pathways. Therapeutic targeting of these pathways by phytomedicines could pave the design and development of natural therapeutics. Phytomedicines targeting these pathways are schematically elucidated in Figure 4. Abrus agglutinin, a lectin, isolated from the seeds of Abrus precatorius, downregulated the CD44 + expression in FaDu cells (oral cancer cells) and inhibited the growth and plasticity of FaDu orospheres. Further, it inhibited the Wnt/β-catenin signaling pathway and suppressed the self-renewal capacity of FaDu-derived CSCs. This compound also induced apoptosis in FaDu CSCs in a dose-dependent manner. The actions were later reconfirmed in FaDu xenografted nude mice, wherein it ceased the tumor growth [63]. Another compound, sulforaphane, isolated from the cruciferous vegetables such as broccoli and cabbage, inhibited the formation of nasopharyngeal tumor spheroids enriched with CSCs. Sulforaphane at the dose of 60 mg/kg reduced the tumor growth in C666-1 cells in a xenografted mice model through the DNA methyltransferase 1/Wnt inhibitory factor 1 axis [64]. It possesses strong anticancer activities. Treatment with sulforaphane also suppressed the expression of miR-19, which regulates the miR-19/GSK3β/β-catenin axis and the traits of lung CSCs. It also downregulated the Wnt/β-catenin pathway and βcatenin/TCF transcriptional activity in lung CSCs. Following treatment with sulforaphane, the lung tumorospheres did not develop and reduced expression markers of lung CSCs [65]. Further, Chelerythrine chloride, a benzophenanthridine alkaloid isolated from Chelidonium majus, downregulated the expression of Sox2, MYC, and β-catenin in SK-LU-1 and NCI-H1703 cells. This showed inhibition of the Wnt/β-catenin pathway in lung cancer cells mediating downregulation of β-catenin and resulting in curtailing the CSC properties and inducing apoptosis [66].
Sanguinarine, a benzophenanthridine alkaloid obtained from Chelidonium majus L. Plant (traditional Chinese medicine celandine), downregulated the Wnt/β-catenin signaling pathway and inhibited the proliferation and invasion of lung CSCs, thereby inducing the apoptosis in lung CSCs [67]. Gomisin M2, a lignan belonging to the class of hydrolysable tannins, also forms an active component of a Chinese medicine Baizuan, and inhibited the proliferation of MDA-MB-231 and HCC1806 cells. Additionally, it suppressed the selfrenewal potential of breast CSCs by downregulating the Wnt/β-catenin signaling pathway. Consequently, it blocked the formation of mammospheres in breast CSCs and induced apoptosis in breast CSCs by altering mitochondrial membrane potential [68]. Furthermore, evodiamine, a natural quinolone alkaloid isolated from Evodia rutaecarpa, inhibited the proliferation of bulk cultured colon cancer cells and arrested cell cycle at G2/M phase, thereby inducing apoptosis in these cells. Further, this compound repressed the expression of several genes regulating the key signaling pathways such as Notch and Wnt of colon CSCs, and eliminated these cells [69]. Moreover, evodiamine also inhibited the proliferation and induced apoptosis in gastric CSCs. This compound also decreased the expression of pluripotent stem cell markers such as Bmi-1, KLF4, Sox2, and Oct4, and EMT markers such as Slug, Zeb1, Twist, and vimentin. The observations demonstrate that evodiamine exerts an inhibitory effect on the Wnt/β-catenin signaling pathway and EMT, thus suppressing the proliferation and stem cell-like properties of gastric CSCs [70].

Phytomedicines Targeting Notch Signaling Pathway
Like the Wnt signaling pathway, the Notch pathway also maintains the stemness of CSCs. A traditional Chinese medicine (TCM), Qingyihuaji formula (QYHJ), composed of different Chinese herbs, including Herba Scutellariae Barbatae, Herba Hedyotdis, Herba seu Radix Gynostemmatis Pentaphylli, Rhizoma Arisaematis Erubescentis, and Fructus Amomi Rotundus, was found to target the Notch signaling pathway. This formulation reduced the CD133 expression on pancreatic CSCs. Additionally, it downregulated the expression of the Notch-4 gene, but in combination with gemcitabine, it significantly suppressed expression of Notch-1, Notch-2, and Notch-3 genes, too. Furthermore, QYHJ, at the dose of 36 g/kg, inhibited tumor growth in SW1990 cells xenografted tumor mice models, suggesting that QYHJ possesses the potential to increase the survival time of patients by reducing pancreatic CSCs [71]. Similarly, another TCM formulation, Xiaotan Sanjie (XTSJ), composed of many herbs, inhibited the cell viability of gastric CSCs in a dose-dependent manner, attributed to the downregulation of Notch-1 expression, i.e., regulating the proliferation of gastric CSCs. Further, XTSJ at different doses (1.46, 2.92, and 5.84 g/mL) reduced the tumor growth in a dose-dependent manner in gastric CSC-transplanted mice models [72]. Another very popular TCM preparation, Pien Tze Huang (PZH), decreased the percentages of side population cells in SW480 cells. Additionally, it reduced the viability of side population cells in a dose-dependent manner and induced apoptosis in these side population cells, as evidenced by fragmented nucleus and condensed chromatin. Subsequently, PZH downregulated the Notch1 gene expression in colon CSCs, demonstrating its action as a potent agent targeting CSC [73]. Next, Psoralidin, a prenylated coumestans derivative isolated from the seeds of Psoralea corylifolia, inhibited Notch-1 signaling in breast CSCs that promoted the inhibition of EMT markers. This resulted in decreased invasion and migration of ALDH+ breast CSCs. Further, it inhibited the growth and induced programmed cell death in breast CSCs [74].

Phytomedicines Targeting Hedgehog Signaling Pathway
The Hh signaling pathway also plays an important role in maintaining the stemness of CSCs. Additionally, numerous studies demonstrated that deregulation of the Hh pathway plays an important role in tumorigenesis as well as drug resistance in a multitude of cancers by driving cancer cell proliferation, malignancy, metastasis, and the expansion of CSCs. The targeting of this signaling pathway by phytomedicines may inhibit the proliferation and growth of CSCs. Withaferin A, a lactone isolated from the leaf extract of Withania somnifera, inhibited the transcriptional activity of the GLI1-DNA complex formed during the Hh signaling pathway in different CSCs. This compound exhibited potent cytotoxicity against PANC1, DU145, and MCF7 cancer cells [75]. One of the popular phytocompounds curcumin, isolated from rhizomes of Turmeric (Curcuma longa), was shown to inhibit the Sonic Hh pathway and reduce the expression of breast CSC markers (ALDH1, CD44, OCT4, and CD133). This causes cessation of the cell proliferation and induces the apoptotic cell death in breast CSCs [76]. Furthermore, another constituent, sulforaphane, isolated from cruciferous vegetables including broccoli, has been shown to block the Sonic Hh pathway (Smo, Gli-1, 2) and reduce the markers of EMT (Zeb-1), pluripotency (Oct4, Nanog), angiogenic (VEGF, PDGFRα), and metastasis in pancreatic CSCs. This leads to the induction of apoptosis in pancreatic CSCs, and thus significantly reduced the tumor growth in pancreatic CSC-transplanted NSG mice [18].
One of the polyherbal plant extract preparations, BRM270 (BRMLife), consisting of seven medicinal plants, including Saururus chinensis, Citrus unshiu Markovich, Aloe vera, Arnebia euchroma, Portulaca oleracea, Prunella vulgaris var. lilacina, and Scutellaria bacicalensis, has been found to inhibit the metastasis and stemness (SALL4, CD133, Nanog, Sox2, and Oct4) in CD44+ pancreatic ductal adenocarcinoma cells (PDACs) via the Sonic Hh pathway. BRM270 at the dose of 5 mg/kg reduced the tumor growth in a CD44+ PDAC-xenografted mice model [77]. In another study, baicalein, a natural bioactive compound predominantly presents in Scutellaria bacicalensis and many other herbal formulations, including QYHJ, was shown to downregulate the pluripotent markers (Sox2, Oct4) and members of the Sonic Hh signaling pathway (SHH, SMO, and Gli-2) in PANC1 CSCs. Baicalein alone at the dose of 60 mg/kg reduced tumor growth in PANC1 CSC-xenografted nude mice. The study results highlighted the therapeutic effect of baicalein against pancreatic CSCs [78]. Another formulation, MSC500, a Korean herbal preparation made up of primarily eight herbs, including Phellinus linteus, Gastrodiaelata, and Mulberry leaf, also showed modulation of Hh signaling pathways. This herbal preparation modulated all three signaling pathways (Notch, Wnt, and Sonic Hedgehog) required for the stemness of glioblastoma stem-like cells. MSC500 suppressed the stemness genes as well as CSC markers (Oct4, Sox2, ABCB5, Gli1, Notch1, and β-catenin) in the side population of GBM8401 cancer cells. This results in reduced percentages of side population cells. It has been reported that MSC500 possesses a potent effect against high-grade glioma, and it could be promising for glioma [79].
In addition to their activity on CSCs, phytomedicines have also been found to improve sensitization of CSCs towards the conventional chemotherapeutic drugs. Ovatodiolide, a macrocyclic diterpenoid isolated from Anisomeles indica (L.) Kuntze, augmented the chemotherapeutic effect of temozolomide for glioblastoma stem-like cells [26]. It also enhanced the therapeutic effects of cisplatin for nasopharyngeal and oral CSCs [23,24]. Similarly, sulforaphane, commonly found in cruciferous vegetables, augmented the therapeutic effect of cisplatin for nasopharyngeal carcinoma [64]. Curcumin, one of the highly studied natural dietary agents, improved the sensitivity of paclitaxel, cisplatin, doxorubicin, and mitomycin C for breast CSCs [48]. Similarly, ginsenoside-Rb1, a popular compound isolated from Panax notoginseng, improved the therapeutic effect of both cisplatin and paclitaxel, commonly used chemotherapeutic drugs for ovarian CSCs [62]. Carnosol, a phenolic diterpene commonly present in Rosemary and Sage, sensitized glioblastoma CSCs to temozolomide for its anti-proliferative effects [57]. Similarly, the combination of aqueous extract of aerial parts of Gynura divaricata and cisplatin/doxorubicin/5-Fluorouracil displayed a high level of synergism for treating hepatocellular carcinoma by enhancing cytotoxicity of liver CSCs [20]. These studies demonstrate that phytomedicines not only help in reducing the CSC's resistance to treatment, but were also shown to synergize the effects of modern chemotherapeutic drugs by improving the sensitivity of cancer cells towards the chemotherapeutic drugs when administered as a combinatorial therapy. Based on the presented studies, more studies are encouraged to investigate the chemo-sensitizing effect of phytomedicines which can be used as adjuvants, and this may help in reducing the dose of modern chemotherapeutic drugs which often cause dose-limiting toxicity, that limit their clinical usage. The targeting of phytomedicinal compounds in the signaling pathways in CSCs has been summarized in Table 3.   --= NA.

Clinical Studies on Phytomedicines
In recent years, few clinical studies have been carried out to evaluate their safety and efficacy focusing on phytomedicines targeting drug-resistant CSCs and cancer cells. In patients with acute myeloid leukemia, Zhebei granules (formulation of three herbs) combined with chemotherapy have been shown to reduce the percentages of CD34 + , CD123 + and CD33 + , CD123 + leukemia stem cells [81]. The clinical status of some phytomedicines targeting CSCs has been synoptically summarized in Table 4.

Conclusions and Future Perspectives
In the present review, the recent preclinical and clinical studies of medicinal plants, their bioactive compounds, and herbal preparations shown to be effective against CSCs have been presented. Phytomedicines targeting Hh, Wnt/β-catenin, and Notch signaling pathways as well as the resistance mechanisms involving the CSCs have been summarized using synoptic tables and figures. Targeting of CSCs with phytomedicines show therapeutic promise to reduce the resistance to chemotherapy. The available data are mostly from experimental studies; therefore, additional investigations are necessary to establish the use of phytomedicines in combination with chemotherapeutic agents. Additionally, drug interaction studies are required to understand whether they affect biotransformation and exert the combination showing synergism, antagonism, or additive effects. In most of the studies, the main purified phytoconstituent which could be druggable has been tested for its activities. However, in some studies, the extract of a particular part of a whole plant was tested for its activities. To ascertain the drug discovery and development, the bioactive compounds present in the plant extract need to be characterized, then its mode of action against CSC should be determined. As it is well-established that the plant extracts have numerous bioactive compounds, isolating an individual compound and its mechanism should be encouraged for establishing the role of plant-derived compounds in regulating CSCs. The role of many anticancer agents has been convincingly shown in the experimental studies, and investigating these for targeting CSCs of a particular type of cancer might be more promising from a therapeutic perspective. Furthermore, high-throughput screening of the plant-derived compounds and their synthetic analogues could be useful in order to develop them for pharmaceutical development. There are numerous issues in clinical drug development for the phytomedicines. To name a few, important ones are the physicochemical properties, including poor solubility, stability, and residence time. Various pharmaceutical technologies, including nanoparticle-based delivery, liposomes, and hydrogel formulations, are currently designed to enhance the stability, aqueous solubility, and residence time of the phytomedicines. More research is required to ascertain preclinical and clinical safety as well as the efficacy of the plant-derived bioactive compounds. Studies assessing pharmacokinetic properties along with the pharmacodynamic activity of the phytomedicines will provide a better rationale for the pharmaceutical development of the phytocompounds. Although there is a long way to go to establish these phytomedicines to develop them as drugs targeting CSCs, the phytomedicines shown as efficacious in preclinical studies are promising for future therapeutics targeting CSCs.

Conflicts of Interest:
The authors declare no competing interests with the work presented in the manuscript.