*Review* **Targeting Cancer Stem Cells by Dietary Agents: An Important Therapeutic Strategy against Human Malignancies**

**Mahshid Deldar Abad Paskeh 1,2,† , Shafagh Asadi 3,†, Amirhossein Zabolian <sup>4</sup> , Hossein Saleki <sup>5</sup> , Mohammad Amin Khoshbakht <sup>5</sup> , Sina Sabet <sup>5</sup> , Mohamad Javad Naghdi <sup>5</sup> , Mehrdad Hashemi 1,2 , Kiavash Hushmandi <sup>6</sup> , Milad Ashrafizadeh 7,8, Sepideh Mirzaei <sup>9</sup> , Ali Zarrabi 8,10 and Gautam Sethi 11,12,\***


**Abstract:** As a multifactorial disease, treatment of cancer depends on understanding unique mechanisms involved in its progression. The cancer stem cells (CSCs) are responsible for tumor stemness and by enhancing colony formation, proliferation as well as metastasis, and these cells can also mediate resistance to therapy. Furthermore, the presence of CSCs leads to cancer recurrence and therefore their complete eradication can have immense therapeutic benefits. The present review focuses on targeting CSCs by natural products in cancer therapy. The growth and colony formation capacities of CSCs have been reported can be attenuated by the dietary agents. These compounds can induce apoptosis in CSCs and reduce tumor migration and invasion via EMT inhibition. A variety of molecular pathways including STAT3, Wnt/β-catenin, Sonic Hedgehog, Gli1 and NF-κB undergo down-regulation by dietary agents in suppressing CSC features. Upon exposure to natural agents, a significant decrease occurs in levels of CSC markers including CD44, CD133, ALDH1, Oct4 and Nanog to impair cancer stemness. Furthermore, CSC suppression by dietary agents can enhance sensitivity of tumors to chemotherapy and radiotherapy. In addition to in vitro studies, as well as experiments on the different preclinical models have shown capacity of natural products in suppressing cancer stemness. Furthermore, use of nanostructures for improving therapeutic impact of dietary agents is recommended to rapidly translate preclinical findings for clinical use.

**Keywords:** medicinal herbs; cancer treatment; cancer stem cells; drug resistance; metastasis; proliferation

**Citation:** Deldar Abad Paskeh, M.; Asadi, S.; Zabolian, A.; Saleki, H.; Khoshbakht, M.A.; Sabet, S.; Naghdi, M.J.; Hashemi, M.; Hushmandi, K.; Ashrafizadeh, M.; et al. Targeting Cancer Stem Cells by Dietary Agents: An Important Therapeutic Strategy against Human Malignancies. *Int. J. Mol. Sci.* **2021**, *22*, 11669. https:// doi.org/10.3390/ijms222111669

Academic Editor: Hidayat Hussain

Received: 23 September 2021 Accepted: 23 October 2021 Published: 28 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

The cancer is the second leading cause of death worldwide after cardiovascular diseases [1]. Based on the new estimates published by Siegel and co-authors, prostate cancer is the most common cancer in males, while breast cancer is the most common cancer in females. Noteworthy, lung cancer is the most aggressive cancer in both sexes and causes highest death among other tumors [2]. Regardless of cancer incidence rate and cell deaths, there have been efforts in developing novel therapeutics for tumor treatment [3–6]. Surgery or tumor resection is beneficial in early stages of cancer and when tumor cells diffuse into various tissues in body, it is impossible to eliminate cancer by using surgery [7,8]. Radiotherapy is less invasive compared to surgery, but it has its own problems including side effects and risk of resistance [9]. Immunotherapy is a new emerging therapeutic modality in cancer and uses checkpoint inhibitors in impairing cancer progression [10–12]. Another strategy is tumor treatment is chemotherapy that is most common compared to other modalities, but resistance and adverse impacts reduce its potential [13,14]. In order to prevent therapy resistance, combination cancer therapy has been utilized. In this strategy, a combination of chemotherapy and radiotherapy, or chemotherapy and immunotherapy are applied to suppress cancer progression and inhibit resistance [15,16]. Other kinds of treatments including photothermal therapy induced by nanoparticles in ablating tumor progression, inducing DNA damage and preventing cell cycle progression [17–19]. Besides, gene therapy using small interfering RNA (siRNA), short hairpin RNA (shRNA) and CRISPR/Cas9 system can be applied in cancer suppression [14,20–22]. These strategies have been partially advantageous in improving overall survival and prognosis of cancer patients. However, cancer is still a challenge for healthcare providers and new attempts should be made in this case [23–30].

The plant derived-natural products have been under attention in recent years in field of cancer therapy [31–33]. These agents have great therapeutic activities that anti-tumor activity is among them [34,35]. Due to the potential of phytochemicals in apoptosis induction, cell cycle arrest, metastasis inhibition and multitargeting capacity, they are able to inhibit cancer progression [20,36–42]. Clinical trials have shown that plant derived-natural products are generally well-tolerated in cancer patients [43,44]. Therefore, they can be extracted for developing commercialized drugs in cancer therapy [45]. Experiments have shown that natural occurring compounds are beneficial in sensitizing tumor cells to therapeutic modalities [46,47]. For instance, curcumin can increase sensitivity of cancer cells to cisplatin, docetaxel, paclitaxel and doxorubicin [31,48,49]. A combination of resveratrol and radiotherapy is advantageous in suppressing cancer progression [50]. Hence, application of natural products alone or in combination with other therapeutic strategies can pave the way towards effective tumor treatment [51–53]. The present review article focuses on using dietary agents in cancer therapy via targeting cancer stem cells (CSCs). For this purpose, we first provide a summary of CSCs, their analysis, markers and metabolism. Then, we show how CSCs can enhance stemness and progression of tumor cells. Finally, we mechanistically discuss how each phytochemical can be beneficial in cancer suppression via targeting CSCs.

#### **2. Cancer Stem Cells**

The stem cells have self-renewal capacity and can develop colonies [54,55]. The stem cells exist in various phases of life from embryonic phase to adulthood and are able to differentiation in forming various organs and tissues of body [56,57]. A kind of cells with characteristics similar to stem cells was found to be involved in carcinogenesis and called CSCs [58–60]. The CSCs are abundantly found in the tumor microenvironment and due to their self-renewal capacity, they can preserve population of tumor cells and mediating tumorigenesis. Besides, CSCs can differentiate into different cell kinds, enhancing cancer progression [61,62]. Overall, there are two concepts for carcinogenesis. At the first model, known as stochastic model, tumor cells are similar and have the same potential in tumorigenesis. Based on this model, the accumulation of mutations has resulted in

carcinogenesis [61,63]. However, upon discovery of CSCs, a new concept of tumorigenesis was introduced, known as hierarchical model that CSCs are responsible for cancer development, maintaining and tumor seeding [64].

The identification of CSCs occurred in nineteenth century, when it was found that there are dormant cells in adult tissues that can be activated by stimuli and have capacity of proliferation and generating large masses of cells [65]. Although this was a great idea showing a special function for stem cells in cancer progression, it was ignored until in 1994 that Lapidot and colleagues isolated CSCs from leukemia cells and confirmed their presence [58]. The isolated CSCs were injected in mice and they showed potential in tumor initiation and development. After the isolation of CSCs from breast cancer in 2003 [60], more investigation was performed to isolated CSCs from other kinds of tumors including brain tumors, colorectal cancer and liver cancer [66–68].

Overall, CSCs have three distinct features from normal cells including differentiation, self-renewal capacity and homeostasis control [69,70]. Regardless of tumor stage, CSCs can be abundantly found in TME and a variety of techniques for isolation and enrichment of CSCs are utilized such as side population detection of cells with ability for Hoechst 33,342 exclusion, sphere forming capacity and aldehyde dehydrogenase (ALDH) measurement [71]. The Oct4, SOX2, Nanog, c-Myc and KLF4 are able to regulate CSC features and related signaling networks are Wnt, Notch, Hedgehog and PI3K/Akt, among others. Furthermore, complicated conditions in TME such as hypoxia, stromal cells, growth factors and extracellular matrix are able to control CSC characteristics in tumor cells [72–77].

One of the important aspects of CSCs is their metabolism. Based on experiments, CSCs rely on glycolysis, mitochondrial oxidative phosphorylation and other metabolic pathways that can be targeted therapeutically for suppressing CSCs [78–80]. It has been reported that CSCs are able to induce glycolysis via upregulating glucose transporters (GLUTs), hexokinases (HKs), monocarboxylate transporters and pyruvate dehydrogenase kinase 1 [81–85]. The CD133+ cells that exist in pancreatic cancer and glioma, have the ability of oxidative phosphorylation and enhancing expression level of genes involved in tricarboxylic acid (TCA) cycle [86,87]. Noteworthy, the levels of reactive oxygen species (ROS) can also affect CSC metabolism. The CSCs that are in dormant conditions can preserve low levels of ROS via glycolysis or stimulating antioxidant defense system. However, CSCs want to proliferate and differentiate, they induce ROS overgeneration via oxidative phosphorylation [88]. Therefore, metabolism, growth and differentiation of CSCs have a close association that should be considered.

#### **3. Cancer Stem Cells in Oncology**

The presence of CSCs is in favor of tumor progression. Thanks to experiments performed recently to shed some light on the role of CSCs. It seems that presence of CSCs in TME results in immune evasion and immunosuppression [89]. The CSCs are able to induce drug resistance feature of tumor cells. It has been reported that STAT3 signaling enhances stemness and CSC features in ovarian tumor cells to mediate their resistance to cisplatin and paclitaxel chemotherapy [90]. An interesting study has provided new insight about process of CSC generation in breast cancer and enhancing carcinogenesis. In this case, adipose-derived stem cells and breast cancer cells (MDA-MB-231 cells) fuse to produce CSCs. This process is mediated by CD44 [91]. As it was mentioned, CSCs involve in triggering drug resistance in tumor cells. Noteworthy, CSCs can also mediate radioresistance features. An experiment on nasopharyngeal cancer demonstrated that hTERT promotes CSCs features in nasopharyngeal cancer and mediates radioresistance. Knockdown of hTERT is correlated with reduced CSC characteristics and enhanced sensitivity to radiotherapy [92].

A variety of molecular pathways can modulate CSC features in tumors. For instance, microRNA (miRNA)-326 is suggested to be a tumor-suppressor in cervical cancer. The miRNA-326 binds to 30 -UTR of transcription factor 4 (TCF4) to diminish its expression. Upon TCF4 down-regulation, a significant decrease occurs in CSC features via down-

regulating CD44 and SOX4 expression levels [93]. On the other hand, tumor-promoting factors pave the way for increasing CSC features in tumors. For instance, DUSP9 undergoes overexpression in triple-negative breast cancer and down-regulates ERK1/2 expression to enhance levels of CSC markers including SOX2, Oct4 and ALDH1 [94]. Therefore, recapitulation of CSC niche can promote tumor progression and mediate drug resistance feature [95]. Identification of such factors and reducing their expression can pave the way to drug sensitivity. For instance, silencing RAD51AP1 is associated with impairment in self-renewal capacity of CSCs and inducing drug sensitivity in colorectal cancer [96]. Another example is musashi-1 that enhances glioblastoma progression. Silencing musashi-1 diminishes CSC features in glioblastoma [97].

Each experiment has focused on a certain molecular pathway that leads to cancer progression via enhancing CSC features. The lung cancer stemness and CSC features can be mediated via JAK2/STAT3 axis. As upstream mediator, aryl hydrocarbon receptor induces JAK2/STAT3 axis to promote lung cancer stemness [98]. Another experiment reveals that long non-coding RNA (lncRNA)-WDFY3-AS2 reduces miRNA-139-5p expression to promote SCD4 expression. Then, ovarian cancer stemness enhances via promoting CSC features and resistance to cisplatin is mediated [99]. The NEDD4 expression undergoes upregulation in breast cancer and preserves stemness via promoting CSC features [100].

One of the important aspects of cancer progression is the role of extracellular vesicles (EVs), especially exosomes. Briefly, exosomes can provide cell–cell communication via transferring proteins, lipids and nucleic acids [101–103]. A recent study has shown that EVs can stimulate generation of CSCs from stem or progenitor cells [104]. The exosomes containing lncRNA UCA1 enhances SOX2 expression via miRNA-122-5p down-regulation to enhance differentiation and self-renewal capacity of CSCs [105]. It is worth mentioning that CSCs can also secrete exosomes in cancer progression. For instance, exosomes derived from CSCs contain lncRNA DOCK9-AS2 that can enhance growth, metastasis and stemness of thyroid tumor via inducing Wnt/β-catenin axis [106]. Overall, experiments highlight the fact that CSCs play a significant role in progression of tumors and their targeting is of importance in cancer therapy. Furthermore, a variety of molecular pathways including PCGF1, circFAM73A, CXCL1 and NUMB are able to affect CSC features. The growth, metastasis and therapy response are mainly regulated by CSC characteristics [107–115].

The interesting point is the role of CSC markers as prognostic factors in tumor [116]. The clinical studies have confirmed this statement. The overexpression of BMI-1 and CD44 as CSC markers occurs in head and neck squamous carcinoma to promote cancer progression and mediate undesirable prognosis [117]. The CD133 and CXCR4 as other CSC markers also demonstrate alterations in osteosarcoma patients. The CD133 upregulation occurs in 26% of patients, while CXCR4 demonstrates overexpression in 36% of cases. The overexpression of aforementioned CSC markers provides undesirable prognosis and survival of osteosarcoma patients [118].

#### **4. Search Strategy**

Various databases including Google scholar, Web of Science and Pubmed were used to search and collect articles. The names of dietary agents discussed in this article and other words including "cancer" and "cancer stem cell" were searched to find the relevant articles. Furthermore, there were many phytochemicals found whose impact on CSCs have not been evaluated yet but can be considered in future studies.

#### **5. Dietary Agents and Cancer Stem Cells**

*5.1. Flavonoids*

5.1.1. Flavones Nobiletin

The nobiletin is a potent anti-tumor agent capable of suppressing tumor migration via EMT inhibition [119]. Nobiletin stimulates apoptosis and DNA damage to impair progression of oral cancer cells [120]. Nobiletin suppresses breast cancer progression in a dose-dependent manner. Nobiletin enhances miRNA-200b expression to elevate apoptosis and pyroptosis in breast cancer cells [121]. Three experiments have shown role of nobiletin in affecting CSCs in tumor therapy. The Wnt/β-catenin signaling is a possible target of nobiletin in impairing CSC characteristics [122]. The invasion and angiogenesis are suppressed by nobiletin via targeting CSCs. Nobiletin (100 and 200 µM) reduces STAT3 expression via binding to CD36 to inhibit NF-κB signaling, leading to a significant decrease in migration and metastasis of CSCs [123]. In order to potentiate efficacy of nobiletin in CSC suppression, its co-administration with xanthohumol is recommended. This combination suppresses migration of CSCs and decreases CD44v6 expression. Furthermore, they induce apoptosis and cycle arrest at G2/M phase. This combination impairs progression of colorectal CSCs and enhances their sensitivity to oxaliplatin and 5-flouroruacil chemotherapy [124].

#### Chrysin

The chrysin is a new emerging anti-tumor agent capable of suppressing growth and invasion of tumor cells, and promoting their sensitivity to chemotherapy [125]. A combination of chrysin and daidzein inhibits colorectal cancer progression via suppressing ERK and Akt molecular pathways [126]. Chrysin-loaded nanostructures are capable of enhancing apoptosis via triggering p53 expression. Furthermore, chrysin-loaded nanoparticles suppress PI3K/JNK axis and inhibit tumor growth in vivo [127]. Chrysin enhances miRNA-let-7a expression, while it reduces H19 and COPB2 expression levels to impair progression of gastric cancer cells [128].

An experiment has applied micellar nanoparticles for co-delivery of chrysin and docetaxel in cancer therapy. The application of micelles promotes therapeutic effect of both docetaxel and chrysin. The micelles were biodegradable and capable of docetaxel and chrysin co-delivery in synergistic cancer chemotherapy. This combination enhanced ROS levels to induce apoptosis in colon CSCs. Furthermore, docetaxel- and chrysin-loaded micelles inhibit migration and invasion of CSCs, impairing colon cancer metastasis [129]. Another study has focused on a derivative of chrysin, known as CHM-04 that has 3.2-fold higher anti-tumor activity compared to chrysin. The CHM-04 suppresses colony formation capacity and invasion of breast CSCs and induces apoptotic cell death [130].

#### Apigenin

Similar to chrysin, apigenin is a potent anti-tumor agent against various cancers including breast cancer, lung cancer and gastric cancer [7]. Apigenin impairs progression of multiple myeloma cells in a dose-dependent manner. Apigenin is able to down-regulate STAT1 expression in suppressing COX-2/iNOS axis [131]. A combination of apigenin and hesperidin prevent DNA repair in breast tumor to potentiate DOX activity in cancer suppression [132]. Furthermore, apigenin reduces activity of ABCG2 and ABCC4 as drug efflux transporters to enhance internalization of doxorubicin in breast cancer and mediate apoptosis [133].

The activation of YAP/TAZ axis is responsible for CSC features in triple negative breast cancer. The apigenin administration (0–64 µM) decreases colony formation and selfrenewal capacity of CSCs. Furthermore, apigenin reduces number of CD44+ cells in breast cancer. These anti-tumor activities were mediated via suppressing YAP/TAZ axis [134]. The presence of CD133 cells decreases potential of cisplatin in lung cancer suppression. The apigenin administration (10–30 µM) induces apoptosis in CSCs via p53 upregulation and enhances cisplatin cytotoxicity against lung tumors [135]. The stimulation of tumorpromoting factors enhances CSC features in tumors. For instance, activation of PI3K/Akt signaling leads to CSC features in prostate cancer via inducing NF-κB signaling. The apigenin administration (0–100 µM) suppresses PI3K/Akt/NF-κB axis to reduce Oct3/4 levels, as CSC markers in prostate cancer. Apigenin reduces survival of CSCs and enhances p21 and p27 upregulation. For decreasing viability of CSCs in prostate cancer therapy, apigenin induces both intrinsic and extrinsic apoptosis. Furthermore, apigenin disrupts

invasion and metastasis of CSCs [136]. When prostate cancer cells obtain stemness and CSC features, they can easily achieve resistance to cisplatin chemotherapy. Similar to previous experiment, apigenin (15 µM) suppressed phosphorylation of PI3K, Akt and NF-κB in impairing CSC features. Furthermore, apigenin inhibited cell cycle progression of CSCs in prostate cancer via upregulating p21, CDK2, CDK4 and CDK6. By reducing Snail expression, apigenin impaired progression and invasion of CSCs. Apigenin enhanced capase-8, Apaf-1 and p53 levels, while it decreased Bcl-2, sharpin and survivin levels in triggering apoptosis in CSCs in prostate cancer. These impacts of apigenin promote sensitivity of prostate cancer cells to cisplatin chemotherapy [137]. For disrupting cancer stemness, apigenin (40 µM) decreases expression levels of CD44, CD105, Nanog, Oct4, VEGF and REX-1 [138]. These studies demonstrate that apigenin is a potent inhibitor of CSCs in tumor treatment that further experiments can focus on revealing more signaling pathways affected by apigenin [139].

#### Baicalein

The baicalein is another anti-tumor agent that can induce apoptosis in tumors via upregulating caspase-3, -8 and -9 levels. Baicalein reduces MMP-2 and MMP-9 expression levels to impair metastasis of cancer cells [140]. The baicalein prevents SNO-induced ezrin tension and reduces iNOS levels to impair progression of lung tumors [141]. By suppressing Akt and Nrf2 molecular pathways, baicalein induce both apoptosis and autophagy in gastric tumor cells [142]. Furthermore, baicalein mediates proteasomal degradation of MAP4K3 to impair progression of lung tumor cells [143].

The members of Sonic Hedgehog signaling including SHH, SMO and Gli2 undergo upregulation to mediate CSC features in pancreatic cancer. The overexpression of Sonic leads to upregulation of SOX2 and Oct4 as CSC markers in pancreatic cancer. The baicalein administration (0–300 µM) suppresses Sonic signaling to reduce SOX2 expression and impair CSC features in pancreatic cancer [144].

#### Wogonin

The wogonin has demonstrated great therapeutic impacts in pre-clinical experiments [145]. The wogonin is able to induce senescent in breast tumors via down-regulating TXNRD2 expression [146]. Wogonin diminishes expression levels of Notch1 at mRNA and protein levels to suppress growth and metastasis of skin cancer cells [147]. This section focuses on wogonin impact on CSCs.

As it was mentioned, natural products can enhance ROS levels to induce apoptosis in CSCs [148]. A same strategy is followed by wogonin in osteosarcoma therapy. For this purpose, wogonin (0–80 µM) enhances ROS levels to reduce expression of factors responsible for CSC features such as STAT3, Akt and Notch1 [149]. In addition to triggering apoptosis and reducing survival of CSCs in osteosarcoma, wogonin is able to affect invasion of CSCs. In this way, wogonin administration (0–80 µM) decreases MMP-9 expression to impair migration and invasion of CSCs in osteosarcoma (Figure 1) [150]. Interestingly, studies have only focused on osteosarcoma and more experiments on other tumor models should be performed to shed more light on anti-tumor activities of wogonin via targeting CSCs. Overall, flavones are potential agents in suppressing stemness and CSC features in tumors that have been summarized in Table 1.

features in tumors that have been summarized in Table 1.

targeting CSCs. Overall, flavones are potential agents in suppressing stemness and CSC

**Figure 1.** The potential of flavones in suppressing CSCs. Abbreviations: CSCs, cancer stem cells; NF-B, nuclear factorkappaB; CDK, cyclin-dependent kinase; Oct4, octamer-4; YAP, Yes-associated protein, SOX2, sex determining region Ybox 2; TAZ, Transcriptional coactivator with PDZ-binding motif; CHM-04, a chrysin derivative ; STAT3, signal transducer and activator of transcription 3; Akt, protein kinase-B; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species. **Figure 1.** The potential of flavones in suppressing CSCs. Abbreviations: CSCs, cancer stem cells; NF-κB, nuclear factorkappaB; CDK, cyclin-dependent kinase; Oct4, octamer-4; YAP, Yes-associated protein, SOX2, sex determining region Y-box 2; TAZ, Transcriptional coactivator with PDZ-binding motif; CHM-04, a chrysin derivative; STAT3, signal transducer and activator of transcription 3; Akt, protein kinase-B; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species.




#### 5.1.2. Flavanones

**Naringenin** administration (100 µM) prevents colony formation, metastasis and EMT in breast tumor, while it induces apoptosis via upregulating p53 and ERα at mRNA level, as tumor-suppressor factors [152].

**Naringin** administration (300 µM) impairs CSC features in esophageal cancer and inhibit viability of CSCs [153].

**Hesperetin** (50–200 µM) also demonstrated potential in triggering apoptosis in breast CSCs. Hesperetin impairs invasion of breast CSCs and stimulates cell cycle arrest in breast CSCs. The hesperetin enhances p53 expression, while it down-regulates Notch1 expression in impairing stemness in breast CSCs [154].

The experiments evaluating role of flavanones in targeting CSCs in tumor suppression are limited and more studies are required to reveal true potential of these natural products in cancer therapy.

#### 5.1.3. Flavonols

Fisetin

The fisetin is a natural flavonol that has demonstrated high anti-tumor activity and capacity in chemoprevention [155,156]. Fisetin mediates histone demethylation to induce DNA damage and impair progression of pancreatic tumor cells [157]. To date, just one experiment has evaluated role of fisetin in targeting CSCs that is included here. The proliferation, metastasis, angiogenesis and carcinogenesis of renal CSCs undergo inhibition by fisetin. The in vitro and in vivo experiments have shown role of fisetin in suppressing renal CSCs. The fisetin is able to decrease expression levels of cyclin Y and CDK16 via inhibiting 5hmC modification in CpG islands. Furthermore, fisetin can reduce TET1 levels in renal CSCs. Therefore, a significant decrease occurs in growth of renal CSCs and their angiogenesis and migration are suppressed [158].

#### Epigallocatechin 3-Gallate

The epigallocatechin 3-gallate (EGCG) is another naturally occurring compound that has demonstrated high potential in cancer therapy. The EGCG enhances Beclin and LC3 levels to induce autophagy in bladder cancer cells. Furthermore, EGCG induces apoptosis in bladder cancer via upregulating Bax, caspase-3 and caspase-9 levels [159]. The EGCG reduces Sonic Hedgehog expression to suppress PI3K/Akt axis, leading to apoptosis in colon cancer cells [160]. The EGCG impairs migration and metastasis of cervical tumor cells via down-regulating TGF-β and subsequent inhibition of EMT mechanism [161]. Therefore, EGCG can be considered as a promising agent in cancer therapy [162]. Noteworthy, EGCG targets CSCs in affecting cancer progression. The lung tumor cells demonstrate high expression level of CLOCK to improve their CSC features. Noteworthy, EGCG (0–40 µM) reduces mRNA and protein levels of CLOCK to impair CSCs features and suppress selfrenewal capacity of CSCs in lung tumor therapy [163]. The miRNAs are considered as important modulators of CSCs in cancer [164–167]. The expression level of hsa-miRNA-485- 5p undergoes down-regulation in serum of lung tumor patients, showing tumor-suppressor role of this miRNA. Restoring miRNA-485-5p expression impairs lung tumor proliferation and stimulates apoptosis via down-regulating RXRα expression. The EGCG administration (0–40 µM) enhances miRNA-485-5p expression to reduce RXRα expression, leading to a decrease in expression levels of CD133 and CD44 as CSC markers [168]. The CSC features in lung cancer mainly depend on upregulation of β-catenin and its inhibition suppresses lung tumor progression. An experiment has shown that EGCG (0–100 µM) inhibits Wnt/βcatenin axis to impair CSC features in lung cancer [169].

Similar to lung cancer, a number of experiments have focused on anti-tumor activity of EGCG in colorectal cancer via targeting CSCs. In this way, EGCG (0–40 µM) suppresses Wnt/β-catenin axis to reduce expression levels of CD133, CD44, ALDHA1, Nanog and Oct4 in impairing CSC features in colorectal tumor [170]. The colorectal tumor cells demonstrate resistance to various chemotherapeutic agents including oxaliplatin and

5-flourouracil [171,172]. The EGCG (0–400 µM) decreases expression levels of Notch1, Bmi1, Suz12 and EZH2, while it enhances expression levels of miRNA-34a, miRNA-145 and miRNA-200c in impairing CSC features in colorectal cancer and enhancing 5 flourouracil sensitivity [173]. The bladder cancer cells rely on Sonic Hedgehog signaling to enhance their CSC features and mediate their progression. Noteworthy, EGCG suppresses Sonic signaling to decrease CD44, CD133, Nanog, Oct4 and ALDH1 in bladder cancer therapy [174]. Therefore, EGCG is a potent inhibitor of CSCs in various cancers and for this purpose, it targets various molecular pathways including Notch and NF-κB signaling pathways (Table 2) [175–177].


#### **Table 2.** Gallate in suppressing CSCs.
