5.1.4. Chalcones Isoliquiritigenin

The isoliquiritigenin (ISL) is derived from licorice root and it is a modulator of molecular pathways in cancer suppression [178]. The ISL suppresses TGF-β signaling to reduce Smad3 expression, resulting in EMT inhibition and decreased endometrial cancer migration [179]. Furthermore, ISL enhances miRNA-200c expression to inhibit β-catenin signaling, leading to EMT suppression in triple-negative breast cancer [180]. A combination of ISL and docosahexaenoic acid induces apoptosis in colorectal cancer via enhancing ROS levels and mediating phosphorylation of JNK and ERK [181]. The ISL-loaded liposomes suppress colorectal tumor progression via inhibiting AMPK-mediated glycolysis [182]. This section focuses on anti-tumor activity of ISL via targeting CSCs. The ISL administration (25 µM) suppresses self-renewal and multidifferential capacities of CSCs in breast cancer. Furthermore, ISL inhibit growth and colony formation of breast CSCs. The ISL suppresses β-catenin signaling and decreases ABCG2 expression to enhance drug sensitivity of breast cancer cells and suppress CSC features [183]. The expression levels of CD44 and ALDH1 undergo down-regulation upon ISL administration (0–50 µM) in oral cancer. The ISL suppresses colony formation and invasion of CSCs in oral tumor. In suppressing CSCs, ISL also decreases expression level of GRP78 at mRNA and protein levels. Furthermore, by reducing ABCG2 expression, ISL enhances sensitivity of oral CSCs to cisplatin chemotherapy [184]. Another experiment reveals that ISL administration (25 and 50 µM) enhances SIF1 expression via demethylation to suppress DNMT1 methyltransferase. Furthermore, ISL simultaneously suppresses β-catenin signaling to eradicate CSCs in breast cancer therapy [185].

#### 5.1.5. Isoflavonoids

#### Daidzein

An experiment has focused on a derivative of daidzein, known as N-t-boc-Daidzein. This derivative is able to suppress CSC features in ovarian cancer in concentration (0–10 µM) and time (0–70 h)-dependent manner. This derivative induces apoptosis in CSCs via upregulating caspase-3, -8 and -9 levels. Suppressing growth and survival of CSCs is mediated by N-t-boc-Daidzein. In addition to triggering caspase cascade, N-t-boc-Daidzein mediates mitochondrial depolarization and decreases Akt expression via degradation to impair CSC features in ovarian tumor [186].

#### Genistein

The genistein is an isoflavonoid compound that has potent anti-tumor activity. The genistein administration decreases chance of mammary cancer recurrence [187]. The glioblastoma cells exposed to radiation, demonstrate enhanced metastasis. The genistein is able to suppress Akt2 pathway in reducing invasion and migration of glioblastoma cells [188]. The genistein administration reduces hsa-circ-0031250 expression to promote miRNA-873-5p expression, leading to lung cancer suppression and decreased proliferation rate [189]. Both inflammation and GSK-3 are suppressed by genistein to inhibit ovarian tumor progression in vivo [190]. In order to improve genistein capacity in cancer suppression, nanoparticles have been developed for its targeted delivery [191,192].

The Sonic Hedgehog signaling enhances CSC features in nasopharyngeal cancer. The administration of genistein (0–100 µM) inhibits sonic signaling and reduces expression level of CSC markers including CD44, ALDH1, Oct4 and Nanog to impair nasopharyngeal progression [193]. A same strategy occurs in renal cancer, so that genistein administration (0–90 µM) suppresses Sonic Hedgehog signaling to impair growth and colony formation of renal CSCs and stimulate apoptotic cell death [194]. The genistein is able to mediate differentiation of CSCs in a paracrine mechanism to suppress cancer progression. An experiment on breast tumor has shown genistein administration (2 µM or 40 nM) stimulates PI3K/Akt and MEK/ERK molecular pathways to induce differentiation of CSCs in breast cancer [195]. Another experiment also reveals that low doses of genistein (15 µM) inhibits

**Cancer Type In Vitro/In** 

Nasopharyngeal cancer

**Vivo**

Renal cancer In vitro 786-O and ACHN cell

Breast cancer In vitro MCF-7 and MDA-MB-

**Cell Line/Animal** 

In vitro CNE2 and HONE1 cells 0–100 μM

231 cells

self-renewal capacity of gastric CSCs in vitro and in vivo [196]. The capacity of genistein in suppressing CSC features in gastric cancer is attributed to down-regulating FOXM1 expression that subsequently, reduces expression levels of CD133, CD44 and Nanog. Furthermore, by inhibiting FOXM1 expression, genistein reduces Twist1 expression that is in favor of inhibiting EMT in CSCs via reducing N-cadherin levels and enhancing E-cadherin levels [197]. In addition to aforementioned molecular pathways, genistein suppresses Gli1 expression to impair CSC features in gastric cancer [198]. Some of the molecular pathways are similar among various cancers. For instance, previous research demonstrated that genistein suppresses Gli1 pathway in reversing CSC features in gastric cancer. Another experiment in prostate cancer reveals that genistein is able to suppress Gli1 pathway in inhibiting CSC characteristics [199]. Furthermore, it was mentioned that genistein suppresses nasopharyngeal CSCs via inhibiting Sonic signaling. A similar study reveals Hedgehog signaling inhibition and subsequent Gli1 down-regulation by genistein in prostate cancer stemness suppression [199]. Genistein also suppresses Hedgehog signaling in impairing CSC features in breast cancer [200]. Overall, studies highlight the fact that genistein is a potent inhibitor of cancer progression via targeting CSCs (Figure 2, Table 3) [200–204]. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 12 of 48

**Figure 2.** The potential of isoflavonoids in suppressing CSCs. **Figure 2.** The potential of isoflavonoids in suppressing CSCs.

**Table 3.** The potential of genistein in cancer suppression via targeting CSCs.

2 μM or 40 nM

lines <sup>0</sup>–<sup>90</sup> μM

Reducing expression level of CSC markers including CD44 and CD133 via suppressing Sonic signaling

Suppressing growth and colony formation capacities of CSCs Triggering apoptosis and reducing CSC marker expression Inhibiting Sonic Hedgehog signaling

Decreasing number of CSCs Inducing PI3K/Akt and MEK/ERK pathways in a paracrine manner and mediating differentiation of CSCs

[193]

[205]

[195]


**Table 3.** The potential of genistein in cancer suppression via targeting CSCs.

#### *5.2. Pomegranate and Its Bioactive Compounds*

#### 5.2.1. Ellagic Acid

Ellagic acid is a polyphenolic compound that can suppress cancer progression. The ellagic acid increases sensitivity of bladder cancer cells via inhibiting drug transporters and EMT mechanism [206]. The p21 and p53 levels undergo upregulation by ellagic acid to induce apoptosis in prostate cancer cells [207]. The ellagic acid stimulates autophagy via Akt down-regulation and AMPK upregulation to reduce viability and survival of ovarian cancer cells [208]. The ellagic acid is a potent agent in suppressing drug resistance via down-regulating P-glycoprotein (P-gp\_levels [209]. This section focuses on anti-tumor activity of ellagic acid based on targeting CSCs.

Ellagic acid is able to target CSCs in tumor eradication. An experiment has shown role of ellagic acid in breast cancer therapy via targeting CSCs. The β-catenin stabilization is responsible for CSC features in breast cancer and ACTN4 functions as upstream mediator. Silencing ACTN4 impairs growth, migration and colony formation capacities in breast cancer. The ellagic acid administration (0–50 µM) reduces ACTN4 expression to inhibit βcatenin signaling in CSCs, impairing breast cancer progression [210]. The ALDH level as an inducer of drug resistance and CSC marker undergoes down-regulation by a combination

of ellagic acid and urolithins in colon CSCs [211]. Although just two experiments have evaluated role of ellagic acid in targeting CSCs, these obviously demonstrate role of ellagic acid in tumor treatment and more studies are required in this case.

#### 5.2.2. Caffeic Acid

Caffeic acid (CA) is a phenolic acid compound that is isolated from natural sources such as tea and has various therapeutic activities. The anti-tumor activity of CA and its derivatives have been under attention, summarized in this review [212]. The CA can mediate ROS overgeneration to induce cell death in cervical cancer cells [213]. Furthermore, CA suppresses P-gp function to mediate chemosensitivity of tumors [214]. Noteworthy, CA derivatives are also able to induce apoptosis via survivin down-regulation and caspase-3 and -9 upregulation [215,216].

The colorectal CSCs that have high ability in mediating radioresistance feature. The CD44+ and CD133+ CSCs in colorectal cancer have self-renewal capacity and carcinogenesis impact. The CA administration inhibits PI3K/Akt axis that is responsible for CSCs features in colorectal cancer, leading to enhanced radiosensitivity and reduced carcinogenesis impact [217]. The keratinocytes demonstrate high expression level of NF-κB that is beneficial for upregulating Snail expression and mediating migration and CSCs features. The CA administration (0–100 µM) enhances p38 expression to reduce potential of NF-κB in binding to Snail. Therefore, invasion and CSC features undergo inhibition [218]. The CA has a naturally occurring derivative, known as caffeic acid phenethyl ester (CAPE) that can suppress self-renewal capacity and progenitor formation in breast cancer in a dosedependent manner [219]. The expression of miRNAs is mainly affected by CA in cancer therapy. The TGF-β/Smad2 axis mediate CSC features in tumors. The CA administration (20 µM) increases miRNA-184a expression via DNA methylation, as a tumor-suppressor factor. Then, overexpressed miRNA-184a inhibits Smad2 expression by binding to its 3 0 -UTR, impairing CSC features [220]. Hence, CA and its derivative CAPE are potential anti-tumor agents in cancer treatment via targeting CSCs.

#### 5.2.3. Luteolin

The luteolin is a natural flavonoid that can suppress cancer progression via regulating autophagy [221]. The luteolin suppresses Akt/mTOR axis to reduce MMP-9 expression, impairing metastasis and invasion of breast cancer cells [222]. The luteolin suppresses EMT mechanism via down-regulating YAP/TAZ, resulting in a significant decrease in breast cancer invasion [223]. Furthermore, lutelon enhances death receptor 5 (DR5) expression and mediates mitochondrial fission to promote TRAIL sensitivity of lung tumors [224]. To date, just one experiment has evaluated role of luteolin in targeting CSCs. Mechanistically, IL-6 stimulates STAT3 signaling to promote CSC features in oral cancer and mediate their resistance to radiotherapy. The luteolin administration (0–40 µM) suppresses IL-6/STAT3 axis to impair self-renewal capacity of CSCs and reduce expression level of CSC markers including ALDH1 and CD44 [225].

#### 5.2.4. Quercetin

Quercetin is a flavonol present in fruits and vegetables such as grape, anion, apple, berries and broccoli [226]. The diet intake of quercetin is estimated to be higher than 70% of all flavonol intake [227]. Quercetin has different pharmacological activities including immunomodulatory, hepatoprotective, neuroprotective and nephroprotective that can be mediated via regulating autophagy [228]. Furthermore, quercetin can be considered as a protective agent against ischemic/reperfusion injury [229]. Regardless of its protective impacts, quercetin is suggested to display significant anti-tumor activity. Quercetin reduces CDK6 expression to impair progression of breast and lung cancer cells [230]. Quercetin enhances reactive oxygen species (ROS) levels to induce ferroptosis. Furthermore, quercetin promotes cell death via lysosome activation [231]. Angiogenesis and metastasis of esophageal cancer cells was suppressed by quercetin via reducing expression

levels of VEGF-A, MMP-2 and MMP-9 [159]. Quercetin reverses multidrug resistance and using nanoparticles for its delivery enhances its potential in cancer suppression [232]. This section focuses on CSC targeting by quercetin in cancer therapy.

The PI3K/Akt/mTOR axis is responsible for growth and progression of CSCs in breast cancer. The quercetin administration (0–200 µM) suppresses PI3K/Akt/mTOR axis in induce cell cycle arrest in G1 phase in breast cancer cells. The quercetin impairs viability, colony formation and mammosphere formation in CD44+ stem cells in breast cancer and triggers apoptosis [233]. Another experiment also reveals role of quercetin in eradicating CSCs in breast cancer. The quercetin administration (0–200 µM) suppresses growth, metastasis and self-renewal capacity of CSCs in breast cancer. For this purpose, quercetin reduces expression levels of ALDH1, CXCR4, mucin 1 and epithelial cell adhesion molecule (EpCAM) [234]. The nuclear translocation of Y-box binding protein 1 (YB-1) is responsible for CSCs features in breast cancer. For reversing multidrug resistance in breast cancer, quercetin decreases expression level of P-gp as an efflux transporter. More importantly, quercetin inhibits nuclear translocation of YB-1 to suppress CSCs features in breast cancer cells and enhance their sensitivity to doxorubicin, paclitaxel and vincristine [235]. It is worth mentioning that by down-regulating expression levels of P-gp, BCRP and MRP1, quercetin (0–2 µM) increases internalization of doxorubicin in breast cancer cells, resulting in eradication of CSCs [236].

Another cancer that can be affected by quercetin is pancreatic cancer. For enhancing potential of quercetin in suppressing CSCs, its combination therapy has been used. For this purpose, a combination of sulforaphane (SFN) (0–10 µM) and quercetin (20 µM) effectively inhibits self-renewal capacity of CSCs [237]. In pancreatic cancer, CSC divisions tend to be symmetric. The low expression of miRNA-200b-3p induces Notch signaling to make daughter cells be symmetric. However, quercetin administration (50 µM) enhances expression level of miRNA-200b-3p as tumor-suppressor to reduce Notch expression, resulting in asymmetric divisions in CSCs [238]. A combination of SFN, quercetin and catechins reduces ALDH1 expression in pancreatic cancer, as CSC marker. For this purpose, SFN, quercetin and catechins enhances miRNA-let-7 expression to down-regulate K-ras expression [239]. Hence, quercetin and its combination with anti-tumor agents synergistically suppress CSC features in pancreatic cancer [240].

The induction of Notch1 signaling and enhanced CSC features lead to radioresistance in colon cancer. The quercetin administration (20 µM) suppresses Notch1 signaling and reduces expression levels of CSC markers including SOX9, CD133 and CD44 [241]. The CD133+ colorectal cancer cells seem to be responsible for triggering drug resistance in colorectal cancer. The quercetin administration (10–100 µM) induces apoptosis and cycle arrest (G2/M phase) in CD133+ cells to enhance sensitivity of colorectal tumor cells to doxorubicin chemotherapy [242]. Therefore, quercetin targets CSCs to increase therapy response of colorectal cancer cells.

The prostate cancer (PCa) is the most common cancer and second malignant tumor in men [2,243]. The various molecular pathways are responsible for PCa progression and various anti-tumor compounds and genetic tools have been utilized for its treatment [20,244–247]. The quercetin has shown capacity in suppressing growth and invasion of PCa stem cells (CD44+/CD133+ cells). The midkin (MK) pathway is responsible for CSC features in PCa. Co-application of quercetin and siRNA-MK enhances potential in suppressing CSCs. Furthermore, this combination induces apoptosis and G1 arrest in CSCs. The molecular pathways including PI3K/Akt, ERK1/2, p38, ABCG2 and NF-κB undergo down-regulation by quercetin and siRNA-MK in inhibiting CSCs in PCa [248]. Furthermore, a combination of quercetin and EGCG induces apoptosis (via caspase-3/7 upregulation and down-regulating Bcl-2, survivin and XIAP) in CSCs and prevents their growth and invasion. The reduced metastasis of CSCs by quercetin and EGCG is attributed to suppressing epithelial-to-mesenchymal transition (EMT) via down-regulating Snail, vimentin, Slug and β-catenin [249]. Therefore, quercetin is a well-known compound in eradicating CSCs in preventing cancer progression (Figure 3, Table 4) [250,251].

Pancreatic cancer In vitro

Pancreatic cancer In vitro AsPC1 and

In vivo

Colon cancer In vitro DLD-1 and HT-

Colorectal cancer In vitro HT29 cells 0–100 μM

BxPc-3 and MIA-PaCa2 cells Nude mice and xenografts

100, 200 and 400 μM 50 mg/kg

29 cells <sup>0</sup>–<sup>50</sup> μM

ARPE-19 cells 40 μM

PANC1 cells 50 μM Suppressing self-renewal capacity of tumor cells

via enhancing miRNA-200b expression [238]

[240]

[241]

[242]

[248]

Synergistic impact between quercetin and sulforaphane Down-regulating NF-κB signaling Impairing tumor growth in vitro and in vivo

Suppressing Notch signaling, impairing CSCs features and increasing sensitivity of tumor cells to radiotherapy

Triggering apoptosis and cell cycle arrest in CSCs Promoting sensitivity of tumor cells to doxorubicin chemotherapy

Queretin and midkine-siRNA co-application suppresses CSC features via dual inhibition of PI3K/Akt and MAPK/ERK molecular pathways

**Figure 3.** The potential of bioactive constituents of pomegranate in suppressing CSCs. **Figure 3.** The potential of bioactive constituents of pomegranate in suppressing CSCs.




#### *5.3. Carotenoids*

#### 5.3.1. Astaxanthin

The astaxanthin is derived from algae and has anti-tumor activity. Astaxanthin The astaxanthin impairs gastric cancer development and progression via suppressing inflammatory storm [252]. Astaxanthin induces apoptosis and promotes PARPγ expression to reduce esophageal cancer progression [253]. The pontin overexpression is responsible for CSC features in breast cancer. Suppressing pontin expression impairs colony formation and CSC characteristics in breast cancer. The astaxanthin administration (80 and 100 µM) reduces pontin expression and impairs CSC features via down-regulating Oct4, Nanog and mutp53 levels [254].

### 5.3.2. β-Carotene

The β-carotene is another member of carotenoids that is able to effectively suppress cancer progression. The β-carotene suppresses IL-6/STAT3 axis and inhibits M2 polarization of macrophages to impair colon tumor progression [255]. β-carotene induces apoptosis and reduces antioxidant markers to impair breast tumor progression [256]. A combination of β-carotene and lycopene suppresses esophageal cancer progression via down-regulating COX-2 and cyclin D1 expression levels [257]. For promoting potential of β-carotene in breast cancer suppression, lipid–polymer nanoparticles have been developed for its targeted delivery [258]. Compared to astaxanthin, more experiments have focused on role of β-carotene in targeting CSCs. The β-carotene administration (20 and 40 µM) reduces growth and colony formation capacities of colon CSCs. The βcarotene reduces DNMT3A expression at mRNA levels and prevents DNA methylation. Furthermore, β-carotene promotes histone H3 and H4 acetylation levels in impairing CSC progression [259]. The self-renewal capacity of CSCs undergoes a decrease by β-carotene in neuroblastoma cells and this leads to enhanced sensitivity of tumor cells to cisplatin chemotherapy [260]. Furthermore, in vivo experiment on xenograft model has shown role of β-carotene in suppressing CSC features in neuroblastoma [261]. The Oct3/4 and DLK1 undergo down-regulation by β-carotene in decreasing CSC features and stemness of

neuroblastoma cells [262]. The potential of β-carotene in reducing DLK1 expression and impairing CSC features is mediated via retinoic acid receptor β [263].

#### *5.4. Sulforaphane*

The sulforaphane (SFN) is an isothiocyanate and is present in various vegetables including cabbage broccoli, cauliflower and Brussels. The SFN is generated upon glucoraphanin hydrolysis. The SNF application in cancer therapy has witnessed a growing increase [264]. A recent experiment demonstrates that SFN reduces expression level of H19, as a lncRNA to suppress growth and invasion of pancreatic cancer cells [265]. The SFN is able to suppress tubulin polymerization in triggering apoptosis and cell cycle arrest in glioblastoma cells [266]. The expression level of FAT-1 undergoes down-regulation by SFN to impair proliferation and invasion of bladder cancer cells [267]. The SFN administration is of interest in increasing sensitivity of breast cancer cells to doxorubicin chemotherapy via inhibiting myeloid-derived suppressor cells [268]. Therefore, SFN is a potent anti-tumor agent [269] and this section focuses on CSC targeting by SFN in cancer treatment.

The activation of Sonic Hedgehog signaling promotes stemness of gastric cancer cells. The SFN administration (0–10 µM) stimulates apoptosis in CSCs and inhibits their growth to suppress gastric cancer proliferation. For this purpose, SFN inhibits Sonic signaling [270]. The upregulation of TAp63α occurs in colorectal tumor spheres with CSC features. Noteworthy, TAp63α enhances self-renewal capacity and CSC markers in colorectal tumor. Investigation of molecular pathways reveals that SFN increases Lgr5 expression by binding to its promoter, leading to β-catenin signaling activation. The SFN administration (0–10 µM) reduces TAp63α expression to inhibit expression of CSC markers such as CD133, CD44, Nanog and Oct4 [224].

Human tumor necrosis factor (TNF)-related apoptosis ligand (TRAIL) is suggested to be involved in triggering apoptosis in tumor cells [271]. However, experiments have shown capacity of tumor cells in obtaining resistance to TRAIL-mediated apoptosis [272–275]. Furthermore, TRAIL can surprisingly enhance progression of cancer cells via inducing NFκB signaling [276,277]. An experiment has shown synergistic impact between TRAIL and SFN in PCa therapy. Both TRAIL and SFN are able to suppress CSCs in PCa, and SFN has higher ability compared to TRAIL. However, TRAIL induces NF-κB signaling and promotes PCa progression. The SFN administration (10 µM) decreases expression level of NF-kB, CXCR4, Jagged1, Notch-1, SOX2, Nanog and ALDH1 to impairs CSC features and stemness in PCa [278]. It is worth mentioning that down-regulation of ALDH1, c-Rel and Nothc-1 as CSC markers by SFN, significantly elevates sensitivity of prostate and pancreas cancer cells to cisplatin, gemcitabine and 5-flourouracil chemotherapy [279]. The stimulation of NF-κB signaling is responsible for ALDH1 upregulation and enhanced CSC features in pancreatic cancer. Therefore, NF-κB inhibition by SFN (20 µmol/L) impairs CSC features in pancreatic cancer cells and enhances their sensitivity to sorafenib chemotherapy [280]. Overall, the experiments advocate the fact that SFN is a potent agent in targeting CSC and suppressing stemness of cancers (Figure 4, Table 5) [281–285].

c-Rel and Nothc-1 as CSC markers by SFN, significantly elevates sensitivity of prostate and pancreas cancer cells to cisplatin, gemcitabine and 5-flourouracil chemotherapy [279]. The stimulation of NF-κB signaling is responsible for ALDH1 upregulation and enhanced CSC features in pancreatic cancer. Therefore, NF-κB inhibition by SFN (20 μmol/L) impairs CSC features in pancreatic cancer cells and enhances their sensitivity to sorafenib chemotherapy [280]. Overall, the experiments advocate the fact that SFN is a potent agent in targeting CSC and suppressing stemness of cancers (Figure 4, Table 5) [281–285].

**Figure 4.** The potential of carotenoids in suppressing CSCs. **Figure 4.** The potential of carotenoids in suppressing CSCs.




#### *5.5. Curcumin*

The *curcuma longa* is a medicinal plant belonging to family of Zingiberaceae family that has a well-known bioactive compound, curcumin that can be isolated from its rhizome [294,295]. The curcumin and other curcuminoids lead to yellow color of rhizome. Although content of curcuminoids in rhizome of *curcuma longa* is various, it seems that curcumin is the main component and comprises 77% [296]. The curcumin has demonstrated various therapeutic activities that among them, anti-cancer potential is of importance [297]. The curcumin administration is beneficial in suppressing proliferation and metastasis of cancer cells. A recent experiment has shown role of curcumin in down-regulating NF-κB, ERK, MMP-2 and MMP-9, while it can enhance p38 and JNK levels to induce cell death and prevent invasion of leukemia cells [298]. Recently, attention has been directed towards chemopreventive role of curcumin in cancer therapy [299]. The curcumin- and docetaxelloaded micellar nanoparticles effectively suppress progression of ovarian cancer cells via suppressing angiogenesis and inducing apoptosis. The curcumin is able to sensitize ovarian cancer cells to docetaxel chemotherapy [300]. Due to poor bioavailability of curcumin, nanostructures are mainly applied for curcumin delivery alone or its combination with other anti-tumor agents to exert synergistic cancer therapy [31,301]. A fibrin matrix has been developed for prolonged release of curcumin and suppressing cancer metastasis. As studies have shown role of the nanoplatforms for promoting therapeutic activity of curcumin, future clinical trials can be performed [302]. The current section focuses on anti-tumor activity of curcumin based on targeting CSCs.

The capacity of curcumin in cancer suppression via targeting CSCs is attributed to affecting various molecular pathways. The activation of Wnt/β-catenin and Sonic Hedgehog molecular pathways results in CSC features in lung cancer and enhancing tumor progression. The curcumin administration (0–40 µM) induces apoptosis in CSCs and diminishes their proliferation. Furthermore, curcumin reduces CSC hallmarks such as Nanog, Oct4, CD133, CD44 and ALDH1A1. Mechanistically, these anti-tumor activities of curcumin are mediated via inhibiting Wnt and Hedgehog signaling pathways [303]. In addition, nuclear factor-kappaB (NF-κB) pathway also participates in enhancing survival of CSCs [304]. The down-regulation of NF-κB signaling impairs CSC features in bladder cancer [305]. In liver cancer cells, curcumin reduces their proliferation and colony formation capacities as well as inhibiting CSCs features. The curcumin capacity in suppressing CSCs features in liver cancer is pertained to inhibiting NF-κB signaling [306]. Hence, identification of molecular pathways affected by curcumin in suppressing CSC features can broaden our understanding towards underlying mechanisms of its anti-tumor activity.

The signal transducer and activator of transcription 3 (STAT3) is another tumorpromoting factor related to cancer progression [307–309]. The upregulation of STAT3 enhances growth and invasion of tumor cells and mediates their therapy resistance [310–316]. The activation of STAT3 signaling promotes CSC features in tumors and its inhibition, for instance by acetaminophen, reduces CSC markers and colony formation capacity [317,318]. The curcumin administration (0–40 µM) decreases levels of CD44, ALDH, SOX2, Nanog and c-Myc as CSC markers to impairs thyroid cancer progression and induce apoptosis. Furthermore, by reducing CSC features, curcumin enhances potential of cisplatin in thyroid cancer suppression [319]. Similar phenomenon occurs in bladder cancer, so that curcumin administration (0–50 µM) decreases CD44, CD133, ALDH1, Nanog and Oct4 to impair CSC characteristics in bladder cancer and induce apoptosis and proliferation inhibition. The investigation of molecular pathways reveals that these anti-tumor activities are mediated via sonic signaling inhibition [320]. The interesting point is the curcumin and CD44 coupling in cancer therapy. An experiment demonstrates that colon cancer cells overexpressing CD44 as CSC marker are more sensitive to curcumin compared to CD44- cells. The curcumin-CD44 coupling stimulates apoptosis in colorectal stem cells via preventing influx of glutamine into cancer cells and reducing its intracellular accumulation [321].

In order to potentiate anti-tumor activity of curcumin, its combination with other tumor-suppressor agents is performed. A combination of curcumin and quinacrine can be beneficial in impairing breast cancer progression via targeting CSCs. The quinacrine is able to induce DNA damage in cancer cells, but its intracellular accumulation should be improved. In this case, curcumin is helpful and by suppressing ABCG2 activity via occupying its ligand-binding site, enhances internalization of quinacrine, resulting in cell death and DNA damage in breast CSCs [322]. Furthermore, curcumin also has capacity of inducing DNA damage in CSCs [323]. The curcumin can negatively affect colony formation capacity of lung cancer cells. It is suggested that curcumin prevents self-renewal capacity of CSCs to suppress colony formation of lung cancer cells [323]. Therefore, curcumin is a potent suppressor of CSCs in lung cancer and for this purpose, it can inhibit JAK2/STAT3 axis to impair CSC features [324]. Based on previously discussed molecular pathways, both STAT3 and NF-κB pathways are suppressed by curcumin in targeting CSCs. Noteworthy, STAT3 can function as upstream mediator of NF-κB and mediates its activation via enhancing IKKα stability [325]. A combination of curcumin (10 µM) and epigallocatechin gallate (10 µM) suppresses STAT3/NF-κB axis to impair CSC features and reduce number of CD44+ cells [326]. In addition to curcumin, its derivatives have been also capable of targeting CSCs. The colon cancer cells positive for ALDH and CD133 demonstrate activation of STAT3 signaling and its phosphorylation. An analogue of curcumin, known as GO-Y030 can suppress STAT3 phosphorylation to impair CSC features in colon cancer and prevent its progression [327]. Therefore, curcumin and its derivatives suppress proliferation and invasion of tumors via targeting CSCs. Furthermore, they can enhance sensitivity of cancer cells to therapies by targeting CSCs [328]. Interestingly, curcumin can be advantageous in enhancing sensitivity of cancer cells to radiotherapy [329]. The breast cancer stem-like cells have the ability of obtaining radioresistance. A combination of curcumin and glucose gold nanoparticles induces apoptosis in CSCs and reduces expression levels of hypoxia inducible factor-1α (HIF-1α) and HSP90 in sensitizing breast cancer cells to radiotherapy [330]. Therefore, curcumin is a versatile agent in suppressing CSC features in cancer and various molecular pathways such as Wnt, Sonic, STAT3 and NF-κB, among others, are affected to reduce CSC markers such as CD44, ALDH and CD133 (Figure 5, Table 6) [331–338].

Pancreatic cancer

Prostate cancer

cancer

Bladder cancer

In vitro UM-UC-3 and EJ

Breast cancer In vitro MCF-10A and MCF-

Breast cancer In vitro SUM159 and MCF7

Breast cancer In vitro MDA-MB-231 and

In vitro In vivo

Brain cancer In vitro U87MG cells -

BxPC3, MiaPaCa2 and Panc1 PDAC cells Nude mice

In vitro DU145 cells -

cells <sup>0</sup>–<sup>50</sup> μM

<sup>7</sup> cells 8 μM

cell lines <sup>0</sup>–<sup>40</sup> μM

cells <sup>0</sup>–<sup>20</sup> μM

0–20 μM 100 mg/kg

Inhibiting Wnt/β-catenin and Sonic signaling pathways

Apoptosis induction Decreasing expression levels of CD44, CD133, Oct4, Nanog and ALDH1 Inhibiting Sonic Hedgehog signaling

A combination of curcumin and quinacrine induces DNA damage and reduces ABCG2 expression to impair cancer progression

Reducing colony formation capacity of CSCs Down-regulating expression levels of CD44, ALDH1, Nanog and Oct4 Inhibiting Wnt/β-catenin axis

The curcumin-loaded nanoparticles efficiently penetrate into BBB to induce apoptosis in CSCs and reduce tumor progression

Suppressing self-renewal capacity of tumor cells Retarding tumor growth in vivo Enhancing sensitivity to gemcitabine chemotherapy

Inhibiting growth and invasion of prostate CSCs Overexpression of miRNA-770-5p and miRNA-1247 [337]

Reducing expression level of CSC markers including CD44, Oct4 and ALDH1 Inhibiting STAT3 signaling

Enhancing sensitivity of breast CSCs to mitomycin C [328]

MCF-7 cells 5 μmol/L Inducing apoptosis via Bcl-2 down-regulation

[320]

[322]

[333]

[332]

[339]

[338]

**Figure 5. Figure 5.** Curcumin in inhibiting CSCs. Curcumin in inhibiting CSCs.



#### *5.6. Resveratrol*

The resveratrol (Res) is a well-known compound in treatment of different ailments including diabetes, cancer and neurological disorders [340,341]. The recent years have witnessed an increase in attention towards Res application in cancer therapy. Res induces apoptosis via impairing mitochondrial function in cancer cells. Furthermore, tumorpromoting factors such as NF-κB, COX-2 and PI3K undergo down-regulation by Res in tumor suppression [342]. A combination of Res and radiation stimulates apoptosis in breast cancer via Bcl-2 down-regulation and Bax upregulation [50]. Res suppresses phosphorylation of STAT3 at tyrosine 705 to impair metastasis of cervical cancer cells [343]. Down-regulation of HPV E6 and E7 by Res leads to apoptosis and cell cycle arrest in cervical cancer [344]. Furthermore, Res inhibits progression and growth of PCa cells via suppressing PI3K/Akt signaling [345]. The c-Myc as downstream target of PI3K/Akt axis is suppressed by Res in lung cancer therapy [346]. Loading Res on nanostructures significantly promotes its potential in thyroid cancer suppression. Furthermore, Res-loaded nanoparticles decrease tumor volume up to 55% in thyroid cancer in vivo [347].

The tumor sphere formation capacity in renal cancer is mediated by CSCs and Sonic signaling plays a significant role in this case. The Res administration (0–30 µM) downregulates Sonic signaling to inhibit CSCs features in renal cancer, leading to apoptosis and proliferation inhibition [348]. As it was mentioned earlier, Res is loaded on nanoparticles to promote its anti-tumor activity [349–351]. Another promising strategy for enhancing bioavailability of Res in synthesizing analogues with superior activities. The pterostilbene is a derivative of Res that has higher bioavailability compared to Res. The pterostilbene is able to reduce expression levels of CD133, Oct4, SOX2, Nanog and STAT3 in impairing CSC features and retarding progression of cervical cancer [352]. Furthermore, Res is able to suppress Wnt/β-catenin in reducing CSC features in breast cancer. It is worth mentioning that Res enhances LC-3II, Beclin-1 and ATG7 expression levels in triggering autophagy in CSCs and reducing breast cancer progression [353]. It is worth mentioning that autophagy has both tumor-promoting and tumor-suppressor roles in cancer [221,354,355] and its induction by Res requires more clarification.

The STAT3 signaling is considered as a tumor-promoting factor in osteosarcoma. The upregulation of STAT3 by VEGFR2 results in metastasis of osteosarcoma cells [356]. Fur-

thermore, induction of JAK2/STAT3 axis by exosomal LCP1 leads to carcinogenesis and migration of osteosarcoma cells [357]. The Res administration (0–3 µM) decreases cytokine generation and suppresses JAK2/STAT3 axis to impair CSCs features in osteosarcoma via CD133 down-regulation [358]. Another molecular pathway that is responsible for CSCs features is nutrient-deprivation autophagy factor-1 (NAF-1). The Res inhibits NAF-1 signaling to reduce CSC features including SOX2, Oct4 and Nanog in pancreatic cancer treatment [359]. The interesting point is the interaction between tumor microenvironment (TME) components and molecular pathways in providing CSCs features. The T lymphocytes and fibroblasts cells present in TME can effectively induce NF-κB signaling and upon nuclear translocation, NF-κB significantly enhances growth, metastasis and survival of CSCs in colorectal cancer. The TNF-β and TGF-β3 are secreted by T lymphocytes and fibroblasts in inducing NF-κB signaling. Noteworthy, Res (0–10 µM) prevents secretion of TNF-β and TGF-β3 by lymphocytes and fibroblasts in reducing CSC features in colorectal cancer [360]. Another experiment reveals that cancer-associated fibroblasts (CAFs) existing in TME are able to reduce SOX2 expression and suppress stemness in breast cancer via reducing CSC features [361].

Another strategy that can be followed by Res, is the induction of endothelial differentiation. For this purpose, Res and sulindac stimulate vascular endothelial cadherin (VE-cadherin) and von Willebrand factor (vWF) in triggering trans-differentiation in CSCs and mediating their transformation into endothelial lineage [362]. Recently, attention has been directed towards using siRNA and natural compounds in synergistic cancer therapy [312]. An experiment has shown that RAD51 has oncogenic role and enhances CSC features in cervical cancer. A combination of Res and RAD51-siRNA induces apoptosis in HeLa cells and prevents cervical cancer progression [363]. Overall, experiments reveal role of Res in suppressing CSCs in cancer progression (Figure 6, Table 7) [364–367]. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 24 of 48

**Figure 6.** Res in suppressing CSCs in tumor therapy. **Figure 6.** Res in suppressing CSCs in tumor therapy.

*5.7. Berberine*

on BBR function in affecting CSCs.

The berberine (BBR) is an isoquinoline alkaloid mainly derived from *Coptis chinesis* with great therapeutic impacts [375]. In addition, BBR can be isolated from other *Berberis*

The pancreatic cancer treatment is an increasing challenge for physicians, as the tumor cells have high growth and migration capabilities [383,384]. Furthermore, they can develop drug resistance and risk of recurrence is present in pancreatic cancer patients. The CSCs play a significant role in aforementioned processes [385–388]. An experiment has shown that BBR (15 µM) reduces expression levels of SOX2, Nanog and POU5F1 as CSC markers to impair resistance of pancreatic cancer cells to gemcitabine and suppress their progression [389]. One of the targets of BBR in cancer therapy is miRNAs. Overall, the expression level of tumor-promoting miRNAs undergoes down-regulation by BBR, while an increase occurs in expression profile of tumor-suppressor miRNAs [390–392]. The oral CSCs have self-renewal capacity and demonstrate high colony formation and migration abilities that are in favor of triggering drug resistance. The miRNA-21 overexpression plays a tumor-promoting role in oral CSCs. The BBR (10 μM) reduces expression

macological activities including antioxidant, anti-inflammatory, anti-diabetes, hepatoprotective and renoproective, among others, much attention has been directed towards its anti-tumor activities [377–380]. The BBR induces apoptosis, autophagy and cycle arrest (G1 phase) in colon cancer cells. Mechanistically, BBR enhances PTEN expression, while it reduces expression of PI3K, Akt and mTOR [151]. Furthermore, BBR suppresses fatty acid metabolism and diminishes extracellular vesicle production in favor of cancer growth suppression [381]. The BBR is able to decrease expression level of IGF2BP3 in triggering cycle arrest (G0/G1 phase) in colorectal cancer cells [382]. The current section emphasizes


**Table 7.** Determining resveratrol potential in targeting CSCs and suppressing tumor progression.

#### *5.7. Berberine*

The berberine (BBR) is an isoquinoline alkaloid mainly derived from *Coptis chinesis* with great therapeutic impacts [375]. In addition, BBR can be isolated from other *Berberis* plants such as Berberis julianae and Scutellaria baicalensis [376]. Although BBR has pharmacological activities including antioxidant, anti-inflammatory, anti-diabetes, hepatoprotective and renoproective, among others, much attention has been directed towards

its anti-tumor activities [377–380]. The BBR induces apoptosis, autophagy and cycle arrest (G1 phase) in colon cancer cells. Mechanistically, BBR enhances PTEN expression, while it reduces expression of PI3K, Akt and mTOR [151]. Furthermore, BBR suppresses fatty acid metabolism and diminishes extracellular vesicle production in favor of cancer growth suppression [381]. The BBR is able to decrease expression level of IGF2BP3 in triggering cycle arrest (G0/G1 phase) in colorectal cancer cells [382]. The current section emphasizes on BBR function in affecting CSCs.

The pancreatic cancer treatment is an increasing challenge for physicians, as the tumor cells have high growth and migration capabilities [383,384]. Furthermore, they can develop drug resistance and risk of recurrence is present in pancreatic cancer patients. The CSCs play a significant role in aforementioned processes [385–388]. An experiment has shown that BBR (15 µM) reduces expression levels of SOX2, Nanog and POU5F1 as CSC markers to impair resistance of pancreatic cancer cells to gemcitabine and suppress their progression [389]. One of the targets of BBR in cancer therapy is miRNAs. Overall, the expression level of tumor-promoting miRNAs undergoes down-regulation by BBR, while an increase occurs in expression profile of tumor-suppressor miRNAs [390–392]. The oral CSCs have self-renewal capacity and demonstrate high colony formation and migration abilities that are in favor of triggering drug resistance. The miRNA-21 overexpression plays a tumor-promoting role in oral CSCs. The BBR (10 µM) reduces expression level of miRNA-21 to impair self-renewal capacity of CSCs and decrease ALDH1 expression. Then, a significant increase occurs in sensitivity of oral CSCs to cisplatin and 5-flourouracil chemotherapy [393].

The glioma-associated oncogene-1 (Gli1) is a tumor-promoting factor that its upregulation enhances growth and invasion of colorectal cancer cells [394]. Noteworthy, Gli1 participates in enhancing CSCs features and mediating tumor progression [395]. Therefore, its inhibition by BBR can exert anti-tumor activities. A recent experiment has shown that chemotherapy enhances ovarian cancer progression via enhancing CSCs features. The Gli1 and its downstream target BMI1 are involved in promoting CSCs features in ovarian cancer. The BBR administration down-regulates Gli1 expression to inhibit BMI1, leading to a decrease in CSC features [396]. Overall, CD133, β-catenin, n-Myc, nestin, SOX2 and Notch2 undergo down-regulation by BBR in suppressing CSC features [397]. Furthermore, BBR co-administration with Dodecyl-TPP (d-TPP) can synergistically suppress CSC features in breast cancer [205]. Moreover, to selectively target CSCs, nanostructures can be utilized [398,399]. In an effort, BBR was loaded on liposomal nanocarriers and results demonstrated potential of these nanocarriers in crossing over CSC membrane. Then, they can reduce expression level of ABCC1, ABCC2, ABCC3 and ABCG2. Furthermore, BBR-loaded liposomes selectively internalize in mitochondria and induce apoptosis via triggering mitochondrial membrane potential loss, enhancing Bax expression and reducing Bcl-2 level. Then, cytochrome C (cyt C) release occurs and promotes caspase-3 and -9 expressions to mediate apoptosis in CSCs and suppress breast cancer progression. Furthermore, in vivo experiment on xenografts in nude mice also has shown potential of BBR-loaded liposomes in suppressing tumor growth (Table 8) [400].


**Table 8.** The BBR in targeting CSCs for tumor suppression.


#### *5.8. Ginseng and Its Derivatives*

The ginsenosides are derivatives from ginseng that have therapeutics impacts including anti-diabetes, anti-inflammatory and neuroprotective, and among them, anti-tumor activity of ginsenosides is of importance [401–403]. The ginsenoside Rg3 is able to suppress progression of colorectal tumor cells and enhance their sensitivity to oxaliplatin and 5-flouroruacil chemotherapy. Mechanistically, ginsenoside Rg3 (200 µM) reduces levels of CD24, CD44 and EpCAM to inhibit stemness and population of CSCs [404]. The ginsenoside Rh2 also suppress skin cancer progression in a concentration-dependent manner (0–1 mg/mL) and induces autophagy to suppress β-catenin signaling and impair CSC features in skin cancer. Autophagy inhibition abrogates capacity of ginsenoside Rh2 in suppressing β-catenin signaling and CSC features in skin cancer. Therefore, autophagy induction by ginsenoside Rh2 is of importance for inhibiting β-catenin-mediated CSC features in skin cancer [405]. However, a special attention should be directed towards autophagy, as it has both tumor-promoting and tumor-suppressor roles in cancer [355]. An experiment demonstrates that ginsenoside F2 (0–120 µM) stimulates apoptosis and autophagy in breast CSCs. Noteworthy, autophagy plays as a pro-survival mechanism and autophagy inhibition increases ginsenoside F2 capacity in mediating apoptosis in CSCs [406]. The ginsenoside Rb1 and compound K are able to suppress self-renewal capacity of CSCs in ovarian cancer. Furthermore, Rb1 and compound K enhance sensitivity of ovarian cancer cells to cisplatin and paclitaxel chemotherapy via suppressing CSCs. The drug sensitivity activity of Rb1 and compound K are mediated via suppressing β-catenin signaling and subsequent down-regulation of ABCG2 and P-gp. Furthermore, Rb1 and compound K suppress CSC migration and invasion via EMT inhibition [407]. For suppressing self-renewal capacity of CSCs, ginsenoside Rg3 (0–100 µM) suppresses Akt signaling. Furthermore, ginsenoside Rg3 reduces HIF-1α expression to down-regulate

SOX-2 and Bmi-1 expression levels, leading to decreased stemness and CSC features in breast cancer [408]. Overall, ginsenosides similar to genistein, are able to suppress cancer progression via affecting CSCs [409–411]. Besides, ginsenosides are derived from ginseng extract and it has been reported that ginseng extract is also capable of suppressing CSC features in cancer (Figure 7, Table 9) [412,413]. Figures 8 and 9 show the chemical structures of selected phytochemicals discussed in the current review. 1 expression levels, leading to decreased stemness and CSC features in breast cancer [408]. Overall, ginsenosides similar to genistein, are able to suppress cancer progression via affecting CSCs [409–411]. Besides, ginsenosides are derived from ginseng extract and it has been reported that ginseng extract is also capable of suppressing CSC features in cancer (Figure 7, Table 9) [412,413]. Figures 8 and 9 show the chemical structures of selected phytochemicals discussed in the current review.

The ginsenosides are derivatives from ginseng that have therapeutics impacts including anti-diabetes, anti-inflammatory and neuroprotective, and among them, anti-tumor activity of ginsenosides is of importance [401–403]. The ginsenoside Rg3 is able to suppress progression of colorectal tumor cells and enhance their sensitivity to oxaliplatin and 5 flouroruacil chemotherapy. Mechanistically, ginsenoside Rg3 (200 μM) reduces levels of CD24, CD44 and EpCAM to inhibit stemness and population of CSCs [404]. The ginsenoside Rh2 also suppress skin cancer progression in a concentration-dependent manner (0–1 mg/mL) and induces autophagy to suppress β-catenin signaling and impair CSC features in skin cancer. Autophagy inhibition abrogates capacity of ginsenoside Rh2 in suppressing β-catenin signaling and CSC features in skin cancer. Therefore, autophagy induction by ginsenoside Rh2 is of importance for inhibiting β-catenin-mediated CSC features in skin cancer [405]. However, a special attention should be directed towards autophagy, as it has both tumor-promoting and tumor-suppressor roles in cancer [355]. An experiment demonstrates that ginsenoside F2 (0– 120 μM) stimulates apoptosis and autophagy in breast CSCs. Noteworthy, autophagy plays as a pro-survival mechanism and autophagy inhibition increases ginsenoside F2 capacity in mediating apoptosis in CSCs [406]. The ginsenoside Rb1 and compound K are able to suppress self-renewal capacity of CSCs in ovarian cancer. Furthermore, Rb1 and compound K enhance sensitivity of ovarian cancer cells to cisplatin and paclitaxel chemotherapy via suppressing CSCs. The drug sensitivity activity of Rb1 and compound K are mediated via suppressing βcatenin signaling and subsequent down-regulation of ABCG2 and P-gp. Furthermore, Rb1 and compound K suppress CSC migration and invasion via EMT inhibition [407]. For suppressing self-renewal capacity of CSCs, ginsenoside Rg3 (0–100 μM) suppresses Akt signaling. Furthermore, ginsenoside Rg3 reduces HIF-1α expression to down-regulate SOX-2 and Bmi-

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 26 of 48

*5.8. Ginseng and Its Derivatives*

**Figure 7.** The berberine and ginsenosides as modulators of CSCs in tumor eradication. **Figure 7.** The berberine and ginsenosides as modulators of CSCs in tumor eradication.




**Figure 8.** The chemical structures of selected phytochemicals discussed in the review. **Figure 8.** The chemical structures of selected phytochemicals discussed in the review.

**Anti-Tumor Agent**

Ginsenoside Rg3

Ginsenoside

Ginsenoside Rb1

Gisnenoside Rg3

Ginsenoside F2

Ginsenoside Rh2

**Cancer Type**

Colorectal cancer

Ovarian cancer

Breast cancer

Breast cancer

Hepatocellular carcinoma

**Figure 9.** Chemical structures of ginsenosides. **Figure 9.** Chemical structures of ginsenosides.

#### **Table 9.** Ginsenosides as potential inhibitors of CSCs in different tumors. **6. Conclusions and Remarks**

**In Vitro/In Vivo Cell Line/Animal Model Study Design Remarks Refs** In vitro In vivo LoVo, SW620 and HCT116 cells Nude mice - Promoting anti-tumor potential of oxaliplatin and 5-flourouracil Suppressing stemness and CSC features [404] Rh2 Skin cancer In vitro A431 cells <sup>0</sup>–1 mg/mL Inhibiting viability of tumor cells Autophagy induction and β-catenin signaling inhibition [405] In vitro SKOV-3 and 0–500 nM Suppressing Wnt signaling and reversing EMT to A variety of factors have been found to be responsible for tumor progression. The CSCs role in tumor progression has been confirmed in different experiments. In addition, molecular pathways participating in CSC feature suppression/induction have been shown. The CSCs enhance stemness of tumor cells and can mediate their growth and migration. Furthermore, CSCs are able to reduce sensitivity of tumor cells to chemotherapy and radiotherapy. The CD44, CD33, ALDH1, Nanog, SOX2 and Oct2 are the most well-known CSC markers. The overexpression of these factors significantly enhances stemness of tumors. Various molecular pathways are able to enhance cancer progression and stemness that include STAT3, NF-κB, Wnt/β-catenin signaling and Sonic Hedgehog, among others. Such molecular pathways were discussed in main text and it was shown that their inhibition can reduce expression of CSC markers and mediate tumor suppression.

In vivo HEYA8 cells Mice 50 mg/kg impair cancer stemness and inhibit drug resistance [407] In vitro MCF-7 and MDA-MB-231 cells 0–100 μM Inhibiting Akt/HIF-1α axis Reducing expression levels of SOX2 and Bmi-1 Inhibiting CSC features and self-renewal capacities [408] In vitro MCF-7 cells 0–140 μM Apoptosis induction in CSCs Inhibiting autophagy promotes potential of ginsenoside F2 in tumor suppression [406] In vitro In vivo HepG2 and Huh7 cells Mice 0–1 mg/mL 1 mg/kg Inhibiting CSC features in a dose-dependent manner Autophagy induction and suppressing β-catenin signaling [409] Different strategies can be employed for suppressing CSCs. One of the potential methods is application of gene therapy. As signaling networks involved in regulating CSC features have been recognized, gene therapy can inactivate/activate molecular pathways and modulate CSC markers. However, there are some drawbacks with gene therapy, such as high cost. Therefore, plant derived-natural products are of importance due to their multitargeting capacity. The present review attempted to reveal the possible role of selected dietary agents in suppressing CSCs. As it was mentioned in main text and Tables, various doses of these anti-cancer compounds have been utilized and most of them are less than 100 µM. However, each natural product has its own IC<sup>50</sup> and based on various experiments performed in this case, it is possible to determine optimal dose for using in cancer therapy. One of the limitations of experiments is lack of using nanostructures for delivery of dietary agents and enhancing their capacity in CSC suppression. Hence, future experiments can focus on this aspect and different nanoparticles such as lipid-based nanoparticles,

carbon-based nanoparticles and polymeric nanoparticles, among others, can be utilized for targeted delivery. Furthermore, nanostructures overcome poor bioavailability of dietary agents in CSC suppression.

Noteworthy, the molecular pathways inducing CSC marker overexpression, can be targeted by natural products. The PI3K/Akt, Wnt, STAT3, NF-κB and Sonic signaling pathways undergo down-regulation by natural products to reduce expression level of CSC markers including CD44, CD133, Notch, Oct4 and Nanog, impairing cancer stemness and progression. The dietary agents induce apoptosis via both extrinsic and intrinsic pathways to reduce viability of CSCs. These compounds decrease expression level of MMPs and suppress EMT to reduce migration and invasion of CSCs. All these impacts are vital for preventing cancer progression. By suppressing CSC features, natural products enhance sensitivity of tumors to radiotherapy and chemotherapy. However, there are limited experiments in this case. Furthermore, there is lack of study evaluating potential of natural products in targeting CSCs and their interaction with immune system. One of the limitations related that was noted in these studies is that most of them have been performed under in vitro settings and have evaluated the potential impact of dietary agents in targeting CSCs in different cellular models. Hence, additional in vivo studies should be performed to analyze the exact potential of the natural products in targeting CSCs, suppressing tumor progression and investigating possible limitations associated with their use. There are no clinical trials performed so far to evaluate the role of natural products in targeting CSCs in cancer patients. However, there are clinical studies on using dietary agents in treatment of cancer patients (NCT03769766, NCT03980509, NCT01042938; clinicaltrials.gov, accessed on 22 October 2021). Therefore, still enormous work remains to be done to analyze the role of natural products in specifically targeting CSCs and providing effective treatment for cancer patients.

**Author Contributions:** M.D.A.P., S.A. and A.Z. (Amirhossein Zabolian), H.S., M.A.K., S.S., M.J.N., M.H., K.H. and M.A. prepared first draft and participated in manuscript writing. S.M., A.Z. (Ali Zarrabi) and G.S. collected articles, supervised and edited English language. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Singapore Ministry of Education Tier 1 Grant [R-184-000-301- 114] to GS.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **Abbreviations**

siRNA: Small interfering RNA; shRNA, short hairpin RNA; CSCs, cancer stem cells; ALDH, aldehyde dehydrogenase; GLUTs, glucose transporters; HKs, hexokinases; ROS, reactive oxygen species; TCA, tricarboxylic acid; miRNA, microRNA; TCF4, transcription factor 4; EVs, extracellular vesicles; EGCG, epigallocatechin 3-gallate; ISL, isoliquiritigenin; P-gp, P-glycoprotein; CA, caffeic acid; CAPE, caffeic acid phenyl ester; DR5, death receptor 5; EpCAM, epithelial cell adhesion molecule; YB-1, Y-box binding protein 1; COX-2, cyclooxygenase-2; STAT3, signal transducer and activator of transcription 3; EMT, epithelial-to-mesenchymal transition; NF-κB, nuclear factor-kappaB; SFN, sulforaphane; TGF-β, transforming growth factor-β; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis ligand; HIF-α, hypoxia inducible factor-α; PCa, prostate cancer; MK, midkin; Res, resveratrol; NAF-1, nutrient-deprivation autophagy factor-1; TME, tumor microenvironment; VE, vascular endothelial; vWF, von Willebrand factor; BBR, berberine; Gli1, glioma-associated oncogene-1; d-TPP, Dodecyl-TPP; cyt C, cytochrome C.

### **References**

