3. Preclinical research on phytomedicines targeting key regulators of anti-cancer drug resistance in CSCs
CSCs contribute to drug resistance by regulation of EMT; elevated expression of ABC transporters, increase in aldehyde dehydrogenase (ALDH) enzymes, resistance to DNA damage and cell death, slow cycling of microRNAs and regulation of tumor microenvironment (Makena et al., 2020) . Phytocompounds targeting either one of these key regulators of anti-cancer drug resistance (as shown in Figure 3 ) can prove to be useful in the elimination of CSCs and in improving the outcome of cancer disease treatment.
Curcumin is a dietary polyphenol extracted from turmeric rhizomes (Curcuma longa ). It has been explored for past many years and considered to be a potential anti-cancer therapeutic agent. Recently, reports have revealed that curcumin targets CSCs in breast, thyroid, and brain cancers. It can act in different ways but converges to a final outcome of reducing the tumor cells. In a study, curcumin was reported to downregulate EMT (Vimentin, Fibronectin, β-catenin,) and stemness (Sox-2, Nanog, and Oct-4) markers whereas in another study it reduced the expression of ABC transporters in breast CSCs (Hu et al., 2019; Zhou et al., 2015) . It dysregulated JAK/STAT3 signaling pathway in papillary thyroid CSCs (Khan et al., 2020) . Also, recently, in order to boost the stability and water solubility, thus improving the drug’s permeability and antitumor activity, curcumin liposome was constructed. As a result, its apoptotic effect on glioblastoma stem cells was established (Y. Wang et al., 2017) .
Ovatodiolide is a macrocyclic diterpenoid isolated from Anisomeles indica, whose effect against different cancers like glioblastoma, nasopharyngeal carcinoma and oral cancer was studied in vitro andin vivo and its potential therapeutic properties were established. It was found that ovatodiolide reduces stemness markers (CD44, CD133, Sox2, Klf4, Nanog and Oct-4) and decreases expression of EMT genes. It modulated JAK2/STAT3 signaling pathway by inhibiting either JAK2 or STAT3; thereby dysregulating transcription of genes. Also, it induced apoptosis of tumor cells. Furthermore, in vivostudies on oral carcinoma mouse xenografts were carried out and promising results were obtained. Treatment of nude mice (previously injected with SAS cells) with 3.6 mg/kg ovatodiolide depicted 2.2-folds lesser tumor growth compared to the untreated mice (Lin et al., 2018; S.-C. Liu et al., 2019; Su et al., 2019) .
Stem extract of Dendrobium venustum containing Lusianthridin downregulated Src-STAT3-c-Myc pathway and suppressed CD133, ABCG2, and ALDH1A1 stemness markers which induced apoptosis in lung CSCs(Bhummaphan et al., 2019) . Polygonum cuspidatum root extract which mainly comprises of 2-Ethoxystypandrone showed inhibition of STAT3 signaling in hepatocellular carcinoma (W. Li et al., 2019) .
Most of the compounds that have been evaluated have shown to target cell death/ apoptosis pathway, while some also contribute to cell cycle arrest. Fruit extract of Alstonia scholaris induced apoptosis in glioma stem cells, owing to the presence of two nor-monoterpenoid indole alkaloids, Scholarisine Q(1) and R(2) (B. Wang et al., 2018) . Similarly, bark extract of Walsura pinnata Hassk and rhizome extract of Costus speciosus induced apoptosis in leukemia and prostate cancer cells respectively (Elkady, 2019; Leong et al., 2017) . Viola odorata, a plant possessing active components such as saponin, salicylic acid derivatives, glycosides, alkaloids, anthocyanidins and cyclotides was shown to induce apoptosis and reduce migration and growth of breast CSC (Yousefnia et al., 2020) .Berberis is a plant possessing bioactive compound berberine, which reportedly causes G0-G1 arrest. It was found to be effective in reducing stemness and cell migration in neuroblastoma and prostate CSCs respectively (El-Merahbi et al., 2014; Naveen et al., 2016) .
Targeting stemness markers and EMT genes thus presents with the hope to decrease CSCs. Cinnamic acid was shown to decrease stemness in colon CSCs (Soltanian et al., 2018) . Carnosol modulated EMT genes and induced apoptosis in glioblastoma CSCs (Giacomelli et al., 2017) . Likewise, N-butylidenephthalide, a bioactive component ofAngelica Sinensis induced apoptosis in human bladder cancer cells and supressed tumor in BFTC-xenograft animal models (100 and 200 mg/kg dose) (Chiu et al., 2017) .
Phenethyl isothiocyanate, a component of cruciferous vegetables like broccoli and water cress promoted oxidative stress and downregulated cancer stemness genes in cervical and colon carcinomas respectively. A study reported that NOD-SCID mice injected with 10μM PEITC pre-treated HeLa CSCs yielded lower tumor volume compared to the control group (untreated HeLa CSCs). In another study, nude mice were treated with 20 mg/kg PEITC after EpCAM+ cell inoculation to determine whether PEITC suppresses CSCs in vivo and reduction in tumor growth was observed (Upadhyaya et al., 2019; Yun et al., 2017) .
Rhizome extract of Atractylodes macrocephala Koidz downregulated AKT/mTOR pathway and brought about alteration in glucose metabolism and stem-like behaviour in colon cancer cell line. Subsequently, 25 mg/kg and 75 mg/kg Atractylenolide-1 inhibited colorectal tumor progression in xenografted nude mice (K. Wang et al., 2020) . Similarly, total flavonoids of Fructus Viticis modulated AKT/mTOR pathway and stemness characteristics in lung CSCs (Cao et al., 2016) .
Allicin commonly found in garlic and it was evaluated as a promising compound for treatment of melanoma cells (Jobani et al., 2018) . Shikonin, a natural derivative of naphthoquinone, found in the root tissues of the traditional Chinese medical (TCM) herb Lithospermum erythrorhizon was proven to be effective against glioblastoma stem cells (J. Liu et al., 2015) . An extract of herbal mixture (H3) consisting of 3 oriental herbal plants (Meliae fructus ,Cinnamon bark and Sparganium rhizome ) was investigated for anticancer activity in vitro and in vivo (200 mg/kg dose of H3 used) and was found to be a promising therapeutic agent(Pak et al., 2016) .