Research Progress on the Effect of Traditional Chinese Medicine Monomers and Derived Compounds on Liver Cancer Stem Cells

Hepatocellular carcinoma (HCC) is the most common malignant tumor in the world with a high mortality rate. Liver cancer stem cells (LCSCs) are a small part of HCC cells with the abilities of self-renewal, proliferation, differentiation and tumorigenesis, which may be the main reasons for the high drug resistance and recurrence rate of HCC. Therefore, targeting LCSCs has become an effective way to cure HCC. A lot of evidences show that traditional Chinese medicine (TCM) plays an extremely important role in the prevention and treatment of HCC as an alternative or complementary therapy. In recent years, an increasing number of studies have shown that TCM monomer extracts and their derived compounds target LCSCs through a variety of pathways, providing new ideas for the treatment of liver cancer.


Introduction
Hepatocellular carcinoma (HCC) is the most common malignant cancer all over the world, accounting for about 75% ~ 85% of primary liver cancer. It is the third leading cause of cancer-related mortality with a poor prognosis [1,2]. HCC can be caused by chronic hepatitis virus (HBV, HCV) infection, aflatoxin exposure, excessive alcohol consumption, obesity and other reasons, whereas in China, chronic HBV infection is one of the key factors for the incidence of HCC [1].
Liver cancer stem cells (LCSCs) are considered to possess stem cell-like properties in HCC. And many surface markers of LCSCs have been identified, such as EpCAM, CD133, CD44, CD13, CD90, CD24, CD47, OV6, K19, C-KIT, ABCG2 and ALDH, etc. LCSCs are also characterized by infinite self-renewal, in vivo tumorigenicity, subsequent generation of differentiated progeny recapitulating the parental tumor phenotype, the ability to form sphere and so on, which are closely related to the stemness, metastasis, recurrence and therapeutic resistance of HCC [3,4]. In addition, Side Population cells (SP) can also be used to identify LCSCs and is associated with the chemotherapy resistance of HCC [5]. They are also regulated by multiple signaling pathways, such as the classical Wnt/β-catenin, Notch, TGF-β, IL-6/STAT3and PI3K/Akt/mTOR signaling pathways [4,6].
Traditional Chinese medicine (TCM), as an important treatment of China, has been used to prevent and treat lots of diseases for thousands of years. With the increasing attention and studies on TCM, researchers have found that TCM monomer extracts and their derivatives can exert outstanding antihepatocarcinogenic effect through myriad mechanisms [7,8]. In recent years, a growing number of studies have shown that TCM monomers and their derivatives play an extremely important role in the regulation of LCSCs, which are potential targets for the treatment of HCC, including inhibiting their proliferation and promoting their apoptosis [9,10]. Here, we have summarized the effects of TCM monomers and their derivatives on LCSCs and their mechanisms in the past ten years, so as to provide new ideas and methods for the treatment of HCC.

Wnt signaling pathway
The classical Wnt signaling pathway enhances the expression of CSCs markers and the ability of self-renewal, which is of great significance for the maintenance of CSCs stemness [11]. Casticin (CAS), which is derived from Fructus Viticis Simplicifoliae，decreased the expression level of β-catenin and its downstream target CyclinD1 in LCSCs, and inhibited cell self-renewal. Moreover, the Wnt/β-catenin agonist lithium chloride could reverse this effect, demonstrating that CAS inhibits LCSCs by blocking the Wnt/β-catenin signaling pathway [12]. Amarogentin, a secoiridoid glycosidic compound which is extracted from Swertia chirata, notably suppressed the expression of β-catenin, phosphorylated β-catenin, Gli1, Sonic Hedgehog ligand as well as SMO, and up-regulated the expression of PTCH1 in CD44+ LCSCs [13]. Additionally, epigallocatechin gallate (EGCG) in green tea could reduce the number of CD44+ LCSCs, and in this process, Wnt/β-catenin, Hh/Gli1 and their related genes Cyclin D1, c-Myc and EGFR up-regulated, while the expression of E-cadherin down-regulated [14]. Amarogentin as well as EGCG may regulate LCSCs by modulating self-renewal Wnt and Hh pathways. Ovatodiolide, a macrocyclic diterpenoid which is isolated from the TCM herb Anisomeles indica, induced the down-regulation of β-catenin and its downstream effect genes and prevented hepatospheres formation, thereby inhibiting liver cancer stem cell-like properties via Wnt/β-catenin pathway [15]. Gynura divaricata (GD) belongs to Compositae family and has long been used in the treatment of liver diseases such as hepatitis and liver cancer. GD extracts down-regulated the activity of Wnt reporter gene and the expression of Wnt target genes Cyclin D1, c-Myc and AXIN2, meanwhile, up-regulated the expression of E-cadherin, then targeted LCSCs through Wnt pathway [16].

PI3K/AKT Signaling Pathway
PI3K/AKT pathway is involved in the occurrence and progression of various types of cancers comprising HCC, and it is also an important signaling mechanism in the regulation of LCSCs [6].ψ-Bufarenogin is a novel bioactive compound which is isolated and identified from toad skin with less side effects that has a strong inhibitory effect on HCC [17]. After pretreated with ψ-Bufarenogin, the phosphorylation of Akt were suppressed in a dose-dependent manner without affecting the expression of STAT3.
Then, the inhibition of stemness regulators MCL1 and SOX2 could be blocked by the overexpression of dominant negative mutant Akt (DN-Akt), indicating that PI3-K/AKT pathway may be involved in the inhibition of proliferation of LCSCs by ψ-Bufarenogin [18]. As a kind of polyphenols existing in Rhizoma curcumae longae, curcumin has a prominent anti-tumor property [19]. It significantly reduced the protein levels of PI3K, p-AKT and p-mTOR, and PI3K/AKT activator reversed the inhibitory effect of curcumin. These results prove that curcumin inhibits the growth of LCSCs through PI3K/Akt/mTOR signaling pathway [20]. Docetaxel, a complex diterpene molecule originally isolated from the needles of the European yew (Taxus baccata), could reduce the expression of p-Akt and SOX2 in LCSCs [21]. But after blocking the PI3K/ Akt signaling pathway with LY294002, its inhibitory effects were partially limited, suggesting that docetaxel may promote the apoptosis of LCSCs by restraining the PI3K/Akt signaling pathway [22]. Sophorine, a quinolizine alkaloid which is extracted from Sophora alopecuroides Linn, could observably suppress the PI3K/ Akt pathway, decrease the expression levels of p-Akt, p-GSK-3β and p-β-catenin in cells, and inhibit LCSCs by regulating the Akt/ GSK-3β/β-catenin axis [23]. Futhermore, some studies have found that as a tumor suppressor gene, PTEN can regulate the activity of ABCG2 through the PI3-K/Akt pathway, thus targeting CSCs [24]. Lupanol, a natural triterpenoid, could increase the PTEN level in LCSCs and continuously decrease the expression levels of phosphorylated Aktser473 and ABCG2. After knocking down the PTEN, the inhibitory effect on the ability of primary spheres to form secondary spheres significantly diminished [25].

JAK2/STAT3 signaling pathway
JAK2/STAT3 signaling pathway can induce the effects of multifarious growth factors and cytokines, which is closely related to the occurrence and development of many kinds of cancers. And the abnormal activation of JAK2/STAT3 signaling pathway in multiple CSCs can promote the occurrence of tumors [26]. Silybinin is a flavonoid which is extracted from silymarin seeds. When combined with sorafenib, silybinin could inhibit the phosphorylation of STAT3 and down-regulate the expression of CSCs-related proteins KLF4, NANOG and β-catenin, and then restrained the formation and self-renewal of LCSCs [27]. Juglone analogues are active constituents with antiviral, anti-inflammatory and anti-cancer effects that can be isolated from many medicinal plants, and have STAT3 pathway inhibitory activity. Li et al. found that a juglone analogue 2-ethoxypyranone, the EtOAc extract of the P. cuspidatum root, inhibited the constitutive activation of STAT3 phosphorylation in HCC cells induced by IL-6, blocked the formation of tumorspheres, and dose-dependently inhibited the proliferation of LCSCs by blocking the activation of STAT3 [28]. Furthermore, cucurbitin B (CuB), a tetracyclic triterpenoid mainly isolated from Cucurbitaceae, had been proved to restrain the expression of phosphorylated p-JAK2 and phosphorylated p-STAT3 in LCSCs and down-regulate the expression of Cyclin B1, CDK1 and CD133 in a concentration-dependent manner, thereby arrested cell cycle and stemness characteristics by suppressing the JAK2/STAT3 signaling pathway [29].

TGF-β signaling pathway
TGF-β regulates cell self-renewal, growth and differentiation, while Smad2 is a receptor for TGF-β. The TGF-β/Smad2 signaling pathway has been shown to be closely related to the stemness of LCSCs [30]. As an isoflavone, glabridin (GLA) is isolated from the root of Glycyrrhiza glabra L., and exhibits notable biological properties including anti-bacterial anti-inflammatory, anti-tumor, anti-oxidation, neuroprotective [31]. GLA could inhibit TGF-βinduced Smad2 phosphorylation and endogenous Smad2 activation, promote the expression of miR-148a in a dose and time dependent manner, finally suppressed LCSCs-like characteristics through the inhibition of miR-148a-mediated TGF-β/Smad2 signaling pathway [32]. Arsenic trioxide (ATO) is considered to be the most toxic compound in TCM and has been proved to inhibit the growth of cancer cells [33]. ATO could reduce the expression of Smad3 by promoting the expression of miR-491 in vivo and vitro. After the knockout of miR-491 or Smad3, inhibition of endogenous TGF-β signaling and CSCs-like properties diminished, demonstrating that ATO targets LCSCs by inhibiting endogenous TGF-β signaling through miR-491-mediated Smad3 silencing [34].

NF-κB signaling pathway
NF-κB signaling pathway is involved in a range of biological processes including cell proliferation and differentiation, it is also believed to be involved in the maintenance of many stem cells as well as CSCs, and contributes to the invasion of CSCs [35]. Curcumin treatment led to selective depletion of LCSCs in susceptible strain, which was evidenced by reduction of SP cells and sphere formation, down-regulation of CSCs markers and diminished tumorigenicity. Alternatively, this process could be enhanced by use of NF-κB inhibitors or blocking of NF-κB signaling [36]. Wang et al. found that the inhibitory effect of ATO on CD133 and EpCAM weakened in the BELMDR cells which were transfected with NF-κB siRNA. Moreover, ATO could activate miR-148A through demethylation, weaken the phosphorylation of P65, and control the NF-κB pathway, thereby inhibiting the phenotype of LCSCs [37]. Further, after ATO intervention, the expression of LIF, JAK1, STAT3 as well as NF-κB signaling pathway members P50, P52, P65, c-Rel, significantly down-regulated in LCSCs, revealing that ATO effectively induces differentiation of LCSCs by synergistic inhibition of LIF/JAK1/ STAT3 and NF-κB signaling pathways [38].

EGF/EGFR signaling pathway
EGF is a ligand that binds to EGF receptor (EGFR), which is a member of the receptor tyrosine kinase (RTK) family. The activation of the EGF/EGFR signaling pathway is closely associated with poor prognosis of HCC [39]. It has been found that the EGF/EGFR signaling pathway plays an important role in the formation and maintenance of many CSCs [40]. Catechol is a kind of natural compounds which can be separated from natural plants [41]. Lim et al. found that the number of mammosphere increased by EGF were decreased after catechol treatment through mammosphere assay. And the same concentration of catechol and EGFR antagonist carnitinib could reduce the expression of CD44 and Nanog. Taken together, catechol can regulate the level of CSCs markers and inhibit stem cell-like characteristics through EGF/ EGFR signaling pathway [42]. Moreover, Brucea javanica (BJ) water extract dose-dependently diminished the levels of EGFR and Akt, and inhibited the phosphorylation of Akts473 in Hep3B cells. It could also promote cell apoptosis and reduce the growth of LCSCs by targeting EGFR [43].

Other relevant signaling pathways
Bis

TCM monomers inhibit LCSCs-related EMT
As a component of Pterocarpus marsupium, Pterostilbene (PT) is a resveratrol analog and has lots of biological activities such as anti-inflammatory, lowering blood lipid and blood glucose [51]. PT could reduce the expression of the stem gene c-Myc in CD133+Mahavu cells, which caused the inhibition of invasion and migration abilities of cells. PT also reduced the expression of vimentin in a dose-dependent manner, down-regulated the expression of chemotaxis related molecules CXCR4 and EMTrelated transcription factor Twist1, and prevented the production of LCSCs [52]. He et al. found that E-cadherin and N-cadherin upregulated but the level of EMT-related transcription factor Twist reduced in CD133+ sphere-forming cells when treated by CAS. The EMT process was reversed, but this effect could be partially restored by overexpression of Twist, suggesting that CAS can inhibit EMT through down-regulating Twist to target LCSCs [53].

TCM monomers up-regulate LCSCs-related miRNAs
The subcytotoxic concentration of CAS inhibited the activity and expression of DNMT1, promoted the expression of miR-148a-3p, reduced the expression of CSCs markers and the ability of sphere formation, and restrained the stemness of HCC cells by blocking the negative regulation between DNMT1 and miR-148a-3p [54]. Furthermore, isovitexin (ISOV), as a natural flavonoid compound in traditional Chinese medicine, such as Fructus viticis and Cucurbitaceae, has been found to have inhibitory effect on CSCs in osteosarcoma [55]. After ISOV treatment of SK-Hep-1 cells, the sphere and colony formation decreased, the expression level of CSCs markers down-regulated, while the level of miR-34a up-regulated. In other words, ISOV inhibits the stemness of HCC cells via regulating miR-34a[56].

TCM monomers inhibit the expression of LCSCs-related oncogenes BMI1
As an oncogene, BMI1 plays a crucial role in the selfrenewal of stem cells and is associated with a lot of tumors

FOXM1
FOXM1 is considered to be an oncogenic protein complex that is involved in tumor angiogenesis, invasion and metastasis. Isovitexin could not only markedly reduce the sphere formation rate and colony formation rate of cultured LCSCs by preventing the overexpression of MnSOD and down-regulating the expression of FOXM1, but also inhibited the expression of CSCs markers in a dose-dependent manner. These results prove that isovitexin can restrain the stemness and tumorigenicity of LCSCs [61].

TCM Derivatives Inhibit LCSCs TCM derivatives regulate LCSCs-related signaling pathways
Wnt/β-catenin signaling pathway: Chrysin, as a natural flavonoid, is abundant in propolis and honey, and has been proved to have anti-proliferative effects on a variety of cancers comprising HCC [65]. 8-bromo-7-methoxychrysin (BrMC) is a synthetic analog of it, which has a stronger anti-tumor effect than chrysin, it plays an anti-HCC role by antagonizing Wnt/β-catenin signaling pathway. When treated with BrMC, the expression of β-catenin in LCSCs decreasd. Nevertheless, down-regulation of β-catenin expression could cooperate with BrMC to inhibit the tumor spherogenesis of LCSCs and the expression of CSCs markers CD133 and CD44 [66]. 6-C-(E-styrene)naringin (6-CEPN) is a new semi-natural derivative of naringenin produced by the reaction of naringenin with phenylacetaldehyde. 6-CEPN could significantly down-regulate the levels of the direct targets SOX2, OCT4, NANOG, KLF4 and c-Myc transcription factors of Wnt/β-catenin pathway. And LCSCs were directly targeted by 6-CEPN through inducing β-catenin degradation and inhibiting its nuclear translocation [67].

STAT3 signaling pathway
BrMC decreased sphere formation and self-renewal abilities of liver cancer stem cell-like cells (LCSLCs) in a dose-dependent manner, and down-regulated the expression of CSCs markers and the phosphorylation of STAT3. The synergistic effect of STAT3 inhibitor JSI-124 and BrMC demonstrated that BrMC reversed the stemness of LCSLCs by inhibiting STAT3 [71]. In a subsequent study, they found that the inhibitory effect of BrMC on LCSLCs dramatically reduced after heterotectomic expression of Twist1 [72]. All above shows that BrMC can inhibit the stemness of LCSLCs by inhibiting the STAT3/Twist signaling axis. Besides, it was found that SHP-1-mediated dephosphorylation of p-JAK2 and p-STAT3 played a vital role in the formation and invasion of tumors. Huang et al. prepared a new type of ZnAs@SiO2 nanoparticles with stronger anti-tumor effect and less side effects than ATO by reverse microemulsion method, which could up-regulate the expression of SHP-1 and down-regulate the expression of p-JAK2 and p-STAT3. It could also notably suppress the formation of tumor spheres and the expression of CSCs markers CD133, SOX-2 and Oct-4 in vitro by modulating the SHP-1/JAK2/STAT3 signaling pathway [73].

TCM derivatives inhibit LCSCs-related EMT
The chrysin derivative BrMC could inhibit the proliferation and self-renewal of CD133+ sphere cells and the expression of CD133 and CD44 markers of CSCs, down-regulate the expression of N-cadherin and Vimentin, and up-regulate the expression of E-cadherin and ZO-1 to suppress the invasion of LCSCs. And the EMT of LCSCs was suppressed by down-regulating the expression of Twist and β-catenin in LCSCs [74]. In addition, when BrMC combined with sorafenib, the expression level of EMT-related key protein Twist1 was reduced in a dose-dependent manner. The self-renewal, cell migration and invasion abilities of LCSCs remarkably diminished, and the cell apoptosis increased [75]. The 8-amino-ICD (d-ICD), a derivative of isocorydine, has stronger anticancer activity than ICD. In order to improve the anticarcinogenic activity of d-ICD, Yan et al. designed and synthesized a new derivative named FICD, which could memorably inhibit the expression of Vementin, the main marker of EMT and target the CSCs of EMT transformation to initiate relapse with the combination of sorafenib [76].

TCM derivatives inhibit LCSCs-related cytokines
IL-6 is closely related to STAT3 and can promote tumor formation in many aspects. HGF is closely related to the growth, migration and morphogenesis of a variety of cells, and plays a key role in tumor invasion and metastasis. The combination therapy with BrMC and chrysin could effectively diminish the sphere formation of LCSCs as well as the expression of biomarkers CD133 and CD44, and also reduced the stemness of LCSCs by restraining the secretion of IL-6 and HGF [77].

TCM derivatives inhibit the expression of LCSCs-related oncogenes
As an oncogene, IGF2BP3 promotes the stemness-like cell phenotype of tumor cells as well as the migration and invasion potential of tumor cells [78]. d-ICD could suppress the expression of drug-resistant genes ABCB1 and ABCG2, as well as CD133, LGR5, IGF2BP3 and IGF2BP1. Besides, the over-expression of IGF2BP3 almost completely eliminated this effect, which proves that d-ICD targets LCSCs by inhibiting the expression of stemness genes through down-regulating IGF2BP3 [79].

Discussion
LCSCs are considered to be the key to the growth, invasion, metastasis and recurrence of HCC, and one of the main reasons for the poor prognosis of HCC. As a traditional treatment method, TCM monomer extracts and derivatives (Table 1) could inhibit the stemness characteristics of LCSCs, such as proliferation, selfrenewal, sphere formation, migration as well as invasion, and promote the apoptosis of LCSCs through a variety of ways, which included the regulation of the classical Wnt signaling pathway, PI3-K/Akt signaling pathway, STAT3 signaling pathway and other eleven LCSCs-related signaling pathways (Figure 1), inhibition of the expression of EMT and related oncogenes in LCSCs. Among them, TCM monomers could also up-regulate miRNAs related to LCSCs and activate the expression of tumor suppressor genes, while TCM monomer derivatives could also inhibit the secretion of cytokines. Broadly speaking, TCM gives full play to the advantages of multi-target, multi-link and multi-level to target LCSCs.  Although a large number of traditional Chinese medicine monomers and derivatives have been studied to inhibit LCSCs through various ways, there are still some compounds, such as including ginsenoside Rh2 (GRh2), Quercetin, 4-methylumbelliferone (4Mu), a coumarin derivative, and 6-O-β-D-2-deoxyglucopyranoside (MAN-6DG) which is a derivative of a natural xanthone named α-mangostin (α-MGT) in mangosteen pericarp, can inhibit the expression of LCSCs markers, self-renewal and differentiation, and reduce the number of LCSCs [80][81][82][83], but the specific mechanisms of their effects on LCSCs remain unclear. There is no doubt that further studies are needed to reveal the mechanisms of the effect of known TCM monomers and derivatives on LCSCs, and discover more TCM monomers