Preventive and Therapeutic Effects of Chinese Herbal Compounds against Hepatocellular Carcinoma

Traditional Chinese Medicines, unique biomedical and pharmaceutical resources, have been widely used for hepatocellular carcinoma (HCC) prevention and treatment. Accumulated Chinese herb-derived compounds with significant anti-cancer effects against HCC have been identified. Chinese herbal compounds are effective in preventing carcinogenesis, inhibiting cell proliferation, arresting cell cycle, inducing apoptosis, autophagy, cell senescence and anoikis, inhibiting epithelial-mesenchymal transition, metastasis and angiogenesis, regulating immune function, reversing drug resistance and enhancing the effects of chemotherapy in HCC. This paper comprehensively reviews these compounds and their effects on HCC. Finally, the perspectives and rational application of herbal compounds for HCC management are discussed.


Introduction
Despite advances in diagnosis and treatment, hepatocarcinoma remains the sixth most common malignancy and the third principal cause of cancer deaths worldwide [1], underscoring the need to develop novel approaches for liver cancer control. Traditional Chinese Medicines (TCMs) play a positive role in the management of hepatocarcinoma [2,3]. TCMs are effective in alleviating clinical symptoms, improving quality of life and immune function, preventing recurrence and metastasis, delaying tumor progression, and prolonging survival of hepatocarcinoma patients [4]. Accumulated Chinese herbal compounds have been identified and demonstrated anti-cancer effects against hepatocellular carcinoma (HCC). Chinese herbal compounds represent an important medical and pharmaceutical resource for the development of new treatments for hepatocarcinoma. These compounds and their effects on HCC are comprehensively reviewed in present work.
The compound Astragalus and Salvia miltiorrhiza extract, a herbal component formula composed of astragalosides, Astragalus polysaccharide and salvianolic acids, has demonstrated efficacy in preventing DEN-induced hepatocarcinoma in a dose-dependent manner, accompanied by down-regulation of glutathione S-transferase placental type (GST-P) and α-SMA [13] (Table 1).

Inhibition of Cell Proliferation
Cancer is characterized by uncontrolled cell proliferation and tumor growth. Inhibition of cell proliferation and tumor growth is one of the primary goals of cancer therapy. Some herbal compounds are effective in inhibiting HCC cell/tumor growth.

Cell Cycle Arrest
Cancer is characterized by uncontrolled proliferation cycle and stopping the cell cycle is an ideal approach for cancer treatment. Some herbs are effective at arresting the cell cycle. Bufothionine inhibits cell proliferation and arrests cell cycle at G2/M phase in liver cancer cells [59]. Rhein, a component of R. palmatum L, R. tanguticum Maxim.ex Balf. or R. officinale Baill. (Da-Huang), is effective in inhibiting cell growth, inducing apoptosis and arresting cell cycle at S phase in HCC cells [53]. Oridonin, a compound from R. rubescens (Dong-Ling-Cao), induces G2/M cell cycle arrest and apoptosis in HepG2 cells through activation of the MAPK and p53 pathways [60] (Figure 1, Table 2).
Tetrandrine, a compound isolated from S. tetrandra (Han-Fang-Ji), inhibits proliferation and arrests cell cycle at G2/M phase in HCC cells [88]. Dihydroartemisinin, a semi-synthetic derivative of artemisinin isolated from Artemisia annua L. (Qing-Hao), induces apoptosis, and G2/M cell cycle arrest through induction of p21 and inhibition of cyclin B and CDC25C in liver cancer cells [101]. Saikosaponin d, a compound isolated from B. chinense (Chai-Hu), has demonstrated potency in inducing apoptosis and G0 cell cycle arrest by up-regulating p53 and p21/WAF1 in HCC cells [62] (Table 2).

Induction of Autophagy
Autophagy, type II programmed cell death, is a process in which organelles and proteins are sequestered and subsequently degraded through fusion with lysosomes, and has been recognized as a target for HCC treatment [121,122]. Tetrandrine, isolated from S. tetrandra (Han-Fang-Ji), promotes ROS generation and activates ERK MAP kinase to induce autophagy in HCC cells [89] (Figure 1). Shikonin, a naphthoquinone from L. erythrorhizon (Zi-Cao), induces autophagy in HCC cells via ROS production and ERK activation [76]. Matrine, a compound from S. flavescens Ait. (Ku-Shen), induces apoptosis and autophagy in HepG2 cells [66] (Table 2).
Bufalin, a component from toad skin, has demonstrated efficacy in the induction of AMPK-dependent autophagy accompanied by enhanced Beclin-1 expression, and decreased p62 expression and mTOR signaling in HepG2 cells [86] (Figure 1). Arenobufagin, a natural bufadienolide from toad venom, is a potent inducer of apoptosis and autophagy by down-regulating PI3K/Akt/mTOR pathway in HepG2/ADM hepatoma cells [90]. Curcumin, a common compound in herbs belonging to the plant genus Curcuma, when combined with Adriamycin, induces apoptosis and autophagy in Hep G2 cells by down-regulating Bcl-2/Bax [79] (Table 2).
However, in a different context, autophagy may promote cell death and contribute to anticancer therapeutic response, or inhibit cell death and thus contribute to drug resistance [123]. Inhibition of autophagy may enhance chemotherapy or target therapy-induced cell death in HCC cells [124,125]. As an autophagy inhibitor, epigallocatechin-3-O-gallate (EGCG) strengthens doxorubicin (DOX)-mediated anticancer effects in hepatoma cells [94]. As an autophagy inducer, elemene injection (a herbal injection) protects hepatoma cells from apoptosis induced by apatinib or serum-free starvation [95]. These observations suggest that autophagy-inducing herbal compounds need to be more thoroughly investigated in combination with other treatments such as chemotherapy and targeted therapy Table 2.

Induction of Cell Senescence
Cell senescence is a stable, irreversible cell cycle arrest triggered by a variety of stimuli. Induction of senescence has been suggested as novel approach for HCC treatment [126]. Ganoderiol F, a tetracyclic triterpene from Ganoderma amboinense, activates ERK and up-regulates p16 to induce cell senescence in hepatoma HepG2 cells [96] (Figure 1, Table 2).
Liver Yin tonifying formula (LYTF) activates caspases-8, -9 and -3 to induce apoptosis, and up-regulates p16 and p21 and down-regulates RB phosphorylation to induce cell senescence in Bel-7402 cells [128]. The known compounds in LYTF include oleanolic acid, ursolic acid and emodin. The compound(s) responsible for inducing cell senescence need further investigation.

Induction of Anoikis
Anoikis, an apoptotic process occurring when a cell detaches from extracellular matrix, is associated with cell survival in suspension and metastasis of HCC [129]. Arecoline, an alkaloid from Areca catechu L. (Bing-Lang), induces anoikis in HA22T/VGH cells by inhibiting STAT3 and increasing RhoA/Rock activation [97] (Figure 1, Table 2). P. cuspidatum (Hu-Zhang), a common herb used for HCC treatment, activates caspase-3 and -9 and induces anoikis in human HCC cells accompanied by ROS generation and FAK down-regulation [130]. The known compounds in Hu-Zhang include resveratrol, emodin, polydatin and physcion. However, the compound(s) contributing to Hu-Zhang-induced anoikis need further study.
Modified Yi Guan Jian (MYGJ) may activate caspase-3, -8 and -9, inhibit the expression and phosphorylation of p38 MAPK, and induce anoikis in human HCC cells [131]. The known compounds in MYGJ include coumarin, glucosides, betane and toosendanin. Further study is needed to identify the compound(s) responsible for MYGJ-induced anoikis.

Inhibition of Epithelial-Mesenchymal Transition (EMT)
EMT is a biological process in which polarized epithelial cells (including epithelial cancer cells) switch to a fibroblastoid or mesenchymal cellular phenotype [132]. During EMT, the epithelial marker E-cadherin (E-cad) is down-regulated and mesenchymal markers, such as N-cadherin (N-Cad) and vimentin, are up-regulated. EMT contributes to cancer metastasis by enhancing cell migration and invasiveness. EMT also contributes to drug resistance [133].
Tanshinone IIA, a compound isolated from S. miltiorrhiza Bunge (Dan-Shen), has demonstrated effects in inhibiting EMT and metastasis in HCC after palliative resection [99] (Figure 1). Curcumin, a common herbal compound in herbs of the plant genus Curcuma, inhibits hypoxia-induced EMT in hepatoma HepG2 cells [80]. Cordycepin, a compound isolated from Cordyceps sinensis (Dong-Chong-Xia-Cao), may up-regulate E-cad and down-regulate integrins and phosphorylation of FAK to inhibit proliferation, migration and invasion in HCC cells [103] (Figure 1 Table 2).
Huaier polysaccharides are effective in inhibiting proliferation, adhesion, migration and invasion in HCC cells accompanied by EMT inactivation as indicated by marker gene expression [104] (Figure 1, Table 2). Songyou Yin, a formula composed of five herbs, has reported efficacy in attenuating EMT and inhibiting metastatic potential in residual HCC resulted from oxaliplatin treatment [134]. The known compounds in Songyou Yin include tanshinone IIA and astragaloside IV.
In addition to single compound, multiple herbal ingredients can be combined and used as a formula. Compound Astragalus and Salvia miltiorrhiza extract (CASE), a herbal component formula comprising astragalosides, astragalus polysaccharide and salvianolic acids, inhibits TGF-β1 mediated invasion in hepatoma HepG2 cells by modulating TGF-β/Smad signaling [109] (Table 2).

Targeting Drug Resistance
Drug resistance contributes to chemotherapy-refractory HCC [136]. The search for effective herbal components to reverse drug resistance has become a research focus area in liver cancer studies. Astragalus membranaceus (Huang-Qi) polysaccharides enhance the anti-tumor effects of adriamycin in H22 hepatocarcinoma by up-regulating IL-1α, IL-2, IL-6 and TNF-α, as well as down-regulating IL-10 and MDR1 [110]. Astragaloside II, another component from A. membranaceus (Huang-Qi), is effective in enhancing cytotoxicity of 5-fluorouracil (5-Fu) in 5-Fu-resistant HCC cells accompanied by down-regulation of P-gp, phosphorylation of ERK1/2, p38 and JNK [137] (Figure 1, Table 2).
In addition to reversing drug resistance, some herbal compounds have direct effects against drug-resistant liver cancer cells. Hedyotiscone A, a compound isolated from H. corymbosa (San-Fang-Hua-Er-Cao), activates caspases-3, -7 and -9 to induce apoptosis in multidrug resistant HCC cells [113]. Polyphyllin D, derived from Paris polyphylla Sm. (Chong-Lou), induces apoptosis in multidrug resistant HepG2 cells via mitochondrial dysfunction [114]. Ursolic acid, a common component in multiple herbs, activates Bak and promotes release of apoptosis-inducing factor to induce apoptosis in doxorubicin-resistant human hepatoma cells [115] (Table 2).
Pseudolaric acid B, a compound present in Pseudolarix kaempferi (Tu-Jin-Pi), may disrupt cellular microtubule networks and inhibit mitotic spindle formation to induce apoptosis and G2/M cell cycle arrest in conventional and P-gp-overexpressing HCC cells [116]. Imperatorin, a compound isolated from Angelica dahurica (Bai-Zhi), induces proteosome-dependent Mcl-1 degradation to release Bak and Bax and triggers apoptosis in multidrug-resistant liver cancer cells [117] (Table 2).

Regulation of Immune Function
The major cell populations for cellular immunity against cancer include CD4+ T helper (Th) and CD8+ T lymphocytes. CD4+ Th1 cells produce cytokines, such as IL-2 and IFN-γ, to evoke cell-mediated immunity or phagocyte-dependent inflammation. CD4+ Th2 cells secrete cytokines, such as IL-4 and IL-6, and are correlated with humoral immunity. Antigen-presenting cells, such as dendritic cells (DCs) and macrophages, processed antigens are necessary to prime CD4+ and CD8+ T cells to elicit antigen-specific immune response.

Inhibition of Angiogenesis
Angiogenesis, the process of new blood vessels generating from existing vessels, plays a crucial role in tumor growth and metastasis and has been recognized as a potential target for HCC treatment [154,155]. Some Chinese herbal components have demonstrated anti-angiogenic effects.
G. chinensis (Tian-Long) inhibits tumor growth, induces apoptosis, and inhibits angiogenesis accompanied by down-regulation of VEGF and bFGF in H22 HCC [156]. Gekko-sulfated glycopeptide decreases bFGF secretion and binding to its low affinity receptor heparin/heparan sulfate, inhibits bFGF stimulated proliferation and migration of endothelial cells, and thus inhibits angiogenesis and tumor growth in liver cancer [148] (Figure 1, Table 3).
Pedicularioside G, a phenylpropanoid glycoside isolated from Pedicularis striata (Ma-Xian-Hao), inhibits proliferation and migration in human umbilical vein endothelial cells (HUVEC), and angiogenesis in chicken embryo chorioallantoic membrane and human hepatoma. Pedicularioside G also inhibited cell proliferation and migration and tumor growth in human hepatoma. The effects of pedicularioside G are partially related to down-regulation of ROS [149] (Figure 1, Table 3).

Herbal Compound-Based Combinational Treatment
Chinese herbs are usually prescribed as formulas guided by TCM theories, i.e., combining multiple herbs in a prescription [157]. It is rational to use herbal components in the same principle.
C. kwangsiensis or C. phaeocaulis or C. wenyujin or C. longa (Yu-Jin) and P. cuspidatum (Hu-Zhang) are frequently used simultaneously as herbal-pairs in liver cancer treatment based on complementary traditional efficacy. Curcumin is a component of Yu-Jin and resveratrol is a compound can be isolated from Hu-Zhang. We have found that curcumin combined with resveratrol may synergistically inhibit XIAP (X-linked inhibitor of apoptosis protein) and survivin expression, up-regulate ROS production, and activate caspase-3, -8 and -9 to induce apoptosis in HCC cells [158] (Table 4).

Conclusions and Future Directions
In summary, Chinese herbal compounds have demonstrated multiple effects against HCC, including prevention of hepatocarcinogenesis, inhibition of cell proliferation, induction of apoptosis, autophagy, cell senescence and anoikis, cell cycle arrest, inhibition of EMT, metastasis and angiogenesis, regulation of immune function, reversal of drug resistance and enhancement of chemotherapeutic effects (Figure 1). These observations provide the basis for further development of new drugs for HCC management.
Chinese herbs are natural products and most herbs are safe for human consumption. One herb may contain multiple anticancer compounds, e.g., C. wenyujin (Er-Zhu or Yu-Jin) contains curcumin, curcumol and β-elemene. One herbal compound may have multiple effects on HCC, such as curcumin, which has demonstrated effects in inhibiting hepatocarcinogenesis and cell proliferation, inducing apoptosis and autophagy, and inhibiting EMT in HCC cells via multiple targets [8,[78][79][80].
Chinese herbs have a long history of clinical use. Thousands years of clinical experiences provide clues for pharmacological studies. Compounds in some clinically-used anticancer herbs have not yet been fully identified, such as S. lyratum Thunberg (Bai-Ying) and Patrinia scabra Bunge (Mu-Tou-Hui). Whether compounds from these herbs possess more potent anticancer effects on HCC are worthy of further study.
Some anticancer compounds containing herbs are neither commonly clinically used nor recognized as anticancer herbs so far, for example, A. pusilla (Jiu-Jie-Long) [21] and P. capitatum (Tou-Hua-Liao) [22]. Whether these herbs possess anticancer effects on HCC warrants more investigation. Some herbal compounds shown significant anticancer effects in HCC, but the corresponding herbs are not clinically used as anticancer herbs or their anticancer effects have not been observed so far, such as A. dahurica (Bai-Zhi) [117], M. officinalis (Hou-Po) [48,49] and A. officinarum Hance (Gao-Liang-Jiang) [92]. The different effects between herb and herbal components may result from relatively insufficient quantities of anticancer compounds in those herbs.
Combinational treatment with multiple herbal compounds is a promising strategy for application of herbal compounds. The combinational principles or compatibility of Chinese herbs provide a theoretical basis for combinational treatment with multiple herbal compounds for HCC. In addition, Chinese herbal formulas or known active herbal-pairs provide clues for the extraction of compounds from multiple herbs and combinational treatment with these compounds.
Current treatments for HCC are less than satisfactory. Anticancer herbal compounds are importent resources for the development of new drugs for liver cancer treatment. These compounds can be developed as single herbal compound drugs with or without chemical modifications, or in combination with other herbal compounds, herbs and even modern drugs guided by the principles of TCM and/or pharmacological interaction.