Hepatocellular carcinoma (HCC) is a malignant tumor with a high incidence and very poor prognosis. Globally, hepatocellular carcinoma has the sixth highest incidence of malignant tumors and the third highest mortality rate [1]. China has a high incidence of hepatocellular carcinoma. According to WHO estimates, the number of new cases of hepatocellular carcinoma worldwide in 2018 was about 8.401 million, accounting for 46.7% in China [2, 3]. The etiology and pathogenesis of primary hepatocellular carcinoma are complex, and it is a process of multi-gene regulation, multiple signaling pathways and multi-pathway interactions [4-6]. Under the effect of certain pathogenic factors such as viral infection, chemical carcinogens or ionizing radiation, abnormal activation of proto-oncogenes (such as ras and c-myc), inactivation of oncogenes (such as P53, P21, P16, Rb, and PTEN), and abnormal activation of signaling pathways (such as Wnt/β-catenin, MAPK, AKT, ERK, Hedgehog, and Notch) and growth factors (HGF, EGF, VEGF, TGF, and IGF) induce hepatocellular carcinoma [7-11]. Treatment options for hepatocellular carcinoma include surgery, radiotherapy, intervention, immunotherapy, targeted therapy and liver transplantation. Most hepatocellular carcinoma patients have no obvious symptoms in the early stage, and most of them are in the middle or late stage when they consult the doctor, and most of the hepatocellular carcinoma patients in China are accompanied by cirrhosis, which limits the efficacy and scope of surgery to a certain extent. For patients with intermediate and advanced stages, radiofrequency ablation, hepatic artery chemoembolization, targeted therapy and immunotherapy have been widely used, but there are problems such as incomplete treatment, high recurrence rate and adverse reactions [12]. In recent years, it has been found that herbs with anticancer activity have significant efficacy in prolonging the survival period and improving the quality of survival, which has become a hotspot of medical research [13]. Berberine, also known as berberine hydrochloride, is an isoquinoline alkaloid extracted from traditional Chinese herbs such as Rhizoma Coptidis and Cyperus rotundus [14]. Berberine has significant anti-inflammatory and antibacterial effects, and is mainly used in the treatment of bacillary dysentery and intestinal diseases. Berberine has a wide range of pharmacological effects, and has unique pharmacological activities on cardiovascular, cerebrovascular, nervous and digestive systems, such as protecting myocardial and vascular endothelial cells, regulating blood lipid and glucose, anti-Alzheimer's disease, anxiolytic and treating peptic ulcer [15-17]. With the in-depth study of the pharmacological effects of berberine, its ex vivo and in vivo antitumor activities have gradually received attention. Studies have shown that berberine has inhibitory effects on nasopharyngeal carcinoma, lung cancer, colorectal cancer, breast cancer, ovarian cancer and hepatocellular carcinoma [18-21]. The anti-hepatocellular carcinoma mechanisms of berberine are mainly induction of apoptosis and autophagic death of hepatocellular carcinoma cells, inhibition of hepatocellular carcinoma cell proliferation, inhibition of hepatocellular carcinoma cell invasion and metastasis, anti-oxidative stress, and modulation of signal transduction pathways [20, 22-25]. In addition, berberine can inhibit the recurrence of hepatocellular carcinoma, and the combination with other drugs can enhance the anti-cancer effect. Berberine derivatives with stronger antitumor activity and higher bioavailability have also been continuously discovered. The research progress of berberine against hepatocellular carcinoma is summarized.

The Mechanism of Berberine Against Hepatocellular Carcinoma

Inducing Apoptosis of Hepatocellular Carcinoma Cells: Apoptosis is a genetically regulated process of cell-autonomous death, and the molecular mechanisms of apoptosis activation include caspase-dependent signaling pathway, caspase-independent signaling pathway, and arachidonic acid metabolic pathway. Novia [26] et al. studied Huh7 hepatocellular carcinoma cells and WRL68 normal hepatocytes and found that berberine could inhibit the activity of hepatocellular carcinoma cell lines and promote apoptosis of hepatocellular carcinoma cells, the mechanism of berberine dose-dependently up-regulated the pro-apoptotic factors Bax, Bid, and p21, and down-regulated the anti-apoptotic factors AKT and Bcl-2, and berberine could also cause the cell cycle to block in the M/G1 phase. Berberine also promotes the activation of caspase-9 and its downstream effectors caspase-3 and caspase-7 thereby inducing apoptosis in the mitochondrial pathway of hepatocellular carcinoma cells [27, 28]. Ou Li[29] found that berberine can also induce apoptosis of hepatocellular carcinoma cells through caspase-independent signaling pathways, which is similar to other scholars' reports [30, 31]. The COX metabolic pathway of arachidonic acid (AA) is closely related to tumorigenesis. cPLA2 and COX-2 are key enzymes in the AA metabolic pathway, and the PLA2-AA signaling pathway can be involved in hepatocellular carcinoma cell apoptosis. Some scholars found [32] that berberine effectively inhibited the expression of cPLA2 and COX2, and dose-dependently increased the ratio of arachidonic acid to prostaglandin E2, and activated apoptosis mediated by apoptosis-inducing factor in hepatocellular carcinoma cells. Thus, inhibition of the AA metabolic pathway is one of the mechanisms by which berberine promotes apoptosis in hepatocellular carcinoma cells. Berberine can activate the AMPK pathway, upregulate the level of death receptor DR5 protein, enhance the sensitivity of hepatocellular carcinoma cells to the death ligand TRAIL, and induce apoptosis of hepatocellular carcinoma cells [33]. Berberine also induces the PHLPP2-Akt-MST1 kinase signaling pathway to promote mitochondrial damage and induces hepatocellular carcinoma cell death by inhibiting the PI3K/AKT pathway [34, 35]. XiaoWu Li[36] et al. used different concentrations of berberine to act on HepG2 hepatocellular carcinoma cell lines and found that berberine could inhibit hepatocellular carcinoma cell viability in a dose- and time-dependent manner, with an inhibition rate of up to 84.25%. Mechanistically, berberine dramatically increased intracellular ROS levels, enhanced the expression of pro-apoptotic protein Bax and activated caspase-3, and induced apoptosis of hepatocellular carcinoma cells through the ROS/Bax/ caspase-3 pathway. Xiao Qiang [28] et al. found that berberine could inhibit Bcl-2, Wnt and β-catenin protein expression, promote Bax protein expression and activate Caspase-3 and Caspase-9 in hepatocellular carcinoma cells, which suggests that the anticancer mechanism of berberine may be related to the inhibition of Wnt-β-catenin pathway and promotion of mitochondrial apoptosis.

Inducing Autophagic Death of Hepatocellular Carcinoma Cells: Autophagic cell death is a form of programmed cell death which is different from apoptosis. To determine the potential of berberine to induce autophagy in hepatocellular carcinoma cells, Qian Hou [37] transfected SMMC7721 hepatocellular carcinoma cells with EGFP-LC3 plasmid. After exposure to berberine for 24 hours, the autophagic level of the cells was observed using transmission electron microscopy, revealing that berberine could increase autophagic cell death of hepatocellular carcinoma cells in a dose-dependent manner. Young [38] showed that berberine inhibited PI3K/Akt/mTOR pathway activation, promoted mitochondrial reactive oxygen species production and mitochondrial phagocytosis-regulating protein recruitment, and ultimately induced hepatocellular carcinoma cell autophagic death. Experiments by La [39] demonstrated that berberine could induce autophagic death of hepatocellular carcinoma cells by increasing GRP78 expression and enhancing the interaction of GRP78 with Vps34. In addition, berberine can activate the AMPK pathway to induce apoptosis and autophagic death in hepatocellular carcinoma cells [40].

Inhibition of Hepatocellular Carcinoma Cell Proliferation: Berberine inhibits hepatocellular carcinoma cell proliferation through multiple pathways [41]. Luo [42] et al. found that miR-22-3p could inhibit the hepatocellular carcinoma cell cycle by directly targeting and down-regulating the expression of methionine adenylyltransferase 2A and 2B (MAT2A, MAT2B), which is a key enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM), and that MAT promotes the increase of intracellular SAM and inhibits the proliferation of hepatocellular carcinoma cells. Chen et al [43] found that berberine inhibited the proliferation of HepG2 hepatocellular carcinoma cells in a time-dose-dependent manner, and the specific mechanism may be related to the up-regulation of miR-22-3p expression and the down-regulation of the expression of the transcription factor SP1 and its downstream cell cycle target proteins CCND1 and BCL2 by berberine. Li [44] et al. found that berberine induced G0/G1 phase cell cycle block in Huh-7 and HepG2 hepatocellular carcinoma cells. The mechanism is that berberine down-regulates Akt phosphorylation, inhibites Skp2 expression, and promotes FOXO3a expression and nuclear translocation in order to promote the up-regulation of the transcription factors p21Cip1 and p27Kip1, and to impede the G1/S transition of the cell cycle. As a nitrogen donor and carbon donor, glutamine metabolism regulates protein synthesis and promotes tumor cell proliferation. Zhang et al [45] found that berberine down-regulated SLC1A5 and c-myc expression to block cellular uptake of glutamine, which in turn inhibited the proliferation of Hep3B and BEL-7404 hepatocellular carcinoma cells. Berberine also promotes cell cycle protein D1 ubiquitin-proteasome-dependent protein hydrolysis to inhibit hepatocellular carcinoma cell cycle and proliferation [46].

Inhibition of Invasive Metastasis of Hepatocellular Carcinoma Cells: Distant metastasis of hepatocellular carcinoma is the main cause of death in patients with advanced hepatocellular carcinoma. Epithelial mesenchymal transition (EMT) is mainly involved in invasive metastasis of tumors, and EMT promotes the metastasis and infiltration of tumor cells into distant tissues. As an EMT-inducing factor, TGF-β1 can induce tumor metastasis by decreasing intercellular adhesion as well as promoting mesenchymal phenotypic transformation [47]. Chunmiao Chen[48] et al. found that berberine inhibited the TGF-β1-induced Smad signaling pathway, as evidenced by the down-regulation of Smad phosphorylation and the expression of epithelial markers (E-cadherin), and the up-regulation of mesenchymal markers (N-cadherin, Vimentin, Snail, and MMP-2), and ultimately berberine inhibited hepatocellular carcinoma cell invasion and metastasis through the TGF-β/Smad signaling pathway. Chemotherapy can alter the tumor microenvironment by activating the LOX pathway and releasing inflammatory factors such as LTB4, which instead promotes invasive metastasis of tumor cells. Zhao [49] et al. found that berberine reversed chemotherapy-induced invasion and metastasis of hepatocellular carcinoma cells by inhibiting LOX-5 and LTB4 expression in the tumor microenvironment. PAI-1 is a tumor suppressor that antagonizes uPA receptors. Berberine was found to upregulate fibrinogen activator inhibitor-1 (PAI-1), inhibit uPA expression, block the uPA receptor pathway, and ultimately reduce the invasive metastasis of hepatocellular carcinoma cells [50].

Anti-Oxidative Stress: Inflammatory stimuli and oxidative stress injury are associated with hepatocellular carcinoma progression. CYP2E1 is considered to be a major source of reactive oxygen species and a major mediator of lipid peroxidation. Some scholars found that berberine could down-regulate the protein levels of endoplasmic reticulum stress markers (ATF6, XBP1, ATF4 and CHOP), inhibit the expression of CYP2E1, and down-regulate the mRNA and protein levels of important regulators of adipogenesis (SREBP-1c, ChREBP, Fas, and C/EBPβ) to inhibit hepatic fat deposition, and berberine could also down-regulate the expression of TNF-α and IL -6 expression and inhibit inflammatory response. This suggests that berberine reverses endoplasmic reticulum stress-activated lipogenesis through the ATF6/SREBP-1c pathway, thereby reducing hepatic inflammation, fibrosis, and lipid peroxidation injury for liver protection [51].

Inhibition of Angiogenesis and Regulation of Metabolism: Angiogenesis promotes hepatocellular carcinoma development, and vascular endothelial growth factor (VEGF) promotes angiogenesis and increases vascular permeability to induce tumor invasion and metastasis. VEGF transcription and expression are regulated by TNF-α, which is closely related to nuclear transcription factor-κB (NF-κB), and the degradation of IκBα and the translocation of NF-κB p65 to the nucleus activate the NF-κB signaling pathway. Studies have shown [52, 53] that berberine inhibits NF-κB activation. Mechanistically berberine reduces VEGF expression and angiogenesis by inhibiting the degradation of IκBα in the cytoplasm, preventing the translocation of NF-κB p65 to the nucleus, and blocking the activation of the NF-κB signaling pathway. Therefore, berberine can inhibit hepatocellular carcinoma development through the NF-κB/TNF-α/VEGF pathway [54, 55]. Luo [56] et al. established a non-alcoholic fatty liver-associated hepatocellular carcinoma mouse model using streptozotocin injected under a high-fat diet, and found that the mRNA transcription of the fat synthesis-related genes FASN, SCD1, and SREBP1c was suppressed in berberine-intervened group of mice, the expression of hepatic fibrosis-associated factors α-SMA, COL1A1, and TIMP-1 was down-regulated, the inflammatory factors IL-6, IL -1 and TNF-α were reduced, Kupffer cell activation was blocked, and hepatic lipid deposition was attenuated. Thus, berberine inhibits hepatic lipid deposition through lipid metabolism pathway.

Combining with Other Treatment Modalities to Increase Anti-cancer Effects

Tumor resistance is a major cause of recurrence and poor prognosis. Berberine enhances anticancer effects when combined with antitumor drugs or radiation therapy. The combination of berberine and rapamycin can improve the prognosis of hepatocellular carcinoma patients by synergistically inhibiting the mTOR signaling pathway [57]. The combination of berberine and vincristine has significant growth inhibitory and apoptosis-inducing effects on human hepatocellular carcinoma cells, and the specific mechanism may be related to the induction of mitochondrial dysfunction and endoplasmic reticulum stress by berberine [58]. Hepatocellular carcinoma has poor sensitivity to radiation therapy, but berberine inhibits the expression of Nrf2 signaling-related proteins (Nrf2, HO-1 and NQO-1) in hepatocellular carcinoma cells to enhance the sensitivity of hepatocellular carcinoma to radiation therapy [59]. It has also been suggested that the specific mechanism by which berberine enhances the sensitivity to radiation therapy may be that berberine activates the p38MAPK pathway and generates ROS to induce apoptosis in hepatocellular carcinoma cells [60]. Wang et al [61] observed that berberine can enhance the inhibition of the activity of rutaecarpine on human SMMC-7721 hepatocellular carcinoma cells. Huang et al. found that berberine combined with the targeted drug sorafenib can inhibit the proliferation of hepatocellular carcinoma cells in a dose-time dependent manner. The specific mechanism may be related to the synergistic effect of berberine with sorafenib to further reduce Bcl-2 and VEGF protein levels, enhance ADP-ribopolymerase expression, and activate caspase-3. This indicates that berberine can enhance the sensitivity of hepatocellular carcinoma cells to sorafenib, playing a synergistic anti-tumor effect. Berberine also increases the killing effect of chemotherapy and targeted drugs regorafenib, regorafenib, oxaliplatin and apatinib mesylate on hepatocellular carcinoma cells [63-66].

Anti-hepatocellular Carcinoma Effects of Berberine Derivatives

The poor lipid solubility of berberine makes it difficult to pass through cell membranes in order to be efficiently absorbed by the gastrointestinal tract, which limits its anticancer potential. Therefore, researchers have modified and altered different sites of berberine to synthesize a series of derivatives with good lipid solubility, high bioavailability and potent antitumour activity [67]. Berberine C9 and C13 site derivatives were found to possess stronger antitumor effects [68, 69]. Targeted drug delivery vehicles constructed from berberine derivatives can deliver drugs to tumor sites in a targeted manner [70, 71]. Wuli Zhao [72] et al. used berberine as the matrix to prepare 13 alkyl derivatives through selective reduction and reaction with fatty aldehydes, and then reacted with acetone and bromobenzyl under alkaline conditions to prepare 13 benzyl derivatives. The results showed that the antitumor activity of both derivatives was significantly enhanced. Li [73] et al. synthesized a 9-O flavin-bile acid conjugate that induced ROS-triggered caspase-dependent and non-caspase-dependent apoptotic pathways in SMMC-7721 hepatocellular carcinoma cells. Compared with berberine, the 9-O berberine-bile acid conjugate inhibits the growth of hepatocellular carcinoma cells more strongly, while hardly affecting the growth and metabolism of normal hepatocytes. Jin [74] et al. synthesized three novel C9-position derivatives of berberine using the "CuAAC click" chemical method, and these compounds showed strong antitumor activity against SMMC-7721 hepatocellular carcinoma cells, and the effect was better than that of berberine, among which 9-O benzyl substituents showed highly effective activity against hepatocellular carcinoma cells. In addition to changing the chemical structure of berberine, Song et al. used berberine 9-O-octadecyl substituted derivative (BD) as raw material to construct mitochondrial targeted nanoparticles using nanoprecipitation method. The nanoparticles were modified with polyethylene glycol (PEG) to improve stability, and then coated with negatively charged hyaluronic acid (HA) to prepare HA/PEG/BD carriers with moderate surface charge and high drug loading to achieve mitochondrial targeted drug delivery. Berberine HA/PEG/BD vector could eliminate mitochondrial membrane potential, up-regulate ROS level, inhibit the anti-apoptotic factor Bcl-2, up-regulate the pro-apoptotic factor Bax, release cytochrome C and activate caspase 9/3 to induce apoptosis of hepatocellular carcinoma cells efficiently.

As a traditional Chinese medicine in China, berberine has a wide range of pharmacological effects, and the role of berberine against hepatocellular carcinoma has been initially explored. The hepatocellular carcinoma cytotoxicity of berberine was found to be a result of the combined action of multiple signaling pathways and multiple molecular mechanisms. Berberine has the advantages of low toxicity, high safety and low price. Continuous research and development of berberine derivatives have corrected the shortcomings of poor water solubility and unfavorable absorption by oral administration, and have a promising future in the prevention and treatment of hepatocellular carcinoma. However, the studies on berberine against hepatocellular carcinoma are mainly focused on cell and animal experiments, and clinical efficacy observation has not yet been carried out. More randomized double-blind controlled, prospective and multicenter clinical trials are needed to prove whether berberine can clinically combat hepatocellular carcinoma or improve the prognosis of patients with hepatocellular carcinoma.

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