Epigallocatechin Gallate Induces Hepatic Stellate Cell Senescence and Attenuates Development of Hepatocellular Carcinoma

Abstract

Introduction

Hepatocellular carcinoma (HCC) incidence has more than tripled since 1980, with 50,000 new cases predicted annually in the United States (1). HCC is a highly morbid condition with a 5-year survival less than 15%. Surgical resection, liver transplant, and radiofrequency ablation are considered the only curative therapies. However, the majority of patients are diagnosed at an advanced stage, and less than 20% of new cases are considered potentially curable. In addition, HCC is minimally responsive to standard chemotherapeutic agents (2). Until recently, sorafenib was the only drug clinically approved by FDA for treatment of advanced-stage HCC, even though sorafenib has been shown to provide only a modest survival benefit (3, 4).

Given the dismal outlook for HCC therapy, there has been increased interest in chemopreventive strategies. HCC arises from a milieu of chronically inflamed and damaged liver tissue, and thus there is a strong association between cirrhosis and HCC risk (5, 6). Typically, cirrhosis onset occurs years before a diagnosis of HCC, therefore, chemoprevention is highly applicable in this context given the large window of time for intervention.

Although HCC derives from hepatocytes, there is a strong association between liver fibrosis and HCC risk; therefore, stromal cells may play a role in mediating hepatocarcinogenesis (7-9). Hepatic stellate cells (HSC), the primary stromal cell type of the liver, transition to an “activated” myofibroblast-like state upon injury characterized by expression of smooth muscle actin and deposition of extracellular matrix components including collagens and matrix metalloproteinases (MMP). Persistent injury, as in the case of cirrhosis, may result in a state of chronic inflammation and tonic HSC activation, resulting in a pathologic fibrotic response (10). In addition, activated HSCs have also been shown to provide feed-forward stimuli by production of cytokines and chemokines that recruit inflammatory cells to the site of injury, raising concern that persistent HSC activation may support a protumorigenic environment (11). Accordingly, it has been predicted that direct inactivation of HSCs, or senescence, beneficially influences the dynamic balance of inflammation, fibrosis progression, and HCC development, and may therefore provide a useful chemopreventive strategy (12).

Experimental and epidemiologic studies suggest tea polyphenols may have chemopreventive effects in variety of cancers including HCC (13, 14). Green tea is a rich source of natural polyphenols, which are commonly known as catechins, and epigallocatechin gallate (EGCG) is the most abundant catechin in green tea (comprising 50%–80% of the total pool). Several studies have reported the inhibitory effect of EGCG on cancer cell proliferation through inhibition of receptor tyrosine kinases and their downstream signaling pathways (15). EGCG has also been reported to induce cell-cycle arrest through expression of p21, p18, and p53, and inhibition of cyclins and cyclin-dependent kinases (15, 16). Finally, EGCG has been shown to reduce liver inflammation and fibrosis (17). Therefore, we hypothesized that EGCG would have chemopreventive effects in chronic liver injury through its negative regulation of inflammation, fibrosis, and cellular proliferation.

Materials and Methods

Results

EGCG prevents HCC development in diethylnitrosamine-injured rats

EGCG treatment grossly limited the development of HCC nodules (diethylnitrosamine 18.75 ± 1.76 vs. DEN + EGCG 9.125 ± 1.922; P < 0.001; Fig. 1A and ​andB),B), and was associated with a significant reduction in the liver/body weight ratio (diethylnitrosamine 6.964% ± 0.5097% vs. DEN + EGCG 5.377% ± 0.4375%; P < 0.05; Fig. 1C). EGCG was well-tolerated and mitigated diethylnitrosamine toxicity–related weight loss (diethylnitrosamine 380.5 ± 13.23 g vs. DEN+EGCG 439.2 ± 15.79 g; P < 0.05; Fig. 1D). Commensurate with these gross findings, EGCG treatment also improved biochemical markers of liver function and injury, including TBIL, ALP, AST, and ALT, which were all decreased (Fig. 1E-​-HH).

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Figure 1.

EGCG inhibits HCC development in diethylnitrosamine (DEN)-injured rats. Male Wistar rats received weekly intraperitoneal injections of diethylnitrosamine for 18 weeks. After 12 weeks, a subset of rats received 0.02% EGCG in their drinking water during weeks 13–18. A and B, Representative rat liver images at the time of sacrifice. Tumors that were greater than 5 mm in diameter were counted. N = 8; **, P < 0.01 compared with untreated diethylnitrosamine controls. Liver/body weight (C) and body weight (D) was measured and compared with diethylnitrosamine-injured rats. *, P < 0.05. Serum levels of TBIL (E), ALP (F), AST (G), and ALT (H) are reported. N = 4 for all groups; ***, P < 0.001 compared with untreated diethylnitrosamine group. I, Gene set enrichment analysis of the 73-gene poor-prognosis signature and 113-gene good-prognosis signature in diethylnitrosamine-injured rats after treatment with EGCG.

To better understand the mechanism of EGCG-mediated HCC chemoprevention, we performed transcriptomic analysis of nontumoral, hepatic tissue from diethylnitrosamine controls and EGCG-treated rats. Hoshida and colleagues previously reported a 186-gene signature derived from cirrhotic liver tissue that can discriminate patients by future HCC risk (24, 25). Diethylnitrosamine treatment resulted in transcriptomic changes that mirror Hoshida's poor prognosis signature. In contrast, EGCG treatment resulted in prevention of this signature toward a “good prognosis” expression profile (Fig. 1I). The significantly upregulated and downregulated gene clusters in EGCG-treated rats are shown in Table 1. Good prognosis genes enriched in EGCG-treated animals involve networks that participate in normal liver function such as organic acid metabolism, lipid and fatty acid metabolism and biosynthesis, amino acid metabolism, catabolic processes, and cellular catabolism. In contrast, the poor prognosis gene networks induced by diethylnitrosamine treatment are primarily involved in wound healing and fibrosis progression, including pathways related to cell adhesion, defense mechanisms, and coagulation. Importantly, these networks were significantly downregulated after EGCG treatment.

Table 1.

Gene ontology (GO) functional enrichment analysis for up- and downregulated genes after treatment with EGCG.

No.	GO accession	Upregulated	NES	FDR	P
1	GO:0006082	Organic acid metabolic process	2.45	0	0
2	GO:0019752	Carboxylic acid metabolic process	2.36	0	0
3	GO:0032787	Monocarboxylic acid metabolic process	2.24	0	0
4	GO:0006631	Fatty acid metabolic process	2.15	0	0
5	GO:2001289	Lipid metabolic process	2.08	0	0.001
6	GO:0008654	Phospholipid biosynthetic process	1.98	0	0.002
7	GO:0044255	Cellular lipid metabolic process	1.97	0	0.003
8	GO:0016042	Lipid catabolic process	1.85	0.002	0.019
9	GO:0006732	Coenzyme metabolic process	1.85	0	0.018
10	GO:0044242	Cellular lipid catabolic process	1.84	0.005	0.017
11	GO:0046467	Membrane lipid biosynthetic process	1.83	0.004	0.018
12	GO:0008610	Lipid biosynthetic process	1.77	0	0.033
13	GO:0015980	Energy derivation by oxidation of organic compounds	1.77	0.005	0.032
14	GO:0044270	Nitrogen compound catabolic process	1.75	0.007	0.033
15	GO:0008202	Steroid metabolic process	1.73	0.002	0.038
16	GO:0006519	Amino acid and derivative metabolic process	1.72	0.002	0.043
17	GO:0006807	Nitrogen compound metabolic process	1.71	0	0.042
18	GO:0009056	Catabolic process	1.71	0	0.04
19	GO:0044248	Cellular catabolic process	1.65	0	0.065
20	GO:0006520	Amino acid metabolic process	1.64	0.015	0.067
No.	GO accession	Downregulated	NES	FDR	P
1	GO:0007599	Hemostasis	−1.91	0	0.063
2	GO:0030155	Regulation of cell adhesion	−1.87	0	0.056
3	GO:0007596	Blood coagulation	−1.83	0.004	0.073
4	GO:0009611	Response to wounding	−1.83	0	0.058
5	GO:0050817	Coagulation	−1.82	0.002	0.052
6	GO:0045597	Positive regulation of cell differentiation	−1.81	0.004	0.051
7	GO:0050878	Regulation of body fluid levels	−1.81	0.002	0.045
8	GO:0009605	Response to external stimulus	−1.73	0	0.1
9	GO:0051707	Response to other organism	−1.72	0	0.1
10	GO:0006952	Defense response	−1.71	0	0.102
11	GO:0042060	Wound healing	−1.7	0.002	0.103
12	GO:0008544	Epidermis development	−1.68	0.003	0.118
13	GO:0009617	Response to bacterium	−1.68	0.015	0.111
14	GO:0009607	Response to biotic stimulus	−1.67	0.005	0.117
15	GO:0045595	Regulation of cell differentiation	−1.67	0.002	0.11
16	GO:0051259	Protein oligomerization	−1.66	0.012	0.113
17	GO:0007398	Ectoderm development	−1.65	0.012	0.12
18	GO:0009888	Tissue development	−1.61	0.003	0.157
19	GO:0016337	Cell–cell adhesion	−1.61	0.009	0.156
20	GO:0050863	Regulation of t cell activation	−1.59	0.017	0.169

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Note: Gene sets with FDR < 0.25 are shown (top 20 when there are >20 gene sets with FDR < 0.25). Abbreviation: NES, normalized enrichment score.

EGCG prevents cirrhosis progression in diethylnitrosamine-injured rats

EGCG treatment has previously been shown to have anti-fibrotic properties in rodent models ofliver injury. In our study, EGCG treatment for 6 weeks was associated with decreased progression to cirrhosis, evidenced by reduced type I collagen staining in liver tissue (EGCG 2.64 ± 0.40 vs. diethylnitrosamine 15.78 ± 1.326; P < 0.001; Fig. 2A and ​andB).B). Pathologic scoring of fibrosis using Masson trichrome staining also demonstrated significant improvement in the median Ishak score in EGCG-treated rats compared with untreated diethylnitrosamine controls [4.4, interquartile range (IQR) 2–6 compared with 5.8, IQR 5–6; P < 0.05; Fig. 2C and ​andDD].

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Figure 2.

EGCG decreases fibrosis progression to cirrhosis. A and B, Immunostaining for collagen I (magnification, 100×) was quantified. C and D, Masson trichrome stains of liver sections from each animal were scored by the method of Ishak. *, P < 0.05; ***, P < 0.001, compared with untreated diethylnitrosamine (DEN) group.

EGCG promotes HSC senescence in vivo

Upon diethylnitrosamine injury, quiescent HSCs transform to activated α-SMA–positive myofibroblasts that proliferate and promote fibrogenesis. Diethylnitrosamine-injured rats that received 0.02% EGCG in their drinking water had significant reductions in immunostaining of α-SMA on paraffin-embedded liver samples compared with diethylnitrosamine controls, suggesting decreased HSC activation (Fig. 3A and ​andB).B). This observation was corroborated by Western blot analysis, in which we again observed less α-SMA protein expression in livers isolated from EGCG-treated animals (Fig. 3C). Given the decrease in stromal activation, we investigated whether EGCG induced HSC senescence by measuring HSC SA-β-gal activity (26-28). SA-β-gal activity was increased in EGCG-treated liver tissue as compared with untreated diethylnitrosamine controls, indicating higher levels of senescence (Fig. 3D and ​andE).E). SA-β-gal–positive cells were specifically located in the fibrotic bands where HSCs are abundant, but not in the surrounding hepatic tissue (Fig. 3D), indicating that EGCG induced senescence in HSCs but not hepatocytes. In addition, proliferating cell nuclear antigen (PCNA), a marker for cellular proliferation, was significantly decreased in EGCG-treated livers compared with untreated diethylnitrosamine controls (Fig. 3F).

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Figure 3.

EGCG induces cellular senescence of activated HSCs. A and B, Immunostaining of α-SMA (magnification, 100×) was quantified. **, P < 0.01. C, Representative Western blot analysis for α-SMA performed on nontumoral surrounding liver tissue lysates. D and E, SA-β-gal staining (turquoise blue) of liver sections of untreated and EGCG-treated diethylnitrosamine (DEN) animals was quantified. ***, P < 0.001. F, IHF staining for PCNA (green) and smooth muscle actin (SMA; red) on liver tissue from untreated and EGCG-treated diethylnitrosamine animals. G, Expression of HSC activation was assessed with a HSC gene signature in diethylnitrosamine-injured rats treated with vehicle (water) or EGCG.

A gene signature for activated HSCs was previously established and verified across multiple human datasets and animal models (29, 30). We performed microarray analysis of liver tissue from diethylnitrosamine and DEN+EGCG-treated animals and compared expression profiles with the activated HSC signature. EGCG treatment was associated with HSC inactivation, evidenced by loss of overlap with the HSC activation signature compared with untreated diethylnitrosamine controls (Fig. 3G). Taken together, these findings suggest that EGCG both reduced HSC activation and induced senescence in the setting of diethylnitrosamine liver injury.

EGCG promotes HSC senescence and senescence-associated secretory phenotype in HSCs in vitro

To gain more insight into EGCG-induced HSC senescence, we treated TWNT4 immortalized human HSCs in vitro with EGCG. EGCG treatment at concentrations above 20 μmol/L significantly reduced cell proliferation compared with untreated controls by MTT assay. This growth reduction did not appear to be a toxic effect, as cells remained viable in the EGCG treatment groups on microscopic evaluation (Fig. 4A and ​andB).B). Other general hallmarks of senescence include the absence of proliferative markers such as Ki67, and increased expression of tumor suppressors (P53) and cell-cycle inhibitors (P16 and P21). Consistent with these observations, 20 μmol/L EGCG treatment was also associated with significant reduction (≈1.5-fold) in Ki67 expression (control 48.59% ± 2.523% vs. EGCG 31.24% ± 2.039%; P < 0.001; Fig. 4C and ​andD),D), and significant increases in cell-cycle inhibitor gene expression, including P16, P21, and P53 (Fig. 4G). Finally, SA-β-gal activity also increased (≈4-fold) in TWNT4 cells after 72 hours of treatment with 20 μmol/L EGCG (control 22.52 ± 1.126 vs. EGCG 62.64 ± 1.255; P < 0.001; Fig. 4E and ​andF).F). These findings corroborate our in vivo observation that EGCG induces HSC senescence.

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Figure 4.

EGCG induces SASP in human HSCs cell line in vitro. TWNT4 cells were treated with different concentration of EGCG (0, 2, 5, 10, 20, 40, 50, and 100 μmol/L) for 72 hours. A, Representative bright field pictures of TWNT4 cells treated with 20, 50, and 100 μmol/L of EGCG. B, MTT cell proliferation assay in TWNT4 cells, 72 hours after treatment with EGCG. C and D, Immunofluorescence staining of Ki67 (red) and DAPI (blue) on TWNT4 cells after 72 hours treatment with 20 μmol/L EGCG. Untreated cells were considered as control, 2,000 cells were counted in each group; ***, P < 0.001 compared with control. E and F, SA-β-gal staining (turquoise blue) of TWNT4 cells after 72 hours treatment with 20 μmol/L EGCG. Untreated cells were considered as control, 2,000 cells were counted in each group; **, P < 0.01 compared with control. G-J, RNA expression of senescence and SASP markers in TWNT4 cells after 72 hours treatment with 20 μmol/L EGCG. Untreated cells were considered as control. N = 4 replicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Finally, previous reports have identified the presence of a unique secretory phenotype specifically associated with senescence in HSCs, which is characterized by reduced expression of extracellular matrix (ECM) proteins, increased expression of ECM-degrading enzymes, and increased secretion of inflammatory cytokines, referred to as the senescence-associated secretory phenotype (SASP; ref. 31). In our study, compared with untreated cells, EGCG-treated TWNT4 cells exhibited a similar secretory profile upon undergoing senescence in vitro, including: reduced expression of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and fibronectin (FN; Fig. 4H); increased expression of MMP-1 (MMP1) and -3 (MMP3); and increased expression of immune surveillance chemokines, IL6 (IL6) and -8 (IL8; Fig. 4I and ​andJ).J). These findings therefore suggest that EGCG-induced senescence in HSCs also leads to initiation of SASP.

EGCG does not promote senescence of human HCC cell lines

To further demonstrate that EGCG-induced senescence is specific to HSCs, we treated the human HCC cell lines HepG2 and Hep3B with 20 μmol/L EGCG for 72 hours. EGCG treatment had no effect on SA-β-gal activity in HepG2 cells under normal growth conditions (vehicle 1.512% ± 0.19% vs. EGCG 1.231% ± 0.20%; Fig. 5A and ​andB).B). In addition, no difference in senescence-associated gene expression was observed (Fig. 5C). Similarly, although Hep3B cells have more SA-β-gal activity (<15%) at baseline; no difference was observed with EGCG treatment (Fig. 5D and ​andE).E). Finally, we did not observe any change in gene expression of the tumor suppressors, P16 or P21, after EGCG treatment (Fig. 5F).

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Figure 5.

EGCG does not alter senescence markers in human HCC cell lines in vitro. HepG2 and Hep3B cells were treated with 20 μmol/L of EGCG for 72 hours. A and B, SA-β-gal staining (turquoise blue) of HepG2 cells. A total of 2,000 cells were counted in each group. C, Expression of cell-cycle-associated genes in untreated and EGCG-treated HepG2 cells. D and E, SA-β-gal staining (turquoise blue) of Hep3B cells. A total of 2,000 cells were counted in each group. F, Expression of cell-cycle-associated genes in untreated and EGCG-treated Hep3B cells.

Discussion

Prior studies have shown a protective effect of the main constituent of green tea, EGCG, against liver injury, oxidative stress, inflammation, and fibrosis in experimental animal models of liver diseases (32-35). In this study, we recapitulate the observed antifibrotic effect in a diethylnitrosamine liver injury model and provide a novel mechanism through stellate cell-specific senescence. In addition, we report that EGCG treatment is also chemopreventive for the development of HCC after diethylnitrosamine injury.

Prior studies evaluating the hepatoprotective effects of EGCG often relied on high drug doses that have not been shown to be safe in humans. High doses of EGCG are hepatotoxic in mice (36), and at least 200 cases of EGCG-related hepatic toxicity have been reported since 1996, likely due to growing consumer interest in herbal supplements in the United States (36, 37). In contrast, the EGCG dose used in this study was designed to be equivalent to a human oral dose of 400 mg per day, which had been demonstrated to be safe and tolerable in a human single-dose ascending study (38). More recent results in humans have demonstrated elevations of ALT and AST after 12 months of green tea extract consumption (1,315 ± 115 mg total catechins including 843 ± 44 mg of EGCG per day) in a placebo-controlled, double-blinded phase II clinical trial assessing breast cancer risk biomarkers in healthy postmenopausal women (39). These elevations occurred in a small proportion of participants and were reversible. Therefore, we believe our results represent the first report showing clinically achievable and safe doses of EGCG can prevent fibrosis progression and prevent HCC development.

Multiple previous studies have demonstrated an association between hepatic fibrosis and HCC risk (18, 40, 41). Although the etiologies of liver disease have specific risks for development of HCC, most patients sequentially develop hepatitis, fibrosis, cirrhosis, and then HCC (42). In 80%–90% of patients, HCCs develops in fibrotic or cirrhotic environment and patients with a high serologic fibrosis index (FIB-4) have an up to 15-fold increased risk for future HCC incidence (43). In addition, one-third of patients with cirrhosis develop HCC within 5 years (42). Fibrogenesis in liver is mediated by HSCs, which deposit ECM molecules and recruit immune cells to the site of injury (44, 45). We previously reported that HSC inactivation prevents fibrosis progression, which is associated with a reduction in HCC development in rodents (18, 46). In this study, EGCG reduced fibrosis progression through induction of senescence in HSCs, which resulted in improved liver function and reduced HCC development

It has previously been shown that senescent HSCs develop a unique secretory phenotype known as the SASP. We observed a similar phenotype in HSCs in vivo and TWNT4 cells in vitro with EGCG treatment, which resulted in reduced proliferation as measured by Ki67 staining and MTT assay, and increased expression of senescence markers including increased SA-β-gal staining, and upregulation of tumor suppressor genes including P16, P21, and P53. TWNT4 cells also increase the expression of immunosurveillance cytokines (IL6 and IL8), which are known to induce recruitment of natural killer cells, regulatory T lymphocytes, and macrophages to remove matrix debris and activated HSC (45, 47).

Interestingly, other reports have shown that the increased SASP from senescent HSC can induce HCC development (48, 49). These studies concluded that while cellular senescence usually provides a barrier to epithelial transformation, senescence in mesenchymal cells, like HSCs, could support cancer growth through the SASP by releasing cytokines like IL6. Most of these studies were performed in the context of obesity-associated HCC in animal models without significant fibrosis. The discrepancy in these studies could be explained by differences in the senescence triggers, the composition of microenvironment, and extent of senescence surveillance in different animal models (48, 50).

Other possible mechanisms could explain the reduction of HCC development after EGCG treatment. This includes reversibility of mutation load that is caused by diethylnitrosamine. In a high-fat diet (HFD) mouse model of HCC, theaphenon E, a standardized green tea extract formulation, blocked HCC formation via prevention of lipid peroxidation–derived mutagenic cyclic DNA-adduct (γ-OHPdG) formation resulting in decreased mutation load particularly G>T, the most common mutation in human HCC (51). Consistent with these results, theaphenon E reduced HCC incidence in xeroderma pigmentosum group A–knockout mice and diethylnitrosamine-injected mice and the reduction in HCC incidence was associated with decreased (γ-OHPdG) levels (52). In addition, using a microarray approach we showed that after EGCG treatment the pathways/genes involved in normal liver metabolic functions such as amino acid and lipid metabolism were markedly upregulated, and at the same time, pathways that are involved in wound healing and immune system response were downregulated. These mechanisms will need to be further evaluated in future studies.

Our data therefore suggests that the cancer prevention effects of EGCG-induced fibrosis regression, including the upregulation of ECM remodeling and inflammatory cytokines that contribute to the clearance of senescent cells and resolution of fibrotic tissue, outweigh any procancer effects of the SASP at least in the setting of cirrhosis. It is also possible that the SASP response needs to be combined with appropriate environmental cofactors, like DCA, which is produced by the gut microbiota in HFD and required for HCC development (48).

Green tea is very popular and mainly consumed in Asia. Several meta-analyses have been performed on Asian populations to investigate the association between green tea consumption and the risk for liver cancer (53-55). While some of these studies have shown no association with cancer risk (56), others have shown a decreased risk of liver cancer with green tea consumption especially in people who drink more green tea for longer periods of time (54-56). Given the slow progression of cirrhosis to HCC development, green tea consumption represents a potentially safe and cheap chemopreventive strategy for HCC.

Acknowledgments

Footnotes

References