Eicosapentaenoic acid attenuates obesity-related hepatocellular carcinogenesis

Abstract

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of obesity, is an emerging risk factor for hepatocellular carcinoma (HCC). Accumulating evidence has shown that chronic inflammation represents a plausible link between obesity and HCC and that the pro-inflammatory cytokine interleukin (IL)-6 contributes to the development of obesity-related HCC. In the present study, we aimed to examine the therapeutic potential of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which exerts anti-inflammatory effects. The results showed that the development of carcinogen-induced HCC was significantly less in mice fed a high-fat diet (HFD) supplemented with EPA than in those fed HFD only, suggesting that EPA attenuates the development of obesity-related HCC. Although EPA did not appear to affect obesity-linked inflammation, it suppressed the activation of the pro-tumorigenic IL-6 effector STAT3, contributing to the inhibition of tumor growth. These findings suggest a clinical implication of EPA as a treatment for obesity-related HCC.

Figure 1.

EPA has no effect on HFD-induced obesity. (A) Experimental protocol of the EPA treatment for the obesity-related HCC mouse model. (B) Growth curve of DEN-injected mice fed SD, HFD or HFD + EPA. Liver weight (C) and the ratio of liver weight to body weight of mice (D) from three dietary groups at 9 months old. All values represent the mean ± SD (n = 10–12). **P < 0.01; ***P < 0.001.

Figure 2.

EPA ameliorates hepatic steatosis in obese mice. ORO staining (A) and hematoxylin and eosin (HE) staining (B) of liver sections from DEN-injected mice fed SD, HFD or HFD + EPA for 37 weeks. (C) Percentage of the area occupied by ORO staining in each field (n = 3–4). (D) Liver triglyceride (TG) and total cholesterol (TCHO) levels in DEN-injected mice in each dietary group were measured at 9 months old (n = 6–8). (E) Plasma concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in DEN-injected mice in each dietary group were measured at 9 months old (n = 9–12). Scale bars = 50 µm (A) and 200 µm (B). All values represent the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

EPA attenuates obesity-enhanced hepatocarcinogenesis. Gross liver morphology (A), tumor numbers (B), tumor maximal sizes (C) and tumor size distribution (D) from DEN-injected mice fed SD, HFD or HFD + EPA for 37 weeks. Arrows indicate tumors (A). Scale bars = 10 mm. All values represent the mean ± SD (n = 10–12). *P < 0.05; ***P < 0.001. n.s. indicates not significant.

Figure 4.

EPA affects obesity-enhanced STAT3 activation and tumor cell proliferation. (A) Ki67 immunostaining of tumor areas in livers from DEN-injected mice fed SD, HFD or HFD + EPA for 37 weeks. (B) The percentage of Ki67-positive cells per field (n = 7). (C) Tumor liver tissues from DEN-injected mice in each of the three dietary groups were analyzed by immunoblotting with the indicated antibodies. (D) Ratio of PCNA expression to γ-tubulin (n = 5). (E) TUNEL staining of tumor areas in livers from DEN-injected mice fed SD, HFD or HFD + EPA for 37 weeks. (F) The number of TUNEL-positive cells per field (n = 3). (G) Tumor liver tissues from DEN-injected mice in each of the three dietary groups were analyzed by immunoblotting with the indicated antibodies. (H) Ratio of tyrosine phosphorylation of STAT3, ERK and JNK to the total amount of each protein (n = 3–7). The mean relative intensity of the phosphorylation of each protein in SD mice was arbitrarily defined as 1. Scale bars = 50 µm. All values represent the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. n.s. indicates not significant.

Figure 5.

EPA does not affect obesity-linked hepatic inflammation. (A) Mac-2 immunostaining of livers from DEN-injected mice fed SD, HFD or HFD + EPA for 37 weeks. (B) The number of Mac-2-positive cells per field (n = 6). (C) mRNA levels of inflammatory markers in livers from each dietary group were assessed by qRT-PCR (n = 6–8). The mean relative amount of each mRNA in SD mice was arbitrarily defined as 1. (D) The concentrations of IL-6 and TNFα in the plasma of mice from each dietary group were assessed by enzyme-linked immunosorbent assay (n = 7–10). N.D. indicates not detected. Scale bars = 100 µm. All values represent the mean ± SD. *P < 0.05; ***P < 0.001. n.s. indicates not significant.