Inhibitory effects of resveratrol on hepatitis B virus X‐protein (HBx)‐induced hepatocellular carcinoma (HCC)

Liver cancer occurs very frequently worldwide and hepatocellular carcinoma (HCC) accounts for more than 80% of total primary liver cancer cases. Here, the anticarcinogenic effects of resveratrol against hepatitis B virus (HBV)‐induced HCC were investigated using HBV X‐protein‐overexpressing Huh7 (Huh7‐HBx) human hepatoma cells. MTT assay showed that resveratrol decreased cell viability. Fluorescence‐activated cell sorter analysis showed that it induced G1 cell cycle arrest without increasing the sub‐G1 phase cell population. Therefore, we evaluated its effect on regulation of cyclin D1, which is critically involved in G1/S transition. Resveratrol lowered cyclin D1 transcription. Western blot analysis of the effects of resveratrol on upstream cyclin D1 transcriptional signaling, extracellular signal‐related kinase (ERK), p90 RSK , Akt, and p70 S6K revealed inhibition of Akt but not ERK signaling pathways. Collectively, these results indicated that resveratrol inhibits Huh7‐HBx proliferation by decreasing cyclin D1 expression through blockade of Akt signaling. We investigated the anti‐carcinogenic effect and mechanism of resveratrol in xenograft model mice implanted with Huh7‐HBx cells. Intraperitoneal injection of resveratrol reduced the tumor size in the mice. Expression of survivin was reduced, whereas cyclin D1 was not affected. These results demonstrated that treatment with resveratrol might be helpful to manage of HBV‐induced HCC by regulating survivin. Support or Funding Information This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2015R1A1A1A05000990). 1 Protein expression of HBx and survivin in Huh7‐HBx cells(A) HBx protein expression was upregulated in Huh7‐HBx cells compared to Huh7‐CMV cells and HBx transgenic mice were verified by PCR using specific primer sets. (B) Survivin protein expressions were upregulated in Huh7‐HBx cells compared to Huh7‐CMV cells and survivin protein expressions was upregulated in HBx transgenic mice compared to wild‐type mice.2 Effects of resveratrol on cell proliferation and survivin expression in Huh7‐HBx cells(A and B) Resveratrol inhibited cell proliferation of Huh7‐HBx and Huh7‐CMV cells incubated with varying concentrations for 48 h ; ** , p < 0.01 comparing control group at 0 and control group at 48 h. ## , p < 0.01 comparing control and resveratrol‐treated groups at 48 h. (C) Resveratrol decreased survivin protein expression in Huh7‐HBx cells treated with varying concentrations for 24 h.3 Effect of resveratrol on cell cycle progression of Huh7‐HBx cells(A and B) Resveratrol blocked G1‐S transition of Huh7‐HBx cells treated with 100 μM for indicated times as shown by FACS analysis. Graphs are quantification of result of at least 3 independent experiments; * , p < 0.05 and ** , p < 0.01 comparing control and resveratrol‐treated groups.4 Effects of resveratrol on cyclin D1 and survivin expression in Huh7‐HBx cells(A) Resveratrol decreased cyclin D1 and survivin protein expression in Huh7‐HBx cells treated with varying concentrations for 8 h. Western blot analysis was conducted with β‐actin as loading control. (B) Resveratrol decreased cyclin D1 mRNA expression in Huh7‐HBx cells treated with varying concentrations for 4 h. ; * , p < 0.05 comparing control and resveratrol‐treated groups.5 Effects of resveratrol on ERK and Akt signaling pathway in Huh7‐HBx cells(A and B) Resveratrol blocked Akt, but not ERK signaling pathway in Huh7‐HBx cells treated with varying concentrations for 4 h. p‐ERK, phosphorylated extracellular signal‐regulated kinase.6 Inhibitory effects of resveratrol in xenograft model mice implanted with Huh7‐HBx cell in vivo(A) Tumor size curve. The size of tumors were measured from day 1 to day 21 each 3‐day. (B) The tumor weight on 21 days at the end of study. (C) Representative photographs of nude mice bearing Huh7‐HBx cell at the end of study. (D) The Inhibition rate of tumor growth (E) Body weight curve. * , significance at p < 0.05 and ** , p < 0.01, between treated group and vehicle group (one‐way ANOVA)7 Effect of resveratrol in tumor specimens(A) Pathologic representative status of tumor specimens. (B) The protein expression of survivin. Survivin/actin expression ratio was significantly reduced at resveratrol treated groups, compared to vehicle. (C) The protein expression of cyclin D1 was determined by western blot analysis. * , significance at p < 0.05 and ** , p < 0.01, between treated group and vehicle group (Student's t ‐test).