J Vet Sci.  2017 Dec;18(4):419-429. 10.4142/jvs.2017.18.4.419.

Inhibitory effects of resveratrol on hepatitis B virus X protein-induced hepatocellular carcinoma

• Park S
• Lim J
• Kim JR
• Cho S

• KMID: 2398500
• DOI: http://doi.org/10.4142/jvs.2017.18.4.419

Abstract

Liver cancer occurs very frequently worldwide and hepatocellular carcinoma (HCC) accounts for more than 80% of total primary liver cancer cases. In this study, the anticarcinogenic effects of resveratrol against hepatitis B virus (HBV)-induced HCC were investigated by 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 resveratrol 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 the ERK signaling pathway. Collectively, the results indicate that resveratrol inhibits Huh7-HBx proliferation by decreasing cyclin D1 expression through blockade of Akt signaling. We investigated the anticarcinogenic effect and mechanism of resveratrol in xenograft model mice implanted with Huh7-HBx cells. Intraperitoneal resveratrol injection reduced tumor size in the mice. Expression of survivin was reduced, but cyclin D1 was not affected. The results demonstrate that resveratrol treatment may help manage HBV-induced HCC by regulating survivin.

Keyword

Akt pathway; hepatitis B virus X protein; hepatocellular carcinoma; resveratrol; survivin

MeSH Terms

Animals
Anticarcinogenic Agents
Blotting, Western
Carcinoma, Hepatocellular*
Cell Survival
Cyclin D1
G1 Phase Cell Cycle Checkpoints
Hepatitis B virus*
Hepatitis B*
Hepatitis*
Heterografts
Humans
Liver Neoplasms
Mice
Phosphotransferases
Ribosomal Protein S6 Kinases, 90-kDa
Anticarcinogenic Agents
Cyclin D1
Phosphotransferases
Ribosomal Protein S6 Kinases, 90-kDa

Figure

• 

  Fig. 1 Protein expression of hepatitis B virus X protein (HBx) and survivin in HBx-overexpressing Huh7 (Huh7-HBx) cells. (A) HBx expression was detected in Huh7-HBx cells but not in cytomegalovirus (CMV)-expressing Huh7 (Huh7-CMV) cells. HBx transgenic mice were verified by polymerase chain reaction using specific primer sets. (B) Survivin protein expressions were upregulated in Huh7-HBx cells compared to that in Huh7-CMV cells, and survivin protein expression was upregulated in HBx transgenic mice compared to that in wild-type mice.

• 

  Fig. 2 Effects of resveratrol on cell proliferation and survivin expression in hepatitis B virus X protein-overexpressing Huh7 (Huh7-HBx) cells. Resveratrol inhibited cell proliferation of Huh7-HBx (A) and cytomegalovirus-expressing Huh7 (Huh7-CMV) cells (B) incubated with varying concentrations for 48 h. Results are mean ± SD of at least 3 independent experiments; **p < 0.01 comparing control group at 0 and control group at 48 h and ††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. Graphs include quantification of results of at least 3 independent experiments; *p < 0.05 comparing resveratrol-treated and control groups.

• 

  Fig. 3 Effect of resveratrol on cell cycle progression in hepatitis B virus X protein-overexpressing Huh7 (Huh7-HBx) cells. Resveratrol blocked G1-S transition of Huh7-HBx cells when treated with 100 µM resveratrol for indicated times (A), as shown by FACS analysis (B). Graphs include quantification of results of at least 3 independent experiments; *p < 0.05 and **p < 0.01 comparing control and resveratrol-treated groups.

• 

  Fig. 4 Effects of resveratrol on cyclin D1 and survivin expression in hepatitis B virus X protein-overexpressing Huh7 (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 the loading control. Results represent at least 3 experiments that produced similar results. (B) Resveratrol decreased cyclin D1 mRNA expression in Huh7-HBx cells treated with varying concentrations for 4 h. Graphs include quantification of results of at least 3 independent experiments; *p < 0.05 comparing control and resveratrol-treated groups.

• 

  Fig. 5 Effects of resveratrol on ERK and Akt signaling pathway in hepatitis B virus X protein-overexpressing Huh7 (Huh7-HBx) cells. Resveratrol blocked Akt (A) but not the ERK signaling pathway (B) in Huh7-HBx cells treated with varying concentrations for 4 h. p-ERK, phosphorylated extracellular signal-regulated kinase.

• 

  Fig. 6 The entire schedule of the in vivo study. Tumor cells were injected subcutaneously into the right flank of each nude mouse. When the tumors' size progress approximated 100 mm3 (at 21 days), resveratrol was injected intraperitoneally daily for 3 weeks. At 21 days after treatment, the experiments were ended and the mice sacrificed. The transplanted tumors were sampled and weighed.

• 

  Fig. 7 Inhibitory effects of resveratrol in xenograft model mice implanted with hepatitis B virus X protein-overexpressing Huh7 (Huh7-HBx) cell in vivo. Huh7-HBx cells (5 × 106/0.2 mL) were inoculated into the right upper flank of BALB/c (nu/nu) mice subcutaneously. Then, the mice were randomly allocated to vehicle-treated group and two treatment groups and sacrificed at 21 days after inoculation. (A) Tumor size curve. Tumor size was measured every 3 days from day 1 to day 21. (B) Tumor weight at 21 days, the end of the study. (C) Representative photographs of nude mice bearing Huh7-HBx cells at the end of the study. (D) Inhibition of tumor growth was calculated by using the formula: inhibition rate (%) = (tumor weight of vehicle group − tumor weight of treated group)/tumor weight of vehicle group × 100%. (E) Body weight curve. Results were expressed as the mean ± SD (n = 3). Significance at *p < 0.05 and **p < 0.01 between resveratrol and vehicle-treated groups. One-way ANOVA was used for analysis with post-hoc comparisons performed by using Tukey's HSD.

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  Fig. 8 Effect of resveratrol in tumor specimens. (A) Pathologic status of representative tumor specimens. Tumor specimens were taken from the xenograft model mice, embedded in paraffin and stained with hematoxylin & eosin (H&E). Expression of survivin was examined by IHC staining in the tumor tissues of vehicle- and 100 mg/kg resveratrol-treated groups. Resveratrol (100 mg/kg treated group) reduced tumor development and intensity of staining of survivin. (B) Protein expression of survivin. Xenograft model mice implanted with Huh7-HBx cells were treated with 0, 50, and 100 mg/kg resveratrol. Survivin:actin ratio was decreased in both resveratrol-treated groups. Survivin:actin expression ratio was significantly reduced in the 50 mg/kg and 100 mg/kg treated groups (0.77 ± 0.06, p < 0.05 and 0.60 ± 0.03, p < 0.01, respectively), compared to the vehicle-treated group (1.0 ± 0.14). (C) Protein expression of Cyclin D1. Cyclin D1:actin ratio did not show any significant changes. Results were expressed as the mean ± SD (n = 3). Significance at *p < 0.05 and **p < 0.01 between resveratrol- and vehicle-treated groups (Student's t-test). 400× (A).

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