The combination of Biochanin A and SB590885 potentiates the inhibition of tumour progression in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the most aggressive and frequently diagnosed malignancy of the liver. Despite aggressive therapy, life expectancy of many patients in these cases is extended by only a few months. Hepatocellular carcinoma (HCC) has a particularly poor prognosis and would greatly benefit from more effective therapies.

Methods: The CCK-8 assay and colony formation assays were used to test the cell proliferation and viability. The effects of combination Biochanin A and SB590885 on apoptosis and cell cycle arrest of HCC cells were analysed by flow cytometry. The expression of ERK MAPK and PI3K/AKT/mTOR signalling as well as apoptosis and cell cycle-related proteins in HCC cells were tested by western blotting. The HCC cell xenograft model was established to test the tumor proliferation. Serum and plasma were tested for liver and kidney safety markers (ALP, ALT, AST, total bilirubin, creatinine, urea nitrogen) by using SpectraMax i3X.

Results: The combination of natural product Biochanin A with the BRAF inhibitor SB590885 synergistically suppressed proliferation, and promoted cell cycle arrest and apoptosis in vitro. Furthermore, we demonstrated that the combination of Biochanin A and SB590885 led to increased impairment of proliferation and HCC tumour inhibition through disrupting of the ERK MAPK and the PI3K/AKT pathways in vitro. The volumes tumors and the weights of tumours were significantly reduced by the combination treatment compared to the control or single treatments in vivo. In addition, we found that there was no significant hepatorenal toxicity with the drug combination, as indicated by the hepatorenal toxicity test.

Conclusion: The results identify an effective combination therapy for the most aggressive form of HCC and provide the possibility of therapeutic improvement for patients with advanced HCC.

Keywords: Biochanin A; Combination therapeutic; ERK MAPK pathway; Hepatocellular carcinoma; SB590885.

Fig. 1

Anti-proliferative effects of combination treatments in HCC cells. After treatment with biochanin A (12.5 μM, 25 μM, 50 μM, 75 μM, and 100 μM) and SB590885 (3 μM, 6 μM, 9 μM, 12 μM, and 15 μM) alone or in combination for 48 h, the CCK‑8 assay was used to determine cell viability in the a Bel-7402 cell line and b SK-Hep1 cell line. The results are the mean ± SD of three independent experiments performed in triplicate. **P < 0.001 versus single treatments (one-way ANOVA followed by a Student–Newman–Keuls test). The combination of 75 μM biochanin A and 12 μM SB590885 significantly inhibited colony formation in HCC cell lines c Bel-7402 and d SK-Hep1. The results are the mean ± SD of three independent experiments performed in triplicate. **P < 0.001 versus single treatments (one-way ANOVA followed by a Student–Newman–Keuls test)

Fig. 2

Effect of the combination treatment on HCC cell apoptosis. Synergistic activity of the combination treatment of biochanin A and SB590885 on apoptosis after 48 h of exposure in the HCC cell lines a Bel-7402 and b SK-Hep1 cell line. The results are the mean ± SD of three independent experiments performed in triplicate. **P < 0.001 versus single treatments (one-way ANOVA followed by a Student–Newman–Keuls test). BCL2, BAX, cleaved caspase-9 and cleaved PARP expression was measured in the c Bel-7402 cell line d, and SK-Hep-1 cell line using western blot analysis

Fig. 3

Effect of the combination treatment on HCC cell cycle arrest. Synergistic activity of the combination treatment of biochanin A and SB590885 on cell cycle arrest after 48 h of exposure in HCC cell lines a Bel-7402 and b SK-Hep1. The results are the mean ± SD of three independent experiments performed in triplicate. **P < 0.001 versus single treatments (one-way ANOVA followed by a Student–Newman–Keuls test). The expression of p21, p27, and cyclinD1 in the c Bel-7402 cell line and d SK-Hep1 cell line were tested using western blot analysis

Fig. 4

Effect of the combination treatment on the ERK MAPK pathway in HCCcell lines. The p-ERK, total-ERK, p-MEK and total-MEK levels in the Bel-7402 cell line and SK-Hep1 cell line were measured using western blot analysis. These results are performed with three independent experiments in triplicate

Fig. 5

Effect of combination treatment on the PI3K/AKT/mTOR pathway in HCCcell lines. The p-AKT, total-AKT, p-P70S6K, total-P70S6K, p-S6 and total-S6 in the Bel-7402 cell line and SK-Hep1 cell line were measured using western blot analysis. These are the results of three independent experiments performed in triplicate

Fig. 6

Anti-proliferative effects of the combination treatments in vivo. HCC xenograft model with treated with control (vehicle-treated mice), biochanin A (25 mg/kg/day), SB590885 (7.5 mg/kg/day) and the combination for 5 weeks. The size (a), volume (b) and weight of the tumours (c) were significantly reduced by the combination treatment compared to those of the control or single treatments. No significant differences in body weight were observed at the end of the treatment period (d). Immunohistochemical analyses of the xenograft tumours revealed that the biochanin A and SB590885 combination effectively inhibited the expression of PCNA, a marker of tumour proliferation (e). Western blot analysis of the xenograft tumours revealed that the combination of biochanin A and SB590885 inhibited activation of the ERK MAPK and PI3K/AKT/mTOR signalling pathways in the in vivo xenograft model (f). The results are the mean ± SD of three independent experiments performed in triplicate. **P < 0.001 versus single treatments (one-way ANOVA followed by a Student–Newman–Keuls test)