The Underlying Mechanism For Enhencement Of Anti-hepatocellular Carcinoma Activity By Sorafenib In Combination With Capsaicin

Hepatocellular carcinoma(HCC)is characterized with high morbidity and mortality,representing a serious threat to human health.In China,because of the prevalence of hepatitis B virus infection,the morbidity and mortality of liver cancer are particularly severe.The current methods for treating HCC include: surgery and medication.Surgical treatment is only applicable to early and non-metastatic conditions,while the onset of occult cancer,most patients have developed to the middle and advanced stages when the first diagnosis,missed the best period of surgical treatment.For advanced and metastatic HCC,medical treatment is an important option.Sorafenib,a small inhibitor of tyrosine protein kinases,is currently the common first line systemic chemotherapy drug for the treatment of advanced HCC.Although sorafenib plays an irreplaceable role in the comprehensive treatment of advanced liver cancer,it only prolongs the median survival of HCC patients to only about 2.8 months,and requires further improvement for efficacy.So finding compounds that can enhance the anti-tumor activity of sorafenib will help treat liver cancer patients.Capsaicin,the peppery ingredient of chili peppers from the genus Capsicum,displays antitumor activity alone and enhances the sensitivity of cancer cells to cytotoxic drugs.It has been shown that capsaicin inhibits STAT3 phosphorylation and activates the RAF/MEK/ERK pathway in multiple myeloma and prostate cancer cells.Because the activation of the RAF/MEK/ERK pathway is positively correlated with the sensitivity of HCC cells to sorafenib.We in this study investigated the synergistic antitumor effect and potential mechanism of combined sorafenib and capsaicin on HCC cells and xenograft tumors.Objective This study was to study the synergistic inhibition of HCC cells and xenograft tumor activity in combination of sorafenib and capsaicin,and to explore the underlying mechanism.Methods The inhibitory effects of sorafenib,capsaicin,and their combination on the proliferation of HCC cells was evaluated using the MTT method.The Chou-Talalay combination index analysis was used to study the synergistic inhibitory effect of combination sorafenib and capsaicin on proliferation of HCC cells.Hoechst 33258 and PI staining and Caspase 3/7 activation assay were used to evaluate the effects of sorafenib,capsaicin,and their combination on the apoptosis of PLC/PRF/5 cells.Detection of expression of cell signaling pathways and apoptosis-related proteins in HCC cells were determined by Western blot.The effects of sorafenib in combination with capsaicin on the growth of xenograft tumors were studied using sorafenib and capsaicin in vivo.Results Capsaicin alone dose-dependently inhibited the proliferation of the HCC cell lines such as PLC/PRF/5,HuH7,and HepG2,with IC50 values of 137.0,108.0,and 140.7 μmol/L,respectively.Sorafenib alone also exerted dose-dependent inhibition of the proliferation of HCC lines including PLC/PRF/5,HuH7,and HepG2 with IC50 of 7.6 μmol/L,6.4 μmol/L,and 5.9 μmol/L,respectively.Combination sorafenib and capsaicin gave rise to a better synergistic inhibition on proliferation of HCC cells such as PLC/PRF/5,HuH7 and HepG2 at a relatively high concentration(3,10,30 μmol/L)of sorafenib.Hoechst 33258 and PI staining results showed that the apoptosis rate of PLC/PRF/5 cells increased by 13.37% compared with 5 μmol/L sorafenib in combination with 100 μmol/L capsaicin and 5 μmol/L sorafenib alone;Hoechst 33258 and PI staining results showed that the apoptosis rate of PLC/PRF/5 cells increased by 30.58% compared with 10 μmol/L sorafenib in combination with 100 μmol/L capsaicin and 10 μmol/L sorafenib alone.The results of the Caspase 3/7 activation experiment demonstrated that the activation level of Caspase 3/7 in PLC/PRF/5 cells was significantly higher in the combination of sorafenib and capsaicin than in the sorafenib alone or capsaicin alone group.Western Blot results showed that the Bax/Bcl-2 ratio was significantly increased in PLC/PRF/5 cells exposed to their combination compared to those exposed to capsaicin alone or sorafenib alone.The results from in vivo test in nude mice showed that tumor growth inhibition(TGI)of capsaicin(intratumor injection,200 μmol/L,100 μL)and sorafenib(oral administration,50 mg/Kg)was 52.2% and 58.9%,respectively.In contrast,combining sorafenib(oral administration,50 mg/Kg)with capsaicin(intratumor injection,200 μmol/L,100 μL)gave rise to a greater suppression of tumor growth with a TGI of approximately 83.3%.Mechanistically,Transient receptor potential vanilloid 1(TRPV1)is a well-known receptor of capsaicin,but western blot results showed that there is no obvious TRPV1 expression on PLC/PRF/5,HuH7 and HepG2 cells,suggesting that inhibition of cell growth by capsaicin is independent of TRPV1 channel.N-acetyl-L-cysteine(NAC),a membrane-penetrating antioxidant that reduces ROS levels,could dose-dependently alleviate the cytotoxic effect of capsaicin on cancer cells.In terms of cell signaling pathway,capsaicin decreased the level of p-STAT3 and increased the level of phosphorylated ERK(p-ERK)in a dose-dependent manner,thus we speculate that the inhibitory effect of capsaicin on STAT3 signaling pathway is related to its anti-tumor effect,but its activation of ERK signaling pathway promotes cell survival,offsetting part of its anti-tumor activity.To verify this hypothesis,we examined the effect of capsaicin in combination with PD032590,a selective inhibitor of ERK phosphorylation.The results showed that PD032590 can enhence the inhibitory effects of capsaicin on proliferation of PLC/PRF/5 cells,and this combination achieved synergism in suppressing the growth of PLC/ PRF/5 cells.We further examined the effects of sorafenib on STAT3 and ERK signaling pathways and found that sorafenib decreased the expression of p-ERK and phosphorylated STAT3(p-STAT3)in PLC/PRF/5 cells in a dose-dependent manner.Combining capsaicin and sorafenib can both inhibit the expression of p-STAT3 and reduce the expression of p-ERK.Collectively,these results suggest that the activation of ERK by capsaicin,in contrast to the attenuation of ERK activation by sorafenib,and the co-suppression of STAT3 by the two agents are likely responsible for the synergistic inhibitory effect of the combined treatment on HCC cell growth.Conclusions1.Sorafenib can inhibit proliferation of HCC cells in a dose-dependent manner.2.Capsaicin can inhibit proliferation of HCC cells in a dose-dependent manner.3.Combining sorafenib and capsaicin exhibits synergistic suppression of cell proliferation and induces cell apoptosis in HCC cells.4.Combination of sorafenib with capsaicin can achieve a robust synergistic antitumor effect in nude mice.5.The inhibition of cell growth by capsaicin is independent of TRPV1 channel activation.6.The activation of ERK by capsaicin and the attenuation of ERK activation by sorafenib,and the co-suppression of STAT3 by the two agents are likely responsible for the synergistic inhibitory effect of the combined treatment on HCC.7.Capsaicin may be a useful strategy for improvement of sorafenib efficacy in suppression of HCC.