Research On Combined Sorafenib And Artesunate Against Hepatocellular Carcinoma In Vitro And In Vivo

Hepatocellular carcinoma?HCC?,the most common type of liver cancer,is extremely harmful to humans.Global Cancer Statistics 2018 shows that the incidence of liver cancer accounts for 4.7%of the total number of cancer cases worldwide,and the mortality rate accounts for 8.2%.China is a country with a high incidence of liver cancer in the world,accounting for more than 50%of new cases worldwide.Early liver cancer usually has no obvious symptoms,but there are obvious symptoms of advanced liver cancer,such as weight loss,liver pain,and gastrointestinal symptoms.Many patients have been in the advanced stage and missed the opportunity for surgery at the time of diagnosis.As an oral small molecule kinase inhibitor,sorafenib is the first molecularly targeted drug approved for the treatment of advanced HCC,which has shown remarkable efficacy in clinical treatment.However,the efficacy of sorafenib in the treatment of advanced HCC is still limited.The occurrence of drug resistance and some adverse reactions affects its efficacy and patient tolerance.In order to improve the therapeutic effect and reduce adverse reactions,researches have been conducted on sorafenib based combined therapy in recent years.Artesunate,a derivative of artemisinin,is a clinically safe and effective antimalarial drug.In recent years,studies have revealed that artesunate can inhibit tumor both in vitro and in vivo,and can produce synergistic anti-tumor effect when combined with other chemotherapy drugs.This study investigated the effect of artesunate combined with sorafenib on the proliferation and apoptosis of liver cancer both in vitro and in vivo,and explored the potential mechanismObjective:The aim of this study is to investigate the effect of combined sorafenib and artesunate on the proliferation and apoptosis of HCC cells and explore the underlying mechanisms.Methods:The MTT method was used to evaluate the effects of sorafenib and artesunate alone or in combination on the proliferation of hepatoma cells.The Chou-Talalay combination index analysis was used to evaluate the synergistic effect of the combined use of sorafenib and artesunate on the proliferation of hepatoma cells.Hoechst/PI staining and Caspase 3/7 kit were used to evaluate the effects of sorafenib and artesunate alone or in combination on apoptosis of hepatoma cells.The effect of artesunate on the production of reactive oxygen species?ROS?in hepatoma cells was detected by DCF method.The effects of sorafenib and artesunate alone or in combination on apoptosis protein and cell signaling pathway were detected by Western blot.The effect of combined use of sorafenib and artesunate on the proliferation of hepatoma cells in vivo was evaluated using tumor-bearing nude mice.Results:Artesunate inhibited the proliferation of hepatoma cells HepG2,PLC/PRF/5,HCCLM3,Hep3B and HuH7 in a dose-dependent manner with IC₅₀ values of 37.4?mol/L,121.2?mol/L and 44.2?mol/L,41.6?mol/L,46.4?mol/L,respectively.Sorafenib inhibited the proliferation of HepG2,PLC/PRF/5,HCCLM3,Hep3B and HuH7 cells in a dose-dependent manner with IC₅₀ values of 6.1?mol/L,7.4?mol/L,and14.7?mol/L,3.6?mol/L,2.6?mol/L,respectively.Hoechst/PI staining showed that sorafenib?10?mol/L?combined with different concentrations?10,30,100?mol/L?of artesunate induced enhanced apoptosis in HCC cells HuH7,HepG2,and PLC/PRF/5compared with sorafenib monotherapy.The apoptosis rate increased by 18.4%,44.6%,and 69.5%in HuH7 cells;by 2%,14%,and 48.1%in HepG2 cells;by 5.9%,22.9%,and51.7%in PLC/PRF/5 cells.Caspase3/7 staining showed that the combination of sorafenib and artesunate significantly increased the level of activated caspase3/7 in HuH7,HepG2,and PLC/PRF/5 cells.The results of Western blot showed that the level of mitochondrial Bax/Bcl-2 in HuH7 cells of the combination group was significantly higher than that of the drug alone group.The results of ROS measurement showed that artesunate increased the levels of ROS in HuH7 and Hep3B cells.The addition of antioxidant NAC dose-dependently attenuated the inhibitory effect of artesunate on the proliferation of HUH7 and Hep3B cells,suggesting that artesunate exerts an anti-tumor effect by producing ROS.We explored the mechanism underlying the synergistic anti-tumor effects of sorafenib plus artesunate.Sorafenib is a protein kinase inhibitor that inhibits tumor growth by inhibiting Raf/MEK/ERK and STAT3 signaling pathways.Western blot results showed that sorafenib could decrease the levels of p-ERK and p-STAT3,while artesunate increased the expression levels of p-ERK and p-STAT3.When used in combination,sorafenib decreased the levels of p-ERK and p-STAT3 which were upregulated by artesunate.We speculate that artesunate exerts anti-tumor effects by promoting the production of reactive oxygen in cells,but its activation of p-ERK and p-STAT3 promotes cell survival to a certain extent.When used in combination,sorafenib counteracts its activation of p-ERK and p-STAT3 to produce synergistic anti-tumor effects.To validate this hypothesis,we employed the ERK signaling pathway-specific inhibitor PD0325901 and the STAT3 pathway-specific inhibitor Stattic,and found that it has a significant synergistic effect with artesunate on the proliferation of HCC cells.To further validate the anti-tumor activity of the combination of sorafenib and artesunate,we used a nude mouse xenograft model to observe its tumor growth inhibition.Results showed that artesunate had a tendency to inhibit tumor growth compared with the normal control group,and sorafenib significantly inhibited tumor growth.However,there was no significant difference between the combination group and the sorafenib group.We speculate that this may be related to the low activity and short half-life of artesunate in the body.To this end,we have structurally modified artesunate to obtain a series of artesunate derivatives,aiming to improve its anti-tumor activity.Conclusions:1.Sorafenib could inhibit the proliferation of HCC cells in a dose-dependent manner.2.Artesunate could inhibit the proliferation of HCC cells in a dose-dependent manner,but its activity is weaker than sorafenib.3.The combination of sorafenib and artesunate could synergistically inhibit the proliferation of HCC cells and induce cell apoptosis in vitro.4.The synergistic anti-tumor mechanism by the combined treatment may be related to the inhibition of artesunate on the activation of ERK and STAT3 signaling pathway by sorafenib.5.The low activity and short half-life of artesunate may affect its antitumor activity in vivo.6.Structural modification of artesunate is necessary to find novel potent artesunate derivatives for treatment of HCC.