Inhibitory Effect Of Sorafenib In Combined With Cisplatin On Hepatocellular Carcinoma

Background and objectionHepatocellular carcinoma(HCC) is the fifth most common cancer in the world and is responsible for＞600,000 deaths annually.The majority of patients with HCC die within 1 year after the diagnosis of their disease.Unfortunately,the disease is often diagnosed at a late stage when potentially curative therapies are least effective. For these patients,medical treatments,including chemotherapy,chemoembolization, ablation,radiotherapy,remain disappointing.Most patients show disease recurrence and their 5-year relative survival rate is less than 5%.The prognosis for HCC patients who have surgically resectable localized tumors is better,but they still have only a 15%to 39%5-year survival rate.Clearly,there is an urgent need for new therapies for this aggressive disease.With the development of the molecular biology of carcinogenesis and tumor progression of HCC,the management of advanced HCC is entering the era of molecular targeting therapy,which is of particular significance for HCC in view of the lack of existing effective systemic therapy for this cancer.Effective agents targeting these molecular abnormalities,such as Raf kinase inhibitors,have been developed and widely used.Several agents have entered clinical trials in HCC patients,and recent data indicated that a multikinase inhibitor targeting Ras kinase and VEGFR-2,sorafenib(Nexavar,BAY 43-9006) is effective in prolonging survival of patients with advanced HCC.Sorafenib is an orally available multikinase inhibitor that targets kinases of wild-type B-Raf,mutant B-Raf and C-Raf thus blocking tumor growth.Furthermore, sorafenib shows potent inhibition of receptor tyrosine kinases(RTKs) involved in angiogenesis,including human vascular endothelial growth factor receptors-2 and -3 (VEGFR-2/-3),the platelet derived growth factor receptor-b(PDGFR-b),FLT3,Ret, and c-KIT.Sorafenib has demonstrated preclinical and clinical activity against several tumor types.At the recent Annual Meeting of the American Society of Clinical Oncology,results of a phaseâ…¢,randomized,placebo-controlled trial were presented in which sorafenib demonstrated improved survival in patients with advanced HCC.This landmark study represents the first agent that has demonstrated an improved overall survival benefit in this disease and sets the new standard for first-line treatment of advanced HCC.FDA has approved sorafenib for patients with inoperable liver cancer in 2007.Cisplatin(DDP) is a DNA-damaging agent that is widely used in cancer chemotherapy.DDP cross-links to DNA,forming intra- and inter-strand adducts, which bend and unwind the duplex and attract high-mobility-group domain and other proteins.Systematic chemotherapies are least effective in HCC,so we try to combine molecular targeting therapy with cytotoxic agents,DDP,to improve efficacy. Biochemotherapy,a new synthesis therapy mode,is the use of biotherapy in conjunction with chemotherapy.Rationally combining biotherapy with cytotoxic agents may be more effective at extending overall survival and quality of life for patients.Our study aimed at further improving efficacy of sorafenib by combination with DDP in HCC and providing preclinical evidence for biochemotherapy of HCC.Partâ… :Coadministration of Sorafenib with DDP inhibits cell proliferation in hepatocellular carcinoma cells HepG2Methods1.HepG2(p53 wild type) human HCC tumor cells were cultured in RPMI 1640 containing 10%fetal calf serum and subcultured every 3 to 5 days.Exponentially growing cells were chosen for experiment. 2.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group, DDP-treated group and combination treatment group.MTT assay included cells treated with Sorafenib(2,4,8,16μmol/L),DDP(3,6,12,24μmol/L) alone and combination(2 + 3,4 + 6,8 + 12μmol/L) for 24,48 and 72 hours respectively.Cell cycle,apoptosis and Western-blot analysis included cells treated with Sorafenib (4μmol/L) and DDP(6μmol/L) alone and combination.3.Group as the above mentioned,the inhibitory of HepG2 cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value.4.Interaction between Sorafenib and DDP was assessed using the q value,where q＞1.15,0.85≤q≤1.15,and q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.5.Group as the above mentioned,collected cells and analyzed cell cycle and apoptosis by flow cytometry.6.Group as the above mentioned,expression of ERK,pERK,Cyclin D1 and p21^WAF1/CXP1 were detected by Western-blot.7.Statistical analysis:Unless otherwise stated,data were expressed as(x±s).The inhibitory rate was analyzed by Repeated Measure followed by LSD multiple comparison test.Data of cell cycle and apoptosis were analyzed by One-way ANOVA followed by LSD multiple comparison test.P-Values were considered to be significant at≤0.05.Results1.Sorafenib and DDP inhibit proliferation of HepG2 cells aloneSorafenib and DDP alone inhibited cell proliferation time- and dose-dependently (P＜0.001).2.Combination of Sorafenib and DDP enhanced inhibition of proliferation in HepG2 cellsInhibitary rate was different among Sorafenib and DDP alone and combination at three concentration level respectively(P＜0.001).Combination group showed better inhibitory effect than single agent group(P＜0.001).3.q value of Sorafenib combined with DDP Coadministration of Sorafenib with DDP showed additive effect of all concentration levels at 72h(0.85≤q≤1.15).Only Sorafenib(4μmol/L) together with DDP(8μmol/L) showed additive effect at 24,48,and 72h(0.85≤q≤1.15).4.Sorafenib and DDP alone and together affected cell cycle of HepG2 cellsSorafenib and DDP alone can induced accumulation of cells in G₁ phase compared with untreated control(P=0.002,0.032).Accordingly,the number of cells in the S-phase decreased(P=0.004,0.022).Combinaton group showed greater increase of G₁(P=0.025,0.002) and decrease of S-phase than single agent group(P=0.036, 0.006).5.Sorafenib and DDP alone and together induced HepG2 cells apoptosisCells apoptosis was detected after treated with Sorafenib and DDP alone(P=0.006, 0.036).Coadministration of Sorafenib with DDP increased cells apoptosis compared with each agent individually(P=0.007,0.001).6.Sorafenib and DDP alone and together affected the expression of ERK and pERKThe expression of ERK showed no difference in all groups.The decrease of pERK expression were seen after treated with sorafenib alone and in combination with DDP for 24h(P＜0.001),and the latter was more obvious.7.Sorafenib and DDP alone and together affected the expression of Cyclin D1 p21^WAF1/CIP1The expression of Cyclin D1 in Sorafenib-treated group and combination group were lower than that in DDP-treated group and control group(P≤0.001).The increase of p21^WAF1/CIP1 were seen in DDP-treated group and combination group(P=0.001), the Sorafenib-treated group and control group showed lower expression of p21^WAF1/CIP1Conclusion1.Sorafenib and DDP alone inhibited cell proliferation time- and dose-dependently.2.Coadministration of Sorafenib with DDP showed additive inhibitory effect.3.Sorafenib and DDP alone or combination induced cell cycle arrest and potentiated apoptosis.4.Sorafenib and DDP induced cell cycle arrest by down regulating the expression of cyclin D1 through RAF/MEK/ERK signal pathway and up regulating the expression of p21^WAF1/CIP1 through p53 pathway respectively,Sorafenib and DDP showed no interaction of cyclin D1 and p21^WAF1/CIP1 expression in combination group, promoted cell cycle arrested and contributed to more cells apoptosis and enhanced antitumor effect.Partâ…¡:Efficacy of Sorafenib combined with DDP against HepG2 hepatocellular carcinoma tumor xenografts.Methods1.Establish HepG2 HCC xenograft models:HepG2 tumors were generated by harvesting cells from mid-log phase cultures.A volume of 0.2 ml(5×10⁶ cells) of the cell suspension was injected subcutaneously into the right flank of each mouse.2.HepG2 HCC xenograft mice(tumors averaging from 130 to 150 mg) were divided into 5 groups:untreated control group,vehicle control group, Sorafenib-treated group,DDP-treated group and combination treatment group,with 10 mice per group.Xenograft mice were killed in day 21 after the treatment of first day.3.Administration:Sorafenib was administered po on a qd×14 schedule at 30mg/kg in the morning of each day.DDP was administered ip on a q4d×3 dose of 4 mg/kg alone or in combination with sorafenib on the same schedule of single agent.DDP was administered in the afternoon approximately 3-5 h after sorafenib.4.Tumor morphous were observed.Tumor dimensions and body weights were recorded twice weekly starting with the first day of treatment.Tumor weights were calculated using the equation(1×w²)/2,where 1 and w represent the largest and smallest dimensions collected at each measurement.5.Evaluated the efficacy and toxicity:Anti-tumor efficacy was evaluated by the incidence of durable complete regressions(CR),partial regressions(PR),and tumor growth inhibition(TGI).Treatments resulting in greater than 20%lethality and/or 20%net body weight loss were considered 'toxic'. 6.Tumor tissues were HE dyed and were viewed under microscope.7.Statistical analysis:Unless otherwise stated,data were expressed as(x±s).The tumor weights were analyzed by Repeated Measure followed by LSD multiple comparison test.P-Values were considered to be significant at≤0.05.Results1.Tumor morphous showed the growth of tumor in combination group was slower than that in other groups.At 21st day after treatment,tumor tissues showed the smallest in the combination group.Discissioed the tumor tissues,and less vessels were seen in Sorafenib-treated group and combination group.2.Sorafenib and DDP alone were efficacious against HepG2 hepatocellular carcinoma tumor xenografts.The efficacy of combination was significantly better than Sorafenib or DDP administered alone(P=0.003,P＜0.001).3.All treatments resulted in less than 20%lethality and/or 20%net body weight loss.1/10 mice died during the treatment period in the vehicle group,DDP-treated group and combination treatment group respectively.2/10 mice died in the Sorafenib-treated group.CR was not observed.1/10 mice achieved PR in Sorafenib-treated group and combination treatment group respectively.TGI of Sorafenib-treated group and DDP-treated group were 46.43%and 38.45% respectively,and combination group(60.42%) was higher than single treatment group.4.HE dyeing showed more necrotic tissue,pyknosis,unclear structure of nuclear, caryoclasis in tumor tissues of combination group than that of other groups.ConclusionSorafenib and DDP alone were efficacious and tolerated in HepG2 hepatocellular carcinoma tumor xenografts.Coadministration of Sorafenib with DDP showed better efficacy than single treatment and well tolerated.