| BackgroundHepatocellular carcinoma(HCC),is the fifth most common cancer in the world and is responsible for＞6000,000 deaths annual.Patients with HCC die withinl year after the diagnosis were conformed.Unfortunately,the disease is often diagnosed at a late stage when curative therapies are least effective.For these patients, medical treatments,including chemotherapy,chemoembolization,ablation,and proton beam therapy,remain disappointing.There is an urgent need for new therapies for this aggressive disease.Both angiogenesis and signaling through the Raf/mitogen-avtivated protein (MAP)/extracellular-signal-regulate-kinase(ERK)-kinase(MEK)/ERK(RAF/MEK/ER K)cascade play critical roles in the development of HCC.Activation of Ras signaling pathways is an important mechanism by which human cancers develop.Raf-1 kinases are serine/threonine protein kinases that function in this path way as downstream effectual molecules of Ras.Raf-1 can transmit the signal to ERK1/ERK2 through MEK,initiates a mitogenic kinase cascade that ultimately modulates gene expression via the phosphorylation of transcription factors,which can have profound effects on cellular proliferation and anti-apoptosis.Recent evidence suggests that Raf-1 can prolong the life of cell through the action with apoptosis protein and anti-apoptosis protein.Studies have found Raf kinase is over-expressed in a high percentage of HCC patient tumors,and the RAF/MEK/ERK pathway can be activated by major etiologic factors such as HBV and HCV infection and mitogenic grown factors.On the other hand,anti-angiogenesis therapies,which inhibit blood vessel formation,may have treatment effect of HCC tumors depend on a rich blood supply.Vascular-endothelial growth factors(VEGF) are major factors for cancer angiogenesis.Professor Li's study found mRNA of VEGF is over-expressed in tumors of HCC patients through RT-PCR.Therefore,inhibition of both angiogenesis and RAF/MEK/ERK signaling may represent an attractive approach for the treatment of HCC.One the other hand, studies found inhibition of apoptosis was important for HCC development.So inducing apoptosis may be a good treatment for HCC.Sorafenib is a multikinase inhibitor that has shown to block proliferation of tumor cells and angiogenesis by inhibiting serine/threonine kinases(c-Raf mutant and wild-type B-Raf),as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2(VEGFR2),VEGFR3,platelet-derived growth factor receptor (PDGFR),FLT3 and C-KIT.A report by Wilhelm et al showed that Sorafenib exhibited the RAF/MEK/ERK pathway and VEGFR2 and PDGFRβ-PTKs phosphorylation which were acted by VEGF and PDGFRβin the cancer cells with mutation of k-ras and/or b-raf.Li Liu et al also found Sorafenib inhibited RAF/MEK/ERK pathway and the phosphorylation of eIF4E and down-regulated antiapoptotic protein Mcl-1,induced tumor cell apoptosis in PLC/PRF/5 xenografts. The phaseⅡandⅢclinical studies(SHARP trial) reported that Sorafenib improved overall survival time in 44%HCC patients.Sorafenib was the first drug improving overall survival time of the HCC patients.Arsenic trioxide(As2O3) has shown remarkable curative effect inducing tumor cell apoptosis in HCC.On the other hand,Hui-Ying Cheng,Hai-qing Hua et al also found As2O3 could inhibit the vascularization and electron microscopy showed that As2O3 could damage primitive mesenchymal cells and vascular endothelial cells and inhibit neovascular formation.It is considered that As2O3 can inhibit the tumor neovascularization.As Sorafenib and As2O3 has different mechanisms of inducing tumor cell apoptosis,inhibiting tumor cell proliferation and inhibiting angiogenesis,it is possible that using two drugs to treat HCC through different ways and targets.This study is designed for using two drugs in vitro and in vivo to confirm that they have synergistic effect for the inhibition of HCC.It also can supply evidence for the clinical study.ObjectiveTo investigate the inhibitory effect and molecular mechanisms of sorafenib combined with Arsenic trioxide(As2O3) on hepatocellular carcinoma cells and neoplasms.Methods1.HepG2(p53 wild type) human HCC tumor cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group,As2O3-treated group and combination treatment group.MTT assay included cells treated with Sorafenib(1.5,3μmol/L),As2O3(2,4μmol/L) alone and combination(1.5+2and 3+4μmol/L) for 24,48 and 72 hours respectively.In other tests cells were treated with Sorafenib(3μmol/L) and As2O3(4μmol/L) alone and combination.2.Groups 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. Interaction between Sorafenib and As2O3 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.3.Groups as the above mentioned,collected cells after 48h and analyzed cell cycle and apoptosis by flow cytometry.4.Groups as the above mentioned,collected cells after 48h and analyzed the changes of mitochondrial membrane potential(Δψm) labeled by Rhodamine123 and examined by flow cytometry.5.Groups as the above mentioned,collected cells after 48h and the proteins were extracted from cells.Western Blot was performed for protein expression of Bcl-2,Mcl-1,pERK 1/2,ERK1/2.6.Construct nude mouse tumor animal model.The nude mouse with HepG2 cell xenografts were randomized into control group,As2O3-treated group, Sorafenib-treated group and combination-treated group.Every group had 10 mice. Evaluate volume of tumor in four groups before and after treatments.Execute the nude mouse at the twenty-three day after treatments,to strip and weigh the neoplasm for evaluating the inhibition of the anti-tumor.Determine tumor tissue VEGF protein expressions and micro-vessel density(MVD,assessed by CD34 immunohistochemistry).7.Statistical analysis:All data were analysed by SPSS 13.0 statistical software.Data were expressed as mean±S.D.Two independent samples were analyzed by T test. The others data were analyzed by One-way ANOVA followed by LSD multiple comparison tests.Rank data were analyzed by 2 Independent Samples Test.P-values were considered to be significant at＜0.05.Results1.The MTT result indicated:Combination of Sorafenib and As2O3 enhanced inhibition of proliferation in HepG2 cells(P＜0.05).They have synergistic effect for the inhibition of HepG2 cells(q＞1.15) except Sorafenib(3μmol/L) together with As2O3(4μmol/L) showed additive effect at 24h(q=1.121).When Sorafenib(3μmol/L) together with As2O3(4μmol/L) effected at 48h,q-value was the biggest(p=1.416). 2 Sorafenib and As2O3 alone and together affected cell cycle of HepG2 cells. Sorafenib alone can induce accumulation of cells in S phase compared with untreated control group(P=0.005).As2O3 alone can induce accumulation of cells in G2/M phase compared with untreated control group(P=0.000).Combinaton group showed greater increase of S and G2/M phases than control group(P-value was 0.001and 0.015 individually).3 Sorafenib and As2O3 alone induced more HepG2 cells apoptosis compared with untreated control(P-value was 0.000and 0.041 individually).Cells apoptosis was increased induced by combinaton of Sorafenib with As2O3 compared with each agent (P-value was 0.001 and 0.000 individually).4 The mitochondrial membrane potential was down-regulated in As2O3,Sorafenib or combination group compared with untreated control group(P＜0.05).The affection by the combination was stronger than that alone(P＜0.05).5.There was no difference of the expression of ERK among four groups.The decrease of pERK expression was seen after treated with sorafenib and As2O3 alone and the combination,and the latter was more obvious.Sorafenib alone and in combination with As2O3 could decrease Mcl-1 expressions,while As2O3 alone and in combination with Sorafenib can decrease Bcl-2 expressions and the one of combination was more obvious. 6.The tumor volume and weight of Sorafenib and As2O3 alone groups were lower than the control groups after treatments(P＜0.05).The group of combination was the lowest among the four groups(P＜0.05).7.The immunohistochemistry results showed:the MVD were decreased in the three groups compared with control group(P=0.000).The one of combination was more obvious compared with Sorafenib and As2O3 alone groups(P=0.000).The expressions of VEGF were obviously decreased in Sorafenib and As2O3 alone and in combination groups(P＜0.05),the one of combination was more obvious compared with Sorafenib and As2O3 alone groups(P＜0.05).ConclusionSorafenib and As2O3 alone or combination induced cell cycle arrest.Sorafenib combined with As2O3 showed synergistic effect on inhibition proliferation of cells and induction apoptosis and they had a synergistic effect.The mechanisms of synergistic effect were closely related to the blockage of cell-cycle progress and the promotion of apoptosis as well as affecting signaling pathway.Sorafenib and As2O3 affected different targets or the same target in deeper degree resulting in enhancing the anti-tumor effect.The inhibition mechanisms were also attributed to inhibition of tumor angiogenesis in vivo.
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