In Vitro Study Of SORAFENIB Influencing Prostatic Carcinoma DU145 Cell

Background Prostatic carcinoma(PC)was most frequent malignnant tumor in western counties,morbility of PC situated the second place and fatality situated the first place in maleness malignant tumors in Western Europe.Morbility of PC in our country was obviously lower than morbility of PC in western counties,but for the past few years, detection rate and morbility of PC zoomed in our country.Morbility of PC situated the third,even second place in urinary system tumor in our country.More than half patients with PC were already partly progressive and metastatic PC,,and missed opportunity of radical cure,only adopted endocrine combined therapy,in which blocking androgen was most important method.Moreover tumor cell of majority patients were to change to androgen independent prostatic carcinoma(AIPC)cell,which response rate to chemotherapy was low.In a word,now ensemble curative effect of patient with AIPC is disaffect.Research mechanism about androgen dependent prostatic carcinoma(ADPC)transformed AIPC and treatment of AIPC were focus.Sorafenib(Nexavar,Bayer-Onyx, BAY 43-9006)is an oral,micromoleecule diplo-aryl Urea.Sorafenib was known as micromolecule kinase inhibitor that initially developed as Raf kinase.Subsequent laboratory characterization of sorafenib demonstrated that it was a multikinase inhibitor of several other targets including vascular endothelial growth factor receptor-2(VEGFR-2), VEGFR-3,and platelet derived growth factor receptor-beta(PDGFR-b), and depressed expression of RET,Mcl and BCL-2,degraded phosphorylation level of eIF4E.Sorafenib would repress tumor cell proliferation and induce tumor cell apoptosis in vitro and vivo research about renal carcinoma,melanoma,pulmonary carcinoma,hepatoma. Sorafenib.as Raf kinase and angiogenesis inhibitor,Sorafenib is regarded as neotype anticancer drug without drug resistance,it can reverse or partly reverse multidrug resistance(MDR)of tumor cell,which is mediated by p-glycoprotein(p-gp).Current study show that ADPC transform AIPC and AIPC low response rate to chemotherapy are significance relation with MDR of PC.In this test,we observed that sorafenib effected DU145(AIPC)cell proliferation and apoptosis in vitro by application light microscope, electron microscope,flow cytometry(FCM)and methyl-thiazolyl tetrazolium(MTT);detected expression of key proteinum relation with DU145 cell proliferation and apoptosis by Western-blot to investigate mechanisms of proliferation and apoptosis;observed DU145/ADM cell proliferation by MTT,respectively detected expression of MDR gene 1 and p-gp by reverse-transcription PCR(RT-PCR)and Western-blot,. Evaluated Sorafenib.to reverse effect of MDR.PartⅠstudy of Sorafenib inhibiting DU145 cell proliferation and inducing apoptosisObjective To survey influence which sorafenib contribute AIPC DU145 cell proliferation,cell cycle and cell apoptosis.Method After DU145 was treated by different concentration sorafenib,we observed change of cell morphology and ultrastructural organization by microscope and transmission electron microscope, viewed cell proliferation by MTT,detected variation of cell cycle and cell apoptosis by FCM.Result 0.5,1,2,5,10,15,20μmol/L concention sorafenib inhibited DU145 cell growth for 24h,48h,72h,except 0.5μmol/L group for 24h did not show significant deviation with control group(P =0.64),other groups showed significant deviation(P＜0.05),and showed time and dosage symbiosis(P＜0.05).Sorafenib could evoke typical apoptosis in DU145 cell,which including cell volume deflating,karyopyknosis,nuclear spallation,nuclear fragmentation, apoptotic body formation.After DU145 cell were treated by 0,2,5,7,10,15 umol/L sorafenib for 48 hours,G1 cell population of DU145 cell were respectively 45.4%,51.7%,55.0%,63.7%,67.0%,74.7%, and apoptosis rate 0.28%,7.6%,11%,18.5%,23.2%,28.4%.Conclusion Sorafenib could inhibit AIPC DU145 cell proliferation,and showed time and dosage dependent.Sorafenib could block DU145 cell cycle in G1 stage,induced DU145 cell apoptosia,and showed dose-dependent.PartⅡMechanism of sorafenib inhibiting DU145 cell proliferation and inducing apoptosisObjective To research mechanism of sorafenib inhibiting AIPC DU145 cell proliferation,blocking cell cycle and inducing apoptosis.Method After 5umol/L,10umol/L,15umol/L sorafenib respectivly treated DU145 for 2 hours and 24 hours,we assaied expression of t-ERK,p-ERK,t-AKT,p-AKT,PTEN,BCL-2,cyclinD1 proteinum in DU145 cell by Western-blot.Result After sorafenib treated DU145 cell for 2 hours,expression of t-ERK,t-AKT,p-AKT was not different with control group,but phosphorylation level of p-ERK gradually degraded along with effective dosage of sorafenib increasing.After sorafenib treated DU145 cell for 24 hours,expression of t-ERK,t-AKT in DU145 cell did not changed compare control group,but phosphorylation level of p-ERK,p-AKT gradually cut down,expression of BCL-2,cyclinD1 proteinum tapered, expression of PTEN increased gradually,with effective dosage of sorafenib increasing.Conclusion Mechanism of action what sorafenib inhibited AIPC DU145 cell proliferation,blocked cell cycle and induce apoptosis was because that phosphorylation level of p-ERK,p-AKT cut down, expression of BCL-2,cyclinD1 proteinum degressed,expression of PTEN increased for sorafenib effection.PartⅢResearching sorafenib influencing chemotherapy sensibility that adriamycin treat DU145/ADM cellObjective To research sorafenib influencing chemotherapy sensibility that adriamycin treat DU145/ADM cell.Method We surveied effection that sorafenib and adriamycin (ADM)simultaneously were applied to inhibit DU145 cell proliferation we evaluated sorafenib reversed MDR of DU145/ADM according to view DU145/ADM cell proliferation by MTT method,detect expression level of MDR-1 gene mRNA in DU145/ADM cell by reverse transcriptionpolymerase chain raction(RT-PCR),observe expression level of p-gp in DU145/ADM by western blot,which before and after was treated by sorafenib.Result Inhibition ratio of sorafenib and ADM simultaneously applied was higher than inhibition ratio of sorafenib and ADM alone applied,the two had statistical difference(P＜0.05).Inhibition ratio of ADM to cell proliferation of DU145/ADM which after sorafenib treated was higher than which before sorafenib treated(P＜0.05).After DU145/ADM was treated by sorafenib,sensibility of DU145/ADM cell to ADM was 2.16 times confer sensibility which before it was treated by sorafenib,expression level of MDR gene 1 mRNA and its production p-gp in DU145/ADM obviously downed regulation.Conclusion Sorafenib would be good for reversed MDR of AIPC which was mediated by p-gp.Reversion mechanism was that sorafenib downed regulation expression of MDR gene 1 mRNA and p-gp,and PTEN,BCL-2,Raf/MER/ERK signal access possible participated reversion process.When sorafenib and ADM simultaneously treated AIPC DU145 cell,the two synergismly inhibited DU145 cell proliferation.