Study Of The Inhibitory Effect Of Src Kinase Inhibitor Ⅱ On Human Transitional Cell Carcinoma Of Bladder Cell Line T24 And The Mechanism

Transitional cell carcinoma of bladder(BTCC) is the most frequentcancer on human urogenital system not only in global but also in china.The incidence rate of BTCC is 8%of all adult cancer,it on male is 4th and female is 8th.BTCC accounts for about 90%of all bladder cancer. The features of BTCC are high incidence rate,high relapse rate,complex of treatment,fickle of biological behaviour.The most important treatment is to reduce relapse of BTCC.Although some progress has been made in the treatment of BTCC,BTCC remains high relapse rate in most countries.Although regional perfusion chemotherapy reduces the rate of relapse to advanced BTCC,disease progression is inevitable.A novel strategy for BTCC in advanced stage is molecularly targeted therapy inhibiting specific pathways and key molecules implicated in tumor growth and progression.Therefore,novel drugs targeting other critical molecules are required for patients with BTCC.Src is the first reported oncogene and has been a prototype in identifying many characteristics of follow on oncogenes.C-Src and its retroviral form(v-Src)are both non-receptor tyrosine kinases,encoding a M60,000 membrane-associated protein tyrosine kinase(PTK).Results from many different studies suggest a role of Src in various aspects of a cell's life,including proliferation,survival,motility,cell-cell and cell-matrix adhesion,and local invasion,all of which are deregulated during cancer progression.C-Src has been found to be over-expressed and highly activated in a variety of human cancers,including colon cancer,breast cancer,and pancreatic cancer.Recent studies showed that the proto-oncogene C-Src was also highly activated in BTCC,especially in adenocarcinomas,suggesting the possibility of molecular targeted therapy for bladder cancer.Src Kinase InhibitorⅡis a novel,available selective inhibitor of Src kinases.It has potential to inhibit migration of the highly metastatic human pancreatic cancer cell Line(L3.6p1)at submicromolar dose levels in vitro.In the present study,we examined the effect of Src Kinase InhibitorⅡon in vitro properties of human bladder cancer cell lines T24 expressing various levels of Src protein.We further investigated the role of Src Kinase InhibitorⅡon Src protein and its downstream signal path.PartⅠExpression and clinical significance of prote in BTCCObjective To investigate the expressions of Src mRNA and protein in BTCC with regard to the clinical significance.Methods Expressions of Src mRNA and protein were detected by semi-quantitative RT-PCR technique in 36 cases of BTCC,22 cases of tissues adjacent to carcinoma and 18 cases of normal bladder tissues as controls.The relevance between the expressions of Src mRNA and the clinical pathology of the patients of BTCC were studied.Results Semi-quantitative RT-PCR demonstrated that the expression ratio of Src mRNA were 0.311±0.014,0.252±0.024 in the tissues of sBTCC and mBTCC,which significantly up-regulated compared with that in tissues adjacent to carcinoma as 0.150±0.032 and normal bladder tissues as 0.144±0.028(P＜0.05).The expression of Src mRNA was 0.329±0.103 in the recurrence patient,which significantly up-regulated compared with that in primary patient as 0.288±0.121(P＜0.05).The expression ratio of Src mRNA were 0.289±0.128,0.302±0.104 and 0.325±0.098 in the tissues of G1,G2,G3 of BTCC(P＜0.05). Conclusion 1.The expressions of Src mRNA and protein were significantly increased in BTCC tissues.2.The expressions of Src mRNA was in high relevance with cell differentiation,infestationand stagingsize of BTCC.PartⅡEffect of Src Kinase InhibitorⅡon the biological behavior of human transitional cell carcinoma of bladder Cell Line T24Objective To study the influence of Src Kinase InhibitorⅡon the biological behavior of human transitional cell carcinoma of bladder Cell Line T24.Methods Hoechst,M'IT assay and flow cytometry was used to examine the effect of Src Kinase InhibitorⅡon T24 cell proliferation.We used Turnout Invasion Assay to assess the effect of Src Kinase InhibitorⅡagainst cellular invasion in T24 in vitro.Results Src Kinase InhibitorⅡcould suppress cell proliferation of T24 cells.The inhibition of T24 cells showed a dose-dependent reduction by Src Kinase InhibitorⅡ.Tumour Invasion Assay certified that Src Kinase InhibitorⅡcould suppress cell migration of T24 cells in vitro.Conclusion:Src Kinase InhibitorⅡcould induce apoptosis and suppress cell proliferation of T24 cells.It also could suppress cell migration of T24 cells in vitro. PartⅢEffect of Src Kinase InhibitorⅡon human transitional cell carcinoma of bladder Cell Line T24 and the molecular biology MechanismObjective To study the the molecular biology mechanism of Src Kinase InhibitorⅡon Cell Line T24.Methods Src expression and activation in T24 cell lines as well as Src Kinase InhibitorⅡmediated inhibition of phosphorylation of Src were assayed with western blot.We examined the level of E-Cadherin,P38,and MMP-9 in T24 cell lines as well as the effect of Src Kinase InhibitorⅡon Src expression,activated by semi-quantitive RT-PCR.Results Phosphorylation Src protein expression was down regulation in T24 cell lines as well as Src Kinase InhibitorⅡdid its work.With Src Kinase InhibitorⅡon T24 cells,expression of c-Src protein,P38 protein and MMP9 protein were down regulation.expression of E-Cadherin protein was up-regulation.Conclusion1.Phosphorylation(activation),but not over-expression,of Src is crucial for the progression of BTCC.Phosphorylation Src protein play a pivotal role in regulating cell migration and invasion in BTCC.2.Src Kinase InhibitorⅡcould reduce the proliferation of NSCLC cells by the inhibition of activated P38,MMP9 and up-regulation E-Cadherin,three of the downstream signaling of Src.