Studies Of The Influence Of Beta-carotene On The Biological Behaviors And The Mechanism Of Molecular Biology To Human Bladder Carcinoma Cell Line T24

Background:In China,as the most common malignant tumor in the genitourinary system,bladder transitional cell carcinoma(BTCC) has been harmful to people's health heavily and there has been obviously increasing trend of incidence in many cities.Despite of the improvement of operational skills and intravesical chemotherapeutic agents,the recurrent rate of bladder cancer still keeps highly at 50-70%after initial treatment.It is urgent in clinic to develop a new effective therapy on BTCC.Gap junction intercellular communication(GJIC) which is the chief mode of signal transduction in adjacent cells play an important role in the control of cell growth,proliferation,differentiation,the maintenance of homeostasis and morphogenesis during normal tissue growth and neoplastic development.The inhibition of GJIC can disrupt the delicate balance of cell homeostasis,leading to increased cell proliferation and inhibiting apoptosis.Beta-carotene is an organic compound-a terpenoid,abundant in plants and fruits which is the most common form of carotene.It has been proved to be no acute toxicity and chronic toxicity,including no-mutagenesis and no-carcinogenesis.Conversely,dietary beta-carotene materially reduces human cancer rates,which has been proved by epidemiologic Studies and experiments in vitro.For few studies of beta-carotene on BTCC,we make a research on beta-carotene on human bladder cancer cell line T24 to develop an anticancer approach and explore the anticancer mechanism of beta-carotene on apoptosis and GJIC. Objective To investigate the expressions of connexin43,Bcl-2 and Bax mRNA in BTCC with regard to the clinical significance.Methods Expressions of connexin43(Cx43),Bcl-2 and Bax mRNA were detected by semi-quantitative RT-PCR in 35 cases of BTCC and 21 cases of para-carcinoma tissue,15 cases of normal bladder tissues as controls.The relevance between the expressions of connexin43,Bcl-2 and Bax mRNA and the clinical pathology of the patients of BTCC were studied.Results Semi-quantitative RT-PCR demonstrated that the expression ratio of connexin43 mRNA was 0.341±0.367 in BTCC,which significantly down-regulated compared with that in para-carcinoma tissues as 0.787±0.697 and normal bladder tissues as 0.806±0.466 (P＜0.01).The expression ratio of Bcl-2 mRNA was 0.345±0.430 in BTCC,which significantly up-regulated compared with that in para-carcinoma tissues as 0.160±0.334 and normal bladder tissues as 0.145±0.225(P＜0.01).No correlation was found between para-carcinoma tissues and normal bladder tissues(P＞0.05).The expression of Cx43 mRNA were significantly negative correlated with the grade of the tumor (P＜0.05),and Bcl-2 mRNA with positive correlation.And no correlation was found between the expression of Cx43 and the sex,age,clinical staging,and the diameter and the growth pattern of BTCC(P＞0.05), Bcl-2,too.But the expression of Bcl-2 mRNA was significantly positive correlated with the recurrence of BTCC.There was no correlation between cancer tissues,para-carcinoma tissues and normal bladder tissues on the expression of Bax mRNA(P＞0.05),And no correlation was found between the expression of Bax mRNA and the sex,age,grade, clinical staging,and the diameter and the growth pattern of BTCC(P＞0. 05).Conclusion1.The expression of Cx43 mRNA was significantly decreased in BTCC tissues,and Bcl-2 mRNA was significantly increased in BTCC tissues.No correlation was found between cancer tissues,para-carcinoma tissues and normal bladder tissues on the expression of Bax mRNA2.The expression of Cx43 mRNA was significantly negative correlated with the grade of the tumor,and Bcl-2 mRNA with positive correlation.Bcl-2 mRNA was in high relevance with the recurrence of BTCC.The expression of Bax mRNA was not relevant with the clinical pathology on BTCC. Objective The present study was undertaken to examine the effects of beta-carotene on biological behavior of T24 cells,including growth inhibition,and cell apoptosis.Methods The effect of beta-carotene on human bladder cancer T24 cells was evaluated with varying concentration of beta-carotene(0,1,5, 10 and 20uM) treatment for 72 hours by MTT assay.The cell apoptosis of beta-carotene on human bladder cancer T24 cells was determined with the indicated concentration of beta-carotene treatment for 72 hours by hoechst staining and flow cytometry,the apoptosis index was detected.Results The inhibition ratio of beta-carotene on human bladder cancer T24 cells was 0.41%,6.32%,28.83%,63.02%respectively by MTT assay,we found that beta-carotene treatment exerted a significant cytotoxic effect upon T24 cells in a dose-dependent manner,especially 10 and 20uM group compare with the control group(P＜0.05).Observed by hoechst staining,the apoptotic cells were detected with varying concentration of beta-carotene,the apoptotic body appeared in high concentration.The cell apoptosis index with beta-carotene treatment at concentrations of 0-20uM after 72 hours ranged from 6.5%-19.0%, increased significantly compared with control group(P＜0.01).Conclusion Our data suggested beta-carotene treatment resulted in a significant dose-dependent Inhibition in the growth of T24 Cells and cell apoptosis induced with a significant dose-dependent. Objective To investigate the effects of beta-carotene on the mechanism of molecular biology of T24 cells.Methods The effect of beta-carotene on human bladder cancer T24 cells was evaluated with varying concentration of beta-carotene(0,5,10 and 20uM) treatment for 72 hours,both Semi-quantitive RT-PCR and western blot were used to detect the expression of Bcl-2,Bax,Cx43mRNA.Results Semi-quantitive RT-PCR demonstrated that the expression ratio of Bcl-2 mRNA decreased and the expression ratio of Cx43 mRNA increased in 10 and 20uM groups significantly compared with control group(P＜0.01).The expression ratio of Bcl-2 mRNA was 0.193±0.031 (20Um),0.443±0.045(10uM),0.623±0.045(5uM),0.670±0.050(0uM) and Cx43 mRNA was 0.647±0.055(20Um),0.443±0.050(10uM), 0.290±0.050(SuM),0.243±0.025(0uM),respectively.No correlation was found in the expression of Bax mRNA(P＞0.05).We found that beta-carotene treatment with Bcl-2 mRNA and Cx43 mRNA upon T24 cells was in a dose-dependent manner.Western blot demonstrated that the expression ratio of Bcl-2 decreased and the expression ratio of Cx43 increased in 10 and 20uM groups significantly compared with control group(P＜0.01),both were in a dose-dependent manner.The expression ratio of Bcl-2 protein was 0.152±0.037(20Um),0.441±0.038(10uM), 0.982±0.157(0uM) and Cx43 protein was 0.416±0.048(20Um), 0.217±0.026(10uM),and 0.133±0.322(0uM),respectively.No correlation was found in the expression of Bax protein(P＞0.05). Conclusion1.The expression of Cx43 mRNA was significantly decreased and Bcl-2 mRNA significantly increased in T24 after beta-carotene treatment for 72 hours.Both were in a dose-dependent manner.No correlation was found in the expression of Bax mRNA.2.The expression of Cx43 protein was significantly decreased and Bcl-2 protein significantly increased in T24 after beta-carotene treatment for 72 hours.Both were in a dose-dependent manner.No correlation was found in the expression of Bax mRNA.