Effects Of Proliferation Of Bladder Cancer Cells Under The Stimulation Of Interferon-γ And Explore The Molecular Mechanism

PART ONE The level of IFN-γ in serum of patients suffered fromTCCB and the expression of hepaCAM protein in TCCB and thecorrelations between themObjective: To detect the level of interferon-gamma（IFN-γ） in serum ofpatients suffer from transitional cell carcinoma of the bladder（TCCB） andthe expression of hepatocyte cell adhesion molecule （hepaCAM） protein inTCCB. And to analyze the correlation between them and the correlationsamong each clinical parameters.Methods: The level of IFN-γ in serum of33patients and15healthyvolunteers was measured by enzyme-linked immunosorbent assay（ELISA）kit.33cancer and26peripheral noncancerous tissue sections wereexamined by paraffin wax immunohistochemistry（PIH） to detect theexpression of hepaCAM protein and ensure the position.Results: IFN-γ was remarkably low in TCCB patients compared withthe healthy volunteers（P<0.01）. The decrease in bladder cancer patients’serum was correlated with the parameters of histological grade and occurrence（P<0.05）.The protein of hepaCAM was highly expressed inperipheral noncancerous tissues, mainly positioned in cytomembrane, asmall percentage in nucleus, little in cytoplasm, but was nearly absent inbladder carcinoma（P<0.01）. Statistical analysis has shown no significantdifferences between hepaCAM expression levels and various clinicalparameters（P>0.05）. The decrease of IFN-γ in serum and low expression ofhepaCAM in specimens has linear correlation（Pearson r=0.899, P<0.001）.Conclusions: The deficiency of hepaCAM in bladder cancer tissues atthe protein level suggests an important connection between hepaCAM andIFN-γ, and provide an evidence to proceed the cytological experimentsabout the possible mechanism of IFN-γ inhibiting the bladder cancer cells. PART TWO Effects of bladder cancer cell lines under the treatment ofIFN-γ and explore the possible molecular mechanismPurpose: To explore the effect of T24and BIU-87cells stimulated byIFN-γ and to probe the possible molecular mechanism involved in IFN-γinhibiting the proliferation of cells.Methods: Both T24and BIU-87cell lines were stimulated by IFN-γ atfive different final concentrations（125U/ml,250U/ml,500U/ml,1000U/mland2000U/ml） and then tested at24h,48h,72h, respectively. Theproliferation inhibition ratio of T24and BIU-87was measured by 3-（4,5-Dimethylthiazol-2-yl）-,5-diphenyltetrazolium bromide （MTT）colorimetric assay. The median concentration,500U/ml, of IFN-γ was thendetermined to stimulate the cells, and set as experiment groups, withoutIFN-γ processing set to the blank control group. Cell cycles were examinedby flow cytometry（FCM）. The expression of hepaCAM mRNA wasdetermined by RT-PCR. Western bolt was used to detected the proteinexpression of hepaCAM and p21^WAF1. Another cytokine tumor necrosisfactor-alpha（TNF-α） was chosen to set as negative control group of IFN-γ,the same concentration as IFN-γ was determined to treat with the T24andBIU-87cells. RT-PCR and western blot were used to detect the hepaCAMat gene or protein level, respectively.Results: IFN-γ inhibited the proliferation of T24and BIU-87cells intime-dose dependent pattern（P<0.01）. Compared with the blank controlgroup, cell cycles were arrested at G0/G1phase（P<0.01）. The hepaCAMmRNA was re-expressed slightly under the stimulation of IFN-γ（P<0.05）.The expression of p21^WAF1was gradually increased（P<0.01）. In contrast,the expression of hepaCAM gene in T24and BIU-87cells did not alter atall under the stimulation of TNF-α. Furthermore, neither IFN-γ nor TNF-αcould induce the re-expression of hepaCAM at the protein level by westernblotting.Conclusions: IFN-γ inhibits the proliferation of T24and BIU-87cellsmay through the gene of hepaCAM via a p21^WAF1-dependent manner and arresting cells at G0/G1phase.