| Part1Effects of parthenolide on proliferation and cell cycle of human5637bladder carcinoma cellsObjective The present study was undertaken to examine the effects of parthenolide on growth inhibition, cell cycle and corresponding molecular mechanism of human5637bladder carcinoma cells.Methods The inhibitory effect of parthenolide on human5637bladder carcinoma cells was determined with varying concentrations of parthenolide (0,2.5,5.0,7.5and10μM) treatment for24and48hours by MTT assay. The anti-proliferative or anti-carcinogenic effect of parthenolide in5637cells was determined by anchorage-dependent colony-forming assay.5637cells were cultured with different concentrations of parthenolide (0,2.5.5.0.7.5and10μM) for24hours and then stained with propidium iodide. The DNA content was analyzed by flow cytometry. The cell cycle modulating proteins Cyclin D1and CDK2, p-CDK2, CDK4were determined by western blotting analysis.Results1. Using the human5637bladder carcinoma cells line, we found that parthenolide could inhibit proliferation and cell viability of5637cells in a dose-dependent (2.5-10μM) and time-dependent (24-48hours) manner.2. Inhibition in5637cells viability with parthenolide treatment at concentrations of2.5-10μM after24hours ranged from9.5%to40%, whereas after48hours ranged from23.9%to79%.3. There was a remarkable decrease in the ability of the5637cells to form colonies with increasing doses of parthenolide (0,2.5,5.0,7.5and10μM). The inhibition rate of forming colonies was87.7%when parthenolide was at dosages of10μM.4. As shown by flow cytometry, parthenolide treatment (0,2.5,5.0,7.5and10μM for24hours) of the5637cells led to significant Gl-phase cell cycle arrest in a dose-dependent and associated with a concomitant decrease in cell population in the S-phase, whereas the population of cells in G2/M phase did not change significantly as compared with the corresponding controls.5. Using western blotting analysis, parthenolide treatment (0,2.5,5.0,7.5and10μM for24hours) of the5637cells resulted in significant dose-dependent down modulation of the protein expression of cyclin D1and p-CDK2, but no change in CDK4levels.Conclusions Our data suggested parthenolide treatment led to a significant dose-dependent and time-dependent inhibition in the growth and colony-forming of human5637bladder carcinoma cells. Our study also suggested that parthenolide also could cause a G1-phase arrest of the cell cycle through the reduction in the protein levels of the cyclin D1and p-CDK2.Part2Parthenolide induced apoptosis of human5637bladder carcinoma cellsObjective The present study was undertaken to examine the effects of parthenolide on apoptosis of human5637bladder carcinoma cells and to identify the altered signaling pathway underlying the response to parthenolide exposure.Methods5637cells exposing to parthenolide for48hours were stained with Hoechst stain (HO33258), visualized by UV microscopy, and quantitated by counting condensed and fragmented nuclei in five randomly selected areas. The extent of apoptosis was quantified by flow-cytometric analysis of parthenolide-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blotting analysis probing for IκBα and phosphorylated IκBα (p-IκBα) proteins, indicative of NF-κB activation. The role of PARP, Bcl-2family in apoptosis was analyzed by western blotting.Results1. Using the human5637bladder carcinoma cells line, we found that parthenolide treatment led to induction of apoptosis in dose-dependent manner (2.5-10μM).2. Changes in nuclear morphology were detected after Hoechst staining using fluorescence microscopy.5637cells exposing to parthenolide (0,2.5,5.0,7.5and10μM for48hours) revealed a dose-dependent decrease in cell density. High concentrations of parthenolide treatment led to significant chromatin condensation or fragmentation, which correlated with the occurrence of apoptosis.3. As shown by PI staining and the annexin V method, we found that parthenolide caused a dosage dependent increase in5637cells apoptosis. It was observed that parthenolide treatment of5637cells with2.5to10μM for48hours increased the number of early apoptotic cells (LR) from8.1%to16.5%respectively. The number of late apoptotic cells (UR) increased from7.0%to70.1%. The total percent of apoptotic cells (UR+LR) increased from15.1%to86.6%.4. Western blotting analysis indicated that parthenolide treatment of5637cells with varying concentration (0-10μM) for48hours resulted in a dose-dependent activation of PARP proteins.5. Parthenolide treatment resulted in an appreciable down-regulation of protein expression of phosphorylated IκBαa (p-IκBα) in a dose-dependent without an effect on IκBα expression in5637cells.6. Parthenolide treatment (0-10μM) for48hours to5637cell lines resulted a significant reduction in anti-apoptotic Bcl-2and phosphorylated Bad (p-Bad) proteins in a dose-dependent manner, which were indicative of induction of apoptosis.Conclusions We found that parthenolide caused an inhibition of NF-κB activation in human5637bladder carcinoma cells, and in turn, resulted in modulations in Bcl-2family proteins in such a way that the apoptosis of5637cells was induced. Based on these studies, we suggested that parthenolide could be developed as an agent for the management of bladder carcinoma. Part3Effects of parthenolide on migration and invasion of human5637bladder carcinoma cellsObjective The present study was undertaken to examine the effects of parthenolide on invasion and metastasis of human5637bladder carcinoma cells and to identify the possible molecular mechanisms.Methods To evaluate whether parthenolide could inhibit invasion and metastasis of5637bladder carcinoma cells in vitro, Matrigel invasion assays were performed to examine whether invasion and metastasis of the5637cells treated with parthenolide were inhibited. After treating human5637bladder carcinoma cells with different concentrations parthenolide (0,2.5,5.0,7.5and10μM) for48hours, the effects of parthenolide on the transcription levels of VEGF, MMP-2and MMP-9were analysed by reverse transcription-polymerase chain reaction (RT-PCR).Results1. Using the human5637bladder carcinoma cells line, the results of the in vitro migration assay displayed that parthenolide was able to inhibit migrative ability of5637cells dose-dependently(0-10μM).2. Using the human5637bladder carcinoma cells line, the results of the in vitro invasion assay also displayed that parthenolide was able to inhibit invasion ability of5637cells dose-dependently(0-10μM).3. RT-PCR analysis suggested that treatment of5637cells with parthenolide resulted in a dose-dependent (0-10μM) reduction in VEGF mRNA, MMP-2mRNA, MMP-9mRNA levels.Conclusions In this study, we demonstrated that parthenolide effectively suppressed invasion and migration in human5637bladder carcinoma cells in vitro. The inhibition of invasion ability of5637cells by parthenolide might be attributed to reduction of the expression of VEGF, MMP-2, MMP-9. Therefore, clinical application of parthenolide may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis. |
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