Research Of The Inducing Effect Of Pirarubicin To Apoptosis And Its Mechanisms In Bladdercancer Cell T24

Objective: To investigate the impact of different concentrations of THP onproliferation, cell cycle and apoptosis in bladder cancer cell line T24, to detectthe expression changes of Survivin mRNA, and to investigate the apoptosisinduction and the mechanisms of THP in bladder cancer cell line. Our researchwill provide a theoretical basis for perfusion treatment in bladder cancer.Methods:1. Use various concentrations of THP (0,0.1,1,10μg/ml) inbladder cancer cell T24and observe the morphological changes of the cellsunder the inverted microscope.2. After treated with various concentrations ofTHP (0,0.1,1,10μg/ml), apply CCK-8assay to detect the proliferation effect ofTHP in bladder cancer cell T24.3. Flow Cytometry was used to detect thechanges of cell cycle of T24which was treated by various concentrations ofTHP after24hours.4. AV/PI was used to detect the apoptosis in T24which wastreated with various concentrations of THP (0,0.1,1,10μg/ml) after24hours.5.Apply RT-PCR to detect the expression changes of Survivin mRNA in bladder cancer cell T24treated with various concentrations of THP (0,0.1,1,10μg/ml).Results:1. T24cells can be observed as polygonal shape, adherentgrowing, merging in adjacent cells under the inverted contrast microscope. AfterTHP treatment, with increasing concentration, cells are becoming rounding, thevolume begins to decrease, karyopyknosis appears, the color of nuclear deepen,refractive enhancements, and some floating and dead cells can be seen.2. Detectthe cell proliferation level through CCK-8method in4groups of cells in24h，48h，72h, and compare the growth of each group by OD values. In thecomparison with the matched group, each group in the concentration of THP in24h，48h，72h can suppress the proliferation of bladder cancer T24. Thedifference was of statistically significance (P <0.05). Calculate the proliferationinhibition rate of the group which added with THP at24h,48h, and72h. Theproliferation ability was inhibited in a time-dependent manner (P <0.05).3.The cycle measured by PI staining test showed: the bladder cancer T24cellswere treated with different concentrations of THP (0,0.1,1,10μg/ml) for24hand the cell percentage in G2/M phase were respectively:(5.45±0.51)%,(7.91±1.33)%,(81.34±1.43)%,(88.97±2.95)%. The cell percentage of the entireexperimental group in G2/M phase was significantly increased and theperformance showed in a concentration-dependent manner (P <0.05), comparingwith the control group.4. Annexin V/PI test showed that THP can induceapoptosis in T24cells (0,0.1,1,10μg/ml). The apoptosis rate of the T24cellsdealing with THP after24h were:(6.18±0.27)%,(15.29±0.98)%,(19.56±1.45)%,(23.81±0.81)%. Comparing to the control group, the apoptosis rate ofthe entire experimental group increased significantly (P <0.05).5. T24cellstreated with different concentrations (0,0.1,1,10μg/ml) of THP can significantly decrease the expression of Survivin mRNA. The performanceshowed in a concentration-dependent manner (P <0.01).Conclusions: Pirarubicin can inhibit the proliferation of bladder cancerT24cells in vitro and in a time and concentration-dependent manner. That iswith the increasing of the concentration and the action duration; the growthinhibition of T24cells will be increasingly obvious. Pirarubicin can inhibit theapoptosis of bladder cancer T24cells and restrict the cells in G2/M phase. Theperformance shows in a concentration-dependent manner. Its mechanism may berelated with the decrease of the expression of Survivin mRNA.