Research About The Apoptosis Inducing Effect And Its Mechanism Of Hydroxycamptothecine To Human Bladder Cancer Cells

ObjectiveCarcinoma of urinary bladder is a common mankind malignant tumor,the life and health of the human being are seriously threatened by it. The incidence rate of carcinoma of urinary bladder occupy the first position among the uropoiesis tumor of China. High recurrence rate is a significant feature of carcinoma of urinary bladder. Therefore,after the tumour-reductive surgery, the majority of the patient need to operate a dabbling chemotherapy. In order to find a more effective dabbling chemotherapeutics, the exterior and interior scholars are making efforts for it. Hydroxycamptothecin is a alkaloid, extracting from camptotheca acuminata.It becomes one of the most frequently used clinically chemotherapeutics, but antitumous effect mechanism is not very clear. Recent research found that inducing apoptosis is an important mechanism of hydroxycamptothecin. Bcl-2 is the capital antiapoptosis gene and the most early found among Bcl-2 gene family.New research found that most apoptosis process accompany down regulation of Bcl-2 expression.The down regulation of Bcl-2 expression may be the important mechanism of apoptosis induction.Using hydroxycamptothecine to human bladder cancer cells T24,we observe the act effect and detect Bcl-2 expression after drug treat. To study the apoptosis inducing effect and its mechanism of hydroxycamptothecine to human bladder cancer cells, possess important significance to the therapy of carcinoma of bladder.MethodsDifferent concentrations of hydroxycamptothecine were applied to human bladder cancer T24 cells, then MTT method was used to assay the changes of survival rate, fluorescent staining with TUNEL method and flow cytometer(FCM) were used to detect cell apoptosis. Changes of Bcl-2 protein were also detected by Westernblotting method.1. The detection of cell survival rate by MTT methodThe logarithmic phase T24 cell were modulated to the density of 1×10⁵ piece/milliliter,then vaccinate in 96 wells of cultivation board.24 hours later, different final concentration hydroxycamptothecin were added to experimental groups and equal volumenutrient solution were added to control group.After cultivation for 48 hours,we detect cell survival rates by MTT method.2. TUNEL methodCell were vaccinated on coverslip, subgroup and drug intervention are the same as above.To operate according to the directions of the kit. To count 500 cells trabantly on every coverslip, and calculate the ratio of Apoptosis cells and total cellular score, namely apoptosis index(%)=(Apoptosis cells score/500)×100%.3. The ratio of apoptosis cells were detected by flow cytometryThe logarithmic phase T24 cell were modulated to the density of 1×10⁵ piece/milliliter,then vaccinate in 25 cm² of cell culture bottle. 24 hours later,cells confluens at 80%, subgroup and drug intervention are the same as above.Experimental groups and control group were gathered,then were simply stained by PI and detected by flow cytometry. We analyze the DNA contents through CELL QUEST software. The apoptosis rate were demonstrated by the ratio of hypodiploid cells before G₀/G₁ hump and total cellular score.4. The detection of Bcl-2 protein changesThe extraction of cellule total protein: experimental groups and control group were gathered,then extract cellule total protein.And the sample of protein were quantitated by coomassie brilliant blue methods.Every sample detected for three times,β-actin is the interior reference. The gray were scanned by the software of Fluorchem v2.0 gelatum analysis system, signal intensity were represented by the multiplication of gray scale and strap area.5. The assessment of result and statistics analysisThe data were demonstrated by(?)±s,the comparison of mean are done by independent sample t test or analysis of variance q test. The percentage sample value are tested after the arc sine transformation of square root.Results1,The impact of hydroxycamptothecin to the survival rate of T24 cellThe survival rates of T24 cells were decreased with the increased concentrations of hydroxycamptothecine, to which the survival rates expressed a negative correlation(r=-0.916,P<0.01).2,The morphology observation of apoptosis by TUNEL methodThrough TUNEL method,we can found atrophia of positive cells,nucleus are stained with Buffy and brown, endochylema were stained with light colour,part of apoptosis cells shrink as globular shape. Rare positive cells can be seen in control group, positive cells obviously increased in experimental gyoups. The apoptotic index of the control group and the group of 1μg/ml of hydroxycamptothecine were 0.56%,22.32%, respectively.The interclass difference has obviously significance(P<0.05).3,The detection of apoptosis rate The apoptosis rates of different concentrations of hydroxycamptothecine were all higher than that of the control group(P<0.01). They were 0.96%,12.17%,19.38%,31.82%,47.12%, respectively.4,The impact of hydroxycamptothecin to the Bcl-2 protein expression changes of T24 cellThe impact of hydroxycamptothecin to the Bcl-2 protein expression changes: During the apoptosis of T24 cells induced by hydroxycamptothecine, the amount of Bcl-2 protein has no obviously changes,it has no significant difference(P>0.05)ConclusionHydroxycamptothecine may induce apoptosis of human bladder cancer T24 cells, its positive apoptosis effectiveness has a dosage dependence.It may suppress the reduplication of T24 cell. The induction of apoptosis is a very important mechanism of hydroxycamptothecine to treat urinary bladder cancer cells.