| Objective:The aim of this study is to research the reversion of the anti-apoptotic action on adriamycin-resistant human bladder carcinoma cell line BIU-87/ADM by tetrandrine in vitro, and to explore its possible molecular mechanisms. To provide some experimental foundation on against bladder cancer for clinical application of Tetrandrine.Methods:(1)Both sensitive cell line BIU-87 and drug-resistant cell line BIU-87/ADM were divided into the control group, the tetrandrine group, the adriamycin group and the combination group. In which the concentration of adriamycin was 1.5μg/ml and tetrandrine was 1μg/ml;(2)Application of AO/EB staining and fluorescence microscopy to detect cell apoptosis;(3) DAPI staining was used to detect morphological changes of apoptosis;(4)The mitochondrial membrane potential was measured using the fluorescent probe JC-1;(5)The expression of Bcl-2 and Bax gene were detected By Semi-quantitative RT-PCR and immunocytochemistry;(6) Caspase-9 Activity Assay Kit was used to detect the activity of Caspase-9.Results:(1)The natural apoptosis rate of adriamycin-resistant human bladder carcinoma cell line BIU-87/ADM and the corresponding sensitive cell line BIU-87 were (4.67±1.37)% and (5.83±1.47)%, respectively. The apoptosis rate of the two cell lines were (6.17±1.47)% and (7.50±2.26)% after 1.0μg/ml tetrandrine treated for 24h,compared with each control group, there were no significant differences(P> 0.05). The apoptosis rate were (57.83±5.64)% in the sensitive cell line and (15.33±2.58)% in the adriamycin-resistant cell line after 1.5μg/ml adriamycin treated for 24h, there was a significant difference (P <0.05); Exposured to 1.0μg/mlTet +1.5μg/mlADM for 24h, the apoptosis rate of the adriamycin-resistant cell line increased to(43.17±3.49)%, which had a higher apoptosis rate than the adriamycin alone group (P <0.05);However, the apoptosis rate of the sensitive cell line was (59.17±5.38)% and there was no significant difference between the adriamycin alone group (P>0.05).(2)In addition, DAPI staining had detected the typical apoptotic morphological changes such as nuclear condensation, nuclear fragmentation and formation of apoptotic body; JC-1 assay showed that compared to the adriamycin alone group, the mitochondrial membrane potential of the combination group decreased significantly (P<0.05).(3) Semi-quantitative RT-PCR and immunocytochemistry showed that the Bcl-2 gene expression decreased and the Bax gene expression increased in the combination group on adriamycin-resistant cell lines (P<0.05); Caspase-9 colorimetric assay showed: the Caspase-9 activity in the combination group cells was significantly enha -nced(P <0.05).Conclusion:(1)Relative to the sensitive cell line, the adriamycin-resistant cell line BIU-87/ ADM has obvious anti-apoptosis action to adriamycin. Tet in the concentration of 1.0μg/ml can not induce apoptosis, but it can increase the sensitivity of chemotherapy to adriamycin on adriamycin-resistant cell line BIU-87/ADM, thus reversing the anti- apoptosis action on adriamycin-resistant cell line BIU-87/ADM effectively.(2)Tetrandrine can reverse the anti-apoptotic action effectively on adriamycin -resistant human Bladder Carcinoma cell line BIU-87/ADM.The reversal mechanisms of tetrandrine might be involved in down-regulation of Bcl-2 gene expression and up-regulation of Bax gene expression, and reduce the mitochondrial membrane potential, the tetrandrine-induced apoptosis might be related to the activation of the caspase cascade and mitochondrial pathway.
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