Morphological hallmarks of apoptosis As shown in Figure 3, normal control cells stained very faintly with the Hoechst stain but treated cells had a stronger blue fluorescence indicative of apoptosis. Strong blue fluorescence indicates highly condensed chromatin, characteristic www.selleckchem.com/products/17-DMAG,Hydrochloride-Salt.html of apoptotic cells. These results are also confirmed by TUNEL assay which detects DNA frag mentation. As shown in Figure 3, increased DNA fragmentation was observed with increasing BT concentrations in all the cell lines tested. Analysis of mitochondrial transmembrane potential BT treatment resulted in slight decrease in mitochon drial potential as early as 6 hrs post treatment. At 24 hrs post treatment, significant mitochondrial loss was ob served in all cell lines as indicated by shifts in peaks be tween untreated, 50 uM BT and 100 uM BT treated cells.

As compared to OVACAR 3 and IGROV 1 and IGROV1 CDDP, loss of mitochondrial potential was greater in SKOV 3, A2780 and A2780 CDDP at 24 hrs post treatment. Mechanism of BT induced cytotoxicity Effect of BT on cell cycle in ovarian cancer cell lines At 24 hrs post treatment, cell cycle analysis of BT treated ovarian cancer cell lines revealed a significant in crease in the G1 phase cell population with a concomi tant decrease in S and G2 phases as compared to untreated control. OVACAR 3 did not show significant change in G2 phase. Western blot analysis of cell cycle regulatory proteins revealed up regulation of both P27 and p21 upon BT treatment. Effect of BT on ROS generation Cells treated with BT showed ROS generation as early as 6 hrs post treatment.

This was more remarkable when treatment was extended up to 24 hrs. As shown in Figure 6A, elevated ROS levels were observed in all cell lines as indicated by shift in peaks between untreated, 50 uM BT and 100 uM BT treated cells. Follow up cell viability assays in the presence of antioxi dant ascorbic acid, demonstrated at least a 20 30% restor ation of cell viability in the presence of 1 mM ascorbic acid in OVACAR 3, SKOV 3, IGROV 1 and A2780 cells. Interestingly, greater restoration of cell viability was observed in cisplatin resistant variants of IGROV 1 and A2780. In IGROV 1CDDP, 47% cell viability was restored and A2780 CDDP showed 40% restoration. Effect of BT on pro survival and pro apoptotic signalling molecules As shown in Figure 7A, western blot analysis revealed significant activation of pro apoptotic marker, p38, when cells were treated with BT for 24 hrs.

However, a cell viability assay using SB203580 pre treatment did not restore cell viability. Western blot analysis http://www.selleckchem.com/products/BI6727-Volasertib.html of pro survival marker pAkt showed decreased expression at 24 hrs post BT treat ment in all cell lines except for OVACAR 3 and IGROV 1 where increased expression was observed at 50 uM but decreased at 100 uM BT.