2). Moreover, sensitization to TX in KF-TX cells by CLU-siRNA was further confirmed after time dependent fashion of TX treatment by FACS analysis (at 36, 48 and 60 h; Figure 5C.1) where dead cells indicated by the sub diploid G0 cells. Further confirmation for differential apoptotic cells

was obtained by Annexin V staining (Figure 5C.2). These data indicate that response to TX administration is enhanced after CLU-siRNA transfection. In addition, a dose dependent enhancement of apoptosis by TX in KF-TX cells after CLU knock-down was verified by DNA laddering experiment (data not shown). On the other hand, cellular viability was studied under experimental conditions similar to this described above SB203580 except that OGX-011 was used to knock down CLU while control oligodeoxynucleotide was used for control transfection. Figure 6A shows significantly less viability of KF-TX cells pre-treated with OGX-011 and TX than those pre-treated with control oligodeoxynucleotide and TX. Similarly, sensitization to TX in KF-TX cells by OGX-011 was further confirmed by FACS analysis (Figure 6B). Further confirmation for check details differential apoptotic cells was obtained by Annexin V staining (Figure 6C). Together, the aforementioned data indicate that silencing s-CLU by specific siRNA or

OGX-011 enhanced TX toxicity in the ovarian cancer cells. Figure 6 Targeting CLU by OGX-011 sensitizes ovarian cancer cells to TX treatment. A.Comparative viability of chemoresistant ovarian cancer cells before and after CLU knock down by OGX-011. Cells were cultured in 96-well plates, then transfected Thiamine-diphosphate kinase either with CLU-siRNA or control siRNA twice. Twenty-four hours after last transfection, cells were treated with TX. Seventy-two hours after drug addition at indicated doses, cell viability was estimated. KF-TX cells showed enhanced

TX-induced toxicity in CLU KD cells versus controls. B. A representative time-dependent DNA histogram (FACS analysis) Selleck BIBW2992 demonstrating that CLU KD by OGX-011 at 1200 nM enhanced TX toxicity in KF-TX cells. KF-TX cells were transfected either with OGX-011 or control Oligonucleotide and then challenged with TX dose of 200 nM at indicated time periods (24 h, 36 h,48 h and 60 h). B. Results of Annexin V staining of cells pre-treated with OGX-011 at different concecntrations (400, 800 and 1200 nM) then treated with TX (200 nM) for indicated time periods (24, 48 and 72 h). Quantification of the relative ratio of apoptotic cells at different time points indicated the significant enhancement of TX toxicity by OGX-011. The maximum enhancement was obtained by 800 nM OGX-011 while the conc. of 1200 nM did not show further significant improvement in toxicity. D. CLU knock down modulates cellular growth rate of ovarian cancer cells. (1) KF-TX cells showed enhanced growth rate when transfected with CLU siRNA with regard to controls.