However, the basal level of cleaved PARP was also higher in SKOV3 cells as compared with the other cell lines. To quantitatively investigate the apoptotic effect of 11a, Annexin V/PI double staining was performed. Consistent with the cleaved-PARP assays, 11a only modestly induced www.selleckchem.com/products/MDV3100.html apoptosis in SKOV3 cells but not in A2780 or OVCAR3 cells using etoposide as the positive control (Figure 4B and Figure S7). Similar effects were observed in MCF7 breast cancer cell line, where both doxorubicin and staurosporine induced significant apoptosis while 11a did not induce apoptosis at the tested concentrations (Figure 4C and 4D). Collectively, these data indicate that apoptosis is not the main mechanism accounting for 11a-induced cytotoxicity. Figure 4 11a induces minimal cell apoptosis in ovarian cancer cell lines and a breast cancer cell line.

11a induces G1/S cell cycle arrest in a p53-dependent manner Since 11a failed to induce significant apoptosis in any of the cell lines tested, we hypothesized that 11a-induced cytotoxicity may be attributed to cell cycle arrest, which could induce growth inhibition as determined by the sulforhodamine B (SRB) colorimetric assay used for the NCI-60 cell line cytotoxicity screening. To further discern whether 11a cytotoxicity correlated with p53 status, colorectal cancer isogenic HCT116 p53+/+ and HCT116 p53-/- cell lines were used [35]. Interestingly, these isogenic cell lines exhibited differential sensitivity to 11a. The p53 wild type cell line (IC50 = 0.0337 ��M) was about 10-fold more sensitive than p53 null cell line (IC50 = 0.

3188 ��M) in a pilot screen performed by the Small Molecule Screening and Synthesis Facility (SMSSF) of University of Wisconsin (Figure 5A). The differential sensitivity was later confirmed using the MTT proliferation assay where p53 wild type cells (IC50 = 0.36 ��M) were more sensitive than p53 null cells (IC50 = 1.76 ��M) (Figure 5B). To further investigate the mechanism by which the two isogenic cell lines showed differential sensitivities to 11a, we assessed the apoptotic effects of 11a in these two cell lines. The cells were treated with increasing concentrations of 11a for 24 hours, and apoptosis was measured using a PARP-cleavage assay, in which the PARP cleavage ratio indicates the apoptotic status. Figure 5C shows that only modest PARP cleavage was observed in either p53+/+ or p53-/- HCT116 cells.

To examine whether PARP played a role in 11a mediated cytotoxicity, we co-treated cells with 11a and 3-aminobenzamide (3-AB), a specific PARP inhibitor [51]. PARP inhibition did not affect the cytotoxicity of 11a (Figure 5D), indicating that 11a mediated Brefeldin_A cytotoxicity was independent of PARP activity. The apoptotic effect of 11a was further examined by Annexin V/PI staining. While staurosporine caused severe apoptosis, 11a did not induce any apoptosis in the isogenic cell lines as compared with DMSO control (Figure 5E).