Fig. 6

Caspase inhibitor Z-VAD.fmk blocks SAHA-induced apoptosis in human breast cancer cells. (A) N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk) prevents suberoylanilide hydroxamic acid (SAHA)-induced cell death. MCF7 and MDA-MB-231 cells were treated with dimethylsulfoxide (DMSO) alone (Control), SAHA (4 µM) alone, or SAHA plus Z-VAD (100 µM) for 72 hr. Z-VAD.fmk was added 1 hr before SAHA. Following treatment, cells were washed with phosphate buffered saline (PBS) after removal of the medium, then fixed and stained with Wright-Giemsa Stain, and examined by microscopy. (B) Z-VAD.fmk prevents SAHA-induced DNA fragmentation. MCF7 and MDA-MB-231 cells were treated with DMSO alone, SAHA (4 µM) alone, or SAHA plus Z-VAD.fmk (100 µM). Except for the sample in lane 2, which was treated for 48 hr, samples from all other lanes were treated for 72 hr. After treatment, cells were analyzed for DNA fragmentation by laddering analysis. (C) Z-VAD. fmk prevents SAHA-induced poly(ADP-ribose)polymerase (PARP) cleavage. MCF7 and MDA-MB-231 cells were treated with DMSO alone (Control), SAHA (4 µM) alone, or SAHA plus Z-VAD.fmk (100 µM) for 24 hr. Cell lysates were analyzed for PARP expression by Western blots analysis. Shown are two PARP signals: the 116 kDa full-length protein, and the 86 kDa large cleavage product. The same blot was probed with actin as a loading control.