Lack of involvement of caspases in SAHA-induced apoptosis.

(A) Immunoblotting for caspases-8, -9, and -3 in MM.1S cells treated with SAHA (5 μM) failed to detect caspase cleavage within the first 24 hours of treatment, arguing against the role of caspases in SAHA-induced apoptosis. Treatment with TRAIL/Apo2L (300 ng/mL for 5 hours) served as a positive control, as previously described.29 SAHA induced cleavage of PARP into an atypical approximate 60-kDa fragment, reminiscent of apoptosis mediated by the protease calpain,33 whereas TRAIL/Apo2L resulted in “classic” cleavage of PARP to an approximate 85-kDa fragment. (B) Quantification of SAHA-induced cell death (average ± SD as indicated by error bars), in the absence or presence of caspase and calpain inhibitors, using the LDH release assay. The pan-caspase inhibitor ZVAD-FMK had no protective effect on SAHA-induced cell death, confirming the lack of involvement of caspases in this model. In contrast, the calpain inhibitor calpeptin attenuated SAHA-induced cell death, further supporting the role of the protease calpain in SAHA-induced apoptotic signaling.