Non-caspase-mediated apoptosis contributes to the potent cytotoxicity of the enediyne antibiotic lidamycin toward human tumor cells

Abstract

Enediyne antibiotics have been reported to be the most potent cytotoxic antitumor agents. The pathway by which these compounds cleave DNA and induce apoptosis of tumor cells may be different from the caspase-mediated pathways that initiate typical apoptosis. In this report, we studied the apoptosis induced by lidamycin (LDM), a member of the enediyne antibiotic family, and compared the characteristics of LDM-induced apoptosis with those of typical apoptosis induced by mitomycin C or etoposide. Chromatin condensation occurred very rapidly and appeared as speckles in human hepatoma BEL-7402 and breast carcinoma MCF-7 cells after treatment with 1 μM LDM. In addition, co-staining the cells with the mitochondria-specific dye Mitosensor™ and the DNA-specific dye Hoechst 33342 enabled the visualization of mitochondria in normal control and LDM-treated cells but not in mitomycin C-treated cells. Neither the caspase inhibitor VAD-fmk nor the caspase-3 inhibitor DEVD-fmk was able to inhibit the DNA ladder patterns caused by LDM in BEL-7042 or MCF-7 cells. Smaller fragments of histone H1 cleaved by LDM were detected by SDS–PAGE, indicating that the site of LDM action is the internucleosomal structure. Although caspase-9, caspase-3/7, and caspase-6 activities were increased in BEL-7402 cells, and caspase-7 activity was increased in MCF-7 cells after treatment with 1 μM LDM, this occurred much later, indicating that chromatin condensation reached the maximal level rapidly while caspase activities still remained low. Taken together, these results demonstrate that LDM induced rapid DNA cleavage and chromatin condensation independently of caspase activities; this may contribute to its highly potent cytotoxicity toward tumor cells.