C-myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in M14 human melanoma cells

Our aim in this work was to define the role of c-Myc in the susceptibility to cisplatin [cis-diamminedichloroplatinum(ll) (CDDP)] inhuman melanoma cel ls. Two M14 melanoma cell clones obtained by transfection and expressing si x to ten times lower c-Myc protein levels than the parental cells and the c ontrol clone were employed. Analysis of survival curves demonstrates an inc rease in CDDP sensitivity in c-Myc [ow-expressing clones if compared with t he control clone and the parental liner The enhanced sensitivity is unrelat ed to the impairment in enzymatic DNA repair activity. cell cycle analysis demonstrates that although the control clone is able to completely recover from the CDDP-induced S-G(2)/M block, this arrest is prolonged in c-Myc low -expressing clones and a fraction of cells undergoes apoptosis. Although: n o changes in P53, Bax, Bcl-2, and Bcl-x(L/S) protein levels are observed, a poptosis is associated with the formation of reactive oxygen species (ROS), activation of caspase-1, caspase-3 and cleavage of the specific caspase su bstrate poly-ADP-ribose polymerase. The use of the antioxidant N-acetyl cys teine and caspase inhibitors prevents CDDP-induced apoptosis in c-Myc low-e xpressing clones, demonstrating that ROS, caspase-1, and caspase-3 are requ ired for apoptotic cell death. Moreover, ROS generation depends on caspase- 1-like activation because the Ac-YVAD-cho inhibitor abrogates CDDP-induced ROS in the c-Myc low-expressing clones.