DC-81-enediyne induces apoptosis of human melanoma A375 cells: involvement of the ROS, p38 MAPK, and AP-1 signaling pathways.

Melanoma is one of the most chemoresistant cancers in patient care. The remission rate of current therapy remains low. DC-81, an antitumor antibiotic produced by Streptomyces species, belongs to pyrrolo[2,1-c][1,4]benzodiazepine (PBD), which is a potent inhibitor of nucleic acid synthesis. An enediyne contains either DNA intercalating groups or DNA minor groove binding functions and these are potent DNA-damaging agents due to their ability to generate benzenoid diradicals. We have previously reported an efficient synthesis and antitumor activity of a series of novel PBD hybrids linked with enediyne. The purpose of this study was to examine the mechanism of the antiproliferative effect of DC-81-enediyne agent on human melanoma A375 cells. DC-81-enediyne induced an increase in Ca(2+) level and reactive oxygen species (ROS) generation as detected by flow cytometric assay. Western blot analysis showed that DC-81-enediyne induced the phosphorylation of p38 and activating transcription factor 2 (ATF-2). By using the luciferase reporter assay, activating protein-1 (AP-1) activity was further enhanced after A375 cells were treated with graded concentrations of DC-81-enediyne. DC-81-enediyne treatment-induced A375 cell apoptosis was significantly abrogated by the addition of Ca(2+), ROS, and p38 inhibitors. Collectively, our studies indicate that DC-81-enediyne induces A375 cell apoptosis through an increased Ca(2+) and ROS generation, which involves p38 phosphorylation and enhanced ATF-2/AP-1 expressions, leading to caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 CytoDeath staining, and subsequent apoptotic cell death.