Studies on the mechanism of action of the chemotherapeutic drug bleomycin on cell lines derived from haemangioma and keloid

Abstract

Bleomycin has been used successfully in the treatment of haemangioma, keloid and vascular malformation; however the mode of action of this chemotherapeutic drug on these non-malignant dysplasias is not well understood. The aim of this study was to investigate the effects of bleomycin on a range of human primary cell lines to improve our understanding of the mechanism by which bleomycin exerts its effects. This may facilitate the subsequent identification of alternative therapeutic strategies, thereby avoiding the well recognised and potentially life threatening side effects associated with the use of bleomycin. Primary cell lines were isolated from excised lesions using an explant culture technique. The cell lines thus obtained, along with other cell lines previously derived from other tissues, were exposed to a range of concentrations of bleomycin to examine dose response. Results showed that at a dose of l00mU/ml bleomycin, all cell lines underwent a G1 arrest after 48 hours in culture. At higher doses, bleomycin induced a dose-dependent increase in the proportion of cells in sub- Gl with all cell lines treated, a specific marker of apoptosis. Bleomycin induced apoptosis was further confirmed by TUNEL assay and results showed that keloid derived cells had a significantly greater level of DNA strand breaks than foreskin fibroblasts. An antibody specific for cleaved caspase-3 was also used to investigate apoptosis initiation. Both keloid derived cells and foreskin fibroblasts showed a bleomycin-induced cleavage of caspase-3, however the level of caspase-3 cleavage was significantly greater in keloid derived cells. In conclusion, results from this study show that bleomycin induces apoptosis of haemangioma, venous malformation and keloid derived cells in a dose dependent manner. In addition, this study reports for the first time that bleomycin induces a caspase-3 mediated apoptotic cell death in keloid derived cells.