Relative Bcl-2 independence of drug-induced cytotoxicity and resistance in 518A2 melanoma cells

Purpose: Inhibition of the function of Bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy. G3139 (Genasense) is a phosphorothioate anti-Bcl-2 antisense oligonucleotide, but its mechanism of action is uncertain. The aim of the present work is to investigate inhibition of Bcl-2 expression in 518A2 melanoma cells, the cell line on which recent phase II and phase III clinical trials employing this agent were based. Experimental Design: We down-regulated the expression of Bcl-2 protein by two different strategies in these cells: one employing G3139 and controls, and the other using a small interfering RNA approach. Cell viability after treatment with oligonucleotides or small interfering RNA and cytotoxic agents including gemcitibine, DDP, docetaxel, and thapsigargin was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. A 518A2 melanoma cell line stably overexpressing Bcl-2 protein was constructed and treated with either these cytotoxic agents or G3139. Results: The cytotoxic effects of either G3139 or small interfering RNA treatment of 518A2 melanoma cells are Bcl-2 independent. In addition, in the Bcl-2-overexpressing cells, only a modest increment in chemoresistance was observed, and treatment with G3139 not only did not down-regulate Bcl-2 expression but produced essentially identical toxicity as was observed in the wild-type or mock-transfected cells. Conclusions: Our results suggest that the mechanism whereby G3139 produces drug-induced cytotoxicity in the 518A2 melanoma line is not dependent on levels of Bcl-2. These findings emphasize the nonsequence specific effects of this phosphorothioate oligonucleotide and call into question the validity of Bcl-2 as a target in this cell line.