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


METHODS
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.
Benimetskaya
Luba
L
Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York 10467, USA.
Lai
Johnathan C
JC
Khvorova
Anastasia
A
Wu
Sijian
S
Hua
Emily
E
Miller
Paul
P
Zhang
Li-Ming
LM
Stein
Cy A
CA
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D000077143:Docetaxel; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D043823:Taxoids / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043823:Taxoids; D013873:Thionucleotides; D003841:Deoxycytidine; D000077143:Docetaxel; D019284:Thapsigargin; C408162:oblimersen; D002945:Cisplatin; D000093542:Gemcitabine