Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen)

Abstract

Purpose: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells.Experimental Design: In this work, we down-regulated the expression of bcl-2 protein by 80 -90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells.Results: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mockinfected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously.Conclusions: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.

Antisense Bcl-2 sensitizes prostate cancer cells to radiation

“…One explanation is that the quantity of endogenous Bcl-2 may not be a critical determinant of AS efficacy (36)(37)(38). However, it is also possible that the early apoptotic response measured represents a point in time which may be discordant with overall cell death (31).…”

Gene profiling study of G3139- and Bcl-2-targeting siRNAs identifies a unique G3139 molecular signature

“…All transfections were performed in a mixture of Opti-MEM and complete media without antibiotics, as described previously. 6 The transfection incubation time for siRNA/Lipofectamine 2000 complexes was 24 h. The total incubation time before cell lysis and protein isolation was 72 h.…”

“…All Western blots were performed as described previously, 6 and quantitated by laser-scanning densitometry.…”

“…While preclinical studies revealed that G3139 induced a robust apoptotic/cytostatic phenotype in LNCaP and PC3 cells, respectively, small interfering RNA (siRNA) targeting the Bcl2 transcript failed to generate similar outcomes. 6 This finding brings into question the mechanism of action of G3139 (and possibly related PS-ODNs), suggesting that knockdown of Bcl-2 alone is unrelated to or insufficient to explain the cell culture phenotypes.…”

Antisense and nonantisense effects of antisense Bcl-2 on multiple roles of Bcl-2 as a chemosensitizer in cancer therapy

“…In fact, a previous study showed that ROS in PC3 and DU145 prostate cancer cells was produced by AS Bcl-2, which contributed to its induction of an antitumor effect. 40,41 Another report indicated that the enhanced antitumor effect by 5-fluorouracil with AS Bcl-2 was less than that of other anticancer drugs such as cisplatin and paclitaxel in gastric cancer cells. 21 Nonapoptotic cell death.…”