Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen) Anthony Raffo, 1 Johnathan C. Lai, 3 C. A. Stein, 1,2 Paul Miller, 4 Steven Scaringe, 5 Anastasia Khvorova, 5 and Luba Benimetskaya 1 Departments of 1 Medicine, 2 Pharmacology, and 3 Biomedical Engineering, Columbia University, New York, New York; 4 Johns Hopkins University, School of Public Health, Baltimore, Maryland; and 5 Dharmacon Research, Inc., Lafayette, Colorado ABSTRACT Purpose: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemother- apy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleo- tide 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 extrapo- lation 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 inter- fering 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 (inhibi- tion of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock- infected 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 inde- pendent. INTRODUCTION Because it is an important regulator of the apoptosis proc- ess, bcl-2 is currently a frequently investigated target for antin- eoplastic therapeutic intervention (1– 4). Elevation of expression of this antiapoptotic protein appears to contribute to resistance to a variety of antineoplastic agents, both in vitro and in vivo, which include cis-platinum, taxanes, mitoxantrone, Adriamycin, and dexamethasone, in addition to radiation (5–10). The bcl-2 protein, whereas expressed in very few normal tissues (e.g., B lymphocytes and colon crypt cells), is in contrast, highly ex- pressed in human tumors as diverse as prostate, colorectal, lung, gastric, non-Hodgkin’s lymphoma, and in many leukemias (re- viewed in Ref. 11). Use of a bcl-2 antisense RNA strategy in MCF-7 cells caused down-regulation of bcl-2 protein expression and sensitization of cells to Adriamycin (12). In HL60 cells, induction of an antisense bcl-2 mRNA caused inhibition of cell growth and autophagy, but not apoptosis (13). Similarly, Zhang et al. (14) demonstrated that introduction of an exogenous antisense bcl-2 mRNA increased the sensitivity of a human gastric adenocarcinoma cell line to phototoxic treatment. In human prostate cancer PC3 cells, attempted down-regulation of bcl-2 protein expression with an adenovirally delivered ri- bozyme (15–17) was shown to induce cellular apoptosis. Antisense oligonucleotides such as G3139, an 18mer phos- phorothioate oligodeoxynucleotide targeted to the initiation codon region of the bcl-2 mRNA (18), have also been used extensively to down-regulate the expression of bcl-2 expression (19 –22), and to induce chemosensitization. The clinical utility of this strategy has been extensively demonstrated by Jansen et al. (23, 24) in a Phase II clinical trial in which 6 of 14 heavily pretreated patients with advanced melanoma responded to treat- ment with an antisense bcl-2 oligonucleotide in combination with dacarbazine. This oligonucleotide, known as G3139, Ge- nasense, or Oblimersen, is currently being evaluated in combi- nation with dacarbazine in Phase III clinical trials in advanced melanoma, as well as in other tumors, including chronic lym- phatic leukemia, myeloma, non-small cell lung cancer, and prostate cancer. Whereas we (25, 26) and others have demonstrated that G3139 can down-regulate, by 90%, the expression of bcl-2 protein and mRNA in several prostate cancer cell lines in tissue culture, we have also demonstrated that the effects of G3139 on the phenotype of treated PC3 cells are complex, and at least partially independent of its effects on bcl-2 expression. G3139 treatment did not produce significant cellular apoptosis in PC3 cells (as determined by lack of procaspase 3 cleavage and increase in cell surface Annexin V expression), as it does in many other cell lines. Concordant with the lack of apoptosis, Received 9/25/03; revised 1/14/04; accepted 1/21/04. Grant support: NIH Grant GM58791 (C. Stein and P. Miller). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: C. Stein and L. Benimetskaya are currently at the Albert Einstein- Montefiore Cancer Center, Department of Oncology, Montefiore Med- ical Center, Bronx, New York. Requests for reprints: C. A. Stein, Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, 111 E. 210 Street, Bronx, NY 10467. Phone: (718) 920-8980; Fax: (718) 652- 4027; E-mail: cstein@montefiore.org. 3195 Vol. 10, 3195–3206, May 1, 2004 Clinical Cancer Research Downloaded from http://aacrjournals.org/clincancerres/article-pdf/10/9/3195/1956991/zdf00904003195.pdf by guest on 15 June 2022