Activity of a novel bcl-2/bcl-xL-bispecific antisense oligonucleotide against tumors of diverse histologic origins

Background: Increased expression of the anti-apoptotic proteins Bcl-2 and B cl-xL is involved in the development and progression of many tumors. We rec ently reported that the bcl-2/bcl-xL-bispecific antisense oligonucleotide 4 625 induces apoptosis in lung carcinoma cells. To further assess the therap eutic potential of oligonucleotide 4625, we investigated its effect on a se ries of human tumor cell lines of diverse histologic origins in vitro and i n vivo. Methods: Oligonucleotide 4625-mediated inhibition of bcl-2 and bcl- xL expression in vitro was measured in breast carcinoma cells with the use of reverse transcription-polymerase chain reaction (PCR), real-time PCR, an d western blotting. Cytotoxicity was assessed in several different cell lin es by measurement of tumor cell growth, propidium iodide uptake, and nuclea r apoptosis. The in vivo activity of oligonucleotide 4625 was determined by the inhibition of growth of established tumor xenografts in nude mice, imm unohistochemical staining of Bcl-2 and Bcl-x proteins in the tumors, and we stern blotting of tumor lysates. Apoptosis in tumor xenografts was detected with the use of in situ TUNEL (i.e., terminal deoxynucleotidyl transferase -mediated deoxyuridine triphosphate-digoxigenin nick end labeling) staining . All statistical tests are two-sided. Results: In breast carcinoma cells, oligonucleotide 4625 treatment reduced bcl-2 and bcl-xL messenger RNA level s in a dose-dependent manner. At 600 mM, oligonucleotide 4625 reduced Bcl-2 and Bcl-xL protein levels to 25% (95% confidence interval [CI] = 16% to 34 %) and 20% (95% CI = 14% to 26%), respectively, of the levels in untreated cells and it decreased viability in all cell lines mainly by inducing apopt osis. In vivo, oligonucleotide 4625 statistically significantly inhibited t he growth of breast and colorectal carcinoma xenografts by 51% (95% CI = 28 % to 74%) and 59% (95% CI = 44% to 74%), respectively, relative to those tr eated with control oligonucleotide 4626; it also reduced Bcl-2 and Bcl-xL p rotein levels and induced tumor cell apoptosis. Conclusion: The bcl-2/bcl-x L-bispecific antisense oligonucleotide 4625 merits further study as a novel compound for cancer therapy.