Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma

Abstract

Objective: Malignant pleural mesothelioma (MPM) is resistant to both conventional chemotherapy and apoptosis. The bcl-2 family proteins are major determinants of apoptotic homeostasis. MPM lines and tumors routinely overexpress the anti-apoptotic protein BCL-XL. We have previously shown that antisense inhibition of BCL-XL in MPM cells leads to apoptosis. We sought to determine whether antisense oligonucleotides directed at the bcl-xl gene product would augment response to a conventional chemotherapeutic agent in human mesothelioma cell lines.

Methods: The human MPM cell lines REN and I-45 were exposed to two bcl-xl antisense oligonucleotides (15999, 16009) and one sense oligonucleotide (113529) construct at varying doses, followed by IC(50) cisplatin. Cellular viability was assessed by a calorimetric assay, and apoptosis was evaluated by Hoechst staining, Annexin V staining, and sub-G(1) fluorescence-activated cell sorter analysis. Western blot analysis of BCL-2 family proteins was performed following single agent and combined treatment. Isobologram mathematical analysis was used to determine whether or not combination therapies were additive or synergistic.

Results: Cell viability was most affected with the 15999 antisense oligonucleotides plus IC(50) cisplatin combination (70% of I-45 and 90% of REN cells killed), and apoptosis was markedly increased with this combination by all measures. Western blot demonstrated 15999 antisense oligonucleotides construct down-regulation of BCL-XL, but no further effect on expression of BCL-2 proteins with cisplatin. Isobologram analysis demonstrated 15999 + cisplatin synergistic effect.

Conclusions: Exposure of human MPM cells to bcl-xl antisense oligonucleotides sensitizes human mesothelioma cells to the conventional chemotherapeutic agent cisplatin. Similar approaches using a combination of molecular and conventional treatment may have clinical utility for this tumor.