Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma.
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.
Ozvaran
Mustafa K
MK
Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Cao
Xiaobo X
XX
Miller
Steven D
SD
Monia
Brett A
BA
Hong
Waun Ki
WK
Smythe
W Roy
WR
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology* / Q000633:toxicity; D015536:Down-Regulation; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D006801:Humans; D008654:Mesothelioma / Q000188:drug therapy* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D010997:Pleural Neoplasms / Q000188:drug therapy* / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism*; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D002945:Cisplatin