Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product.
OBJECTIVE
Malignant pleural mesothelioma is resistant to conventional therapies and to apoptosis. The bcl-2 family genes are major determinants of apoptotic homeostasis. Malignant pleural mesothelioma lines and tumors rarely express the antiapoptotic Bcl-2 protein but routinely express the antiapoptotic protein Bcl-xl and the proapoptotic proteins Bax and Bak. We have previously shown pharmacologic inhibition of bcl-xl expression in malignant pleural mesothelioma can lead to apoptosis, so we sought to determine whether antisense oligonucleotides directed at bcl-xl messenger RNA would engender apoptosis, possibly through a "forced imbalance" of bcl-2 family proteins.


METHODS
Malignant pleural mesothelioma lines REN (epithelial) and I-45 (sarcomatous) were exposed to modified bcl-xl antissense oligonecleotides directed near the messenger RNA initiation sequence with and without a liposomal delivery system. Untreated cells and bcl-xl sense oligonucleotides were controls. Cell viability was measured by colorimetric assay, and apoptosis was evaluated with Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis.


RESULTS
Bcl-xl protein expression after antisense oligonucleotides was downwardly regulated in both cell lines relative to sense oligonucleotides (>65%). Significant cellular killing in both the I-45 and REN cell lines was achieved with antisense oligonucleotides (compared with sense oligonucleotides) without (P =.003 and.006, respectively) and with (P =.006 and.0005, respectively) liposomal delivery. Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis demonstrated apoptosis to be the mechanism of cellular death. Use of a liposomal delivery system increased therapeutic effect and allowed lower doses of antisense oligonucleotides.


CONCLUSIONS
Antisense oligonucleotides directed at the bcl-xl gene product engender apoptosis in mesothelioma cell lines. The therapeutic potential of inhibiting expression of this protein in mesothelioma should be evaluated.
Smythe
W Roy
WR
Department of Thoracic and Cardiovascular Surgery, Section of Thoracic Molecular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, 77030, USA. rsmythe@mdanderson.org
Mohuiddin
Imran
I
Ozveran
Mustafa
M
Cao
Xiaobo X
XX
D017209:Apoptosis; D003124:Colorimetry; D015536:Down-Regulation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008654:Mesothelioma / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D010997:Pleural Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051020:bcl-X Protein