Related Experiment Videos
Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product
W Roy Smythe1, Imran Mohuiddin, Mustafa Ozveran
1Department of Thoracic and Cardiovascular Surgery, Section of Thoracic Molecular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, 77030, USA. rsmythe@mdanderson.org
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.
Conclusion:
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.
Insights
Antisense oligonucleotides targeting bcl-xl messenger RNA induce apoptosis in malignant pleural mesothelioma cells. This approach shows therapeutic potential for treating this therapy-resistant cancer.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Malignant pleural mesothelioma exhibits resistance to conventional therapies and apoptosis.
- The bcl-2 gene family regulates apoptosis; mesothelioma cells express antiapoptotic Bcl-xl and proapoptotic Bax/Bak.
- Previous studies indicated pharmacologic inhibition of Bcl-xl can induce apoptosis.
Purpose of the Study:
- To investigate if antisense oligonucleotides targeting bcl-xl messenger RNA can induce apoptosis in mesothelioma.
- To determine if this approach creates an "forced imbalance" in bcl-2 family proteins.
- To assess the efficacy of antisense oligonucleotides with and without liposomal delivery.
Main Methods:
- Malignant pleural mesothelioma cell lines (REN and I-45) were treated with bcl-xl antisense oligonucleotides.
- Controls included untreated cells and sense oligonucleotides.
- Apoptosis was assessed via Hoechst staining and flow cytometry; cell viability was measured colorimetrically.
Main Results:
- Antisense oligonucleotides significantly downregulated Bcl-xl protein expression (>65%) in both cell lines.
- Significant cell killing was observed with antisense oligonucleotides compared to sense controls (P < .006).
- Apoptosis was confirmed as the mechanism of cell death; liposomal delivery enhanced efficacy and allowed lower doses.
Conclusions:
- Antisense oligonucleotides targeting bcl-xl effectively induce apoptosis in mesothelioma cell lines.
- Inhibiting Bcl-xl expression presents a promising therapeutic strategy for mesothelioma.
- Further evaluation of this therapeutic potential in mesothelioma is warranted.