Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product

Our plain-English summary of Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product, by W.R. Smythe, I. Mohuiddin, M. Ozveran, et al (The Journal of Thoracic and Cardiovascular Surgery, 2002).

The study

• Authors: W.R. Smythe, I. Mohuiddin, M. Ozveran, X.X. Cao
• Journal: The Journal of Thoracic and Cardiovascular Surgery
• Published: 2002;123(6):1191-8
• Cited by: 39 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

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.

In plain English

This was a laboratory study using two human malignant pleural mesothelioma cell lines grown in dishes: REN (epithelial type) and I-45 (sarcomatous type). No patients, animals or asbestos exposures were involved. The authors tested short man-made DNA strands, called antisense oligonucleotides, designed to block production of Bcl-xl, a protein that helps cells avoid programmed cell death (apoptosis). Matching 'sense' strands served as the comparison. Compared with sense strands, antisense strands lowered Bcl-xl by 78% in REN and 67% in I-45 cells. Without a fat-based (liposomal) carrier, about 40% of REN and 50% of I-45 cells were killed at the highest dose tested. With the carrier, at a much lower dose of 100 nmol/L, about 60% of REN and 90% of I-45 cells were affected. However, the carrier given with sense strands also killed more than 20% of cells. These antisense-versus-sense differences were statistically significant. Staining and cell-sorting tests indicated that the cells died by apoptosis. The authors concluded that the therapeutic potential of blocking Bcl-xl in mesothelioma should be evaluated.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment on human mesothelioma cell lines
• Who was studied: Two human malignant pleural mesothelioma cell lines: I-45 (sarcomatous type, p53 wild-type, donated from the Fox Chase Institute) and REN (epithelial type, p53 mutant, developed by author W.R.S.). Work was done at the University of Texas M.D. Anderson Cancer Center, Houston; no patients were studied.
• Exposure: No asbestos exposure was studied. The cells were treated with modified 20-unit bcl-xl antisense oligonucleotides, with matching sense oligonucleotides and untreated cells as controls, with and without a liposomal delivery system (Oligofectamine).
• Main results:
  • Compared with sense oligonucleotide exposure, antisense exposure decreased Bcl-xl protein by 78% in REN cells (22% of control) and by 67% in I-45 cells (33% of control). Actin protein, used as a control, was not affected.
  • Without liposomes, about 40% of REN cells and 50% of I-45 cells were killed at the highest antisense dose tested (printed as 200, with the unit prefix symbol lost). The difference from sense oligonucleotides was significant by analysis of variance (REN P .006; I-45 P .003). Sense oligonucleotides alone had little effect.
  • With liposomal delivery, much lower doses worked. At 100 nmol/L, about 60% of REN cells and 90% of I-45 cells were affected (REN P .0005, I-45 P .006, by analysis of variance).
  • The liposome carrier itself was toxic: more than 20% of cells were killed by the sense oligonucleotide-liposome combination in both REN and I-45 cells.
  • Hoechst staining showed fragmentation consistent with apoptosis in antisense-exposed cells (data not shown). Sub-G1 cell-sorting analysis, shown for the I-45 line, found apoptosis increased with antisense relative to sense oligonucleotides, and more so with liposomal delivery.
• The authors' conclusion: The authors concluded that antisense oligonucleotides directed at the bcl-xl gene product cause apoptosis in mesothelioma cell lines, and that the therapeutic potential of inhibiting this protein in mesothelioma should be evaluated.
• Limitations the authors state:
  • The liposomal delivery system caused nonspecific toxicity, killing more than 20% of cells even with sense oligonucleotides.
  • In the published meeting discussion, the author stated that the effect peaked between 24 and 48 hours, after which cells began to regrow, and that the cells resistant to antisense had not been examined.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma, Pleural Mesothelioma, Apoptosis, Mesothelioma.

Related studies

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• Downregulation of Bcl-xL and Mcl-1 is sufficient to induce cell death in mesothelioma cells highly refractory to conventional chemotherapy — E. Varin, C. Denoyelle, et al. (2010)
• Process analysis of carcinogenesis: concept derivation of the tissue function "preservation of a homogeneous gene expression" — T. Löser (2018)
• Exosomal miR-16-5p as a target for malignant mesothelioma — P.B. Munson, E.M. Hall, et al. (2019)
• Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells — Y. Miura, Y. Nishimura, et al. (2006)
• Use of Anti-Noxa Antibody for Differential Diagnosis between Epithelioid Mesothelioma and Reactive Mesothelial Hyperplasia — K. Kushitani, V.J. Amatya, et al. (2016)

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