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Chemosensitization of bladder carcinoma cells by bcl-xL antisense oligonucleotides
1Departments of Medicine and Pharmacology, Columbia University, New York, New York, USA.
Purpose:
We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines.
Materials And Methods:
A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test.
Results:
Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent.
Conclusions:
Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.
Insights
Antisense therapy targeting anti-apoptotic proteins like bcl-xL can sensitize bladder cancer cells to chemotherapy. This approach enhances the effectiveness of cytotoxic agents in treating bladder carcinoma.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Anti-apoptotic proteins, including B-cell lymphoma-extra large (bcl-xL) and B-cell lymphoma 2 (bcl-2), play a crucial role in cancer cell survival.
- Dysregulation of these proteins contributes to chemoresistance in various cancers, including bladder carcinoma.
- Targeting these proteins offers a potential strategy to overcome drug resistance.
Purpose of the Study:
- To investigate the efficacy of antisense inhibition of bcl-xL and bcl-2 proteins in enhancing chemosensitization in bladder carcinoma cell lines.
- To evaluate the impact of antisense oligonucleotides on bcl-xL protein expression and subsequent chemosensitivity.
Main Methods:
- Construction of a T24 bladder carcinoma cell line overexpressing bcl-xL.
- Treatment of T24 and 5637 cells with C5-propynylated and 2'-O-methylribo-oligonucleotides to down-regulate bcl-xL expression.
- Assessment of apoptosis using cell cycle analysis and Annexin V binding.
- Quantification of protein and messenger RNA levels via Western and Northern blot analysis.
- Measurement of cell viability after combined treatment with oligonucleotides and cytotoxic agents using the MTT assay.
Main Results:
- Overexpression of bcl-xL conferred resistance to cytotoxic agents in T24 cells.
- Antisense oligonucleotides effectively reduced bcl-xL protein expression in both T24 and 5637 cell lines.
- Down-regulation of bcl-xL by antisense therapy significantly increased the sensitivity of bladder carcinoma cells to cytotoxic agents.
- The efficiency of antisense-mediated down-regulation was influenced by the delivery agent used.
Conclusions:
- Antisense inhibition of bcl-xL protein is a viable strategy to sensitize bladder carcinoma cells to cytotoxic chemotherapy.
- The observed chemosensitization may involve a combination of sequence-specific antisense effects and non-specific effects related to delivery agents and oligonucleotide properties.