Background Deregulation of apoptosis is one of the causes of cancer developing. The Bcl-2 family are central regulators of apoptosis. They are subdivided into two classes, the proapoptotic members (which include Bim) and antiapoptotic members (like Bcl-2). The overexpression of Bcl-2 is generally associated with many cancers and resistance to chemotherapy, including microtubule-targeting agents (MTAs). Therefore several anti-Bcl-2 strategies are in development. Unexpectedly, several studies show that a decrease in Bcl-2 may be associated with resistance to MTAs. This paradoxical role of Bcl-2 has not yet found a clear explanation.
Purpose To show that overexpression of Bcl-2 leads to overexpression of Bim, which is responsible for increasing sensitivity to MTAs. Bim is a potential biomarker which may be included in tests to predict the response to paclitaxel treatment in human lung carcinoma. Our work also enables a better understanding of how Bim regulates genes.
Materials and Methods The techniques used to study the sensitivity of cells to MTAs are the Western Blot and immunofluorescence. To study Bim’s regulation of genes, we used the technique of a reporter gene.
Results Firstly, we showed that overexpression of Bcl-2 in human lung carcinoma cells (A549 Bcl-2) in turn triggers the overexpression of Bim. Apoptosis is detected after treatment with paclitaxel at 20 nM, after 24 hours. For this, we used the anti-caspase 9 antibody to show it was being cleaved and to signal the release of the apoptosis mitochondrial pathway. To confirm this, we used immunofluorescence staining to objectify the release of cytochrome c from the mitochondria. So we showed that the overexpression of Bim in cells that overexpress Bcl-2 accounts for their increased sensitivity to paclitaxel.
We also conducted a study of gene regulation by Bim in A549 cells overexpressing Bcl-2. We highlighted the increasing transcriptional activity of Bim promoter by a factor of 2.3 ± 0.2 compared to control cells. The Bim protein level seems to be a better determinant of MTAs sensitivity than Bcl-2 status in pulmonary epithelial tumours. Thus, it appears that Bim expression may be an effective biomarker in predicting the efficiency of MTA treatment. We are currently evaluating the involvement of various transcription factors, especially by DNA microarray.
Conclusions These data suggest that Bim is a more reliable marker of the sensitivity to MTAs than Bcl-2. A test showing the level of Bim expression may be able to predict therapeutic efficacy and/or resistance based on molecular profiling of the tumours. However, the induction of Bim alone cannot be sufficient for significant cell death. Indeed, it is more likely that Bim acts in unison with the other pro-apoptotic proteins. So the development of targeted therapies, on the Bcl-2 family in particular, must await a better understanding of the molecular mechanism involved in the regulation of apoptosis.
No conflict of interest.
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