Molecular mechanism of aven in taxane induced apoptosis in ER± breast cancer cell lines: MCF-7 and MDA-MB-231 cells
Verim, Aysegül (2006) Molecular mechanism of aven in taxane induced apoptosis in ER± breast cancer cell lines: MCF-7 and MDA-MB-231 cells. [Thesis]
In this study, the molecular mechanism of Aven in taxane induced apoptosis were studied in ER(+) MCF-7 and ER(-) MDA-MB-231 cell lines. Taxanes, paclitaxel and docetaxel, are cytotoxic agents which act by promoting deformation of stable microtubules, inhibiting the normal dynamic reorganization of microtubule networks required for mitosis. First the dose kinetics of apoptotic response towards the above mentioned drugs with these two cell lines were established. Cytotoxicity was determined with MTT assay after cells were treated with the drugs for 24 hours. 50nM Paclitaxel treatment for 24h inhibited growth of MCF-7 by 58 percent and MDA-MB-231 by 44 percent. Whereas 50nM Docetaxel caused growth inhibiton by 55 percent and 70 percent for 24h in MCF-7 and MDA-MB-231 cell lines respectively. Then Aven protein levels in response to these drugs were studied by western blot analysis. The western blot results showed that Aven protein levels were not changing in response to taxanes in these cell lines. After that the effect of the drugs on protein-protein interaction was investigated with CoIP experiments. Endogeneous Bcl-xL protein was immunoprecipitated with specific Bcl-xL antibody and then immunoblotting was done with anti-Aven antibody. The interaction of Aven with Bcl-xL was not abolished with taxane treatment. The phosphorylation of Bcl-xL with taxane treatment was also shown by western blot analaysis. The overexpression of Aven by transfection of both cell lines with pSG-HA-Aven plasmid was performed and then Aven overexpression was checked by western blot analysis. As a control, immunoprecipitation of total protein extract with anti-HA antibody was made. But results showed non-specific binding of endogeneous Aven to HA antibody. As a second strategy, gene silencing was used. Aven expression was downregulated by Hs_Aven_3HP siRNA and protein level. The mRNA level of Aven was determined first with one step RT PCR and then with quantitative real time RT PCR. The downregulation of Aven was 2.3 fold in MCF-7 cells and 1.7 fold MDA-MB-231 cells. After that, taxane induced apoptotic cell death was investigated by M30 Apoptosense ELISA Kit. The overexpression study showed that taxane induced apoptosis in MCF-7 cells increased by 2 fold. Whereas downregulation of Aven protects MCF-7 cells from apoptosis induced by taxanes. For cisplatin, Aven overexpression resulted in less apoptotic cell death in MCF-7 cells. In MDA-MB-231 cells, overexpression of Aven sensitizes cells for apoptotic cell death. When Aven was downregulated, drugs induced less apoptotic cell death.
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