Date

2008

Author

Demirel Kars, Meltem

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Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

Subject Keywords

General biology., Genetics.

URI

http://etd.lib.metu.edu.tr/upload/3/12610241/index.pdf
https://hdl.handle.net/11511/18165

Collections

Graduate School of Natural and Applied Sciences, Thesis