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.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12610241/index.pdf |
| Date | 01 December 2008 |
| Creators | Demirel Kars, Meltem |
| Contributors | Gunduz, Ufuk |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | Ph.D. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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