Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein and MRP1, encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by impairing the function of P-gp, they exhibit severe toxicities limiting their clinical relevance. Consequently, selective blocking of the expression of P-gp/MDR1 specific mRNA through RNA interference strategy may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy.
Aim of this study was re-sensitizing doxorubicin resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. The effect of the selected MDR1 siRNA and MRP1 expression after MDR1 silencing was determined by qPCR analysis. XTT cell proliferation assay was performed to
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determine the effect of MDR1 silencing on doxorubicin sensitivity.Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA or other MDR modulators / verapamil or promethazine. The role of P-gp in migration characteristics of resistant cells was evaluated by wound healing assay.
The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However the level of MRP1 mRNA did not change after MDR1 downregulation. Introduction of siRNA resulted in about 70% re-sensitization to doxorubicin. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and re-localization of the drug to the nuclei. Despite the considerably high concentration of the modulators, verapamil and promethazine were not as effective as siRNA for reversal of the drug efflux. According to wound healing assay, MDR1 silencing did not have any effect on migration characteristics of resistant cells, that is, P-gp expression does not seem to affect the motility of the cells.
Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemo-sensitivity of resistant cells, which makes it a suitable future candidate for therapeutic applications.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12611496/index.pdf |
| Date | 01 February 2010 |
| Creators | Donmez, Yaprak |
| Contributors | Gunduz, Ufuk |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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