Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells.
OBJECTIVE
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 (P-gp) and multidrug resistance-associated protein-1 (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 interfering functioning of P-gp, their toxicities limit their usage in clinics. Consequently, RNA interference mediated sequence specific inhibition of the expression of P-gp/MDR1 mRNA may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was resensitizing doxorubicin-resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA.


METHODS
The effect of the selected MDR1 siRNA, and MRP1 expression after MDR1 silencing was determined by qPCR analysis. Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA. XTT cell proliferation assay was performed to determine the effect of MDR1 silencing on doxorubicin sensitivity.


RESULTS
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. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and relocalization of the drug in the nuclei. Introduction of siRNA resulted in about 70% resensitization to doxorubicin.


CONCLUSIONS
Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemosensitivity of resistant cells, which makes it a suitable candidate for therapeutic applications.
Middle East Technical University, Department of Biological Sciences, 06531 Ankara, Turkey.
Yaprak
Y
Dönmez
Ufuk
U
Gündüz
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000493:pharmacokinetics / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004317:Doxorubicin