Abstract
BackgroundHigh resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes. ABCC2, an ATP-binding cassette multidrug resistance transporter, is found to be expressed in a variety of human cancers. In this study the effect of a RNAi construct targeting ABCC2 on the chemosensitivity of NPC cell line CNE2 against cisplatin was investigated.MethodsLentiviral vectors were constructed to allow an efficient expression of anti-ABCC2 siRNA. The effective target sequence comprised nucleotides 1707–1727 of the human ABCC2 mRNA. The cell clones expressing the construct were picked and expanded, followed by identification using qRT-PCR and western blot method. As control, lentiviral vector containing invalid RNAi sequence was transfected to CNE2 cells. In vitro, cellular accumulation of cisplatin was detected by HPLC. The capacity of cellular growth and sensitivity of cells against cisplatin were detected by MTT assay. In vivo, the sensitivity of the tumor tissues against cisplatin were evaluated by transplanted CNE2 nude mice model.ResultsTwo CNE2 cell clones with reduced expression of targeted ABCC2 mRNA and protein for more than 70% by qRT-PCR and western blot were established, and no differences were shown in proliferation rates compared to control CNE2 cells by growth curves analysis. In vitro the accumulation of intracellular cisplatin in these CNE2 cell clones with reduced expression of ABCC2 increased markedly, accompanied by increased sensitivity against cisplatin. In vivo, the growth of CNE2 solid tumors with a stably transfected anti-ABCC2 siRNA construct was significantly inhibited by cisplatin in transplanted nude mice model.ConclusionOur investigation demonstrated that lentivirus-mediated RNAi silencing targeting ABCC2 might reverse the ABCC2-related drug resistance of NPC cell line CNE2 against cisplatin.
Highlights
High resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes
ABCC2 mRNA is highly expressed in CNE2 cell line ABCC2 is normally expressed on the apical membrane of hepatocytes, and encodes a major organic anion transporter in the canalicular membrane of hepatocytes [11]
The expression of ABCC2 mRNA was found in Nasopharyngeal carcinoma (NPC) cell lines, with the highest expression in CNE2 cell line compared to human immortalized nasopharyngeal epithelial cell line NP69 by quantitative RT-PCR method (Fig. 1A), which indicated that CNE2 cell line is a suitable cell model for RNAi targeting ABCC2 mRNA
Summary
High resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes. ABCC2, an ATP-binding cassette multidrug resistance transporter, is found to be expressed in a variety of human cancers. Of all the chemotherapy drugs, cisplatin is the most effective cytotoxic agent used in NPC treatments. Reduction in cellular accumulation of cisplatin is one of the principal mechanisms of resistance, which may be ascribed to an increase in drug efflux. The adenosine triphosphate binding cassette (ABC) transporter families, whose products represent membrane proteins, have the capability to use energy to drive the transporters of various molecules across the cellular membrane, and are confirmed to be associated with anticancer drug transporter [5,6]
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