RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells

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Abstract

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.

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Materials and methods

Cell lines and cell culture. The human ovarian carcinoma cell line A2780 and its cisplatin-resistant subline A2780RCIS were cultivated in supplemented Leibovitz L-15 medium as described previously [4]. To ensure maintenance of the cisplatin-resistant phenotype, medium of A2780RCIS contained 10 μg/ml cisplatin (GRY-Pharma, Kirchzarten, Germany). Medium used for cultivation of stably transfected cell clones of A2780RCIS contained 400 μg/ml zeocin (Invitrogen, Carlsbad, CA, USA).

Cell proliferation

Modulation of ABCC2 mRNA expression by siRNAs

Two different siRNA constructs, ABCC2-A and ABCC2-B, were used to decrease the mRNA expression of ABCC2 in the cisplatin-resistant human ovarian cancer cell line A2780RCIS. ABCC2-A was designed according to general recommendations of siRNA selection, the target sequence of ABCC2-B was chosen to be homologous to a well-characterized hammerhead ribozyme cleavage site within the ABCC2-specific mRNA. Quantitative real-time RT-PCR experiments were performed to quantify ABCC2 mRNA expression values

Conclusions

In conclusion, RNAi technology is effective to modulate the ABCC2 expression in human ovarian carcinoma cells. The RNAi technology showed a comparable gene-silencing efficiency compared to hammerhead ribozymes targeting the identical site of the ABCC2 mRNA sequence in these cells. Thus, the data demonstrate the utility of the analyzed siRNAs and shRNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing

Acknowledgments

This work was supported by Grant LA 1039/2-3 of the “Deutsche Forschungsgemeinschaft” (DFG), by the “RNA-network” funded by the “Bundesministerium für Bildung und Forschung” (BMBF) and Berlin, and oligene GmbH.

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