Inhibition of multi-drug resistance of ovarian carcinoma by small interfering RNA targeting to MRP2 gene.

AIMS: To investigate the inhibiting mechanism of multi-drug resistance (MDR) using expression vectors of short hairpin RNA (shRNA) in a MDR human ovarian cancer cell line (A2780/cp70).
METHODS: Two shRNA expression vectors were constructed and introduced into A2780/cp70 cells. Expression of MRP2 mRNA was assessed by RT-PCR, and Mrp2 expression was determined by Western blot and immunocytochemistry. Apoptosis and sensitization of the cells to cisplatinum were quantified by flow cytometry and methyl-thiazol-tetrazolium (MTT) assays. Cellular cisplatinum accumulation was assayed by laser scanning confocal microscopy (LSCM).
RESULTS: In A2780/cp70 cells transfected with MRP2-A and MRP2-B shRNA expression vectors, RT-PCR showed that MRP2 mRNA expression was reduced by 41.8% (P < 0.05), 30.9% (P < 0.01) (transient transfection) and 39.6% (P < 0.05), 29.4% (P < 0.01) (stable transfection), respectively. Western blot and immunocytochemistry showed that Mrp2 expression was significantly and specifically inhibited. Resistance against cisplatinum was decreased from 173- to 119-fold (P < 0.05), 64-fold (P < 0.01) (transient transfection) and to 117-fold (P < 0.05), 60-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular cisplatinum accumulation. The combination of shRNA vectors and cisplatinum significantly induced the apoptosis of cells.
CONCLUSIONS: shRNA expression vectors effectively reduce MRP2 expression and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.