MRP-1 is the dominant MDR protein in ESFT cells and protects cells from chemotherapy-induced cell death.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 3253 Multi-drug resistance (MDR) is frequently associated with over-expression of the MDR transporter proteins MRP-1and PGP, which function as ATP-dependent pumps to transport anticancer drugs from cells. We have recently shown that MRP-1 is expressed in 92% of primary ESFT (Abstract BACR3, NCRI 2007). The aim of this study was to examine the expression and functional significance of MRP-1 and PGP. MDR protein expression was examined in six ESFT cell lines by flow cytometry, immunoblot and qRT-PCR. The biological significance of MDR proteins was explored using Calcein-AM flow cytometry in combination with the MRP-1 competitive inhibitor MK571. The effect of inhibitor alone and in combination with vincristine, doxorubicin, actinomycin D or etoposide was investigated. Viable cell number was measured using the trypan blue exclusion assay. Expression of MRP-1 protein in ESFT cell lines was high compared to the expression of PGP. There was no correlation between MRP-1 mRNA and protein expression detected by either flow cytometry (r=0.166) or immunoblot (r=0.506). MRP-1 protein was detected in both the mitochondria and membrane fractions of ESFT cell lines. Expression of MRP-1 in the mitochondria was inversely proportional to expression in the membrane. Expression of PGP was not identified in the mitochondria. Inhibition of MRP-1 with MK571 (10-50μM) decreased ESFT viable cell number at 48h; MK571 (50μM) in low (TC-32) and high (SK-N-MC) MRP-1 expressing cells decreased cell viability to 37% and 69% respectively. Pretreatment of ESFT cells for 1h with MK571 (30μM) prior to treatment with vincristine (p=0.003), actinomycin D (p=0.008), fenretinide (p=0.074) and etoposide (p=0.081) resulted in enhanced cell death at 48h. Cells expressing high levels of MRP-1 effluxed more fluorescent calcein than low expressing cell lines (r= 0.93), consistent with the hypothesis that expression of MRP-1 protein correlates with functional activity. In conclusion, MRP-1 is the dominant MDR protein in ESFT and is expressed in the membrane and mitochondria. The functional significance of mitochondrial MRP-1 is currently under investigation. Knockdown of MRP-1 enhances the responsiveness of ESFT cells to common chemotherapeutic agents and consequently may be of therapeutic benefit to ESFT patients.