PAK-104P

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.