Kiyotomo Seto

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells.

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. Int. J. Cancer 91:126–131, 2001.

Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximine in combination with PAK-104P.

The mechanism of multidrug resistance protein (MRP)-mediated multidrug resistance (MDR) is still unclear. MRP reportedly transports some GSH conjugates. Recently, we demonstrated that a pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4 -(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P), that reversed P-glycoprotein (P-gp)-mediated MDR directly interacted with MRP and completely reversed the vincristine (VCR) resistance in MRP-mediated MDR C-A120 cells. We investigated the reversing effect of PAK-104P in C-A120 cells in combination with buthionine sulfoximine (BSO), another MDR-reversing agent with a different reversing mechanism. In immunoblots, MRP was overexpressed in C-A120 cells. The level of ATP-dependent [3H]VCR uptake was high in membrane vesicles from KB-C2 cells, but low in those from C-A120 and parental KB-3-1 cells. The sensitivity to VCR of C-A120 cells, but not of KB-C2 cells, was considerably increased by 100 microM BSO. VCR accumulation in C-A120 cells, but not in KB-C2 cells, was also enhanced by BSO. BSO did not inhibit ATP-dependent [3H]LTC4 uptake in C-A120 vesicles. The combination of BSO with PAK-104P at their low concentrations resulted in complete reversal of VCR resistance in C-A120 cells. These findings suggested that BSO might not directly interact with MRP and reversed resistance in MRP-mediated MDR cells by reducing the intracellular glutathione (GSH) level that was needed for the transport of drugs by MRP and suggested a role for the combination of drug resistance-modulating agents with different reversing mechanisms in the reversal of MRP-mediated MDR.

Potentiation of the Vincristine Effect on P388 Mouse Leukemia Cells by a Newly Synthesized Dihydropyridine Analogue, PAK‐200

A newly synthesized dihydropyridine analogue, 2‐[benzyl(phenyl)amino]ethyl 1,4‐dihydrb‐2,6‐dimethyl‐5‐(5,5‐dimethyl‐2‐oxo‐l,3,2‐dioxaphosphorinan‐2‐yl)‐l‐(2‐morpholinoethyl)‐4‐(3‐nitrophen‐yl)‐3‐pyridinecarboxylate (PAK‐200), at 1 μM completely reversed the resistance to vincristine in vincristine‐resistant P388 mouse leukemia cells (P388/VCR), in vitro. PAK‐200 at 2 μM inhibited the efflux of [3H]vincristine from P388/VCR and increased the accumulation of [3H]vincristine in P388/VCR to a level similar to that in P388 cells. P‐Glycoprotein in membrane vesicles from P388/ VCR cells was photolabeled with [3H]azidopine. The labeling was completely inhibited by 10 μM PAK‐200. The calcium antagonistic activity of PAK‐200 was about 1000 times lower than that of another dihydropyridine analogue, nicardipine. Experiments with P388 and P388/VCR‐bearing mice showed that PAK‐200 enhanced the effect of vincristine on both leukemia cells in vivo. These results suggest that PAK‐200 interacts with P‐glycoprotein and reverses drug resistance in P388 mouse leukemia cells in vitro, and that PAK‐200 has an ability to potentiate the effect of vincristine on P388 mouse leukemia cells in vivo.