Xiao-Qin Ren

Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function

We previously isolated agosterol A (AG‐A) from a marine Spongia sp. and found that it completely reversed colchicine resistance in P‐glycoprotein (Pgp)‐over‐expressing KB‐C2 cells and vincristine resistance in multidrug‐resistance protein (MRP)1‐over‐expressing CV60 cells. However, a tri‐deacetylated derivative of AG‐A (IAG‐A) showed almost no activity in reversing Pgp‐ or MRP1‐mediated drug resistance. In this study, we examined the mechanisms by which AG‐A reverses MRP1‐mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1‐over‐expressing human KB carcinoma (KB/MRP) cells. [3H]‐Leukotriene C4 (LTC4), [3H]‐2,4‐dinitrophenyl‐S‐glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [3H]‐vincristine accumulation and efflux in KB/MRP cells were measured with or without AG‐A and/or inactive IAG‐A. AG‐A reduced MRP1‐mediated [3H]‐LTC4 transport in a dose‐dependent manner, but IAG‐A did not. Inhibition by AG‐A was competitive, with a Ki value of 31 μM. AG‐A at 10 μM enhanced the accumulation of [3H]‐vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent. Likewise, ATP‐dependent efflux of [3H]‐vincristine from KB/MRP cells was enhanced compared with KB‐3‐1 cells and inhibited by AG‐A. In addition, AG‐A reduced intracellular levels of glutathione, a compound required for MRP1‐mediated transport of some anti‐cancer drugs. These findings suggest that AG‐A reverses MRP1‐mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs. In addition, the capacity of AG‐A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1.

Thymidine phosphorylase inhibits apoptosis induced by cisplatin.

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.

Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.

Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C‐terminal half of MRP1 with a photoanalog of agosterol A (azido AG‐A). In this study, we further characterized the GSH‐dependent photolabelling site of azido AG‐A on MRP1. An epitope‐inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG‐A in a GSH‐dependent manner. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti‐HA antibody suggested that the GSH‐dependent azido AG‐A photolabelling site on MRP1 resides in the region within TM14‐17 and the cytoplasmic region proximate to the C‐terminus of TM17. Arg1210 in human MRP2 that corresponds to Arg1202 in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C4 (LTC4) transport activity and conferred Vincristine resistance in LLC‐PK1 cells. In summary, this study demonstrated that the GSH‐dependent azido AG‐A photolabelling site on MRP1 resides in the region within TM14‐17 and the cytoplasmic region proximate to the C‐terminus of TM17. The charged amino acid Arg1202 proximate to TM helix 16 is of critical importance for the GSH‐dependent photolabelling of MRP1 with azido AG‐A. Arg1202 itself or the region nearby Arg1202 may be involved in azido AG‐A photolabelling.

Binding site(s) on P-glycoprotein for a newly synthesized photoaffinity analog of agosterol A.

Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.