Investigations on the role of efflux pumps in drug resistance of human cancer cells

The toxicity of a specific group of chemotherapeutic drugs was demonstrated to be enhanced in the presence of non-toxic concentrations of a number of nonsteroidal anti-inflammatory drugs (NSAIDs). This was found to occur in the drug sensitive A549 human lung adenocarcinoma and D L K P human lung squamous carcinoma cell lines. The chemotherapeutic drugs for which the toxicity enhancement effect was found to occur (adriamycin, daunorubicin, epirubicin, vincristine and VP-16) share the property of being potential substrates for the multidrug resistance associated protein (MRP). The toxicity of drugs which were not MRP-substrates, was unaffected by NSAID treatment. The toxicity of non-chemotherapeutic drug substrates for MRP, such as the heavy metal sodium arsenate was also found to be enhanced in the presence of a suitable NSAID. NSAIDs are believed to exert their anti-inflammatory, analgesic and antipyretic effects in vivo as a result of their ability to inhibit the enzyme prostaglandin H 2 synthase (PGHS), which is also known as cyclooxygenase (COX). The only NSAIDs found to possess chemotherapeutic drug toxicity enhancing abilities were: indomethacin, sulindac, sulindac sulfide, sulindac sulfone, tolmetin, acemetacin, mefenamic acid and zomepirac. The mechanism by which these NSAIDs enhanced the toxicity of a suitable chemotherapeutic drug was demonstrated to be independent of any effects of NSAIDs on PGHS. This was conclusively demonstrated by the ability of sulindac sulfone (a metabolite of sulindac which is devoid of inhibitory effects on PGHS) to enhance chemotherapeutic drug toxicity to the same extent as achieved using sulindac sulfide (a metabolite of sulindac with potent inhibitory effects on PGHS). MRP protein was demonstrated by Western blotting to be present in low levels in DLKP plasma membrane-derived inside-out vesicles (10Vs). The MRP in DLKP IOVs was demonstrated to actively transport the M R P substrate leukotriene C 4 (LtC4). This transport was shown to be inhibited by NSAIDs with drug toxicity enhancing abilities such as indomethacin, but was unaffected by NSAIDs which were without influence on chemotherapeutic d m g toxicity. These results suggested that NSAID-mediated enhancement of chemotherapeutic drug toxicity was dependent on the ability of an NSAED to interfere with the drug efflux activity of MRP. The presence of M R P protein in 10 Vs facilitated investigation into the cytotoxic drug transporting characteristics of MRP. The LtC4 transport activity of MRP in IOVs from the MRP-overexpressing cell line H L 6 0 - A D R was found to be significantly inhibited by vincristine (an MRP substrate) but not by Taxol or cisplatin (non-MRP substrates). This inhibition in LtC4 transport with vincristine was synergistically enhanced in the presence of non-inhibitory concentrations of reduced glutathione (GSH). These results suggested that M R P substrate drugs such as vincristine may be actively transported by M R P in the presence of GSH. Demonstration of the presence of active MRP protein in DLKP suggested that M R P may also have been present in resistant variants of DLKP. Active MRP protein was shown to be present in a variety of DLKP-related drug resistant cell lines namely DLKP-A10, DLKPA2B, DLKPA 1 IB, DLKPA 6 B and DLKPA5F. Preliminary evidence suggested that MRP may be involved in certain aspects of the cytotoxic drug accumulation characteristics of these drug resistant variants of DLKP