Enhancement of the cytotoxicity of MRP substrates by indomethacin and related compounds

Certain non-steroidal anti-inflammatory drugs (NSAIDs), including indomethacin and sulindac, at non-toxic concentrations, were found to enhance the toxicity of a range of chemotherapeutic drugs, such as doxorubicin, epirubicin, vincristine and V P -16. This effect appeared to be mo st significant in MRP-expressing cell lines such as DLKP and A549, and was not evident in Pgp-overexpressing cell lines such as DLKPA. Analogues o f indomethacin were subsequently generated to investigate the structure-activity relationship (SAR) of indomethacin-mediated toxicity enhancement. An important goal of this research was to identify an analogue of indomethacin, capable of potentiating the toxicity of anticancer drugs to the same degree as indomethacin but without the toxic side effects observed after prolonged use of indomethacin. It is believed that these side effects are mediated through inhibition of the constitutively expressed form of the Cyclooxygenase enzyme, Cyclooxygenase-1 (COX-1). A number the positive indomethacin analogues (BRI 138/1, BRI 153/1 and BRI 60/1) were found to have the ability to potentiate the toxicity of a number of anticancer drugs while h aving little or no COX-1 inhibitory activity rendering these compounds less likely to cause gastrointestinal toxicity. BRI 60/1 was also found to be a good COX-2 inhibitor. These results for BRI 60/1 suggest a potential clinical application due to reduced toxic side effects and in addition, increased ability as a tumour suppresser due to inhibition o f COX-2. Most of the active indomethacin analogues were found to have very little Glutathione S-transferase inhibitory activity and hence their mode of action was not by inhibiting the conjugation of glutathione to the anticancer drug. Inside-out Membrane Vesicles (IOVs) were utilised to demonstrate the ability of the analogues to directly inhibit the MRP pump by measuring the uptake of the MRP substrate, LTC4 in to the vesicle. Surprisingly, BRI 138/1, which was quite active in the combination toxicity assay, was a weak inhibitor of LTC4 transport as compared to indomethacin and other positive indomethacin analogues suggesting, due to structural variations, reduced ability of BRI 138/1 to bind to the active site on the MRP molecule and compete with LTC4. Results from drug efflux studies suggested th at the active NSAIDs are competitive substrates for MRP1. Several of these analogues are as effective as indomethacin at potentiating the toxicity of certain anticancer drugs but some are less potent (on a molar basis) th an indomethacin. An analogue of (e.g. BRI 138/1, BRI 153/1, BRI 60/1) with similar potentiation ability, but with out the side effects caused by the inhibition of COX-1, may be a promising candidate for future cancer therapy. The ability of indomethacin, indomethacin analogues and sulindac to potentiate the toxicity of chemotherapeutic drugs in cell lines expressing MRP2-6 has not previously been investigated. The results from the combination toxicity assays in the ovarian carcinoma cell line, 2008, transfected with MRP2 or MRP3, suggest th at indomethacin may have the ability to potentiate adriamycin toxicity in both 2008 MRP2 and MRP3. However, a basal level of MRP 1 was found in all the 2008 cell lines which makes it difficult to distinguish if the potentiation was simply as a result of the expression of MRP1 in the cells. The toxicity of methotrexate was not potentiated in the 2008 MRP2-transfected cell line (which is MTX-resistant in short-term toxicity assays) suggesting that indomethacin is not active in MRP2-overexpressing cell lines. In contrast, sulindac had a small, but significant, potentiation effect on methotrexate in the 2008 MRP2 cells. The toxicity of taxol and taxotere was potentiated by indomethacin and sulindac in a number of cell lines and the effect appears to be MRP-related. However, the synergy between piroxicam (which was unable to enhance the toxicity of other MRP1 substrate chemotherapeutic drugs) and taxol suggests an alternative or additional mechanism of taxane toxicity enhancement may also be present. Enhancement of taxol and taxotere toxicity was not observed in A549 cells which overexpress MRP1 but were also found to highly overexpress MRP4. In contrast, the toxicity of cisplatin was decreased in the presence of indomethacin in a number of cell lines including DLKP, DLKPC 14, HepG2 and the 2008 cell lines. BRI 138/1 did not potentiate the toxicity of cisplatin but the effect was additive not antagonistic. Therefore, it is possible that the antagonistic effect on cisplatin toxicity is indomethacin specific. Perhaps indomethacin actually enhances the efflux (or inhibits efflux) of certain anticancer drugs, including cisplatin, from particular cancer cell lines. Pulse selecting DLKP cells with 300(.ig/ml indomethacin increased the resistance of the cells to adriamycin, vincristine, V P -16, cisplatin, indomethacin and, in particular, 5-FU. RT-PCR analysis demonstrated an increase in MRP1, 2 and 4 mRNA expression in the pulsed cells relative to the parental DLKP cell line