Enhancement of the Cytotoxicity of MRP Substrates by Indomethacin and Related Compounds A thesis submitted for the degree of PhD b y Samantha Touhey, B.Sc. Hons The experimental work described in this thesis was carried out under the supervision of Professor Martin Clynes PhD a t th e National Cell and Tissue Culture Centre, School of Biological Sciences, Dublin City University, Glasnevin, Y Dublin 9, Republic of Ireland. I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of PhD, is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed ¥1> number: A bstract Enhancement of the cytotoxicity of MRP substrates by indomethacin and related compounds. Samantha Touhey 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 VP-16. This effect appeared to be most significant in MRP-expressing cell lines such as DLKP and A549, and was not evident in Pgp-overexpressing cell lines such as DLKPA. Analogues of 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 having 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 of 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, LTC 4 in to the vesicle. Surprisingly, BRI 138/1, which was quite active in the combination toxicity assay, was a weak inhibitor of LTC 4 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 LTC 4. Results from drug efflux studies suggested that 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) than indomethacin. An analogue of indomethacin (e.g. BRI 138/1, BRI 153/1, BRI 60/1) with similar potentiation ability, but without 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 that 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 MRP 1 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, VP-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. Acknowledgements I would like to express my very sincere thanks to Professor Martin Clynes, firstly, for giving me the opportunity to do a PhD and secondly, for his kindness, encouragement and support over the last four years. I wish to express my gratitude to Dr. Anita Maguire and Dr. Stephan Plunkett for synthesising the indomethacin analogues. I wish to acknowledge Prof. Piet Borst and Dr. Marcel Kool for kindly donating the 2008 cell lines. A big thank you to Dr. Robert O' Connor for the enormous amount of help, advice and guidance he gave to me throughout the course of my PhD. Thanks to all my colleagues, past and present, in the NCTCC for all the help and advice given to me over the years. Thank you to everyone, too numerous to mention (especially those in Toxicology), who helped me through the last year especially, and for putting up with my "not so good" moods and my stressed out states!!! Thanks to Paudi for all his much appreciated help with RT-PCR - I was really, really grateful. To Conor for all his help in the early days and for always helping out with any problems throughout the four years. To Mary for teaching me the art of cell culture and for all the help and advice she gave to me. Thanks to Carol and Yvonne for all the help and thanks to John for helping me get the thesis together. In particular, I would like to thank "The Tragedy Girls" Dee and Sharon, for being great friends this last year and for some really great, much needed, de-stressing nights out!! Thanks also to Colette for her friendship (You were much missed during the last year! Thank God for Emails!!). To Rasha, Mags and Sarah for the lunchtime laughs and the help. To Rois for all the encouraging Emails from afar. Thanks to Eugene and Carol for their kindness when I was writing my thesis. To "Paulo Rossi" for making me laugh with those Emails during the toughest months!! Special thanks to my great life-long friend Sinead, who drank numerous bottles of wine with me and let me cry on her shoulder many times during the course of this PhD. Thanks for always being there for me! To Deirdre, my "Thursday night friend", thank you for great food, chats, encouragement and support, To my parents John and Phyllis, who are the best in the world, thank you both so much for everything you have ever done for me. Hope I have done you proud. And to my sisters, Linda, Dympna, Geraldine, Patricia and Sharon, and my brother John who had to put up with a lot from their "big sister" this last while. I wish to acknowledge a very special and kind person, Iggy Galvin, who died in 1996. He had huge belief and faith in my ability and gave me tremendous encouragement and financial support to undertake a PhD. Thank you Iggy, I will never forget your kindness. And last but definitely not least, thanks to Joe for all his love, patience, tolerance, support and encouragement over the last few years. Thank you for helping me through the toughest times and for always being there for me whenever I needed you. I owe you so much. This thesis is decCicatecC to the memory of my Nana Chrissie and my Nana LiCy Table of Contents Section 1.0: Introduction 1 1.1 Multidrug resistance in cancer 2 1.1.1 Multidrug transporters from bacteria to man. 5 1.2 Secondary multidrug transporters 5 1.3 ABC Transporters 10 1.4 Pgp? LRP and MRP in Multidrug Resistance 11 1.5 P-glycoprotein (Pgp) 12 1.5.2 Structure o f the gene encoding P-glycoprotein 14 1.5.3 P-glycoprotein protein structure 14 1.6 Multidrug Resistance Protein - MRP 15 1.6.1 Homologues of MRP 17 1.6.2 Chromosome location of MRP and homologues 19 1.6.3 Cellular location o f MRP 20 1.6.4 MRP expression in cell lines 21 1.6.5 MRP expression in tissues 23 1.6.6 MRP Protein structure 24 1.6.7 Function /Transport by MRP 1 29 1.6.8 MRP 1 and clinical multidrug resistance 34 1.6.9 Function/Transport properties of the MRP Analogues 36 1.6.9.1a MRP2(cMOAT) 36 1.6.9.1 b:Proposed working model for MRP 1 and MRP2 37 1.6.9.1c MRP2 and Drug Resistance in Cancer 38 1.6.9.2 MRP3 40 1.6.9.3 MRP4 41 1.6.9.4 MRP5 42 1.6.9.5 MRP6 43 1.6.10 Circumvention of chemotherapeutic drug resistance 43 1.6.11 Influence of Drug influx and accumulation on multidrug resistance 47 1.7 Nonsteroidal anti-inflammatory drugs (NSAIDs) 50 1.7.1 COX-1 and COX-2 54 1.7.2 NSAIDs and inhibition of COX-1 and COX-2.