ABSTRACT
We report the screening of analogues of indomethacin to investigate the structure-activity relationship (SAR) of indomethacin-mediated multidrug resistance associated protein-1 (MRP-1) inhibition. By examining the activities of compounds with minor variations of the parent structure, we were able to separate MRP-1, glutathione-S-transferase (GST), cyclooxygenase (COX)-1 and COX-2 inhibitory activities. Combination cytotoxicity assays were utilised to identify agents which possess synergistic potential in MRP-1-expressing cell lines. MRP-1 Inside Out Vesicles (IOVs) were utilised to demonstrate the ability of the indomethacin analogues to inhibit the pump directly. Most of the indomethacin analogues active as MRP-1 inhibitors were poor GST inhibitors when compared with the GST-inhibitory activity of indomethacin. Two of the MRP-1 inhibitory analogues were found to have no COX-1 inhibitory activity and low COX-2 inhibitory activity, suggesting potentially reduced clinical toxicity. One MRP-1 inhibitory indomethacin analogue was also found to have low COX-1 inhibitory activity, but significant COX-2 inhibitory activity, making this analogue again interesting in terms of low potential toxicity, but with the possibility of direct inhibitory effects on tumour growth.
Subject(s)
Cyclooxygenase Inhibitors/therapeutic use , Indomethacin/analogs & derivatives , Isoenzymes/antagonists & inhibitors , Multidrug Resistance-Associated Proteins , Neoplasms/drug therapy , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Drug Resistance, Multiple , Drug Screening Assays, Antitumor/methods , Humans , Membrane Proteins , Prostaglandin-Endoperoxide Synthases , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Synthesis of a range of indomethacin analogues, required for investigation in combination toxicity assays, bearing both N-benzyl and N-benzoyl groups, is described.
Subject(s)
ATP-Binding Cassette Transporters/drug effects , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Cyclooxygenase Inhibitors/chemical synthesis , Indomethacin/analogs & derivatives , Indomethacin/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cyclooxygenase Inhibitors/chemistry , Humans , Indomethacin/chemistry , Multidrug Resistance-Associated Proteins , Neoplasm Proteins/drug effectsABSTRACT
An in vitro model that might be relevant to cancer cell chemoresistance in vivo was generated by exposing the human lung carcinoma clonal cell line DLKP-SQ to 10 sequential pulses of pharmacologically attainable doses of doxorubicin. The resistant variant, DLKP-SQ/10p, was found to be cross-resistant to doxorubicin (10x), vincristine (43x), etoposide (3x), sodium arsenate (3x), paclitaxel (38x) [which could imply overexpression of P-glycoprotein (P-gp) and possibly increased multidrug resistance-associated protein activity] and 5-fluorouracil (4x), but slightly sensitized to carboplatin. Analysis of mRNA levels in the resistant variant revealed overexpression of mdr1 mRNA without significant alteration in mrp, Topo. IIalpha, GSTpi, dhfr or thymidylate synthase mRNA levels. Overexpression of the anti-apoptotic bcl-xL transcript and the pro-apoptotic bax mRNA was also detected but no alterations in bcl-2 or bag-1 mRNA levels were observed. Resistance to a P-gp-associated drug, doxorubicin, could be reversed with P-gp circumventing agents such as cyclosporin A and verapamil, but these substances had no effect on resistance to 5-fluorouracil. Overexpression of the pro-apoptotic bcl-xS gene in the DLKP-SQ/10p line partially reversed resistance not only to P-gp-associated drugs but also to 5-fluorouracil, indicating that the ratio of bcl family members may be important in determining sensitivity to chemotherapeutic drug-induced apoptosis.
