Biological evaluation of bismuth non-steroidal anti-inflammatory drugs (BiNSAIDs): stability, toxicity and uptake in HCT-8 colon cancer cells
Recent studies showed that the metal-coordinated non-steroidal anti-inflammatory drug (NSAID), copper indomethacin, reduced aberrant crypt formation in the rodent colon cancer model, while also exhibiting gastrointestinal sparing properties. In the present study, the stability and biological activity of three BiNSAIDs of the general formula [Bi(L)3]n, where L = diflunisal (difl), mefenamate (mef) or tolfenamate (tolf) were examined. NMR spectroscopy of high concentrations of BiNSAIDs (24 h in cell medium, 37 °C) indicated that their structural stability and interactions with cell medium components were NSAID specific. Assessment of cell viability using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium]bromide (MTT) assay showed that the toxicity ranking of the BiNSAIDs paralleled those of the respective free NSAIDs: diflH < mefH < tolfH. While the IC50 values of the BiNSAIDs (ranging between 16 and 81 μM) were lower than the free NSAIDs, it was apparent that the toxicity of the BiNSAIDs was due to the molar ratio of the three NSAID molecules contained in the BiNSAIDs, with the exception of [Bi(difl)3]. The highest cellular bismuth content was observed following treatment with [Bi(tolf)3]. Since NMR studies indicated that [Bi(tolf)3] was the most stable BiNSAID and that cellular uptake of bismuth correlated with structural stability it appears that bismuth uptake is assisted by the NSAID. Microprobe SR-XRF imaging showed that the intracellular fate of bismuth was independent of the specific BiNSAID treatment whereby all BiNSAID-treated cells showed bismuth accumulation in the cytoplasm within 24-h exposure. The size and location of the hot spots (0.3-5.8 μm2), were consistent with cellular organelles such as lysosomes.