Cancer develops as a result of a defective apoptotic system. Anticancer drugs mainly aim to modulate apoptosis. Multiple publications strongly support the link between the elevation of the intracellular calcium concentration and the induction of apoptosis. Cisplatin was one of the pioneers of the metal based anticancer drugs. Many tumors have been treated with cisplatin and other platin-based anti-cancer drugs like carboplatin, and oxaliplatin. Over the decades metal-based anti-cancer treatment has come a long way with the introduction of newer drugs with less cytotoxicity and different biologic targets. A gold-based complex, Auranofin was introduced to treat rheumatoid arthritis but is also a successful anti-cancer drug. Auranofin is currently undergoing clinical trials in treating patients with epithelial ovarian, primary peritoneal, or fallopian tube cancer. Here we test the induction of apoptotis by Auranofin on MCF-7 breast cancer cell-line and compare this results with the elevation of the intracellular calcium concentration. Methods Breast cancer cells obtained from ATCC were grown in DMEM containing 10% FBS at 370C in a carbon dioxide incubator. Varying concentrations of the compound ranging from 0.1µM to 10µM were tested. Intracellular calcium was recorded in cells treated with different concentration of the drug and a dose response for the apoptosis and cell viability was established. Calcium channel modulators were applied to see the source of calcium upon drug treatment which may help in improving the efficiency of the drug. Cell viability was tested by a standard MTS assay after 24 hours of treatment with Auranofin. Parallel, apoptosis and necrosis was measured by flow cytometry, here the cells were stained with 7 AAD and Sr Flica. Cells for calcium imaging were loaded with the calcium sensitive dye Fluo-4 AM. Fluorescence of the selected cells were recorded every 1min for 2 to 3 hours. To see the source of intracellular calcium rise, calcium channel modulators 2-APB, caffeine, cyclosporine a mitochondrial permeability transition pore inhibitor were used. ROS was monitored by spectrophotometry using fluorescent dye di hydro calcein, Results MTS assay showed a concentration-dependent decrease in cell viability. IC50 was below 6.25µM. The flow cytometry analysis showed four different population of cells, viable, early apoptotic, late apoptotic and necrotic. 30% higher or more apoptosis were observed in concentration above 6.25µM. Apoptosis was 98% when higher concentrations were used. Necrotic cells remained below 16% in all the concentrations. Calcium imaging experiments showed a time and concentration dependent elevation of the intracellular calcium concentration. After 2 hours of application fluorescent intensity corresponding to intracellular calcium increased for concentrations 0.1µM, 1µM, 5µM and 10µM respectively (given 100% as control). Viability proportionally decreased when intracellular calcium was elevated. Blockers targeting PM, ER and mitochondria such as caffeine, cyclosporine 2-APB, nimodipine showed no significant changes. It was noticed that 1mM melatonin was able to decrease the ROS production induced by Auranofin. Conclusion: Auranofin induces apoptosis and cell death. The triggered concentration dependent increase of intracellular calcium might be a crucial factor for the induction of apoptosis. More experiments are required to establish the source of calcium.


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