oa Cisplatin's Modulation of Intracellular Calcium Concentration is Related to the Viability of the Breast Cancer Cells, MCF-7

Background: Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is a highly effective antitumor drug. However, tumors can acquire resistance to CDDP. CDDP elevates the intracellular calcium concentration ([Ca²⁺]i), in various cell lines, leading to the activation of apoptotic pathways and cell death. Using cultured breast cancer (MCF-7) cells we: (1) investigated the effects of CDDP on [Ca²⁺]i, (2) compared these results to a cell line that has been desensitized to CDDP (“resistant”), (3) investigated the source of [Ca²⁺]i by modulating calcium channels and transport mechanisms, and (4) correlated these findings to cytotoxicity. Methods: Changes in the [Ca2+]i were recorded using fluorescence microscopy and Ca2+-binding fluorescent dye, Fluo-4AM. CDDP (1nM-10μM) was administered via a bath perfusion system to the sensitive and CDDP-”resistant” MCF-7 cells over a period of 1.5–2.5h. The [Ca²⁺]i modulators, (caffeine; 10mM, nimodipine; 10μM, ionomycin; 10μM, thapsigargin; 500nM, and 2-APB; 50μM) were administered. MTT assays and trypan blue cell viability tests were performed using CDDP at concentrations of 100pM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, and 10mM after 4, 8, and 24h of incubation. Results: CDDP induced a concentration-dependent increase of [Ca²⁺i. A concentration of CDDP 0.1μM triggered the largest elevation of [Ca²⁺]i with 120% increase (n=19). Induction of cytotoxicity was most likely directly correlated to the increase of [Ca²⁺]i, and was significantly lower in CDDP-”resistant” cells (P←0.05). Preapplication of the calcium channel blocker, nimodipine as well as the IP3 receptor blocker 2-APB significantly reduced this elevation (46.6%; n=26, 71.4%; n=52, increase, respectively) (p←0.05). Surprisingly, when [Ca²⁺]i was elevated due to the pre-application of caffeine, ionomycin or thapsigargin, the subsequent application of CDDP was also significantly reduced compared to control conditions (37.8%; n=15, 34.9%; n=32, 53.7%; n=21, increase, respectively) (p←0.05). Conclusion: CDDP concentration-dependently elevates [Ca²⁺]i by Ca²⁺ entry and Ca²⁺ release from the stores. These mechanisms, however, seem to be less effective in CDDP-”resistant” cells since they show increased restriction on Ca2+ elevation. The pre-elevation of [Ca²⁺]i, through releasing Ca²⁺ from the stores, reduces this elevation significantly. The exact mechanisms remain unclear and further investigations are required to determine the mechanisms and pathways that are involved in the disruption of [Ca²⁺]i.