Calcium homeostasis. We next examined whether activation of cell

Calcium signaling regulates many signals involved in cellular processes suchas proliferation, differentiation and apoptosis. Given reports that disturbanceof calcium homeostasis has been described in many apoptotic paradigms19,we first examined the effects of doxorubicin and simvastatin on cytoplasmic Ca2+signaling in breast cancer MDA-MB-231 cells.

Thus, we used a Ca2+imaging technique to study the dynamic changes of intracellular calcium and observedthat, addition of both doxorubicin and simvastatin evoked an increase inintracellular free Ca2+ concentrations, Ca2+i,in these cells (Fig. 1A and 1D, respectively). The doxorubicin- (Fig. 1B) andsimvastatin- (Fig. 1E) induced increases in Ca2+i weresignificantly greater in Ca2+ medium as compared with Ca2+-freemedium.

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Meanwhile, the Ca2+i elevation was alsodetermined after exposure of cultured MDA-MB-231 cells to both doxorubicin andsimvastatin at various concentrations. As shown in Fig. 1C and 1F, doxorubicinand simvastatin, respectively, induced changes in Ca2+ilevel in MDA-MB-231 cells in a dose-dependent manner. Wenoticed that these two drugs failed to increase cytosolic Ca2+ilevel when cells were pretreated with Ca2+ chelators, EGTA (a extracellularCa2+ chelator) and BAPTA-AM (an intracellular Ca2+chelator), respectively (Fig. 1G), suggesting that both simvastatin anddoxorubicin increase cytoplasmic Ca2+ from both extracellular andintracellular pools. As cytosolic Ca2+ overload during apoptosis,results in calcium uptake by mitochondria, we also investigated the effects of bothdoxorubicin and simvastatin on mitochondrial accumulation of calcium in MDA-MB-231cells in the absence or presence of Ca2+chelators (BAPTA-AM and EGTA), respectively.

Consistent with this, we observedthat treatment with both doxorubicin and simvastatin significantly increasedmitochondrial calcium levels, which were notably abolished by intra-orextra-cellular Ca2+ chelators (SupplementaryFig. S1A). It has been well described that influx ofextracellular Ca2+ across the plasma membrane calcium channels is anessential component of cytosolic Ca2+ homeostasis. We next examinedwhether activation of cell membrane calcium channels contributes to doxorubicin-and simvastatin-induced both cytosolic and mitochondrial Ca2+elevation. In most nonexcitable cancer cells, store-operated calcium (SOC) entryis the principal Ca2+ entry pathway20. Recent studieshave identified that stromal interaction molecule 1 (STIM1), Orai proteins andcanonical transient receptor potential cation (TRPC) channels are involved inthe SOC complex21, 22, some of which are expressed in breast cancercells23, 24. To examine the potential involvement of these channels,we analyzed their expression levels by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) after drugs stimulation. We found that theexpression level of TRPC1, TRPC3 and STIM1 genes were upregulated by both doxorubicinand simvastatin treatments, whereas, TRPC4, TRPC5, TRPC6, TRPC7, Orai2 andOrai3 expressions were not observed and the treatment had no effect on the expressionlevel of Orai1 gene in these cell lines (Fig.

1H).To confirm that these Ca2+ channels are involved in doxorubicin- andsimvastatin-induced Ca2+ signaling, we conducted experiments in Ca2+medium in the presence of SKF96365 and 2-aminoethoxydiphenyl borate (2-APB), the SOC channel blockers25, 26 andU73122, an inhibitor of phospholipase C (PLC). Wefound that pretreatment with these inhibitors prevented doxorubicin- andsimvastatin-induced both cytosolic (Supplementary Fig.S1B) and mitochondrial (Supplementary Fig. S1C) calciumlevels in breast cancer MDA-MB-231 cells, respectively. To gain further insights into the implication of TRPC1 or TRPC3 channels, we checked whether silencing of TRPC1 orTRPC3 could diminish simvastatin- or doxorubicin-induced increases in Ca2+iin these cells.Western blotanalysis revealed that MDA-MB-231 cells also expressed these channel componentsand STIM1 and the transfection of these cells with siRNA downregulated theirexpression (Supplementary Fig.

S1D). The silencing of TRPC3 and TRPC6 genes diminishedsignificantly drugs-induced cytosolic (Fig. 1I) and mitochondrial (SupplementaryFig.

S1E) calcium influx. Interestingly, the silencing of both TRPC1 and TRPC3 genes, significantly, butpartially, decreased the drugs-induced increases in Ca2+iin these cells when compared with channel silencing alone (Fig. 1I and SupplementaryFig. S1E).

Moreover, the drugs-evoked Ca2+ responses were alsodiminished in cells that were transfected by STIM1 siRNA (Fig.1I and Supplementary Fig. S1E). Together, theseresults suggest that doxorubicin- and simvastatin-induced cytosolic Ca2+,leads to mitochondrial calcium overload through calcium channels activation, mediatedpartially by TRPC1 and TRPC3 in these cancer cells.