Increased lines. In line with this, Ca2+ channel

Increased cytosolic Ca2+ levels have been reported in severalexperimental models of apoptosis19, 29. We next investigated how Ca2+ homeostasis regulates human breast cancer cellssensitivity to doxorubicin and simvastatin and elucidate the underlyingmechanism. The BH3-only proapoptotic protein BIM (BCL-2interacting mediator of cell death), is a key element mediated cells apoptosis inresponse to stimuli such as cytokine deprivation, deregulated calcium flux or certainstress stimuli30.

We therefore treated MDA-MB-231 breastcarcinoma-derived cells with or without doxorubicin and simvastatin for 24 h followingpre-incubation with or without Ca2+ chelators, for 30 min, andanalyzed the levels of BH3-only proteins by flow cytometry. Interestingly,treatment with doxorubicin or simvastatin caused a substantialincrease in BIM protein levels in MDA-MB-231cells, which were remarkably inhibited by both Ca2+ chelators (Fig. 3B). We next examined whether doxorubicin- and simvastatin-inducedbreast cancer cell death was mediated by Ca2+-dependentcaspase-3 enzymatic activity, which signals cell death by apoptosis. As shownin Fig. 3C and supplementary Fig.

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S3B, pretreatmentwith both BAPTA-AM and EGTA prevented simvastatin or doxorubicin induced caspase-3activation, respectively, in MDA-MB-231 and MCF-7 cell lines. In line with this,Ca2+ channel inhibitors and phospholipase C inhibitor also preventeddrugs-induced caspase-3 activation in MDA-MB-231 cells (SupplementaryFig. S3C). To further confirm the role of doxorubicin- andsimvastatin-elevated cytosolic Ca2+ in caspase-3 activation, weextended our studies using immunocytochemistry with antibodies against cleavedactive caspase-3. The results showed that Ca2+ chelators profoundly preventedboth doxorubicin- and simvastatin-induced significant cleaved (active) caspase-3(Fig. 3D) in MDA-MB-231 cells. Since reactive oxygenspecies (ROS) might be implicated in caspase-3 activity, the role of ROS in doxorubicin-and simvastatin-induced caspase-3 activity was explored.

To this end, wetreated MDA-MB-231 cells with N-Acetyl-L-cysteine (NAC), a ROS inhibitor. We observedthat blockage of ROS with NAC suppressed drugs-induced ROS elevation (SupplementaryFig. S4) and caspase-3 activation (Fig. 3E). In addition, chelating bothextra- or intra-cellular Ca2+ also prevented the release ofcytochrome c, measuring by enzyme-linked immunosorbent assay, duringexposure to doxorubicin and simvastatin (Fig. 3F)in this cell line.

It is well known that the release of cytochrome c into thecytoplasm is one of the most characterized events of theintrinsic apoptotic cascade. Thus, we examined whether Ca2+chelators blocked or prevented doxorubicin- and simvastatin-inducedapoptotic cell death. As shown in Fig. 3G, significantamount of sub-G1 apoptotic cell population triggered by both doxorubicin andsimvastatin was markedly alleviated by Ca2+ chelators in MDA-MB-231cells. The effects of doxorubicin and simvastatin with or without BAPTA-AM orEGTA, respectively, on the progress of the other phases of the cell cycle were shownin Supplementary Fig. S5.

To confirm the role ofCa2+ signaling in doxorubicin- and simvastatin-induced breast cancercells apoptosis, we analyzed the apoptotic status in MDA-MB-231 cells usingAnnexin V-FITC/PI staining. The percentages of early and late apoptotic cellswere significantly increased with both doxorubicin and simvastatin alonetreatment, but significantly decreased by co-treatment with Ca2+chelators (Figure 3H, 3I). Collectively, these results demonstrate that humanbreast cancer cells require Ca2+ signal transduction for cells apoptosiselicited by simvastatin and doxorubicin stimuli.