To modified by a 48-hours treatment with BAPTA-AM (?

To decipher whether Ca2+ signaling is the initial event
leading to cell death following doxorubicin and simvastatin treatments, we
performed an MTT assay, to quantify their effects on cell
survival. MDA-MB-231 breast cancer cells were first treated with drugs
alone for 24, 48 and 72 h. We found that, doxorubicin and simvastatin induced
dose-dependent (Fig. 2A and 2B; Supplementary Fig. S2A and S2B, respectively) and time-dependent
(Fig. 2C and 2D,
respectively) inhibition of cell viability. We
next determined whether these antigrowth effects
were mediated through modulating the extracellular signal-regulated kinases
(ERKs) MAP kinases. Indeed, it has been well established that ERK1/2 signaling promotes
cell survival and proliferation in differentiated cells27. Thus, we monitored
ERK1/2 phosphorylation following MDA-MB-231 breast cancer cells treatment with
different concentrations of doxorubicin and simvastatin for 48 h. We observed
that both doxorubicin and simvastatin treatment significantly decreased ERK1/2
phosphorylation in a dose-dependent manner in these cells (Fig. 2E and 2F, respectively). We next treated MDA-MB-231
breast cancer cells with or without drugs (doxorubicin and simvastatin) for 48
h in the presence or absence of Ca2+ chelators (BAPTA-AM and EGTA). As
illustrated in Supplementary Fig. S2C and S2D, the
dose-response graph of these Ca2+ chelators indicated that the
viability of MDA-MB-231 cell line was not significantly modified by a 48-hours
treatment with BAPTA-AM (? 10 ?M; Supplementary Fig.
S2C) or EGTA (? 100 µM; Supplementary Fig. S2D).
However, the dose-response graph of doxorubicin (Fig. 2G)
or simvastatin (Fig. 2H) indicated that
concentration of 5 ?M BAPTA-AM or 100 µM EGTA in association with concentration
as high as 5000 nM doxorubicin or 100 nM simvastatin significantly attenuated
cell death. Association of 1 µM doxorubicin (Fig. 2I)
or 100 nM simvastatin (Fig. 2J) with BAPTA-AM (5
?M) or EGTA (100 µM) markedly decreased cell death when compared to drug alone
treatment in MDA-MB-231 cell line as well as in MCF-7 cell line (Supplementary Fig. S2E).  These positive effects on cell viability were
also observed with Ca2+ channel inhibitors and phospholipase C inhibitor
in both MDA-MB-231 (Supplementary Fig. S2F) and MCF-7
(Supplementary Fig. S2G) cell lines. To
determine whether addition of extracellular Ca2+ affects the sensitivity
of breast cancer cells to both doxorubicin and simvastatin, we cultured MDA-MB-231
cells for 48 h with drugs in the presence or absence of 1.2 mM CaCl2,
and confirmed that the increase in extracellular Ca2+ improves drugs
sensitivity (data not shown). Additionally, treatment of MDA-MB-231 breast
cancer cells with doxorubicin and simvastatin induced morphological changes
were prevented with Ca2+ chelators, consistent with the results obtained
by the MTT assay on cell growth and death (Fig.  2K). To allow a better comparison between the cytotoxicity of
simvastatin and doxorubicin, the concentrations of both drugs necessary to
decrease cell viability by 50% (IC50) were calculated. We have
observed that simvastatin exhibited superior cell toxicity potential in
comparison with doxorubicin in MDA-MB-231 cells with the 15- to 30-fold difference in IC50
values determined, respectively, after 72 and 48 h incubation (Fig. 2L). Individually,
the toxicity of simvastatin and doxorubicin after 72 h of treatment was 2-fold
and 4-fold higher, respectively, compared to the 48-h treatment (Fig. 2L). We
next examined whether simvastatin will have synergistic or additive effect on
cell death when it is used with doxorubicin. Isobologram analysis28
was used to analyze effects of drug combinations. Combination index (CI) value
<1, =1 or >1 indicates that the drugs are synergistic, additive or
antagonistic, respectively. The combination of 50 nM simvastatin with 500 nM of
doxorubicin produced a significant synergistic effect on MDA-MB-231 cell
viability reduction (Fig. 2M) and apoptosis measuring by caspase-3 activation
(Supplementary Fig. S3A) with a combination index less than 1 (CI value= 0.71).