Calcium homeostasis. We next examined whether activation of cell

Calcium signaling regulates many signals involved in cellular processes such
as proliferation, differentiation and apoptosis. Given reports that disturbance
of 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 observed
that, addition of both doxorubicin and simvastatin evoked an increase in
intracellular free Ca2+ concentrations, Ca2+i,
in these cells (Fig. 1A and 1D, respectively). The doxorubicin- (Fig. 1B) and
simvastatin- (Fig. 1E) induced increases in Ca2+i were
significantly greater in Ca2+ medium as compared with Ca2+-free
medium. Meanwhile, the Ca2+i elevation was also
determined after exposure of cultured MDA-MB-231 cells to both doxorubicin and
simvastatin at various concentrations. As shown in Fig. 1C and 1F, doxorubicin
and simvastatin, respectively, induced changes in Ca2+i
level in MDA-MB-231 cells in a dose-dependent manner. We
noticed that these two drugs failed to increase cytosolic Ca2+i
level when cells were pretreated with Ca2+ chelators, EGTA (a extracellular
Ca2+ chelator) and BAPTA-AM (an intracellular Ca2+
chelator), respectively (Fig. 1G), suggesting that both simvastatin and
doxorubicin increase cytoplasmic Ca2+ from both extracellular and
intracellular pools. As cytosolic Ca2+ overload during apoptosis,
results in calcium uptake by mitochondria, we also investigated the effects of both
doxorubicin and simvastatin on mitochondrial accumulation of calcium in MDA-MB-231
cells in the absence or presence of Ca2+
chelators (BAPTA-AM and EGTA), respectively. Consistent with this, we observed
that treatment with both doxorubicin and simvastatin significantly increased
mitochondrial calcium levels, which were notably abolished by intra-or
extra-cellular Ca2+ chelators (Supplementary
Fig. S1A). It has been well described that influx of
extracellular Ca2+ across the plasma membrane calcium channels is an
essential component of cytosolic Ca2+ homeostasis. We next examined
whether 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) entry
is the principal Ca2+ entry pathway20. Recent studies
have identified that stromal interaction molecule 1 (STIM1), Orai proteins and
canonical transient receptor potential cation (TRPC) channels are involved in
the SOC complex21, 22, some of which are expressed in breast cancer
cells23, 24. To examine the potential involvement of these channels,
we analyzed their expression levels by quantitative reverse transcription
polymerase chain reaction (qRT-PCR) after drugs stimulation. We found that the
expression level of TRPC1, TRPC3 and STIM1 genes were upregulated by both doxorubicin
and simvastatin treatments, whereas, TRPC4, TRPC5, TRPC6, TRPC7, Orai2 and
Orai3 expressions were not observed and the treatment had no effect on the expression
level of Orai1 gene in these cell lines (Fig. 1H).
To confirm that these Ca2+ channels are involved in doxorubicin- and
simvastatin-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 and
U73122, an inhibitor of phospholipase C (PLC). We
found that pretreatment with these inhibitors prevented doxorubicin- and
simvastatin-induced both cytosolic (Supplementary Fig.
S1B) and mitochondrial (Supplementary Fig. S1C) calcium
levels 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 or
TRPC3 could diminish simvastatin- or doxorubicin-induced increases in Ca2+i
in these cells.
Western blot
analysis revealed that MDA-MB-231 cells also expressed these channel components
and STIM1 and the transfection of these cells with siRNA downregulated their
expression (Supplementary Fig. S1D). The silencing of TRPC3 and TRPC6 genes diminished
significantly drugs-induced cytosolic (Fig. 1I) and mitochondrial (Supplementary
Fig. S1E) calcium influx. Interestingly, the silencing of both TRPC1 and TRPC3 genes, significantly, but
partially, decreased the drugs-induced increases in Ca2+i
in these cells when compared with channel silencing alone (Fig. 1I and Supplementary
Fig. S1E). Moreover, the drugs-evoked Ca2+ responses were also
diminished in cells that were transfected by STIM1 siRNA (Fig.1I and Supplementary Fig. S1E). Together, these
results suggest that doxorubicin- and simvastatin-induced cytosolic Ca2+,
leads to mitochondrial calcium overload through calcium channels activation, mediated
partially by TRPC1 and TRPC3 in these cancer cells.