BIM BIM accumulation. Consistent with prior results,

BIM accumulation results in the release of BAK (active
form) from anti-apoptotic molecules (BCL-XL and MCL-1) and release of
cytochrome c from mitochondria48. Collectively, the findings, to our
knowledge molecularly described here for the first time, involve a
calcium-based mechanism as a crucial event for execution of the cell-death
process triggered by doxorubicin and simvastatin in human breast cancer cells. Because
recent studies suggest that the lipid-lowering simvastatin holds great promise
as cancer therapeutics, the data presented here may help understand the
molecular basis proof for simvastatin and provide supporting rationale for
future its trials in breast cancer patients. degradation is negatively regulated by ERK
activity30, 46, 47, the inhibition of ERK activity after doxorubicin
or simvastatin treatment may also contribute to increase the level of BIM. Flow cytometry
analysis of BIM revealed that treatment of breast tumor cells with both doxorubicin
and simvastatin promote BIM accumulation. Consistent with prior results, chelation
of Ca2+ signal obviously inhibited both doxorubicin and simvastatin induced
BIM accumulation45. The proapoptotic activity of BIM can be
regulated by ERK-mediated phosphorylation, which promotes its degradation30,
46, 47. Importantly, we were able to show that both drugs decrease ERK1/2
activation, while only doxorubicin induced inhibition of ERK1/2 pathway is attributed
to cytosolic Ca2+ elevation. Therefore, as BIM The molecular
mechanisms that regulate cell apoptosis are complex, but the ability of the
pro-apoptotic Bcl-2 family member BIM to regulate apoptosis has been
established44.

The disruption of the ubiquitous second messenger Ca2+
homeostasis has been proposed to be critical in cell apoptosis33.
The antitumor properties of both simvastatin and doxorubicin have been associated
with alterations of calcium homeostasis4, 9, 18. However, the exact mechanism
of these antitumor effects is not fully elucidated. We report herein that Ca2+
signaling is absolutely required for these antitumor properties and that it is
mediated by both intracellular Ca2+ component and Ca2+
influx from the extracellular store. Both doxorubicin and simvastatin induced a
strong increase in Ca2+i that was significantly reduced
in Ca2+-free medium in MDA-MB-231 cell line, suggesting that both
drugs initially stimulate Ca2+ from intracellular pool, possibly via
PLC, since drugs-evoked Ca2+ release was significantly curtailed in
the presence of U73122, a selective inhibitor of PLC. Henceforth, we wanted to clarify
the nature of the Ca2+ channels that are opened by doxorubicin and
simvastatin. SOC entry is the principal Ca2+ entry pathway in most
nonexcitable cancer cells20. Although, there is few information
regarding the types of Ca2+ channels that are expressed in breast
cancer cells, it has been reported a significant alteration of the expression
of TRPC channels in these tumor cells34. As reported recently24,
34 in breast tumor cells and tissues, the present study confirms that TRPC1
and TRPC3 are the predominant TRPC channels expressed in the highly metastatic MDA-MB-231
cell line. Recent evidences have identified that TRPC channels, STIM1 and Orai proteins are involved in the SOC
complex21, 22. In our study, TRPC1, TRPC3 and STIM1 expressions are upregulated
by doxorubicin and simvastatin. However, by using the
siRNA experiment, we have observed that the silencing of the expression of
TRPC1 and TRPC3 genes diminished significantly, but failed to completely
abolish, the drugs-induced Ca2+ rise in these cells. These
observations suggest that other channels could be involved in Ca2+ signaling
of simvastatin and doxorubicin. Indeed, expression of other TRP subfamilies, such as TRPV and TRPM, has been
reported in breast cancer tissue (for review: ref. 34). Our results strongly suggest that the upregulation of
these TRPC sub-type channels and Stim1 might sufficiently disrupt Ca2+
homeostasis leading to robust apoptosis observed
in this study. In fact, Shi et al.35
have shown in vascular smooth muscle cells that store-operated interactions
between plasmalemmal STIM1 and TRPC1 proteins stimulate PLC to induce SOC Ca2+
influx via TRPC1 channel activation.  Importantly,
Ca2+ chelation with EGTA or BAPTA-AM or inhibitors of SOC (SKF96365,
2-APB) completely blocked the effects of both doxorubicin and simvastatin,
suggesting that cytoplasmic Ca2+ elevation is critical for inducing tumor
cell death. These results are consistent with the
findings of Giorgi et al.36 and Borahay et al.18 showing,
respectively, that doxorubicin or simvastatin modulates calcium homeostasis,
and as this modulation is necessary to promote cell death. Aberrant
cytosolic Ca2+ elevation has been shown to contribute in multiple
models to cell death by promoting the uptake of mitochondrial Ca2+ (ref.
37, 38). This ultimately leads to the accumulation of reactive oxygen species
(ROS), the loss of ??m and opening of permeability transition pores,
with consequent release of proapoptotic factors, such as cytochrome c which
activates caspase-3 and ultimately result in cell apoptosis37, 39 (Fig.
7F). Our results (Fig. 7) support the idea that cytosolic (and hence
mitochondrial) Ca2+ overload acts as an upstream event and a trigger
for ??m collapse, ROS generation, cytochrome c release, caspase-3 activation and
subsequent breast tumor cells apoptosis induced by both doxorubicin and
simvastatin. This is supported by the findings that chelating or inhibition of
Ca2+ signal markedly blocked these events. Recently, it has been documented
by others that Ca2+ signaling mediated by SOC channels is critical
for breast tumor cell migration and metastasis40, 41. We show that doxorubicin
and simvastatin induce breast tumor cell migration in wound-healing assays, which
was dramatically blocked by Ca2+ chelators, SOC inhibitors or PLC inhibitor.
Both doxorubicin and simvastatin have been shown to induce the stress of endoplasmic
reticulum (ER) 42, 43, which is involved in the enhancement of breast
cancer cells migration42. Thus, it is likely that induction of cell
migration after 6 h of drug treatments might be due to the activation of ER
stress caused by aberrant Ca2+ entry, which may also result in
metastatic MDA-MB-231 cells migration via modulating focal adhesion turnover41.
The observation that abrogation of drugs-stimulated Ca2+ signaling
led to decreased inhibition of invasion (data not shown) and colony formation in vitro tridimensional matrix (HA hydrogels)
and reduction of drugs efficacy against breast tumor xenografts in vivo suggests that Ca2+
signaling is a central regulator for the antitumor properties
of simvastatin and doxorubicin.

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