Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072, Australia.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2012; 287(34):28598-608. DOI: 10.1074/jbc.M112.364737
Source: PubMed


Plasma membrane calcium ATPases (PMCAs) actively extrude Ca2+ from the cell and are essential components in maintaining intracellular Ca2+ homeostasis. There are four PMCA isoforms (PMCA1–4), and alternative splicing of the PMCA genes creates a suite of calcium
efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance
of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed
the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca2+ signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca2+ signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca2+, which was more evident at higher Ca2+ loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had
distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis
mediated by the Ca2+ ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing
was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4
silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation
of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some
PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.

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    • "there were 16 potential Ca2+- pumps (Ca2+-ATPases), while none of CAX homologue could be found in the present genomic data of P. capsici. PMCA has been showed associated with inhibition of NFκB nuclear translocation and to promote cell death by regulating calcium signaling [36]. Taken together, the new pattern involving the antifungal action of CA could be elucidated that CA stimulate a transient Ca2+ efflux via Michael additions with PMCAs of P. capsici, finally leading to its growth inhibition. "
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    ABSTRACT: As a destructive fungus-like plant pathogen, the oomycete Phytophthoracapsici is unable to synthesize its own ergosterol as the potential target of fungicide cinnamaldehyde (CA). In this study, CA exerted efficient inhibitory effects on both mycelial growth (EC50=0.75 mM) and zoospore germination (MIC=0.4 mM) of P. capsici. CA-induced immediate Ca(2+) efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca(2+) content determined by using Fluo-3 AM and the increase in extracellular Ca(2+) concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca(2+) influx with ruthenium red and verapamil led to a higher level of CA-induced Ca(2+) efflux, suggesting the simultaneous occurrence of Ca(2+) influx along with the Ca(2+) efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca(2+) gave rise to the impaired vitality of P. capsici while the addition of exogenous Ca(2+) could suppress the growth inhibitory effect of CA. These results suggested that Ca(2+) efflux played an important role in CA-induced growth inhibition of P. capsici. The application of 3-phenyl-1-propanal, a CA analog without α,β- unsaturated bond, resulted in a marked Ca(2+) influx in zoospores but did not show any growth inhibitory effects. In addition, exogenous cysteine, an antagonist against the Michael addition (the nucleophilic addition of a carbanion or another nucleophile) between CA and its targets, could attenuate CA-induced growth inhibition of P. capsici by suppressing Ca(2+) efflux. Our results suggest that CA inhibits the growth of P. capsici by stimulating a transient Ca(2+) efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.
    Full-text · Article · Oct 2013 · PLoS ONE
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    • ". Imaging was performed using an ImageXpress Micro automated epifluorescence microscope (Molecular Devices Corporation). Criteria for viable and propidium iodide positive cells (PI+) were defined as previously described [12] "
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    ABSTRACT: The mitochondrial calcium uniporter (MCU) transports free ionic Ca(2+) into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca(2+) levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca(2+) levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.
    Full-text · Article · Apr 2013 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Plasma membrane Ca2+ ATPases (PMCAs) are highly regulated transporters responsible for Ca2+ extrusion from all eukaryotic cells. Different PMCA isoforms are implicated in various tasks of Ca2+ regulation including bulk Ca2+ transport and localized Ca2+ signaling in specific membrane microdomains. Accumulating evidence shows that loss, mutation or inappropriate expression of different PMCAs is associated with pathologies ranging from hypertension, low bone density and male infertility to hearing loss and cerebellar ataxia. Compared to Ca2+ influx channels, PMCAs have lagged far behind as targets for drug development, mainly due to the lack of detailed understanding of their structure and specific function. This is rapidly changing thanks to integrated efforts combining biochemical, structural, cellular and physiological studies suggesting that selective modulation of PMCA isoforms may be of therapeutic value in the management of different and complex diseases. Both structurally informed rational design and high-throughput small molecule library screenings are promising strategies that are expected to lead to specific and isoform-selective modulators of PMCA function. This short review will provide an overview of the diverse roles played by PMCA isoforms in different cells and tissues and their emerging involvement in pathophysiological processes, summarize recent progress in obtaining structural information on the PMCAs, and discuss current and future strategies to develop specific PMCA inhibitors and activators for potential therapeutic applications. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
    Full-text · Article · Jan 2013 · Journal of Pharmacy and Pharmaceutical Sciences
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