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Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

ABSTRACT: Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities.

SUBMITTER: Hwang JY

PROVIDER: S-EPMC4857610 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

Hwang Jae Youn JY Yoon Chi Woo CW Lim Hae Gyun HG Park Jin Man JM Yoon Sangpil S Lee Jungwoo J Shung K Kirk KK

Ultrasonics 20150626

Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ...[more]

PMID: 26150401

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Project description:BackgroundPhosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study.MaterialsMDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R.ResultsThe EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate.ConclusionIn summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear. Video Abstract.

Project description:BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

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Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

Publications

Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

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