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Resveratrol sensitizes A549 cells to irradiation damage via suppression of store-operated calcium entry with Orai1 and STIM1 downregulation.

ABSTRACT: Resveratrol is a natural polyphenol with multiple positive biofunctions and was found to have potential as a radiosensitizer with an intricate molecular mechanism. Store-operated calcium entry (SOCE) is a novel intracellular calcium regulatory pattern that is mainly mediated by iron channels, such as by the stromal interaction molecule (STIM) and calcium release-activated calcium channel protein (Orai) families. SOCE was recently reported to be suppressed via the downregulation of STIM or Orai families for the promotion of tumor cell death induced by resveratrol. In the present study, resveratrol combined with irradiation treatment were found to induce more evident cell damage compared with irradiation treatment alone, as shown with Cell Counting Kit-8 assay and mitochondrial membrane potential detection with rhodamine 123. Additionally, resveratrol combined with irradiation treatment decreased the expression of STIM1 and Orai1, while it had no effects on STIM2, Orai2 and Orai3. Moreover, resveratrol combined with irradiation treatment lead to alleviated thapsigargin-induced SOCE. In addition, overexpression of STIM1 and Orai1 reversed resveratrol-induced SOCE inhibition and reduced death in A549 cells under irradiation. In summary, the present results revealed that resveratrol can significantly enhance the effect of irradiation damage on lung adenocarcinoma A549 cells, and this effect may be mediated by suppression of SOCE with reduced expression of both STIM1 and Orai1.

SUBMITTER: Lele W

PROVIDER: S-EPMC8027717 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation.

Project description:Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

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Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1.

Project description:In the present study, we have applied ratiometric measurements of intracellular Ca2+ concentrations ([Ca2+]i) to show that extracellularly applied ATP (adenosine triphosphate) (100?µM) stimulates store-operated Ca2+ entry (SOCE) in 3T3-L1 adipocytes. ATP produced a rapid increase in [Ca2+]i consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+ Gene expression data and [Ca2+]i recordings with uridine-5'-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+]i elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si)RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.

| S-EPMC5813502 | biostudies-literature

The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by preventing STIM1-Orai1 interaction.

Project description:The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca(2+) entry (SOCE), a ubiquitous Ca(2+) entry pathway regulating many cellular functions, is unknown. Here we show that NAGly, but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic β-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca(2+) depletion of the endoplasmic reticulum, whereas it had no effect on Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Enhanced Ca(2+) entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca(2+) depletion of the endoplasmic reticulum. In contrast, independently from its slow depolarizing effect on mitochondria, NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings revealed the STIM1-Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.

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Project description:Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca(2+) entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca(2+)-independent phospholipase A(2) β (iPLA(2)β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA(2)β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA(2)β but not STIM1. These data confirm the role of iPLA(2)β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.

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