Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells

Background/Aims: Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions. Methods: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4. Results: We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice. Conclusion: Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells

Background/Aims: Istaroxime is a validated inotropic Na + /K + ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca 2+ and Western blotting for FAK/pFAK measurements. Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development. © 2017 The Author(s). Published by S. Karger AG, Basel