Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes In adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

Orai1 Expression Is Closely Related with Favorable Prognostic Factors in Clear Cell Renal Cell Carcinoma

Store-operated calcium (Ca2+) entry (SOCE) is the principal Ca2+ entry route in non-excitable cells, including cancer cells. We previously demonstrated that Orai1 and STIM1, the molecular components of SOCE, are involved in tumorigenesis of clear cell renal cell carcinoma (CCRCC). However, a clinical relevance of Orai1 and STIM1 expression in CCRCC has been ill-defined. Here, we investigated the expression of Orai1 and STIM1 in CCRCC, and compared their expression with clinico-pathological parameters of CCRCC and the patients' outcome. Immunohistochemical staining for Orai1 and STIM1 was performed on 126 formalin fixed paraffin embedded tissue of CCRCC and western blot analysis for Orai1 was performed on the available fresh tissue. The results were compared with generally well-established clinicopathologic prognostic factors in CCRCC and patient survival. Membrane protein Orai1 is expressed in the nuclei in CCRCC, whereas STIM1 shows the cytosolic expression pattern in immunohistochemical staining. Orai1 expression level is inversely correlated with CCRCC tumor grade, whereas STIM1 expression level is not associated with tumor grade. The higher Orai1 expression is significantly associated with lower Fuhrman nuclear grade, pathologic T stage, and TNM stage and with favorable prognosis. The expression level of STIM1 is not correlated with CCRCC grade and clinical outcomes. Orai1 expression in CCRCC is associated with tumor progression and with favorable prognostic factors. These results suggest that Orai1 is an attractive prognostic marker and therapeutic target for CCRCC.

STIM1 gene silencing suppresses tumor formation of human hypopharyngeal carcinoma cell line FaDu in nude mice / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To evaluate the inhibition effect of STIM1 gene silencing on tumor growth of human hypopharyngeal carcinoma cell lines FaDu in nude mice.</p><p><b>METHODS</b>STIM1 gene in FaDu was silenced by lentiviral infection, and the effect of inhibition was detected by Real-time PCR and Western blot after lentiviral infection. Nude mice were divided into 2 groups, 5 mice in each group. Inhibition group: subcutaneous inject FaDu cells which STIM1 expression was inhibited.</p><p><b>CONTROL GROUP</b>subcutaneous inject FaDu cells infected with negative control siRNA-expressing lentivirus. Tumor volumes were measured by calipers, and small animal imaging was detected by NightOWL system on the day 10, 14, 18 and 22 after tumor inoculated. Tumor weights were evaluated in the day 22 after tumor inoculated. Statistical analysis was performed using standard student test(P value threshold was 0.05).</p><p><b>RESULTS</b>The expressions of human STIM1 gene and protein in FaDu cells were suppressed effectively after STIM1-siRNA lentiviral infection. The mean tumor volumes of control group and inhibition group were (51±25) mm3 and (40±35) mm3, respectively, on the day 10, (262±107) and (106±41) mm3 on the day 14, (716±226) and (340±158) mm3 on the day, (1 682±592) mm3 and (917±252)mm3 on the day 22 (P<0.05). On the day 22, the tumor weight was (1.22±0.41) g in control group and (0.66±0.26) g in STIM1-siRNA group (P<0.05). Small animal imaging showed that the tumors had a smaller fluorescence range with lower signal intensity in STIM1-siRNA group than in control group on the day 14, 18 and 22.</p><p><b>CONCLUSION</b>The expression of STIM1 in human hypopharyngeal carcinoma cell lines FaDu can be inhibited effectively by lentiviral infection, causing the inhibition of tumor formation and growth.</p>

Stromal interaction molecule 1 silencing attenuates the proliferation and migration capacities of endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>The purpose of this study is to explore the impact of stromal interaction molecule 1 (STIM1) knockdown on the proliferation and migration capacities of endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The rat bone marrow derived EPCs were obtained and divided into three groups: adenovirus negative control (NSC) group, rat STIM1 adenovirus vector transfection (si/rSTIM1) group and rat and human recombinant STIM1 adenovirus transfection (si/rSTIM1+hSTIM1) group. The STIM1 expressions in each group were detected by reverse transcription PCR after transfection. The cell proliferation was tested by [(3)H] thymidine incorporation assay ((3)H-TdR). Cell cycle was analyzed by flow cytometry. The cells migration activity was detected by Boyden assay. Calcium ion concentration was detected by confocal laser scanning microscopy.</p><p><b>RESULTS</b>48 h after transfection, the expression level of STIM1 in si/rSTIM1 group was significantly lower than that in NSC group (0.21 ± 0.12 vs. 1.01 ± 0.01, P < 0.05), and number of EPCs at G1 phase in si/rSTIM1 group ((93.31 ± 0.24)%) was significantly higher than that in NSC group ((78.03 ± 0.34)%, P < 0.05), and EPCs' migration activity in si/rSTIM1 group (10.03 ± 0.33) was significantly lower than that in NSC group (32.11 ± 0.54, P < 0.05), and EPCs calcium ion concentration in EPCs in si/rSTIM1 group (38.03 ± 0.13) was significantly lower than that in NSC group (98.11 ± 0.34, P < 0.05), while there was no significant difference between si/rSTIM1+hSTIM1 group and NSC group on the above four indexes.</p><p><b>CONCLUSION</b>Silencing STIM1 could attenuate EPCs proliferation and migration capacities by modulating the calcium ion concentration in EPCs.</p>

Silence of STIM1 attenuates the proliferation and migration of EPCs after vascular injury and its mechanism

OBJECTIVE@#To investigate the effect of stromal interaction molecule 1(STIM1) knockdown on the proliferation and migration of endothelial progenitor cells (EPCs) after vascular injury and its mechanism.@*METHODS@#The rat bone marrow derived EPCs were divided into three groups: adenovirus negative control (group NSC), rat STIM1 adenovirus vector transfection group (group si/rSTIM1) and rat &human recombinant STIM1 adenovirus transfection group (group si/rSTIM1+hSTIM1). The STIM1 expressions in each group were detected by reverse transcription PCR after transfection; the cell proliferation was tested by [(3)H] thymidine incorporation assay ((3)H-TdR); Cell cycle was analyzed by flow cytometry; the cells' migration activity was detected by Boyden assay; Calcium ion concentration was detected by using laser confocal method.@*RESULTS@#48 h later after transfection, the expression level of STIM1 in si/rSTIM1 cells was significantly lower than that in NSC group (0.21 ± 0.12 vs 1.01 ± 0.01, P<0.05); EPCs that stayed in G1 phase in si/rSTIM1 group [(93.31 ± 0.24)%] were significantly more than that in NSC group [(78.03 ± 0.34)%, P<0.05]; EPCs' migration activity in si/rSTIM1 group (10.03±0.33) was significantly lower than that in NSC group: (32.11 ± 0.54, P<0.05); EPCs calcium ion concentration changes in EPCs in si/rSTIM1 group (38.03 ± 0.13) was significantly lower than that in NSC group (98.11 ± 0.34, P<0.05). While there was no significant difference between si/rSTIM1+hSTIM1 group and NSC group on the four indexes above.@*CONCLUSIONS@#Silence of STIM1 attenuates EPCs proliferation and migration after vascular injury, by mediating the calcium ion concentration in EPCs.

Inhibition of stromal interaction molecule 1 and the expression of apoptosis-related proteins in prostate cancer PC-3 cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the effects of stromal interaction molecule 1 (STIM1) on the expression of apoptosis-related proteins in prostate cancer PC-3 cells.</p><p><b>METHODS</b>We transfected the lentivirus vector STIM1-pGCSIL-GFP carrying STIM shRNA into human hormone-independent prostate cancer PC-3 cells, and 3 days later observed the transfection efficiency by fluorescence microscopy. At 7 days after transfection, we determined the expression of STIM1 in the PC-3 cells by RT-PCR and Western blot and those of apoptosis-related proteins Bcl-2, Bax, survivin and activated Caspase-3 by Western blot.</p><p><b>RESULTS</b>At 3 days, inverted microscopy revealed a transfection efficiency of > 80%. At 7 days, the STIM1 expression was significantly inhibited at both mRNA and protein levels. The Bcl-2/Bax rate was remarkably decreased as compared with that of the control group (0. 31 vs 1.24 ) , and the survivin expression was markedly reduced, 0. 14 times that of the relative expression in the control. However, the Caspase-3 cleavage was significantly activated, 1.52 times that of the control (P <0.05).</p><p><b>CONCLUSION</b>STIM1 can be regarded as an oncogene in prostate cancer PC-3 cells. Inhibition of its expression can induce PC-3 cell apoptosis by reducing the Bcl-2/Bax rate, decreasing the survivin expression, and activating the Caspase-3 pathway.</p>

Role of store-operated Ca2+ channels in primary hepatocytes under conditions of calcium overload and ethanol-induced injury / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury.</p><p><b>METHODS</b>The in vitro model of chronic ethanol-induced hepatocyte injury was established using primary hepatocytes isolated from Sprague-Dawley rats. Ethanol-induced changes (24, 48 and 72 h; 50, 100, 200, 400 and 800 mmol/L) in expression of the SOCs proteins stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Oria1) were detected by qualitative PCR analysis (mRNA) and western blotting (protein). The possible role of these two SOCs proteins in the ethanol-induced extracellular calcium influx and related liver cell injury was determined by treating the cell system with various channel blockers (EGTA, La3+, and 2-APB). Cell viability was determined by MTT assay and cytosolic free calcium ion concentration was determined by flow cytometry.</p><p><b>RESULTS</b>After 24 h of exposure to 0 (untreated) to 800 mM/L ethanol, the cell viability was reduced in a concentration-dependent manner. The 400 mmol/L concentration of ethanol decreased cell viability by 57.34% +/- 2.34%. and was chosen for use in subsequent experiments. Compared with the untreated control cells, the ethanol-treated cells showed significantly up-regulated mRNA and protein expression of both STIM1 and Orai1 at all times examined, suggesting that the ethanol-stimulated expression of STIM1 and Orai1 could persist for at least 72 h. The ethanol treatment induced increase in cytoplasmic calcium levels was significantly (and similarly) reduced by co-treatment with any of the three channel blockers.</p><p><b>CONCLUSION</b>Chronic ethanol exposure can increase the expression of STIM1 and Orai1 in primary liver cells, suggesting that ethanol may increase extracellular calcium influx by up-regulating expression of these SOCs protein molecules, ultimately aggravating liver cell damage.</p>

Effect of sodium butyrate on apoptosis and stromal interaction molecule and Orai1 activity in human colon cancer HCT-116 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism underlying sodium butyrate (NaB)-induced apoptosis of a human colon cancer cell line HCT-116.</p><p><b>METHODS</b>The apoptosis of HCT-116 cells induced by NaB was confirmed by hoechst33342 staining and AnnexinV+ PI assay. The changes in the intracellular localization of stromal interaction molecule (STIM1) and Orai1 following NaB treatment were detected by immunofluorescence technique. Western blotting was used to investigate the protein expression levels of STIM1 and Orai1.</p><p><b>RESULTS</b>NaB induced apoptosis and caused translocation and colocalization of STIM1 and Orai1 in HCT-116 cells.</p><p><b>CONCLUSION</b>The apoptosis of human colon cancer cells induced by NaB is correlated to the redistribution of STIM1 and Orai1.</p>

Role of protein kinase C in the activation of store-operated Ca(2+) entry in airway smooth muscle cells / 华中科技大学学报（医学）（英德文版）

Store-operated Ca(2+) channels (SOCs) are plasma membrane Ca(2+) permeable channels activated by depletion of intracellular Ca(2+) store. Ca(2+) entry through SOCs is known as store-operated Ca(2+) entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca(2+) fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ɛ isoforms in rat ASMCs. PKCα-selective inhibitor Gö6976 and PKCɛ-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.

Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of stromal interaction molecule 1 (STIM1) silencing on EPCs cell cycle.</p><p><b>METHODS</b>Rat bone marrow derived endothelial progenitor cells (EPCs) were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2+ was measured using LSCM. Co-immunoprecipitation was performed to examine the interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay.</p><p><b>RESULTS</b>Forty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated (0.37 +/- 0.02 vs. 1.00 +/- 0.02, P < 0.05) and intracellular free Ca2+ level was significantly reduced (34.07 +/- 4.10 vs. 86.51 +/- 14.12, P < 0.05) in Ad-si/rSTIM1 group compared with control group. The cell cycle was arrested at G1 phase [(90.91 +/- 1.10)% vs. (77.10 +/- 0.56)%, P < 0.05] and the store-operated channel entry was strikingly inhibited in EPCs after treatment with Ad-si/rSTIM1. However, cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 significantly reversed these responses. Interestingly, co-immunoprecipitation study showed that STIM1 co-precipitated with TRPC1, and IP3 levels measured by ELISA were similar among three groups (P > 0.05).</p><p><b>CONCLUSION</b>siRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.</p>