Metiner Tosun

Cancer is considered the second leading cause of death worldwide, following cardiovascular diseases. It is believed that the effect of estrogen is not only limited to tumors of hormonally regulated tissues such as the breast, endometrium, and ovary, but also plays a role in the development of gastric and colorectal cancer. Therefore, this study aimed to investigate the apoptotic and antiproliferative effects of G-Protein-Coupled Estrogen Receptor (GPER)-Specific Agonist G-1 on gastrointestinal adenocarcinoma cells. G-1 was applied to human gastric cancer (AGS) and human colon cancer (HT-29) cells at different concentrations. Real-time cellular analysis (RTCA) and MTT assay were used to evaluate cell proliferation. Flow cytometry was employed for the detection of early and late apoptosis using the Annexin-V FITC Kit, Hoechst staining, and analysis of Caspase-3 activation. siRNA was used to knockdown GPER1 expression at the transcriptional level. G-1 exhibited concentration- and time-dependent antiproliferative effects on AGS and HT-29 cells. At the highest concentration, G-1 induced apoptosis in both cell lines. G-1 showed similar antiproliferative effects on cells with and without siRNA treatment. The data suggest that the antiproliferative effects of G-1 appeared to be independent of GPER1 activation G-1 appears to be an attractive tool for suggesting novel targets in the treatment of gastric and colorectal adenocarcinomas.

CBU-SBED: Celal Bayar University-Health Sciences Institute Journal | Volume: 11 Issue: 1

Objectives: Inflammation can be endogenously modulated by the cholinergic anti-inflammatory pathway via calcium (Ca ²⁺ )-permeable alpha-7 nicotinic acetylcholine receptor (α7nAChR) ion channel expressed in immune cells. α7nAChR agonist choline and tryptophan metabolite kynurenic acid (KYNA) produces immunomodulatory effects. This study aimed to determine the effects of the choline and KYNA on the lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 pathway. Methods: In vitro inflammation model was produced via LPS administration in macrophage cells. To determine the effective concentrations, choline and KYNA were applied with increasing concentrations and LPS-induced inflammatory parameters investigated. The involvement of nAChR mediated effects was investigated with the use of non-selective nAChR and selective α7nAChR antagonists. The effects of choline and KYNA on COX-2 enzyme, PGE ₂ , TNFα, NF-κB and intracellular Ca ²⁺ levels were analyzed. Results: LPS-induced COX-2 expression, PGE ₂ TNFα and NF-κB levels were decreased with choline treatment while intracellular calcium levels via α7nAChRs increased. KYNA also showed an anti-inflammatory effect on the same parameters. Additionally, KYNA administration increased the effectiveness of choline on these inflammatory mediators. Conclusions: Our data suggest a possible interaction between the kynurenine pathway and the cholinergic system on the modulation of LPS-induced inflammatory response in macrophages.

Elevated levels of STIM1, an endoplasmic reticulum Ca²⁺ sensor/buffering protein, appear to be correlated with poor cancer prognosis in which microRNAs are also known to play critical roles. The purpose of this study is to investigate possible HBV origins of specific microRNAs we identified in a stem cell-like subpopulation of Huh-7 hepatocellular carcinoma (HCC) cell lines with enhanced STIM1 and/or Orai1 expression that mimicked poor cancer prognosis. Computational strategies including phylogenetic analyses were performed on miRNome data we obtained from an EpCAM- and CD133-expressing Huh-7 HCC stem cell-like subpopulation with enhanced STIM1 and/or Orai1 expression originally cultured in the present work. Results revealed two putative regions in the HBV genome based on the apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions identified critical human genes, of which their transcripts are among the predicted targets of miR3653, which was increased significantly by STIM1 or Orai1 enhancement. Briefly, this study provides phylogenetic evidence for a possible HBV-driven epigenetic remodeling that alters the expression pattern of Ca²⁺ homeostasis-associated genes in STIM1- or Orai1 overexpressing liver cancer stem-like cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the viral genotype would have a clinical impact on prognosis of HBV-induced liver cancers.

The cholinergic anti-inflammatory pathway plays an important role in controlling inflammation. This study investigated the effects of varenicline, an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, on inflammatory cytokine levels, cell proliferation, and migration rates in a lipopolysaccharide (LPS)-induced inflammation model in RAW 264.7 murine macrophage cell lines. The cells were treated with increasing concentrations of varenicline, followed by LPS incubation for 24 h. Prior to receptor-mediated events, anti-inflammatory effects of varenicline on different cytokines and chemokines were investigated using a cytokine array. Nicotinic AChR–mediated effects of varenicline were investigated by using a non-selective nAChR antagonist mecamylamine hydrochloride and a selective α7nAChR antagonist methyllycaconitine citrate. TNFα, IL-1β, and IL-6 levels were determined by the ELISA test in cell media 24 h after LPS administration and compared with those of dexamethasone. The rates of cellular proliferation and migration were monitored for 24 h after drug treatment using a real-time cell analysis system. Varenicline decreased LPS-induced cytokines and chemokines including TNFα, IL-6, and IL-1β via α7nAChRs to a similar level that observed with dexamethasone. Varenicline treatment decreased LPS-induced cell proliferation, without any nAChR involvement. On the other hand, the LPS-induced cell migration rate decreased with varenicline via α7nAChR. Our data suggest that varenicline inhibits LPS-induced inflammatory response by activating α7nAChRs within the cholinergic anti-inflammatory pathway, reducing the cytokine levels and cell migration.

Background: Elevated levels of STIM1, an endoplasmic reticulum Ca²⁺ sensor/buffering protein, appear to be correlated with poor cancer prognosis where microRNAs are also known to be involved. We investigated a possible viral origin of specific microRNAs identified in Huh-7 human liver cancer stem cells with enhanced STIM1 expression. Methods: Computational strategies including phylogenetic analyses were performed on miRNome data obtained from Huh-7 liver cancer stem cells with enhanced STIM1 and/or Orai1 expression originally cultured in the present study. Results: Results revealed two putative regions in HBV genome based on apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions revealed critical human genes of which their transcripts are among the predicted targets of miR3653 which was increased significantly by STIM1 enhancement. Conclusion: This study presents a phylogenetic evidence for an HBV-driven epigenetic remodeling to alter gene expression pattern associated with Ca²⁺ homeostasis in STIM1-overexpressing liver cancer stem cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the HBV genotype would have a clinical impact on prognosis.

The endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule1 (STIM1) activates the plasma membrane (PM) channel Orai1 in order to mediate store-operated Ca2+ entry (SOCE) in response to ER store depletion. Enhanced expression of STIM1 in cancer tissue has been associated with poor patient prognosis. Therefore, this study investigated the functional consequences of enhanced expression of STIM1 and Orai1 in a tumor-initiating subpopulation of Huh-7 hepatocellular carcinoma (HCC) cells that express epithelial cell adhesion molecule (EpCAM) and Prominin 1 (CD133). We performed qRT-PCR, intracellular Ca2+ monitoring, protein analyses, and real-time cell proliferation assays on EpCAM(+)CD133(+) subpopulation of tumor-initiating Huh-7 HCC cells expressing high levels of STIM1 and/or Orai1. Statistical significance between the means of two groups was evaluated using unpaired Student’s t-test. Enhanced STIM1 expression significantly increased ER Ca2+ release and proliferation rate of EpCAM(+)CD133(+) cells. STIM1 overexpression may facilitate cancer cell survival by increasing ER Ca2+-buffering capacity, which makes more Ca2+ available for the cytosolic events, on the other hand, possibly preventing Ca2+-dependent enzymatic activity in mitochondria whose Ca2+ uniporter requires much higher cytosolic Ca2+ levels.

Real-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the real-time changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well−1, respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h. TRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well−1 seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well−1 density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well−1 for 72 h. Data suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells.

TRPC1 and store-operated Ca²⁺ (SOC) entry have previously been associated with hepatocellular carcinoma cell proliferation. The aim of the study was to determine genes and processes associated with TRPC1 down-regulation and the resulting increase of SOC entry and decrease in hepatocellular carcinoma cell proliferation. For this purpose, transcriptome analysis was performed to determine differentially expressed genes in TRPC1-silenced Huh7 cells. SOC entry- and proliferation-related genes correlated with TRPC1 down-regulation were also examined. Changes in SOC entry and cell proliferation were monitored in the TRPC1-silenced and parental cells and found to be significantly increased and decreased, respectively, in TRPC1-silenced cells. A total of 71 genes were significantly differentially expressed (40 up- and 31 down-regulated), including four mitogen-activated protein kinase (MAPK) signalling-associated genes. STIM1 levels were significantly up-regulated and negatively correlated with TRPC1 levels. In addition, expression of two cell cycle regulation genes, CDK11A/11B and URGCP, was observed to decrease, whereas ERBB3 and FGFR4, pro-survival genes, increased significantly in TRPC1-silenced cells. In conclusion, these results suggest reciprocal alterations in TRPC1 and STIM1 levels and a role for STIM1 in the regulation of SOC entry in TRPC1-silenced Huh7 cells. In addition to TRPC1, STIM1 may participate in Huh7 cell proliferation by regulating SOC entry. Alterations in MAPK signalling genes may be involved in diminished cell proliferation in TRPC1-silenced Huh7 cells. Similarly, changes in cell cycle regulating genes in TRPC1-silenced cells indicate possible cell cycle arrest along with compensatory up-regulation of ERBB3 growth factor receptor—amongst others—to maintain hepatocellular carcinoma cell proliferation.

The purpose of our study was to investigate whether endothelium-derived relaxations induced by store depletion are altered in aging rat thoracic aorta. Vascular responses were measured in aortic segments isolated from young (2–4 month) and old (20–24 month) male Sprague-Dawley rats. In phenylephrine-contracted intact tissues, receptor-mediated and receptor-independent endothelium-derived relaxations were induced by acetylcholine (ACh) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) blocker cyclopiazonic acid (CPA), respectively. In addition, CPA-induced changes in intracellular calcium levels were monitored in fura-2-loaded endothelium-denuded tissues. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis were performed to determine the transient receptor potential canonical (TRPC) 4 mRNA and protein levels. Endothelial TRPC4 mRNA levels were apparently decreased in aging rats. Immunoblot analysis showed that TRPC4 protein levels significantly decreased in intact aorta from 20- to 24-month-old rats compared to that from 2- to 4-month-old rats. ACh- and CPA-induced endothelium-dependent relaxations decreased in old rat aorta without any change in direct vasodilation induced by sodium nitroprusside. Store-operated Ca²⁺ entry (SOCE) induced by CPA was significantly decreased, whereas sarcoplasmic reticulum Ca²⁺ release was unaffected in endothelium-denuded aging rat aorta. In conclusion, TRPC4 downregulation could be associated with decreased endothelium-dependent vasorelaxations. As endothelial nitric oxide synthase is activated by SOCE-induced caveolar internalization, tracking the expression levels of SERCA, ion channels, and/or associated proteins involved in SOCE would lead to the development of novel therapeutics for age-related vasospastic disorders with dysfunctional endothelium.