ObjectiveSeveral transient receptor potential (TRP) channels are expressed in pancreatic beta cells and have been proposed to be involved in insulin secretion. However, the endogenous ligands for these channels are far from clear. Here, we demonstrate the expression of the transient receptor potential ankyrin 1 (TRPA1) ion channel in the pancreatic beta cells and its role in insulin release. TRPA1 is an attractive candidate for inducing insulin release because it is calcium permeable and is activated by molecules that are produced during oxidative glycolysis.MethodsImmunohistochemistry, RT-PCR, and Western blot techniques were used to determine the expression of TRPA1 channel. Ca2+ fluorescence imaging and electrophysiology (voltage- and current-clamp) techniques were used to study the channel properties. TRPA1-mediated insulin release was determined using ELISA.ResultsTRPA1 is abundantly expressed in a rat pancreatic beta cell line and freshly isolated rat pancreatic beta cells, but not in pancreatic alpha cells. Activation of TRPA1 by allyl isothiocyanate (AITC), hydrogen peroxide (H2O2), 4-hydroxynonenal (4-HNE), and cyclopentenone prostaglandins (PGJ2) and a novel agonist methylglyoxal (MG) induces membrane current, depolarization, and Ca2+ influx leading to generation of action potentials in a pancreatic beta cell line and primary cultured pancreatic beta cells. Activation of TRPA1 by agonists stimulates insulin release in pancreatic beta cells that can be inhibited by TRPA1 antagonists such as HC030031 or AP-18 and by RNA interference. TRPA1-mediated insulin release is also observed in conditions of voltage-gated Na+ and Ca2+ channel blockade as well as ATP sensitive potassium (KATP) channel activation.ConclusionsWe propose that endogenous and exogenous ligands of TRPA1 cause Ca2+ influx and induce basal insulin release and that TRPA1-mediated depolarization acts synergistically with KATP channel blockade to facilitate insulin release.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) regulate diverse cellular processes, including cell growth, differentiation, apoptosis, and cancer progression. However, the function of lncRNAs in the progression of hepatocellular carcinoma (HCC) remains largely unknown. We performed a comprehensive microarray analysis of lncRNA expression in human HCC samples. After validation in 108 HCC specimens, we identified a differentially expressed novel tumor suppressive lncRNA termed amine oxidase, copper containing 4, pseudogene (AOC4P). The level of AOC4P expression was significantly downregulated in 68% of HCC samples and negatively correlated with advanced clinical stage, capsule invasion and vessel invasion. Low AOC4P expression correlated with poor prognostic outcomes, serving as an independent prognostic factor for HCC. In vitro functional assays indicated that AOC4P overexpression significantly reduced cell proliferation, migration and invasion by inhibiting the epithelial-mesenchymal transition (EMT). RNA immunoprecipitation assays demonstrated that AOC4P binds to vimentin and promotes its degradation. Animal model experiments confirmed the ability of AOC4P to suppress tumor growth and metastasis. Taken together, our findings suggest that AOC4P lncRNA acts as an HCC tumor suppressor by enhancing vimentin degradation and suppressing the EMT. By clarifying the mechanisms underlying HCC progression, these findings promote the development of novel therapeutic strategies for HCC.

Early diagnosis of bacteremia for patients with suspected sepsis is 1 way to improve prognosis of sepsis. Systemic inflammatory response syndrome (SIRS) has long been utilized as a screening tool to detect bacteremia by front-line healthcare providers. The value of SIRS to predict bacteremia in elderly patients (≥65 years) with suspected sepsis has not yet been examined in emergency departments (EDs).We aimed to evaluate the performance of SIRS components in predicting bacteremia among elderly patients in EDs.We retrospectively evaluated patients with suspected sepsis and 2 sets of blood culture collected within 4 hours after admitting to ED in a tertiary teaching hospital between 2010 and 2012. Patients were categorized into 3-year age groups: young (18–64 years), young-old (65–74 years), and old patients (≥75 years). Vital signs and Glasgow Coma Scale with verbal response obtained at the triage, comorbidities, sites of infection, blood cultures, and laboratory results were retrieved via the electronic medical records.A total of 20,192 patients were included in our study. Among them, 9862 (48.9%) were the elderly patients (young-old and old patients), 2656 (13.2%) developed bacteremia. Among patients with bacteremia, we found the elderly patients had higher SIRS performance (adjusted odds ratio [aOR]: 2.40, 95% confidence interval [CI]: 1.90–3.03 in the young-old and aOR: 2.66, 95% CI: 2.19–3.23 in the old). Fever at the triage was most predictive of bacteremia, especially in the elderly patients (aOR: 2.19, 95% CI: 1.81–2.65 in the young-old and aOR: 2.27, 95% CI: 1.95–2.63 in the old), and tachypnea was not predictive of bacteremia among the elderly patients (all P > 0.2).The performance of SIRS to predict bacteremia was more suitable for elderly patients in EDs observed in this study. The elderly patients presented with more fever and less tachypnea when they had bacteremia.

BackgroundCombination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination.MethodsThe cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity.ResultsThe efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27.ConclusionIn cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-3034-2) contains supplementary material, which is available to authorized users.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo , and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.