S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway.

Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

ERK/MAPK signalling pathway and tumorigenesis.

Mitogen-activated protein kinase (MAPK) cascades are key signalling pathways that regulate a wide variety of cellular processes, including proliferation, differentiation, apoptosis and stress responses. The MAPK pathway includes three main kinases, MAPK kinase kinase, MAPK kinase and MAPK, which activate and phosphorylate downstream proteins. The extracellular signal-regulated kinases ERK1 and ERK2 are evolutionarily conserved, ubiquitous serine-threonine kinases that regulate cellular signalling under both normal and pathological conditions. ERK expression is critical for development and their hyperactivation plays a major role in cancer development and progression. The Ras/Raf/MAPK (MEK)/ERK pathway is the most important signalling cascade among all MAPK signal transduction pathways, and plays a crucial role in the survival and development of tumour cells. The present review discusses recent studies on Ras and ERK pathway members. With respect to processes downstream of ERK activation, the role of ERK in tumour proliferation, invasion and metastasis is highlighted, and the role of the ERK/MAPK signalling pathway in tumour extracellular matrix degradation and tumour angiogenesis is emphasised.

Microbulbifer flavimaris sp. nov., a halophilic Gammaproteobacteria isolated from marine sediment of the Yellow Sea, China.

A Gram-stain-negative, aerobic, non-flagellated, non-motile bacterium, designated strain WRN-8T, was isolated from marine sediment of the Yellow Sea, China (36° 5' 33'' N, 121° 20' 37'' E). Colonies of strain WRN-8T were 0.2-0.3 µm wide, 2.1-2.8 µm long, catalase-positive and oxidase-positive. Colonies on marine agar solid media were circular, wet, smooth, light yellow and approximately 1.3 mm in diameter. Growth occurred optimally at 33-37 °C, pH 7.0-7.5 and in the presence of 2-4 % NaCl (w/v). Phylogenetic analysis of the 16S rRNA gene indicated that strain WRN-8T is a member of the genus Microbulbifer within the family Microbulbiferaceae, and the closest described neighbour in terms of 16S rRNA gene sequence identity is Microbulbifer aestuariivivens KCTC 52569T (98.1 %). The major respiratory quinone of strain WRN-8T is Q-8, its predominant fatty acids are iso-C15 : 0, iso-C17 : 0, C16 : 0, iso-C11 : 03-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and its major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, glycolipid, an unidentified phospholipid and an unidentified lipid. The draft genome obtained in this study was 3 643 020 bp, and the G+C content was 59.2 mol%. DNA-DNA hybridization (<46.3 %) and average nucleotide identity (<86.7 %) values between strain WRN-8T and the closest-related recognized Microbulbifer species confirmed the novelty of this new species. Therefore, we propose a novel species in the genus Microbulbifer to accommodate the novel isolate: Microbulbifer flavimaris sp. nov. (type strain WRN-8T=KCTC 42989T=ACCC 19926T).

DAXX promotes ovarian cancer ascites cell proliferation and migration by activating the ERK signaling pathway.

BACKGROUND: The death-domain-associated protein (DAXX) was originally identified as a protein that binds to the transmembrane death receptor FAS and enhances both FAS-induced and transforming growth factor-ß-dependent apoptosis. In a previous study, we found that nude mice injected with DAXX-overexpressing cells (ES-2-DAXX) accumulated large concentrations of first-generation ascites cells (I ascites cells). The role of DAXX in the development of ascites is unknown. The aim of this study was to analyze the effect of DAXX on proliferation and migration of ascites cells in ovarian cancer in vitro and in vivo. METHODS: Nude mice were housed in cages with a 14:10 h light:dark cycle; water and food were provided ad libitum. ES-2-DAXX cells (1×106) were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, I ascites cells were collected. The I ascites cells were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, II ascites cells were collected and cultured. Ascites cell survival, migration, and colony formation were measured using colony formation and cell growth assays. Immunofluorescent staining revealed the co-localization of DAXX and promyelocytic leukemia protein (PML) in ascites cell nuclei. Western blotting and immunohistochemistry showed that extracellular signal-related kinase (p-ERK) 1/2 and CEBP-ß were highly expressed in tumor tissues formed by II ascites cells. Through immunoprecipitation, we also found that DAXX can interact with CEBP-ß. RESULTS: DAXX enhanced ascites cell survival, migration, and colony formation. DAXX and PML nuclear foci dramatically increased in a passage-dependent manner in ascites cells, DAXX promoted the tumor growth of ascites cells in vivo, increased ascites cell proliferation in vivo, and enhanced ascites cell survival and migration by activating the ERK signalling pathway and integrating with CEBP-ß. CONCLUSIONS: DAXX can interact with CEBP-ß. DAXX can induce ovarian cancer ascites formation by activating the ERK signal pathway and binding to CEBP-ß.

Anti-Depressant-Like Effect of Sinomenine on Chronic Unpredictable Mild Stress-Induced Depression in a Mouse Model.

BACKGROUND Sinomenine (SIN) is an extract of the Chinese medicinal herb Sinomenium acutum; it has various pharmacological properties, including immunosuppression and anti-inflammation. The present study aimed to investigate whether SIN has an anti-depressant-like effect in a mouse model of depression induced by chronic unpredictable mild stress (CUMS), and to explore the underlying molecular mechanisms. MATERIAL AND METHODS A mouse model of depression was established and treated with different concentrations of SIN (30, 100, or 300 mg/kg). Then, behavioral tests, including sucrose preference test (SPT), forced swimming test (FST), and the tail suspension test (TST), were performed. The levels of norepinephrine (NE), 5-hydroxytryptamine (5-HT), and proinflammatory cytokines (interleukin-1ß [IL-1ß] interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) in the hippocampus of mice were detected by ELISA assay. The levels of p-p38, p-p65, NLRP3, ASC, and caspase-1 were measured by Western blot or/and qRT-PCR. RESULTS The results showed that SIN significantly relieved CUMSinduced depressive-like behaviors. Compared with the model mice, SIN treatment significantly increased the sucrose preference of the mice, and the immobility time in the forced swimming and the tail suspension test were shortened. In addition, SIN decreased CUMS-induced reduction in the concentrations of NE and 5-HT in the hippocampus of mice. SIN reduced CUMS-induced increases in the levels of IL-1ß, IL-6, and TNF-α in the hippocampus of mice. Furthermore, activation of the p38MAPK-NF-κB pathway and the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome induced by CUMS were inhibited by SIN treatment. CONCLUSIONS In conclusion, our results indicate the antidepressantlike effects of SIN on chronic unpredictable mild stress-induced depression in a mouse model.

Bafilomycin A1 alleviates depression­like symptoms in chronic unpredictable mild stress rats.

Major depression is a multifactorial disease. Emerging evidence has suggested that autophagy is involved in the pathological process of depressive disorders. Bafilomycin A1 (Baf A1), is an inhibitor of vacuolar H+­ATPase that is frequently used at high concentrations to block late­phase autophagy. However, whether Baf A1 has antidepressant effects remains to be elucidated. The current study aimed to evaluate the antidepressant effects of Baf A1 in rats with chronic unpredictable mild stress (CUMS) and its potential mechanism. The CUMS animal model was established. The sucrose preference test, open­field test (OFT) and forced swim test (FST) were applied to evaluate the depressive behavior. Synaptic plasticity­associated proteins synaptophysin and postsynaptic density protein 95 were measured by western blotting and immunofluorescence. Apoptosis­ and autophagy­associated proteins in addition to pro­inflammatory cytokines, including interleukin­1ß and tumor necrosis factor­α, were detected by western blotting, reverse transcription­quantitative polymerase chain reaction or ELISA. A 4­week treatment period with Baf A1 markedly ameliorated CUMS­induced behavioral abnormalities, including increasing sucrose intake, improving locomotor activity in the OFT, and decreasing immobility time in the FST. In addition, treatment with Baf A1 restored the dysregulation of synaptic plasticity and inhibited neuroinflammation in rats exposed to CUMS. Furthermore, Baf A1 decreased the levels of apoptosis­ and autophagy­associated proteins induced by CUMS. The present study demonstrated that Bafilomycin A1 resulted in antidepressant effects in rats, which may be mediated by the reversal of apoptosis, autophagy and neuroinflammation in the hippocampus.

Association between COMT Polymorphism Val158Met and Opioid Consumption in Patients with Postoperative Pain: A Meta-Analysis.

BACKGROUND/AIMS: Several factors influencing postoperative pain and the effect of opioid analgesics have been investigated on an individual level. The aim of this study was to clarify the impact of catecholamine-O-methyltransferase (COMT) gene Val158Met on opioid consumption in postoperative patients. METHODS: A systematic review and meta-analysis of the literature up to September 30, 2017, were performed by using PubMed, Cochrane Library, ISI Web of Science, and Chinese National Knowledge Infrastructure (CNKI) database. The meta-analysis examined all studies involving the association between genetic polymorphisms of COMT Val158Met and opioid consumption during the acute postoperative period. RESULTS: Of the 153 identified studies, 23 studies were retrieved for systematic review and 10 studies were retrieved for meta-analysis. However, it was impossible to conduct meta-analysis on the association between COMT Val158Met polymorphism and postoperative pain because of heterogeneity of the data. Overall, meta-analysis showed that COMT Val/Met carriers consumed less opioid for analgesia within the first 24 hours after surgery (SMD = 0.14, 95% CI = [0.03, 0.25], P = 0.01) but not within 48 hours (SMD = 0.14, 95% CI = [0.08, 0.36], P = 0.21). There was no significant difference in opioid consumption between Val/ Val and Met/Met patients. CONCLUSION: Patients with Val/Met but not Met/Met allele variant consumed less opioid, though larger and better-designed studies are required to obtain an exclusive conclusion about the correlation between postoperative pain and COMT Val158Met polymorphism.

TET1 inhibits cell proliferation by inducing RASSF5 expression.

Tet methylcytosine dioxygenases (TETs) catalyze the oxidative reactions of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). However, TET1 roles in ovarian cancer cell growth are unknown. Here, we show that ectopic expression of TET1 increased 5hmC levels, and inhibited proliferation and colony formation in ovarian cancer cell lines. Furthermore, in vitro and in vivo functional studies demonstrated that TET1 overexpression is necessary for the suppression of ovarian cancer growth, whereas depletion of TET1 expression had the opposite effect. Furthermore, the results of RNA-seq and qRT-PCR analyses identified a tumor suppressor, Ras association domain family member 5 (RASSF5), as the key downstream target of TET1. TET1 promotes RASSF5 expression by demethylating a CpG site within RASSF5 promoter. Up-regulated RASSF5 expression leads to the suppression of ovarian cancer cells growth. Additionally, we demonstrated that inhibition of CUL4-DDB1 ubiquitin ligase complex decrease 5hmC levels in ovarian cancer cells. These results provide new insights into the understanding of how ovarian cancers develop and grow, and identify TET1 as a key player in this process.

PML silencing inhibits cell proliferation and induces DNA damage in cultured ovarian cancer cells.

The promyelocytic leukemia (PML) gene is a tumor suppressor gene. It was first identified in acute promyelocytic leukemia, in which it is fused to retinoic acid receptor α by the (15;17) chromosomal translocation. The function of the PML protein is frequently lost or aberrant in human solid tumors. In human ovarian carcinoma tissue, PML detected by immunohistochemistry was highly expressed. A PML-silencing vector, pSRG-shPml, was constructed and used to transfect human ovarian cancer cells. Cells were cultured and selected with puromycin for 10-15 days, and then the PML mRNA expression levels were detected by RT-qPCR and immunofluorescence. Proliferation and clone number of PML-depleted cells were detected using MTT assay and colony-forming assay. The protein expression associated with DNA damage and apoptosis was assessed in PML-depleted cells using western blot analysis and immunofluorescence. The results showed that PML was highly expressed in human ovarian tissue. The proliferation and colony formation of ovarian cancer cells were significantly inhibited after PML was depleted. Western blot analysis and immunofluorescence revealed that p-H2AX and cleaved caspase-3 expression significantly increased after PML silencing. PML was located in the nucleus, and it formed foci after X-ray irradiation. PML foci increased significantly with increasing irradiation doses.

In vitro and in vivo characterization of a novel H1N1/2009 influenza virus reassortant with an NS gene from a highly pathogenic H5N1 virus, isolated from a human.

The triple-reassortant H1N1/2009 influenza A virus, which caused the first influenza pandemic of the 21st century, is generally associated with mild disease and a relatively low mortality rate comparable to that of seasonal influenza virus outbreaks. There is a growing concern about the potential for reassortment between the low-mortality H1N1/2009 and other high-mortality influenza viruses. Here, we describe and characterize a novel reassortant H1N1/2009 influenza virus, isolated from a human sample, that contained an NS gene from a highly pathogenic H5N1 virus. We evaluated the effect of the acquired NS gene on viral virulence both in vitro and in vivo and found that the novel NS-reassorted influenza virus replicated well in different cell lines and several organs of BALB/c mice without prior adaption and induced a cytokine imbalance. Therefore, there is a continued risk for further reassortment of the H1N1/2009 virus, and therefore, systematic surveillance should be enhanced to prepare for the next possible pandemic.

Loss of autophagy leads to failure in megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis in mice.

During hematopoiesis, megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis are regulated at multiple stages, which involve successive lineage commitment steps and proceed with polyploidization, maturation, and organized fragmentation of the cytoplasm, leading to the release of platelets in circulation. However, the cellular mechanisms by which megakaryocytes derive from their progenitors and differentiate into platelets have not fully been understood. Using an Atg7 hematopoietic conditional knockout mouse model, we found that loss of autophagy, a metabolic process essential in homeostasis and cellular remodeling, caused mitochondrial and cell cycle dysfunction, impeding megakaryopoiesis and megakaryocyte differentiation, as well as thrombopoiesis and subsequently produced abnormal platelets, larger in size and fewer in number, ultimately leading to severely impaired platelet production and failed hemostasis.

Autophagy regulates the cell cycle of murine HSPCs in a nutrient-dependent manner.

Autophagy is implicated in hematopoiesis, but its role in the regulation of the hematopoietic stem and progenitor cell (HSPC) cycle remains obscure. Here, we show that autophagy is essential to maintain and regulate the cell cycle of HSPCs in a nutrient-dependent manner. The loss of autophagy via conditional deletion of its essential gene atg7 ablated the cell cycle of HSPCs. Under physiologic or nutrient-rich conditions, the in vivo activation of autophagy promoted the cell cycle entry of hematopoietic stem cells and upregulated cyclin D3 expression in HSPCs. In contrast, under poor nutrient conditions, the ex vivo inhibition of early, but not late, autophagy signaling events enhanced the G1/S transition of HSPCs. Cyclin D3 was downregulated in HSPCs in response to nutrient stress. A knockdown of cyclin D3 blocked the G1/S transition in HSPCs. Surprisingly, the ex vivo inhibition of starvation-induced early, but not late, autophagy signaling inhibited the ubiquitin-mediated degradation of cyclin D3 in HSPCs. Furthermore, a conditional autophagy defect also had this effect, leading to an elevated cyclin D3 level in these cells. This suggested a dependency of the proteasomal degradation of cyclin D3 on early, but not late, events in autophagy signaling under nutrient stress in HSPCs. Our results thus indicate a dual role of autophagy in the nutrient-dependent modulation of the cell cycle entry of hematopoietic stem cells and G1/S transition of HSPCs via the regulation of cyclin D3 to maintain a proper cell cycle in HSPCs and normal hematopoiesis during adult life.

Robo3.1A suppresses slit-mediated repulsion by triggering degradation of Robo2.

Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit-triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans-Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome- and lysosome-dependent degradation pathway.