Corticosteroids reduce pathologic interferon responses by downregulating STAT1 in patients with high-risk COVID-19.

We do not yet understand exactly how corticosteroids attenuate hyperinflammatory responses and alleviate high-risk coronavirus disease 2019 (COVID-19). We aimed to reveal the molecular mechanisms of hyperinflammation in COVID-19 and the anti-inflammatory effects of corticosteroids in patients with high-risk COVID-19. We performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from three independent COVID-19 cohorts: cohort 1 was used for comparative analysis of high-risk and low-risk COVID-19 (47 PBMC samples from 28 patients), cohort 2 for longitudinal analysis during COVID-19 (57 PBMC samples from 15 patients), and cohort 3 for investigating the effects of corticosteroid treatment in patients with high-risk COVID-19 (55 PBMC samples from 13 patients). PBMC samples from healthy donors (12 PBMC samples from 12 donors) were also included. Cohort 1 revealed a significant increase in the proportion of monocytes expressing the long noncoding RNAs NEAT1 and MALAT1 in high-risk patients. Cohort 2 showed that genes encoding inflammatory chemokines and their receptors were upregulated during aggravation, whereas genes related to angiogenesis were upregulated during improvement. Cohort 3 demonstrated downregulation of interferon-stimulated genes (ISGs), including STAT1, in monocytes after corticosteroid treatment. In particular, unphosphorylated STAT-dependent ISGs enriched in monocytes from lupus patients were selectively downregulated by corticosteroid treatment in patients with high-risk COVID-19. Corticosteroid treatment suppresses pathologic interferon responses in monocytes by downregulating STAT1 in patients with high-risk COVID-19. Our study provides insights into the mechanisms underlying COVID-19 aggravation and improvement and the effects of corticosteroid treatment.

Staufen1 and UPF1 exert opposite actions on the replacement of the nuclear cap-binding complex by eIF4E at the 5' end of mRNAs.

Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.

Targeting ODC1 inhibits tumor growth through reduction of lipid metabolism in human hepatocellular carcinoma.

Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.

Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma.

PURPOSE: Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells. MATERIALS AND METHODS: Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42 °C/60 min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice. RESULTS: mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice. CONCLUSIONS: These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.

Melatonin enhances arsenic trioxide-induced cell death via sustained upregulation of Redd1 expression in breast cancer cells.

Melatonin is implicated in various physiological functions, including anticancer activity. However, the mechanism(s) of its anticancer activity is not well understood. In the present study, we investigated the combined effects of melatonin and arsenic trioxide (ATO) on cell death in human breast cancer cells. Melatonin enhanced the ATO-induced apoptotic cell death via changes in the protein levels of Survivin, Bcl-2, and Bax, thus affecting cytochrome c release from the mitochondria to the cytosol. Interestingly, we found that the cell death induced by co-treatment with melatonin and ATO was mediated by sustained upregulation of Redd1, which was associated with increased production of reactive oxygen species (ROS). Combined treatment with melatonin and ATO induced the phosphorylation of JNK and p38 MAP kinase downstream from Redd1 expression. Rapamycin and S6K1 siRNA enhanced, while activation of mTORC1 by transfection with TSC2 siRNA suppressed the cell death induced by melatonin and ATO treatment. Taken together, our findings suggest that melatonin enhances ATO-induced apoptotic cell death via sustained upregulation of Redd1 expression and inhibition of mTORC1 upstream of the activation of the p38/JNK pathways in human breast cancer cells.

Electro-hyperthermia inhibits glioma tumorigenicity through the induction of E2F1-mediated apoptosis.

PURPOSE: Modulated electro-hyperthermia (mEHT), also known as oncothermia, shows remarkable treatment efficacies for various types of tumours, including glioma. The aim of the present study was to investigate the molecular mechanism underlying phenotypic changes in oncothermic cancer cells. MATERIALS AND METHODS: U87-MG and A172 human glioma cells were exposed to mEHT (42 °C/60 min) three times with a 2-day interval and subsequently tested for growth inhibition using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to mEHT, global changes in gene expression were examined using RNA sequencing. For in vivo evaluation of mEHT, we used U87-MG glioma xenografts grown in nude mice. RESULTS: mEHT inhibited glioma cell growth through the strong induction of apoptosis. The transcriptomic analysis of differential gene expression under mEHT showed that the anti-proliferative effects were induced through a subset of molecular alterations, including the up-regulation of E2F1 and CPSF2 and the down-regulation of ADAR and PSAT1. Subsequent Western blotting revealed that mEHT increased the levels of E2F1 and p53 and decreased the level of PARP-1, accelerating apoptotic signalling in glioma cells. mEHT significantly suppressed the growth of human glioma xenografts in nude mice. We also observed that mEHT dramatically reduced the portion of CD133(+) glioma stem cell population and suppressed cancer cell migration and sphere formation. CONCLUSIONS: These findings suggest that mEHT suppresses glioma cell proliferation and mobility through the induction of E2F1-mediated apoptosis and might be an effective treatment for eradicating brain tumours.

Long-term Outcome of Extranodal NK/T Cell Lymphoma Patients Treated With Postremission Therapy Using EBV LMP1 and LMP2a-specific CTLs.

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

Selective inhibition of histone deacetylase 2 induces p53-dependent survivin downregulation through MDM2 proteasomal degradation.

In the present study, we found that selective inhibition of histone deacetylase 2 (HDAC2) with small inhibitory RNA (siRNA) induced survivin downregulation in a p53-dependent manner. Interestingly, suberoylanilide hydroxamic acid (SAHA) or knockdown of HDAC2 induced downregulation of Mdm2, a negative regulator of p53, at the protein level. SAHA and/or HDAC2 siRNA increased Mdm2 ubiquitination, and MG132, an inhibitor of proteosome function, prevented HDAC2 inhibition-induced degradation of Mdm2. Clinically, the mRNA levels of HDAC2 and survivin were prominently overexpressed in lung cancer patients compared to normal lung tissues. Silencing of HDAC2 enhanced the cell death caused by ionizing radiation in lung cancer cells. Collectively, our results indicate that selective inhibition of HDAC2 causes survivin downregulation through activation of p53, which is mediated by downregulation of Mdm2. They further suggest that HDAC2 may exert a dominant effect on lung cancer cell survival by sustaining Mdm2-survivin levels.

Maintenance of the viral episome is essential for the cell survival of an Epstein-Barr virus positive gastric carcinoma cell line.

While Epstein Barr virus (EBV) is associated with about 10% of gastric carcinomas worldwide, the role of the virus in the tumorigenesis of EBV-associated gastric carcinoma (EBVaGC) is unclear. Previously, we reported that a gastric cancer cell line, SNU-719, that is naturally infected with EBV closely resembles EBVaGC. Here, we attempted to eliminate the EBV genome from SNU-719 cells to ascertain the influence of EBV in EBVaGC. Southern blotting and fluorescence in situ hybridization (FISH) showed that EBV genomes were maintained as episomes in SNU-719 cells. To remove EBV episomes, SNU-719 cells were first cultured in a hydroxyurea (HU)-containing medium for up to 6 months. Real-time polymerase chain reaction and FISH results revealed no evidence of HU-mediated EBV genome reduction, although cell growth was reduced by acute HU treatment in dose- and time-dependent manners. Two small interfering RNAs against Epstein Barr nuclear antigen 1 (EBNA1) abrogated over 90% of the ectopic EBNA1 expression in HeLa cells, but only 40% of endogenous EBNA1 expression in SNU-719 cells. Together, our data suggest that maintenance of latent EBV infection is essential for the viability of EBVaGC cells, avoiding elimination of EBV episomes from the cells.

Induction of efficient differentiation and survival of porcine neonatal pancreatic cell clusters using an EBV-based plasmid expressing HGF.

Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein-Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.

Establishment and characterization of an in vivo model for Epstein-Barr virus positive gastric carcinoma.

Research regarding the role of the Epstein-Barr virus (EBV) in gastric carcinogenesis has been hampered by the absence of a suitable model system. SNU-719 is a gastric carcinoma cell line naturally infected with EBV. This cell line developed tumors in nude mice approximately 40-56 days after inoculation. SNU-719 also showed low serum dependency and anchorage independent growth in vitro. The developed tumors expressed EBERs, EBNA1, and LMP2A but not other EBV latent genes. Additionally, Qp was active and either mono- or bi-clonal EBV genome was observed in the tumor tissues. Because the developed tumors retained characteristics of EBV-associated gastric cancer, this cell line could serve as a useful in vivo system to investigate the tumorigenesis mechanism and treatment methods for this type of tumor.

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.

Mechanism of prolonged gene expression by Epstein-Barr virus-based plasmid in porcine cells.

BACKGROUND: We previously showed that an Epstein-Barr virus (EBV)-based plasmid, pEBVGFP, exerts prolonged gene expression in porcine neonatal pancreatic cell clusters (NPCCs). In this study, the mechanism underlying this was investigated. METHODS: GFP expression was analyzed in porcine cells transfected with pEBVGFP by FACS analysis and confocal microscopy. The possible integration of pEBVGFP into the chromosomal DNA was analyzed by Southern blot. Self-replication of the EBV-based plasmid in porcine cells was investigated by PCR. The NPCCs were immunostained to characterize cells transfected with pEBVGFP. RESULTS: The EBV based plasmid provided prolonged GFP expression in porcine cells and duct cells were the main cells transfected among NPCCs. Southern blot showed that the transfected pEBVGFP stayed for a long time as an episome rather than integrating into the chromosomal DNA. pEBVGFP isolated from the transfected porcine cells had methylated CpG suggesting that they self-replicated in those cells. CONCLUSIONS: The EBV-based plasmid may be useful for genetically manipulating porcine cells to enhance their value as xenotransplantation sources.

Patients with systemic lupus erythematosus have abnormally elevated Epstein-Barr virus load in blood.

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein-Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean +/- standard deviation: 463 +/- 570 EBV genome copies/3 microg PBMC DNA versus 30 +/- 29 EBV genome copies/3 microg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.

A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer.

In a process seeking out a good model cell line for Epstein-Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis.

Prolonged gene expression in primary porcine pancreatic cells using an Epstein-Barr virus-based episomal vector.

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.

Preparation and characterization of alpha-D-glucopyranosyl-alpha-acarviosinyl-D-glucopyranose, a novel inhibitor specific for maltose-producing amylase.

A novel inhibitor against maltose-producing alpha-amylase was prepared via stepwise degradation of a high-molecular-weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be alpha-D-glucopyranosyl-alpha-acarviosinyl-D-glucopyranose (GlcAcvGlc) by (13)C NMR and MALDI-TOF/MS. Progress curves of PNPG2 hydrolysis by various amylolytic enzymes, including MGase, ThMA, and CDase I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. Analytical ultracentrifugation and X-ray crystallography analyses revealed that the presence of GlcAcvGlc increased the dimerization of ThMA. The formation of dimer complexed with GlcAcvGlc might induce a conformational change in ThMA, leading to a two-step inhibition process. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.