A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

PD-1 inhibitors-associated myocarditis in non-small cell lung cancer patients.

Background: Immune checkpoint inhibitors (ICIs)-associated myocarditis remains a rare but fatal adverse event. The authors sought to provide a comprehensive clinical description of ICI-associated myocarditis by analyzing symptoms, laboratory indicators, imaging features, and management of ICI-associated myocarditis in patients with non-small cell lung cancer (NSCLC). Methods: A retrospective study was conducted to analyze 14 ICI-associated myocarditis cases and 45 control patients to clarify clinical features of ICI-associated myocarditis. Detailed laboratory tests and imaging examinations were performed in 14 cases, and the rescue process and follow-up after the onset of myocarditis were recorded. Results: A total of 14 (2.08%) NSCLC patients developed ICI-related myocarditis, with a median time of onset of 34 days (interquartile range, 12 to 146 days) after ICI initiation. The most common concurrent adverse events in cases were myositis (P<0.001) and peripheral neuritis (P<0.001). Among cases, cardiac troponin I (cTnI) levels were abnormally elevated in 92% of patients, and electrocardiogram (ECG) showed abnormal in all cases. Steroid therapy was used in 92.9% of patients with ICI-associated myocarditis, of which the response rate to steroids was 76.9% and the mortality rate was 7.1%. A dose of 1 g/d of glucocorticoid supplemented by immunoglobulin was observed to be effective for severe myocarditis. Conclusions: Early identification and treatment are essential for managing myocarditis caused by ICI. Routine monitoring of cTnI level and ECG is most sensitive for the early diagnosis of ICI-related myocarditis. High-dose of glucocorticoids can effectively relieve the symptoms of ICI-associated myocarditis and stabilize the condition, especially for fulminant myocarditis.

Histone Deacetylase Inhibitor Sensitizes ERCC1-High Non-small-Cell Lung Cancer Cells to Cisplatin via Regulating miR-149.

Resistance to platinum-based chemotherapy becomes a major obstacle in non-small-cell lung cancer (NSCLC) treatment. Overexpression of the excision repair cross-complementing 1 (ERCC1) gene is reported to negatively influence the effectiveness of cisplatin-based therapy for NSCLC cells. In this study, we confirm that high ERCC1 expression correlates with cisplatin resistance in NSCLC cells. Importantly, histone deacetylase inhibitors (HDACis) re-sensitize ERCC1-high NSCLC cells to cisplatin both in vitro and in vivo. Mechanistically, the HDACi induces the expression of miR-149 by acetylation and activation of E2F1, which directly targets ERCC1 and inhibits ERCC1 expression. Inhibition of miR-149 reverses the promotion effect of HDACis on cisplatin-induced DNA damage and cell apoptosis in ERCC1-high NSCLC cells. In conclusion, this study reveals a novel mechanism by which HDACis re-sensitizes ERCC1-high NSCLC cells to cisplatin via regulation of the E2F1/miR-149/ERCC1 axis, and we propose that combination of HDACis and cisplatin might hold promise to be a more effective therapeutic paradigm for ERCC1-high NSCLCs.

Implication for alphavirus host-cell entry and assembly indicated by a 3.5Å resolution cryo-EM structure.

Alphaviruses are enveloped RNA viruses that contain several human pathogens. Due to intrinsic heterogeneity of alphavirus particles, a high resolution structure of the virion is currently lacking. Here we provide a 3.5 Å cryo-EM structure of Sindbis virus, using block based reconstruction method that overcomes the heterogeneity problem. Our structural analysis identifies a number of conserved residues that play pivotal roles in the virus life cycle. We identify a hydrophobic pocket in the subdomain D of E2 protein that is stabilized by an unknown pocket factor near the viral membrane. Residues in the pocket are conserved in different alphaviruses. The pocket strengthens the interactions of the E1/E2 heterodimer and may facilitate virus assembly. Our study provides structural insights into alphaviruses that may inform the design of drugs and vaccines.

FOXC2 promotes epithelial-mesenchymal transition and cisplatin resistance of non-small cell lung cancer cells.

PURPOSE: Platinum-based drugs, particularly cisplatin (DDP), are used in the treatment of non-small cell lung cancer (NSCLC). However, development of drug resistance remains the major therapeutic barrier in NSCLC. METHODS: The potential cisplatin resistance-related genes were identified from the global transcriptomes of cisplatin-resistant A549/DDP cells using microarray analysis. Gain- and loss-of-function assays were performed to analyze the effects of Forkhead Box Protein C2 (FOXC2) on the in vitro and in vivo sensitivity of NSCLC cells to cisplatin and its possible molecular mechanisms. RESULTS: Using global transcriptome analysis, we found that FOXC2 was one of the most upregulated molecules in A549/DDP cells compared with A549 cells. We further confirmed that the expression of FOXC2 was significantly increased in cisplatin-resistant NSCLC tissues. FOXC2 knockdown significantly increased the in vitro and in vivo sensitivity of A549/DDP cells to cisplatin, whereas overexpression of FOXC2 increased cisplatin resistance in cisplatin-sensitive NSCLC cells. Moreover, we found that FOXC2 promoted cisplatin resistance by induction of epithelial-mesenchymal transition (EMT) in NSCLC cells. Furthermore, FOXC2 activated the AKT/GSK3ß signaling pathway, and then increased the protein expression of EMT-related transcription factor Snail. Inhibition of AKT or knockdown of Snail reversed FOXC2-induced EMT and cisplatin resistance of NSCLC cells. CONCLUSION: FOXC2 enhanced cisplatin resistance of NSCLC cells through activating AKT/GSK3ß/Snail/EMT signaling pathway, which may be a potential novel therapeutic target for overcoming drug resistance in human NSCLCs.

Rapid generation of a novel DPP-4 inhibitor with long-acting properties: SAR study and PK/PD evaluation.

Drug compliance is critical for patients with chronic diseases such as diabetes. In our continuous effort to find better glucose-lowering agents, an exploration for long-acting DPP-4 inhibitors had been launched. Based on our previously reported compounds bearing a pyrrolopyrimidine scaffold, the lead compound 4a (IC50 = 2.3 nM, t1/2(rat) = 5.46 h) with pharmacokinetic superiority was rapidly determined. Further SAR study indicated that the pyrrole ring was generally tolerable for variation, in which a ß-substitution gave a better DPP-4 affinity. In depth evaluation of the pyrrole ring ß position identified a highly potent compound 12a (IC50 = 0.76 nM, t1/2(rat) = 7.89 h). In vivo pharmacodynamics tests demonstrated similar or even slightly better sustained DPP-4 inhibition for compounds 4a and 12a compared with the first marketed once-weekly drug trelagliptin in this category, indicating that improvements to DPP-4 inhibitory activity or pharmacokinetic profile might be feasible ways to rapidly generate long-acting DPP-4 inhibitors.

Suberoylanilide hydroxamic acid (SAHA) promotes the epithelial mesenchymal transition of triple negative breast cancer cells via HDAC8/FOXA1 signals.

Inhibitor of histone deacetylases (HDACIs) have great therapeutic value for triple negative breast cancer (TNBC) patients. Interestingly, our present study reveals that suberoyl anilide hydroxamic acid (SAHA), one of the most advanced pan-HDAC inhibitor, can obviously promote in vitro motility of MDA-MB-231 and BT-549 cells via induction of epithelial-mesenchymal transition (EMT). SAHA treatment significantly down-regulates the expression of epithelial markers E-cadherin (E-Cad) while up-regulates the mesenchymal markers N-cadherin (N-Cad), vimentin (Vim) and fibronectin (FN). However, SAHA has no effect on the expression and nuclear translocation of EMT related transcription factors including Snail, Slug, Twist and ZEB. While SAHA treatment down-regulates the protein and mRNA expression of FOXA1 and then decreases its nuclear translocation. Over-expression of FOXA1 markedly attenuates SAHA induced EMT of TNBC cells. Further, silence of HDAC8, while not HDAC6, alleviates the down-regulation of FOXA1 and up-regulation of N-Cad and Vim in MDA-MB-231 cells treated with SAHA. Collectively, our present study reveals that SAHA can promote EMT of TNBC cells via HDAC8/FOXA1 signals, which suggests that more attention should be paid when SAHA is used as anti-cancer agent for cancer treatment.

Prognostic value of ERCC1, RRM1, and TS proteins in patients with resected non-small cell lung cancer.

PURPOSE: Recent clinical trials showed that expression of excision repair cross-complementation group 1 (ERCC1), ribonucleotide reductase M1 (RRM1), and thymidylate synthase (TS) proteins was able to predict the effects of non-small cell lung cancer (NSCLC) to chemotherapy. However, it remains unknown whether the adjuvant chemotherapy based on expression of the three proteins has survival significance in Chinese NSCLC patients. METHODS: We investigated 128 Chinese patients receiving chemotherapy after tumor resection for expression of these proteins using immunohistochemistry. Based on protein expression, patients were assigned to two groups for different adjuvant chemotherapy regimes. The disease-free survival (DFS) data were collected and analyzed using Kaplan-Meier curves and Cox models. RESULTS: We found that DFS of these patients with carboplatin and a third-generation agent (gemcitabine or pemetrexed) stratified by protein expression showed no statistical difference between individual treatment versus non-individuation treatment analyzed using Kaplan-Meier method (P = 0.143, median 23.9 vs. 30.8 months). Furthermore, the multivariate analysis showed that histology and tumor stages were independent predictors for DFS in these patients. CONCLUSIONS: The results suggest that chemotherapy based on ERCC1, RRM1, and TS expression did not have significant impact on DFS of patients with resection of NSCLC.

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells.

AIMS: Indoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction. MAIN METHODS: IDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay. KEY FINDINGS: We found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression. SIGNIFICANCE: These results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.

Sodium butyrate down-regulation of indoleamine 2, 3-dioxygenase at the transcriptional and post-transcriptional levels.

The clinical outcomes of most immunotherapeutic strategies have been less effective than anticipated partially because of the tumor immune tolerance induced by many immune tolerance factors, which originate from the tumor and tumor microenvironment. Indoleamine 2, 3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-inducible enzyme and is one of main immune tolerance factors during tumor development. Sodium butyrate (NaB) has received much attention as a potential chemopreventive agent for cancer treatment due to its protective action against intracellular events including IFN-γ-mediated signaling transduction. Therefore, the question remains whether IDO is a target of the anti-tumor action of NaB. In this study, we demonstrate for the first time that NaB down-regulated IDO via both transcriptional and post-transcriptional mechanisms. NaB repressed the activity of STAT1 to inhibit STAT1-driven transcriptional activity of IDO. These mechanisms included inhibiting STAT1 701 tyrosine phosphorylation, nuclear translocation, and repression of STAT1 binding to γ-activated sites (GAS). Moreover, immunoprecipitation and immunoblotting assays showed that treatment of cells with NaB caused dramatic ubiquitination of total intracellular proteins, including IDO. Blocking 26S proteasome activity by addition of its specific inhibitor, bortezomib, reversed the ubiquitination and down-regulation of IDO. These results suggest that NaB-induced STAT1 activity inhibition and ubiquitin/proteasome-dependent proteolysis are involved in the down-regulation of IDO. The discoveries in this study represent a new mechanism in the anti-tumor action of NaB and may have implications for development of clinical cancer immunotherapy.

Prevention of spontaneous tumor development in a ret transgenic mouse model by ret peptide vaccination with indoleamine 2,3-dioxygenase inhibitor 1-methyl tryptophan.

The present study investigated an immunotherapeutic strategy for rearranged during transfection proto-oncogene (ret)-associated carcinomas in a transgenic MT/ret 304/B6 mouse model in which spontaneous tumors develop due to overexpression of the ret gene. A Ret peptide vaccine comprising an extracellular fragment of Ret protein and Th1-polarized immunoregulator CpG oligonucleotide (1826) induced strong and specific cellular and humoral immune responses in wild-type C57BL/6 mice, showing that the Ret peptide has a strong immunogenic potential as part of an antitumor vaccine. In MT/ret 304/B6 mice, however, the vaccine was only modestly effective as an inducer of the humoral immune response, and it failed to elicit a T-cell response. An immunohistochemical analysis revealed marked indoleamine 2,3-dioxygenase expression after immunization with Ret peptide vaccine in the lymph nodes and spleens of MT/ret 304/B6 mice. The systemic administration of the potent inhibitor of indoleamine 2,3-dioxygenase 1-methyl tryptophan (1MT) along with Ret vaccine produced a significant increase in tumor-specific cytotoxic activity. A delay in spontaneous tumor development was also observed in the MT/ret 304/B6 mice to which the Ret vaccine and 1MT were administered. These results indicate that an improved Ret vaccine composed of Ret peptide plus CpG oligonucleotide plus 1MT is a potential therapeutic strategy for treatment of ret-associated carcinomas.

[Curcumin inhibiting the expression of indoleamine 2,3-dioxygenase induced by IFN-gamma in cancer cells].

OBJECTIVE: To investigate the effect of curcumin on indoleamine 2, 3 -dioxygenase (IDO) expression induced by IFN-gamma in cancer cells. METHODS: A431, HeLa, HepG2, CNE2 cells were treated with curcumin for 24 hours, then the cell proliferation was detected by methyl thiazolyl tetrazolium (MTF) assay. The effects of curcumin on IDO expression induced by IFN-gamma in these cancer cells were demonstrated by Western blot. The transcription of interferon responsive factor-1 (IRF-1), which was a key transcription factor regulating IDO expression, was analyzed by reverse transcription polymerase chain reaction (RT-PCR) under the treatment of curcumin. RESULTS: Curcumin inhibited the expression of IDO in these cancer cells. However, curcumin did not inhibit the transcription of IRF-1 in cancer cells. CONCLUSION: Curcumin could inhibit the expression of IDO in cancer cells.

[Proliferation cycle changes of Epstein-Barr virus in nasopharyngeal carcinoma cell line CNE after infection].

BACKGROUND & OBJECTIVE: The strategy of gene therapy on nasopharyngeal carcinoma (NPC) is determined by the characteristics of proliferation cycle of Epstein-Barr virus (EBV) in different histological types of NPC cells. This study was to investigate the characteristics of proliferation cycle of EBV in NPC cell line CNE2. METHODS: CNE2 cells were co-cultivated with marmoset lymphocyte line B95-8, which was EBV-positive. Then B95-8 cells were removed by cytotoxic test of complement activation. The initial infection state of EBV in CNE2 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization. RESULTS: CNE2 cells were infected with EBV by cell-to-cell contacting. The transcription of BZLF1, the symbol gene for EBV lytic phase, maintained 12 days on average. The transcription of EBV-encoded small RNA (EBER), the symbol gene for EBV infectious phase, maintained 22 days on average. CONCLUSIONS: After invading CNE2 cells, most EBV initially might be at lytic phase, then switch to the relatively stable latent phase, and finally be lost due to cell culture and passage.

Interfering with cellular signaling pathways enhances sensitization to combined sodium butyrate and GCV treatment in EBV-positive tumor cells.

The combination of sodium butyrate (NaB) and ganciclovir (GCV) was considered to be a noteworthy therapeutic strategy in Epstein-Barr virus (EBV)-associated cancers. However, clinical studies have indicated that an extremely high dose of NaB is required to obtain the expected curative efficacy. This obviously limits the practical clinical application of the two drugs combined. In this study, we investigated the possibility of sensitizing tumor cells to NaB and GCV mediated cytotoxicity by modulating intracellular signal pathways. The results showed that the disruption of Ras/Raf activity by expressing dominant negative forms of both Ras and Raf-1 did not alter the potency of the NaB and GCV combination in the EBV-positive cell line, B95-8. However, blocking Akt activity by expressing its dominant negative form remarkably promoted NaB and GCV-mediated cytotoxicity via a thymidine kinase (TK)-independent mechanism. Interestingly, it was found that the constitutive activation of mitogen-activated protein kinase kinase kinase 1 (MEKK1) dramatically enhanced the sensitization of the cells to the combination of NaB and GCV, accompanied with an increase in TK expression in B95-8 cells. These results suggest that interfering with either the Akt or MEKK1 signaling pathway may be a useful therapeutic strategy to increase the sensitivity of EBV-positive tumor cells to the combination of NaB and GCV.

Enhancement of dendritic cell-tumor fusion vaccine potency by indoleamine-pyrrole 2,3-dioxygenase inhibitor, 1-MT.

PURPOSE: Dendritic cell (DC)-based cancer vaccines are currently being evaluated as novel anti-tumor vaccination strategies, but in some cases, they are demonstrated to have poor clinical efficacies than anticipated. A potential reason is immune tolerance due to the immunosuppressive enzyme, indoleamine-pyrrole 2,3-dioxygenase (IDO). The aim of this study was to determine whether blocking the activity of IDO might improve the anti-tumor efficacy of DC/Lewis lung carcinoma (LLC) fusion vaccine applied to the mouse LLC model. METHODS: To prepare the DC/LLC fusion vaccine, DCs were fused with LLC using polyethylene glycol (PEG) as described. The IDO expression in the DC/LLC fusion vaccine and in the vaccinated mice was detected by western blot (WB) and/or immunohistochemical (IHC) analysis. This fusion vaccine, as a single agent or in combination with 1-methyl-tryptophan (1-MT, an IDO inhibitor), was administered to LLC mice. The anti-tumor efficacy in different treatment was determined by regular observation of tumor development and the level of splenic cytotoxic T lymphocyte (CTL) response, which was examined by lactate dehydrogenase (LDH) release. RESULTS: In the LLC mice, we observed that IDO-positive cells were extensively accumulated in tumor draining lymph nodes (TDLNs). Furthermore, WB and IHC analysis results showed that vaccination with fusion DC/LLC cells alone caused significant up-regulation of IDO in spleens. 1-MT enhanced the anti-tumor efficacy elicited by DC/LLC fusion vaccine via delaying the tumor development and inducing stronger splenic CTL responses. CONCLUSIONS: Our results indicate an IDO-mediated immunosuppressive mechanism might be involved in weakening the anti-tumor efficacy elicited by DC/LLC fusion vaccine, and specific inhibition of IDO activity might be required for development of cancer vaccines.