Tumor necrosis factor-alpha (TNF-α) may disrupt the extracellular matrix components comprising the blood-retinal barrier (BRB) in patients with posterior uveitis, such as Behçet's disease. We investigated changes in the mRNA expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human BRB cells in the presence of TNF-α in vitro and examined the effect of infliximab addition.Cells were cultured in the presence or absence of TNF-α, and TNF-α-exposed cells were treated with or without infliximab. We measured the expression levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 mRNA in human retinal microvascular endothelial ACBRI181 cells and retinal pigment epithelial ARPE-19 cells by real-time polymerase chain reaction. The cell-derived proteins degraded by MMP were observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Expression of MMP-3 increased and TIMP-1 decreased in the presence of 10 ng/mL TNF-α in ACBRI181 cells. Expression of MMP-1 increased and TIMP-2 decreased in the presence of 10 ng/mL TNF-α in ARPE-19 cells. These altered levels of expression were reversed by the addition of infliximab. The cell-derived proteins degraded by MMP-1 and -3 were detected in each set of cells.The presence of TNF-α altered expression of MMPs and TIMPs in cells comprising the BRB, and infliximab counteracted this alteration.
Abstract Epithelial ovarian cancer is a deadly disease due to late detection and lack of targeted therapies. Novel therapies for ovarian cancer are clearly needed and epigenetic agents have emerged as promising new therapies for ovarian cancer and other solid tumors. Clinical success observed for patients with lung cancer suggests that the actions of the DNA methyltransferase inhibitor 5-azacytidine (AZA) may stimulate the immune response and target immune cells to clear tumors. Ovarian cancer is a good candidate for immune therapy, as infiltrating lymphocytes predict longer time to recurrence. Therapies that upregulate the immune system have been shown to be effective in mouse and human ovarian cancers. To determine how epigenetic therapy affects ovarian cancer cells, we utilized genome-wide methylation and expression profiling on 17 ovarian cancer cell lines treated with low-dose AZA. We discovered upregulation of immunomodulatory pathways including viral defense, type I interferon signaling, antigen processing and presentation, and immune evasion via Gene Set Enrichment Analysis. In addition, we observed strong upregulation of cancer testis antigens and molecules that attract and activate natural killer cells. Endogenous retroviruses were also increased; increased transcription of these elements caused by reversal of DNA methylation may trigger the viral defense/ interferon response. This was a specific, not an off-target, effect of AZA, as a colon cancer cell line (DKO) genetically haploinsufficient for DNMT1 and lacking DNMT3b showed a similar upregulation of immune genes. Treatment with the chemotherapeutic agent carboplatin did not upregulate the same immune response genes. The cell lines that had the highest upregulation of immune genes after AZA treatment were often the best responders to AZA when grown as xenografts in NOD/SCID mice. We hypothesize that AZA activates the immune response in cancer cells, resulting in tumor cell killing. Validation of immune target genes showed that AZA treatment activates the interferon response, including transcription of interferon beta. Media transferred from AZA-treated cells to naïve cells was sufficient to cause an interferon response in the target cells, as evidenced by increased levels of interferon-stimulated genes. FACS staining confirmed the upregulation of antigen-presenting MHC Class I molecules on the cell surface of AZA-treated ovarian cancer cells. Adding the histone deacetylase inhibitor MS275 (entinostat) to AZA treatment increased AZA-induced expression of interferon-stimulated genes IRF7 and IFI27. This combination treatment also caused an increase in the activating chromatin mark H3Ac at the IRF7 promoter. AZA increased transcript levels and cell surface expression of the PD-L1 molecule, ligand for PD-1 on lymphocytes and responsible for evasion of the host immune system by tumors, in ovarian cancer cell lines. We predict that AZA plus anti-PD-L1 (or PD-1) treatment, which has shown success in non-small cell lung cancer, might be effective for ovarian cancers. Future work will involve combination treatment of AZA and anti-PD-1 in immune competent ovarian cancer mouse models. The immune pathways upregulated by AZA were used to query hundreds of primary ovarian cancer samples from the Cancer Genome Atlas project (TCGA). Ovarian tumors classified clearly into “high” and “low” interferon gene expression subsets. The “high” interferon group was associated with higher levels of antigen presentation as well as an expression signature associated with better prognosis. The “low” interferon group indicates a group of solid tumors that could be targeted by AZA to upregulate levels of immunomodulatory pathways, and thus apoptosis or targeting by host immune cells. These preliminary results point to an “immune priming” role for the DNA methyltransferase inhibitor 5-azacytidine and could lead to clinical trials with combined AZA and immune therapies in epithelial ovarian cancer. This abstract is also presented as Poster B79. Citation Format: Katherine B. Chiappinelli, Ray-Whay Chiu Yen, Huili Li, Michael Topper, Cynthia A. Zahnow, Stephen B. Baylin. Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR12.
<p>Global and promoter CpG island demethylation in available paired pre- and post-treatment (8 weeks) hormone-resistant and triple-negative tumor biopsies.</p>
<p>Kaplan-Meier curves for survival after disease progression among women with advanced hormone-resistant breast cancer by optional continuation (blue curve) or event monitoring (red curve). The 95% confidence limits are shaded; Tick marks indicate censored data.</p>
Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.
Enterovirus D68 (EV-D68) is a globally re-emerging respiratory pathogen implicated in outbreaks of severe respiratory illnesses and associated with acute flaccid myelitis. However, effective vaccines or treatments for EV-D68 infections remain scarce. We demonstrated that the active constituent of blueberries, pterostilbene (Pte), and its major metabolite, pinostilbene (Pin), facilitated innate immune responses in EV-D68-infected human respiratory cells. Pte and Pin treatment clearly relieved EV-D68-triggered cytopathic effects. Importantly, both Pte and Pin disrupted viral RNA replication (EC 50 rank from 1.336 to 4.997 µM) and infectious virion production in a dose-dependent manner, without cytotoxicity at virucidal concentrations. Pte- or Pin-treated respiratory cells did not show any influences on EV-D68 entry but showed substantially decreased viral RNA replication and protein synthesis. Finally, we showed that Pte and Pin broadly suppressed the replication capacity of circulating EV-D68 strains isolated from recent pandemics. In summary, our results suggest that Pte and its derivative, Pin, enhance host immune recognition of EV-D68 and suppress EV-D68 replication, which represents a promising strategy for antiviral drug development.
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