ROS-dependent Manner Research Articles

Identification of Deubiquitinase OTUD1 As a Novel Player in Resistance of Multiple Myeloma to Bortezomib

Introduction Multiple myeloma (MM) being one of the most widely spread haematological malignancies remains an incurable disease. As malignant plasma cells produce abnormally large amounts of immunoglobulins they particularly rely on the ubiquitin-proteasome system (UPS) to avoid aberrant protein overload. This unique feature is targeted by proteasome inhibitors (PI) that induce MM cell death especially by increasing levels of reactive oxygen species (ROS). Despite the high efficacy of PI most of the MM patients eventually relapse and expansion of drug resistant clones makes the treatment ineffective. Therefore, uncovering new molecular mechanisms of drug resistance is a crucial task. UPS is a very complex system that involves hundreds of proteins. While the roles of the proteasome and E3 ligases in PI resistance are well established, the third UPS component, deubiquitinating enzymes (DUBs), is much less explored. In this work, we performed a comprehensive search for DUBs with impact on MM pathogenesis and PI resistance, and further investigated the underlying molecular mechanisms. Methods Gene expression and survival For analysis of DUB genes (n = 101) expression in blood cells, expression dataset Gds3997, DICE database and data from (Jourdan et al., J Immunol. 2011 Oct 15;187(8):3931-41., Jourdan et al., Blood. 2009 Dec 10;114(25):5173-81.) submitted to http://www.genomicscape.com were used. For survival analysis MM patients were divided into two groups by median of gene expression for each DUB (datasets GSE2658, GSE4581 and GSE9782). Cell line models RPMI8226 and HEK293 cells were used as model cell lines. Cells with OTUD1 knockdowns and overexpression were generated by lentiviral infection using vectors containing doxycycline-inducible shRNA's and different versions of OTUD1 gene, respectively. Proliferation and cell death MTT assay was used for the analysis of cell proliferation and viability. Cell death was also evaluated by flow cytometry by staining with Annexin V and 7-AAD. Cell migration Cells were labelled with calcein-AM and placed into the upper chamber of transwell insert with 8-µM pores. After 16 hours of migration towards SDF-1α gradient, cells were counted by fluorescence detection. ROS analysis ROS was detected by labelling cells with 2′,7′-dichlorodihydrofluorescein diacetate and measuring fluorescence intensity using flow cytometry. Immunoprecipitation HEK293 cells were co-transfected with HA-OTUD1 and FLAG-KEAP1, and reciprocal co-immunoprecipitation and western blot analyses were performed. Results We analysed the expression of all human DUBs in different blood cell types and identified OTUD1 as the most differentially expressed DUB between B-cell lineage and other haematopoietic cells. During B-cell maturation OTUD1 expression reaches the maximum in the bone marrow plasma cells. MM patients with low OTUD1 expression had significantly worse prognosis in OS based on three large datasets (p value= 0,035; 0,008; 2.4e−06. HR=0,55; 0,19; 0,41). Expression of shRNA targeting OTUD1 in MM cell line RPMI8226 did not affect cell proliferation and migration but dramatically increased survival under oxidative stress (high ROS) conditions induced by bortezomib. Treatment with bortezomib promoted expression of OTUD1 in the wild type MM cells in a ROS-dependent manner. Additionally, we identified oxidative stress regulator, the E3 ligase KEAP1 as a novel direct interaction partner of OTUD1 that regulates OTUD1 stability under high ROS conditions. Conclusion Based on the gene expression analysis, OTUD1 was identified as a novel, potentially important player in MM pathogenesis. Low levels of OTUD1 expression in MM patients correlate with significantly worse OS. Knocking down OTUD1 in MM cells causes resistance to bortezomib. Mechanistically, bortezomib-induced ROS promotes transcription of OTUD1 mRNA and further induces stabilization of OTUD1 on protein level via disruption of OTUD1-KEAP1 complex. Our collective data suggest on a crucial role of OTUD1 in bortezomib-mediated MM cytotoxity. Further mechanistic studies delineating the role of OTUD1 in MM pathogenesis and PI resistance are ongoing. Disclosures Hajek: Janssen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Amgen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Celgene: Honoraria, Other: Consultant or advisory relationship, Research Funding; AbbVie: Other: Consultant or advisory relationship; Bristol-Myers Squibb: Honoraria, Other: Consultant or advisory relationship, Research Funding; Novartis: Other: Consultant or advisory relationship, Research Funding; PharmaMar: Honoraria, Other: Consultant or advisory relationship; Takeda: Honoraria, Other: Consultant or advisory relationship, Research Funding.

Polybrominated Diphenyl Ethers Quinone Induces NCOA4-Mediated Ferritinophagy through Selectively Autophagic Degradation of Ferritin.

Polybrominated diphenyl ethers (PBDEs) have been detected ubiquitously in biological and environmental samples. Growing epidemiological data suggested the obvious correlation of PBDEs exposure with adverse health outcomes toward human beings, but exact molecular mechanism(s) are limited. Especially, the toxicological information regarding PBDEs metabolites is missing. Thereafter, this study intends to explore unidentified cell death modalities caused by PBDEs reactive quinone-type metabolite, PBDEQ. We found that PBDEQ induces autophagy in an ROS-dependent manner. Interestingly, the results indicated that PBDEQ degraded ferritin and activated a selective autophagy (termed as ferritinophagy) by using NCOA4 as its cargo receptor. These processes may further promote the release of iron and ROS. These results suggested the incidence of ferritinophagy induced by PBDEQ, which may contribute to PBDE exposure-caused diseases and dysfunctions.

ALA-PDT alleviates the psoriasis by inhibiting JAK signalling pathway.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is a well-known treatment for non-hypertrophic actinic keratoses and superficial basal-cell carcinomas. In this study, we first revealed that ALA-PDT treatment effectively ameliorated the psoriasis-like lesion in imiquimod (IMQ)-induced mouse model and further explored the potential molecular mechanism and related signalling pathways during the treatment. Besides, we also confirmed a significant alleviation of ALA-PDT therapy on IFN-γ-induced over-proliferation of keratinocytes. H&E staining was conducted to reveal the histological changes of mice in different groups. The different expression levels of RNA were illustrated by using QRT-PCR. Western blot was performed to confirm the various expression levels of protein in mice. In vitro, cell proliferation and cell cycle were evaluated by cell counting kit-8 and flow cytometry assay, respectively. The result showed that ALA-PDT's anti-proliferation effect and regulation on Socs1/3, JAK1/2 and K17 in IFN-γ-induced keratinocytes were largely weakened by NAC, indicating that ALA-PDT attenuated the proliferation of IFN-γ-induced keratinocytes by enhancing ROS level. These results demonstrated that ALA-PDT largely activated the productivity of Socs1/3 in a ROS-dependent manner. Socs1/3 is a potential blocker in JAK signalling pathway and inhibits the proliferation and keratinization of keratinocytes in psoriasis.

Koumine Promotes ROS Production to Suppress Hepatocellular Carcinoma Cell Proliferation Via NF-κB and ERK/p38 MAPK Signaling.

In the past decades, hepatocellular carcinoma (HCC) has been receiving increased attention due to rising morbidity and mortality in both developing and developed countries. Koumine, one of the significant alkaloidal constituents of Gelsemium elegans Benth., has been regarded as a promising anti-inflammation, anxiolytic, and analgesic agent, as well as an anti-tumor agent. In the present study, we attempted to provide a novel mechanism by which koumine suppresses HCC cell proliferation. We demonstrated that koumine might suppress the proliferation of HCC cells and promote apoptosis in HCC cells dose-dependently. Under koumine treatment, the mitochondria membrane potential was significantly decreased while reactive oxygen species (ROS) production was increased in HCC cells; in the meantime, the phosphorylation of ERK, p38, p65, and IκBα could all be inhibited by koumine treatment dose-dependently. More importantly, the effects of koumine upon mitochondria membrane potential, ROS production, and the phosphorylation of ERK, p38, p65, and IκBα could be significantly reversed by ROS inhibitor, indicating that koumine affects HCC cell fate and ERK/p38 MAPK and NF-κB signaling activity through producing excess ROS. In conclusion, koumine could inhibit the proliferation of HCC cells and promote apoptosis in HCC cells; NF-κB and ERK/p38 MAPK pathways could contribute to koumine functions in a ROS-dependent manner.

Hepatic encephalopathy is linked to alterations of autophagic flux in astrocytes.

BackgroundHepatic encephalopathy (HE) is a severe neuropsychiatric syndrome caused by various types of liver failure resulting in hyperammonemia-induced dysfunction of astrocytes. It is unclear whether autophagy, an important pro-survival pathway, is altered in the brains of ammonia-intoxicated animals as well as in HE patients.MethodsUsing primary rat astrocytes, a co-culture model of primary mouse astrocytes and neurons, an in vivo rat HE model, and post mortem brain samples of liver cirrhosis patients with HE we analyzed whether and how hyperammonemia modulates autophagy.FindingsWe show that autophagic flux is efficiently inhibited after administration of ammonia in astrocytes. This occurs in a fast, reversible, time-, dose-, and ROS-dependent manner and is mediated by ammonia-induced changes in intralysosomal pH. Autophagic flux is also strongly inhibited in the cerebral cortex of rats after acute ammonium intoxication corroborating our results using an in vivo rat HE model. Transglutaminase 2 (TGM2), a factor promoting autophagy, is upregulated in astrocytes of in vitro- and in vivo-HE models as well as in post mortem brain samples of liver cirrhosis patients with HE, but not in patients without HE. LC3, a commonly used autophagy marker, is significantly increased in the brain of HE patients. Ammonia also modulated autophagy moderately in neuronal cells. We show that taurine, known to ameliorate several parameters caused by hyperammonemia in patients suffering from liver failure, is highly potent in reducing ammonia-induced impairment of autophagic flux. This protective effect of taurine is apparently not linked to inhibition of mTOR signaling but rather to reducing ammonia-induced ROS formation.InterpretationOur data support a model in which autophagy aims to counteract ammonia-induced toxicity, yet, as acidification of lysosomes is impaired, possible protective effects thereof, are hampered. We propose that modulating autophagy in astrocytes and/or neurons, e.g. by taurine, represents a novel strategy to treat liver diseases associated with HE.FundingSupported by the DFG, CRC974 “Communication and Systems Relevance in Liver Injury and Regeneration“, Düsseldorf (Project number 190586431) Projects A05 (DH), B04 (BG), B05 (NK), and B09 (ASR).

Abstract 259: Interplay between Bcl-2 and cFLIP in lymphoma disease progression is a function of an altered redox milieu

Abstract Bcl-2 is associated with chemoresistance and despite the preponderance of evidence linking its anti-apoptotic function to hematologic cancers, it’s now emerging that overexpression of Bcl-2 is a feature shared by a host of cancers. Canonically, the death inhibitory activity of Bcl-2 is associated with its ability to prevent mitochondrial outer membrane permeabilization. However, recent evidence has highlighted a novel facet in the biology of Bcl-2, whereby enforced expression of Bcl-2 resulted in an increase in mitochondrial oxygen consumption and elevated levels of the reactive oxygen species (ROS), superoxide (O2-.). Alleviating the levels of intracellular O2-. sensitized Bcl-2 overexpressing cells to death receptor induced apoptosis via a robust increase in upstream caspase 8 activation. Using a variety of functional mutants of the NOX-activating small GTPase Rac1, we show a clear link between Bcl-2 induced apoptosis resistance and intracellular O2-.. In the search for the molecular target of this inhibitory signal, we now show that Bcl-2 overexpression is associated with an increase in the transcription and protein levels of the receptor inhibitory protein, cFLIP. We employed pharmacological and genetic approaches to modulated intracellular O2-. and present evidence cFLIP is transcriptionally regulated by O2-.; increase in cFLIP promoter activity and mRNA levels. Interestingly, inhibiting glycolysis also resulted in downregulation of cFLIP in a ROS-dependent manner. Notably, primary cells derived from patients with lymphoma also showed a significant downregulation of cFLIP when subjected to conditions that decrease O2-. and elicited enhanced sensitivity to TRAIL-induced apoptosis. Importantly, TCGA and Affy meta-cohorts’ analyses revealed consistent correlation trend: Bcl-2 and cFLIP (CFLAR) were positively correlated; Bcl-2 and SOD1 (superoxide dismutase 1) were negatively correlated. A strong negative correlation was also observed between CFLAR and SOD1. Furthermore, Kaplan-Meier analyses of overall survival (OS) were performed in the in-house High expression of either Bcl-2 or CFLAR conferred poor OS outcome. When stratified by CFLAR and Bcl-2 expressions, cases with higher expression of the two genes had the worst prognosis with the shortest median survival. Notably, DLBCL with high Bcl-2 and low SOD1 had the worst OS outcome, compared against expression levels of Bcl-2 and SOD1. Together, these findings point to a concordance between cFLIP and Bcl-2, which negatively correlates with SOD1 in driving lymphoma progression. Citation Format: Jayshree L. Hirpara, Shazib Pervaiz. Interplay between Bcl-2 and cFLIP in lymphoma disease progression is a function of an altered redox milieu [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 259.

NOX1-Dependent mTORC1 Activation via S100A9 Oxidation in Cancer Stem-like Cells Leads to Colon Cancer Progression

Cancer stem cells (CSCs) are associated with the refractory nature of cancer, and elucidating the targetable pathways for CSCs is crucial for devising innovative antitumor therapies. We find that the proliferation of CSC-enriched colon spheroids from clinical specimen is dependent on mTORC1 kinase, which is activated by reactive oxygen species (ROS) produced by NOX1, an NADPH oxidase. In the spheroid-derived xenograft tumors, NOX1 is preferentially expressed in LGR5-positive cells. Dependence on NOX1 expression or mTOR kinase activity is corroborated in the xenograft tumors and mouse colon cancer-derived organoids. NOX1 co-localizes with mTORC1 in VPS41-/VPS39-positive lysosomes, where mTORC1 binds to S100A9, a member of S100 calcium binding proteins, in a NOX1-produced ROS-dependent manner. S100A9 is oxidized by NOX1-produced ROS, which facilitates binding to mTORC1 and its activation. We propose that NOX1-dependent mTORC1 activation via S100A9 oxidation in VPS41-/VPS39-positive lysosomes is crucial for colon CSC proliferation and colon cancer progression.

Abstract 1007: Reactive oxygen species are essential for neurotensin receptors to regulate EGFR and HER2 transactivation

Abstract Neurotensin (NTS) is a 13 amino acid peptide which is present in many lung cancer cell lines (Moody et al., Life Sci 1985; 36: 1727). High expression of the NTS receptor 1 (R1) is associated with poor survival of non-small cell lung cancer (NSCLC) patients (Alfano et al., Clin Cancer Res 2010; 16: 4401). NTS stimulates but the NTSR1 antagonist SR48692 inhibits the proliferation of NSCLC cells (Moody et al., Life Sci 2014; 100: 25). NTSR1 regulates NSCLC proliferation by transactivation of the EGFR. The cellular signaling mechanisms of the NTSR1 were investigated using NSCLC cells. Using NCI-H838 or A549 cells, addition of NTS or NTS(8-13) but not NTS(1-8) increased tyrosine phosphorylation of P-EGFR, P-HER2 and P-ERK 4-, 3- and 2-fold, respectively. The increase in EGFR and HER2 transactivation caused by NTS addition to NSCLC cells was blocked by SR48692, gefitinib (EGFR tyrosine kinase inhibitor (TKI)), lapatinib (EGFR and HER2 TKI), N-acetyl cysteine ((NAC) is an antioxidant), tiron (reactive oxygen species (ROS) scavenger) or diphenyleneiodonium (DPI is an inhibitor of NOX and DUOX enzymes). NTS increased ROS in NSCLC cells which was inhibited by NAC, tiron or DPI. NTS or NTS(8-13) stimulate NSCLC colony growth but SR48692, gefitinib, lapatinib or DPI inhibit growth. The results indicate that the NTSR1 regulates in a ROS-dependent manner the formation of EGFR homodimers or EGFR-HER2 heterodimers in NSCLC cells. Citation Format: Terry W. Moody, Lingaku Lee, Robert T. Jensen. Reactive oxygen species are essential for neurotensin receptors to regulate EGFR and HER2 transactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1007.

A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring low concentrations of ROS in nonphagocytic cells. We found that dissolving luminol in NaOH, rather than DMSO, increased the H2O2-induced CL signal and that the addition of 4-iodophenylboronic acid (4IPBA) further increased CL intensity. Our new system also increased the rate and intensity of the CL signal in phorbol 12-myristate 13-acetate- (PMA-) treated HT-29 colon cancer cells compared to those in luminol only. We were able to quantify ROS levels from both cells and media in parallel using an H2O2 standard. A significant benefit to our system is that we can easily measure stimulus-induced ROS formation in a real-time manner and also investigate intracellular signaling pathways from a single sample simultaneously. We found that PMA induced tyrosine phosphorylation of protein tyrosine kinases (PTKs), such as focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), and Src, and increased actin stress fiber formation in a ROS-dependent manner. Interestingly, treatment with either N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) reduced the PMA-stimulated phosphorylation of these PTKs, implicating a potential role in cellular ROS signaling. Thus, our newly optimized CL assay using 4IPBA and a chemiluminescent imaging method provides a simple, real-time, and low-cost method for the quantification of low levels of ROS.

Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC

BackgroundChemotherapy combined with immunotherapy becomes the main trend in lung cancer intervention; however, how chemotherapy promotes the immune function remains elusive. Therefore, we sought to determine how chemotherapy promotes the immune function.MethodsWe determined in 100 NSCLC patients the expression of CD8, functional markers (IFN-γ, Granzyme B, and Perforin) and specific chemokines by quantitative real-time reverse transcriptase-PCR. Functional experiments were carried out to check whether docetaxel (DOC), a chemotherapeutic agent, modifies the expression of HMGB1 and CXCL11, and influences the infiltration properties of CD8+ T cells to the tumor microenvironment. The mechanism of the release of HMGB1 and CXCL11 was determined by flow cytometry, immunofluorescence and western blotting. In in vivo experiment, we confirmed how DOC enhanced the recruitment of HER2-CAR T cells to tumor sites.ResultsWe found that DOC upregulated the expression of chemokine receptor ligand CXCL11 in tumor microenvironment and subsequently enhanced CD8+ T cell recruitment. DOC treatment significantly increased HMGB1 release in an ROS-dependent manner. Recombinant protein HMGB1 stimulated the secretion of CXCL11 via NF-κB activation in vitro. Tumors from DOC-treated mice exhibited higher expression of HMGB1 and CXCL11, more HER2-CAR T cell infiltration, and reduced progression, relative to control. Increased HMGB1 and CXCL11 expressions were positively correlated with prolonged overall survival of lung cancer patients.ConclusionsOur results demonstrate that DOC induces CD8+ T cell recruitment to the tumor microenvironment by enhancing the secretion of HMGB1 and CXCL11, thus improving the anti-tumor efficacy, indicating that modulating the HMGB1-CXCL11 axis might be helpful for NSCLC treatment.

Curcumin Induces Neural Differentiation of Human Pluripotent Embryonal Carcinoma Cells through the Activation of Autophagy.

Curcumin is a natural polyphenolic compound, isolated from Curcuma longa, and is an important ingredient of Asian foods. Curcumin has revealed its strong activities of anti-inflammatory, antioxidant, and anticancer. The efficient amount of curcumin could induce differentiation of stem cells and promoted the differentiation of glioma-initiating cells; however, the mechanisms underlying neural induction of curcumin have not yet been revealed. In this study, neural-inducing ability of curcumin was explored by using human pluripotent embryonal carcinoma cells, NTERA2 cells. The cells were induced toward neural lineage with curcumin and were compared with a standard neutralizing agent (retinoic acid). It was found that, after 14 days of the induction by curcumin, NTERA2 cells showed neuronal morphology and expressed neural-specific genes, including NeuroD, TUJ1, and PAX6. Importantly, curcumin activated neurogenesis of NTERA2 cells via the activation of autophagy, since autophagy-related genes, such as LC3, LAMP1, and ATG5, were upregulated along with the expression of neural genes. The inhibition of autophagy by chloroquine suppressed both autophagy and neural differentiation, highlighting the positive role of autophagy during neural differentiation. This autophagy-mediated neural differentiation of curcumin was found to be an ROS-dependent manner; curcumin induced ROS generation and suppressed antioxidant gene expression. Altogether, this study proposed the neural-inducing activity of curcumin via the regulation of autophagy within NTERA2 cells and underscored the health beneficial effects of curcumin for neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease.

D-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species.

d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.

Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

In order to use stem cells as a resource for tissue regeneration, it is necessary to induce expansion in vitro. However, during culture, stem cells often lose functional properties and become senescent. Increasing evidence indicates that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in vitro. The purpose of the current study was to test the hypothesis that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in culture by reducing oxidative stress. In vitro studies were performed in primary human dental pulp cells (hDPCs). Reduced levels of oxidative stress and increased cellular “stemness” in response to physiological hypoxia were dependent upon the expression of reactive oxygen species (ROS). Subsequently, RNA-sequencing analysis revealed the increased expression of phosphoinositide 3-kinase (PI3K)/Akt signaling in culture, a pathway which regulates oxidative stress. Furthermore, we found evidence that PI3K/Akt signaling might affect intracellular ROS production by negatively regulating expression of the downstream protein Forkhead Box Protein O1 (FOXO1) and Caspase 3. Collectively, our data show that the PI3K/Akt pathway is activated in response to hypoxia and inhibits oxidative stress in a ROS-dependent manner. This study identified redox-mediated hypoxic preconditioning regulatory mechanisms that may be significant for tissue regeneration.

Hypoxia-driven paracrine osteopontin/integrin αvβ3 signaling promotes pancreatic cancer cell epithelial-mesenchymal transition and cancer stem cell-like properties by modulating forkhead box protein M1.

Pancreatic stellate cells (PSCs), a key component of the tumor microenvironment, contribute to tumor invasion, metastasis, and chemoresistance. Osteopontin (OPN), a phosphorylated glycoprotein, is overexpressed in pancreatic cancer. However, OPN expression in PSCs and its potential roles in tumor–stroma interactions remain unclear. The present study first showed that OPN is highly expressed and secreted in activated PSCs driven by hypoxia, and this process is in a ROS‐dependent manner; in addition, OPN was shown to be involved in the PSC‐induced epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC)‐like properties of pancreatic cancer cells (PCCs). Mechanistically, OPN from activated PSCs interacts with the transmembrane receptor integrin αvβ3 on PCCs to upregulate forkhead box protein M1 (FOXM1) expression and induce malignant phenotypes of PCCs. Moreover, the Akt and Erk pathways participate in OPN/integrin αvβ3 axis‐induced FOXM1 expression of PCCs. Our further analysis showed that OPN and FOXM1 are significantly upregulated in pancreatic cancer tissues and are associated with poor clinical outcome, indicating that OPN and FOXM1 might be considered as diagnostic and prognostic biomarkers for patients with pancreatic cancer. In conclusion, we show here for the first time that OPN promotes the EMT and CSC‐like properties of PCCs by activating the integrin αvβ3‐Akt/Erk‐FOXM1 cascade in a paracrine manner, suggesting that targeting the tumor microenvironment represents a promising therapeutic strategy in pancreatic cancer.

Modulation of reactive oxygen levels and gene expression in sensitive and resistant tumoral cells by C-phyocyanin.

In this work we investigated the possible cellular changes induced by C-phycocyanin (C-PC) in erythroleukemic cells with (K562-Lucena and FEPS) and without (K562) the multidrug resistance (MDR) phenotype. The reactive oxygen levels (ROS) (evaluated by fluorimetry) were increased relative to the control in K562 and K562-Lucena cells treated with 100µg/mL and were not increased in FEPS cells. The expression of the following genes related to resistance was evaluated by real-time PCR: COX2, ALOX5, ABCB1 and ABCC1. Treatment with 100µg/mL of C-PC increased COX2 and ABCB1 expression in K562-Lucena cells and reduced expression of ALOX5 in K562-Lucena and FEPS cells. ROS levels appear to be involved in biological responses to C-PC in K562 and K562-Lucena cells, although expression of the genes studied here was only modified by C-PC in the K562-Lucena cell line. Thus, it is possible to suggest that C-PC modulates the expression of COX2 and ABCB1 for the K562-Lucena in a ROS-dependent manner and the expression of ALOX5 for the FEPS in a ROS-independent manner; however, more studies are needed to elucidate these mechanisms.

Effect of different surface functional groups of graphene on oxidative stress in <italic>Daphnia magna</italic>

Graphene could be modified by functional groups through a series of transformation processes after being released into environment. Meanwhile, the properties of graphene, such as solubility and biotoxicity, could be altered by surface modification. It is important to investigate the aquatic toxicity of graphene and its surface functionalized derivatives for assessing their ecological risks. Oxidative stress is one of the main toxicity mechanisms of graphene nanomaterials, but the effect mechanism of different surface functional groups (amino and thiol) on the oxidative stress induced by graphene is still unclear. In order to illuminate the toxicity mechanism and evaluate the extent of oxidative stress, a series of graphene nanomaterials including unfunctionalized grapheme (u-G), carboxylated grapheme (G-COOH), aminated grapheme (G-NH2), hydroxylated grapheme (G-OH) and sulfydryl grapheme (G-SH) were selected to determine the levels of reactive oxygen species(ROS), antioxidant enzymes, antioxidant and lipid peroxidation in Daphnia magna induced by these graphene nanomaterials. Daphnia magna were exposed to graphene and its surface functionalized derivatives during 24 h period. Following treatment, the parameters reflecting oxidative stress such as ROS, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde in Daphnia magna were measured. The results showed that malondialdehyde, a marker of lipid peroxidation in Daphnia magna , was significantly increased by u-G, G-COOH and G-OH, respectively. The level of malondialdehyde was significantly higher under u-G exposure than that under G-COOH or G-OH exposure. It is indicated that the degree of oxidative damage induced by u-G in Daphnia magna was more serious than that of G-COOH and G-OH. In addition, the three nanomaterials also caused the changes in ROS and glutathione (GSH) levels, and activities of superoxide dismutase (SOD) and catalase (CAT) were decreased by u-G and G-OH. However, G-SH and G-NH2 did not cause oxidative damage within 24 h exposure period. Marked inactivation of antioxidant enzymes in an ROS-independent manner was observed in response to G-SH, suggesting that G-SH may cause a structural change in enzymes, leading to functional inactivity. G-NH2 did not affect the levels of ROS, antioxidant enzymes, antioxidant and lipid peroxidation. At 24 h after stopping exposure, Daphnia magna can alleviate the oxidative damage induced by u-G, G-COOH and G-OH to a certain extent by its own regulation. The ability of graphene nanomaterials to cause oxidative stress in Daphnia magna is as follows: u-G>G-COOH≈G-OH>G-SH>G-NH2. The main reasons of oxidative stress in Daphnia Magna induced by u-G, G-COOH and G-OH were the generation of ROS, which led to the imbalance of antioxidant defense system and lipid peroxidation. G-SH mainly inhibited the activities of SOD and CAT antioxidant enzymes and affected the antioxidant defense system. However, there were no significant changes in ROS levels and antioxidant enzymes in Daphnia magna under G-NH2 exposure.

Reactive Oxygen Species Are Necessary for Bleomycin A5-Induced Apoptosis and Extracellular Matrix Elimination of Nasal Polyp-Derived Fibroblasts.

The pathology of chronic rhinosinusitis with nasal polyp (CRSwNP) is characterized by the infiltration of a large number of fibroblasts, resulting in extracellular matrix (ECM) deposition. Intralesional bleomycin A5 (BLE) injection has proved to be effective and safe, providing a novel treatment for CRSwNP. However, the mechanism is not clearly understood. The aim of this study is to explore the possible mechanism of BLE-induced apoptosis in nasal polyp-derived fibroblasts (NPDFs). Dichloro-dihydro-fluorescein diacetate probe, cell migration assays, and cell cycle analysis were used to detect the growth characteristics and basal reactive oxygen species (ROS) traits of NPDFs. Annexin V/propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to detect BLE-induced apoptosis. As a control, the antioxidant glutathione (GSH) was used to abrogate ROS induced by BLE. Western blot analysis was used to evaluate the effects of BLE on apoptosis and the ECM proteins of NPDFs. The results showed that NPDFs had more active growth characteristics and higher basal ROS levels than normal nasal mucosa fibroblasts (NMFCs). NPDFs were more sensitive to BLE-induced apoptosis and ROS accumulation. GSH abrogation inhibits BLE-induced ECM degradation and apoptosis in NPDFs through a mitochondrial-mediated pathway. BLE induced NPDF apoptosis and ECM degradation through a mitochondrial-mediated pathway and in a ROS-dependent manner.

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival.

Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

Inhibiting TrxR suppresses liver cancer by inducing apoptosis and eliciting potent antitumor immunity.

Liver cancer is one of the most common malignant tumors worldwide. Thioredoxin reductase (TrxR) is highly expressed in liver cancer cells. The present study aimed to investigate the effect of inhibiting TrxR on liver cancer and to better understand the underlying molecular and immunological mechanisms associated with inhibition. It was demonstrated that targeting TrxR inhibited the growth and induced apoptosis of liver cancer cells, which was accompanied by activation of the mitogen associated protein kinase pathway. This inhibition was dependent on the production of reactive oxygen species (ROS). Blockage of ROS production reversed TrxR inhibitor-induced antitumor effects. Blocking the Trx/TrxR system activated the mammalian target of rapamycin pathway and inhibited autophagy, which occurred in a ROS-independent manner. TrxR inhibition led to lesions in the mitochondrial membrane, indicated by alterations in membrane potential. Mouse xenograft experiments were highly consistent with in vitro studies. Most importantly, blocking the Trx/TrxR system improved the tumor immune microenvironment. Together, these data demonstrated that TrxR is a potential target for liver cancer therapy, which could inhibit hepatocarcinogenesis and progression, and improve the antitumor immune response.

SAHA (vorinostat) facilitates functional polymer-based gene transfection via upregulation of ROS and synergizes with TRAIL gene delivery for cancer therapy

Non-viral gene delivery is an attractive approach for the treatment of many diseases including cancer, benefiting from its safety and large-scale production concerns. However, the relatively low transfection efficacy compared with viral vectors restricts the clinical applications of non-viral gene vectors. Reactive oxygen species (ROS) triggered charge reversal polymers (named B-PDEAEA) presented improved transfection efficacy, because of fast release of plasmid DNA responding to enhanced oxidative stress in cancer cells. But inadequate dissociation can still occur owing to the insufficient intracellular ROS generation. Here, we report SAHA (vorinostat), which is a clinical histone deacetylase inhibitor and anticancer drug, induces the ROS accumulation in cancer cells, and facilitates the charge reversal process of B-PDEAEA and the cellular dissociation of the delivered gene from the vectors. As a result, SAHA remarkably increases the gene transfection efficacy in an ROS-dependent manner. Importantly, SAHA synergizes with B-PDEAEA mediated therapeutic gene TNF-related apoptosis-inducing ligand (TRAIL) delivery in inducing apoptosis of cancer cells. These findings support the first concept of improving the gene delivery efficacy of stimuli-responsive vectors through upregulating the cellular ROS via an FDA approved anticancer agent. Additionally, combination of SAHA and TRAIL gene therapy could be a potential strategy for cancer treatment.