Elevated ROS Production Research Articles

ITRAQ-based proteomics and in vitro experiments reveals essential roles of ACE and AP-N in the renin–angiotensin system-mediated congenital ureteropelvic junction obstruction

ObjectiveUreteropelvic junction obstruction (UPJO) is a common renal obstructive disorder, but its pathogenic mechanisms remain largely unclear. We aimed to investigate the potential involvement of the renin–angiotensin system in congenital UPJO pathogenesis. MethodsDifferentially expressed proteins in exosomes isolated from amniotic fluid of patients with congenital UPJO were characterized using iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics. The expressions of angiotensin-converting enzyme (ACE) and aminopeptidase N (AP-N) in HK2 cells were inhibited by quinapril and siRNA, respectively. Cell proliferation and reactive oxygen species were measured by EdU staining and flow cytometry, respectively. Gene expression was detected by Western blot or qRT-PCR. The inflammatory factors were measured through ELISA. Mice that underwent unilateral ureteral obstruction were used as the animal model. ResultsThe identity of exosomes from amniotic fluids was confirmed by the expression of CD9 and CD26. In total, 633 differentially expressed proteins were identified in the amniotic fluid-derived exosomes from patients with UPJO, including 376 up- and 257 down-regulated proteins associated with multiple biological processes. Of them, ACE and AP-N were significantly decreased in the amniotic fluid exosomes. Inhibition of ACE and AP-N resulted in suppressed cell proliferation; repressed IARP, AT1R, and MAS1 expression; elevated ROS production; and increased IL-1β, TNF-α, and IL-6 levels in HK2 cells. Decreased ACE expression and elevated IL-1β levels were also observed in the mouse model. ConclusionSuppression of ACE and AP-N expression mediates congenital UPJO pathogenesis by repressing renal tubular epithelial proliferation, promoting ROS production, and enhancing inflammatory factor expression.

Lymphotoxin-beta receptor expression by neutrophils prevents metabolic activation and colitis pathogenesis

Abstract Inflammatory bowel disease, while multifactorial, is largely characterized by exacerbated intestinal immune activation. However, the critical mechanisms regulating immune activation remain to be fully defined. We have previously reported that a member of the TNF superfamily, TNFSF14 (LIGHT), and its interaction with the lymphotoxin beta receptor (LTβR), is required for preventing severe disease in a mouse model of dextran sulfate sodium (DSS) induced colitis. Thus, we aimed to determine the cell type and mechanism critical to LTβR mediated suppression of DSS-induced colitis. Employing the use of LTβRfl/fl mice and tissue-specific Cre-mediated deletion, we found that neutrophils were the critical LTβR expressing cell type. Deletion of LTβR expression in neutrophils (LtbrΔneut)resulted in exacerbated DSS-induced colitis and drastically increased colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis of neutrophils revealed transcriptional alterations in genes associated with cellular metabolism, and mitochondrial function. Ex vivo studies of bone marrow neutrophils from LtbrΔneut, compared to Cre- controls, revealed increased mitochondrial mass and number, as well as elevated mitochondrial ROS production in neutrophils lacking the receptor. Subsequent metabolic studies demonstrated that LTβR deficient neutrophils exhibited elevated mitochondrial respiration and glycolysis. Interestingly, these increased levels of mitochondrial mass, ROS production and glycolytic activity persist in LtbrΔneut colonic neutrophils during DSS-induced colitis. In sum, our results demonstrate that neutrophil LTβR signaling plays a critical role in the immune activation associated with DSS-induced colitis.

Cross-talk between redox signalling and protein aggregation.

It is well established that both an increase in reactive oxygen species (ROS: i.e. O2•−, H2O2 and OH•), as well as protein aggregation, accompany ageing and proteinopathies such as Parkinson's and Alzheimer's disease. However, it is far from clear whether there is a causal relation between the two. This review describes how protein aggregation can be affected both by redox signalling (downstream of H2O2), as well as by ROS-induced damage, and aims to give an overview of the current knowledge of how redox signalling affects protein aggregation and vice versa. Redox signalling has been shown to play roles in almost every step of protein aggregation and amyloid formation, from aggregation initiation to the rapid oligomerization of large amyloids, which tend to be less toxic than oligomeric prefibrillar aggregates. We explore the hypothesis that age-associated elevated ROS production could be part of a redox signalling-dependent-stress response in an attempt to curb protein aggregation and minimize toxicity.

Accelerated Bronchiolitis Obliterans Development after Lung Transplant Promoted by the ATG16l1 rs2241880 Mutation is Coupled to Mitochondrial Damage and Metabolic Alterations in Monocyte-Derived Alveolar Macrophages

Bronchiolitis obliterans (BOS) remains a major cause of death for lung transplant recipients, and mechanisms that drive BOS remain poorly understood. Genetically encoded deficiencies in mitophagy, a specialized autophagy that targets the removal of damaged mitochondria, have been shown to promote Parkinson's disease, but it is unclear if they play a role in other chronic diseases. Recent work has shown that the rs2241880 mutation in the autophagy regulator ATG16L1 leads to protein instability resulting in deficiency of ATG16L1 in monocyte-derived macrophages. We previously observed that human lung recipients that are carriers of rs2241880 had faster BOS development. Here, we analyzed the effects of ATG16L1 deficiency in monocyte-derived alveolar macrophages (Mo-AM) in a mouse orthotopic lung transplant model of BOS. CD11cCre ATG16L1flox/flox (ATG16L1Δ/Δ) and control CD11cCre recipients received major MHC-mismatched FVB lungs and were immunosuppressed to induce acceptance. Following induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) for up to 28 days. Intragraft Mo-AM were analyzed for bulk RNA sequencing and by flow cytometry (FACS) for mitochondrial mass and ROS production. Mitophagic flux was visualized by confocal microscopy using Mt-kiema mitophagy reporter mice. Mo-AM were characterized metabolically using a Seahorse XF analyzer. When compared to controls, CD11cCre ATG16L1flox/flox recipients showed severe and accelerated OB development. RNAseq analysis of ATG16L1Δ/Δ Mo-AM demonstrated a loss of transcripts that encode several subunits of mitochondria electron complex I, II and V. Confocal and FACS analysis revealed attenuated mitophagic flux, high mitochondrial mass and elevated ROS production in ATG16L1Δ/Δ Mo-AM. In line with these observations were decreased maximal mitochondrial respiratory capacity, lower mitochondrial ATP production and increased utilization of glycolysis- consistent with the metabolic adaptation of an M1 inflammatory phenotype. Collectively, these data reveal a new role for ATG16L1 as a regulator of mitochondrial quality control with implications for lung recipients that carry the ATG16L1 rs2241880 mutation.

Antifilarial efficacy of green silver nanoparticles synthesized using Andrographis paniculata

The currently available antifilarial drugs are mostly effective against microfilariae. Antifilarial drugs have disadvantages such as toxicity and the development of resistance due to the continuous use. Therefore alternative drugs are required for the control of disease. At present, nanoparticles are used for developing anti-parasitic therapy for their special properties such as smallest in size, bio-availability, bio-compatibility and penetration capacity into a cell. In the present study green nanoparticles were biosynthesized by using leaf extract of Andrographis paniculata to evaluate its antifilarial efficacy. Biosynthesized nanoparticles were characterized by UV–Vis spectroscopy, FTIR, XRD, SEM, EDAX, TEM, HRTEM, AFM and found to be homogenous with average size of 11 nm. The antifilarial efficacy of green nanoparticles was based on adult filarial parasite motility and viability assay. These green nanoparticles (NPs) were found to have antifilarial activity with LC50 of 11.6 μg/ml against adult female filarial parasites. The green nanoparticles induced oxidative stress as evidenced by elevated ROS production and decline of parasitic GST, GR, TRxR and GSH levels in the parasite. The activation of ced-3 gene, a homolog of mammalian caspase 3, reduced expression of ced-9 and decreased activity of cytochrome c oxidase suggested induction of mitochondrial mediated apoptosis in parasite. These green NPs are more effective than the plant extract. This is the first report where green nanoparticles synthesized from A. paniculata showed antifilarial efficacy against adult filarial parasites.

A photo catalyst of cuprous oxide anchored MXene nanosheet for dramatic enhancement of synergistic antibacterial ability

Ti3C2Tx (MXene) as a two-dimensional material has attracted numerous attentions in photocatalysis and antibacterial, and the effectiveness of MXene for photocatalysis triggered antibacterial is in urgent need of development. Herein, we synthesized a novel cuprous oxide (Cu2O) anchored MXene nanosheet. Cu2O nanospheres can be uniformly anchored on the surface of MXene firmly due to the electrostatic effect between single-layer MXene and Cu2O nanospheres. In comparison with MXene, Cu2O and mixture (MXene and Cu2O), Cu2O/MXene nanosheet exhibits an excellent antibacterial activity against S. aureus and P. aeruginosa, whose bacteriostasis efficiencies sharply increased to 97.04% and 95.59%, respectively. Photoluminescence spectroscopy reveals that the incorporation of MXene and Cu2O can effectively prevent the recombination of electron-hole pairs of Cu2O and present a high photocatalytic disinfection. Single-layer MXene with large surface area accepts electrons from Cu2O, leading to abundant electrons on the surface of MXene, which provide better charge transfer between bacteria and Cu2O/MXene nanosheets. In addition, density functional theory calculations were used to investigate an optimized band structure of Cu2O-MXene as well as its strong electronic interactions, which are essential for photocatalysis and ROS generation. Finite element method further revealed that MXene promoted an enhancement of surface plasmon resonance to generate ROS on the surface of Cu2O/MXene. Furthermore, Cu2O/MXene shows a synergistic antibacterial effect of MXene accelerating photoelectron transportation, Cu2O antimicrobial and photocatalysis, elevated ROS production ability, and surface plasmon resonance action on the surface of Cu2O/MXene.

Biochemical thresholds for pathological presentation of ATP synthase deficiencies

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15–80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10–30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

Molecular mechanism of antimicrobial activity of chlorhexidine against carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes hospital-acquired infections, especially in those with impaired immune function. Biocides or disinfectants are widely used antibacterial agents used to eradicate the effect of A. baumannii on inanimate objects and health care environments. In the current study, the antimicrobial activity of chlorhexidine has been investigated against carbapenem-resistant (RS-307, RS-7434, RS-6694, and RS-122), and sensitive (ATCC-19606 and RS-10953) strains of A. baumannii. We have determined growth kinetics, antimicrobial susceptibility, ROS production, lipid peroxidation, cell viability using flow cytometry assay (FACS), and membrane integrity by scanning electron microscope (SEM). The effect of chlorhexidine on the bacterial membrane has also been investigated using Fourier transform infrared (FTIR) spectroscopy. The present study showed that 32μg/ml chlorhexidine treatment results in the decreased bacterial growth, CFU count and cell viability. The antibacterial activity of chlorhexidine is due to the elevated ROS production and higher lipid peroxidation. These biochemical changes result in the membrane damage and alteration in the membrane proteins, phospholipids, carbohydrates, nucleic acids as evident from the FTIR and SEM data. Therefore, chlorhexidine has the potential to be used in the hospital setups to remove the spread of A. baumannii.

Targeting a moonlighting function of aldolase induces apoptosis in cancer cells

Muscle fructose-1,6-bisphosphate aldolase (ALDOA) is among the most abundant glycolytic enzymes in all cancer cells. Here, we show that the enzyme plays a previously unknown and critical role in a cancer cell survival. Simultaneous inhibition of ALDOA activity and interaction with F-actin cytoskeleton using ALDOA slow-binding inhibitor UM0112176 leads to a rapid cofilin-dependent loss of F-actin stress fibers which is associated with elevated ROS production, inhibition of ATP synthesis, increase in calcium levels, caspase activation and arrested cellular proliferation. These effects can be reproduced by silencing of ALDOA. The mechanism of pharmacological action is, however, independent of the catalytic function of the enzyme, specific to cancer cells, and is most deleterious to cells undergoing the epithelial–mesenchymal transition, a process facilitating cancer cell invasion. Our results demonstrate that the overabundance of ALDOA in cancer cells is associated with its moonlighting rather than catalytic functions. This may have significant implications for development of novel broad-based anti-cancer therapies.

Regulation of Staphylococcus aureus-induced CXCR1 expression via inhibition of receptor mobilization and receptor shedding during dual receptor (TNFR1 and IL-1R) neutralization.

Our earlier studies proposed a radically new idea suggesting interdependency between TNF-α/TNFR1 and IL-1β/IL-1R pathways in modulation of Staphylococcus aureus-induced CXCL8/CXCR1 axis. However, the effects of inhibition of cytokine receptor mobilization at intracellular level and surface TNFR1 and IL-1R shedding on S. aureus-induced CXCR1 expression have not been studied so far in peritoneal macrophages. This study aimed to investigate the role of inhibition of receptor mobilization from the intracellular pool (using brefeldin A) and surface receptor shedding (using TAPI-1) on CXCR1 expression during dual receptor (TNFR1 plus IL-1R) neutralization in peritoneal macrophages isolated from wild-type Swiss Albino mice. Release of superoxide anion, nitric oxide, and hydrogen peroxide was measured and cytokine production was done by ELISA. Expression of surface receptors (TNFR1, IL-1R, and CXCR1) and inflammatory mediators was studied by Western blot. It was observed that S. aureus-infected macrophages showed elevated ROS production, secretion of TNF-α, IL-1β, and CXCL8, along with increased expression of surface receptors (TNFR1, IL-1R, and CXCR1), and inflammatory markers (iNOS and COX-2) compared with control or treated groups (p < 0.05). However, prior treatment of macrophages with BFA or TAPI-1 in the presence of anti-TNFR1 antibody and IRAP during S. aureus infection showed significant reduction of all these parameters (p < 0.05). We can conclude that targeting of TNFR1 and IL-1R (with major focus on surface expression study) either through blockage of intracellular receptor trafficking pathway or via surface receptor shedding diminishes TNFR1/IL-1R interaction and consequently downregulates CXCR1 expression along with inflammatory signalling pathways during bacterial infections.

Prolonged activation in CD4+ T cells results in extensive mitochondrial remodeling despite the metabolic dominance of aerobic glycolysis

Abstract Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for four days following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Despite elevated ROS production, it was successfully counterbalanced by antioxidant enzymes, hence the mitochondria did not display signs of oxidative stress as evidenced by the healthy cristae structure and overall morphology as well as the maintained mitochondrial reserve. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Lymphotoxin receptor beta expression by neutrophils prevents metabolic activation and colitis pathogenesis

Abstract Inflammatory bowel disease, while multifactorial, is largely characterized by an exacerbated intestinal immune activation. However, the critical mechanisms regulating immune activation remain to be fully defined. We have previously reported that a member of the TNF superfamily, TNFSF14 (LIGHT), and its interaction with the lymphotoxin beta receptor (LTβR), is required for preventing severe disease in a mouse model of dextran sulfate sodium (DSS) induced colitis. Thus, we aimed to determine the cell type and mechanism critical to LTβR mediated protection from DSS-induced colitis. Employing the use of LTβRfl/fl mice and tissue-specific Cre-mediated deletion, we found that neutrophils were a critical LTβR expressing cell type. Mrp8-Cre driven deletion of neutrophil LTβR expression resulted in exacerbated DSS-induced colitis and drastically increased colonic neutrophil accumulation. Mechanistically, RNASeq analysis of neutrophils revealed transcriptional alterations in genes associated with cellular metabolism, and mitochondrial function in particular. Ex vivo studies of bone marrow neutrophils from LTβRfl/fl x Mrp8-Cre mice, compared to Cre-controls, revealed increased mitochondrial mass and number, as well as elevated mitochondrial ROS production. Subsequent metabolic studies demonstrated that LTβR deficient neutrophils exhibited elevated mitochondrial respiration and glycolysis. Interestingly, these increased levels of mitochondrial mass, ROS production and glycolytic activity persist in LTβR deficient colonic neutrophils during DSS-induced colitis. In sum, our results demonstrate that neutrophil LTβR signaling plays a critical role in the immune activation associated with DSS-induced colitis.

Life-time exposure to waterborne copper III: Effects on the energy metabolism of the killifish Poecilia vivipara

Copper ions (Cu) are essential to life maintenance, nonetheless, elevated concentrations can be hazardous. Acute and sub-chronic toxic effects of this metal are well known and are usually related to enzymatic inhibition, elevated ROS production and dysfunction of energy metabolism. Despite that, chronic studies are extremely rare. Therefore, the aim of this study was to assess the effects of chronic exposure to 5, 9 and 20 μg/L Cu (28 ad 345 days) on the energy metabolism and survival of the killifish Poecilia vivipara. To accomplish that, we evaluated the activity of enzymes related to aerobic (pyruvate kinase (PK); citrate synthase (CS)) and anaerobic metabolism (lactate dehydrogenase (LDH)) in whole-body (28 days) or in gills, liver and muscle (345 days) of exposed fish. Additionally, whole-body oxygen consumption was evaluated in fish exposed for 28 days and hepatic and muscular expression of genes involved in mitochondrial metabolism (cox I, II and III and atp5a1) was assessed in animals exposed for 345 days. Finally, final survival was evaluated. Following 28 days, Cu did not affect survival neither enzyme activities. However, increased whole-body oxygen consumption was observed in comparison to control condition. After 345 days, 76.8%, 63.9%, 60.9% and 0% survival were observed for control, 5, 9 and 20 μg/L groups, respectively. Animals exposed to 5 and 9 μg/L had a significant reduction in branchial and muscular LDH activity and in hepatic PK activity. Also, exposure to 9 μg/L significantly increased hepatic CS activity. For gene expression, Cu down-regulated muscular cox II (9 μg/L) and III (5 and 9 μg/L), and up-regulated hepatic atp5a1 (9 μg/L). Findings reported in the present study indicate that chronic exposure to Cu induces tissue-specific responses in key aspects of the energetic metabolism. In gills and muscle, Cu leads to reduced energy production through inhibition of anaerobic pathways and mitochondrial respiratory chain. This effect is paralleled by an increased ATP consumption in the liver, characterized by the augmented CS activity and atp5a1 expression. Finally, reduced PK activity indicate that oxidative stress may be involved with the observed outcomes.

Abstract TP556: Role of Vascular Smooth Muscle Cells in Diabetes-related Vascular Cognitive Impairment

The incidence of vascular cognitive impairment is increased 2-3 folds in type 2 diabetes, but the underlying mechanisms have not been fully elucidated. The present study examines whether chronic hyperglycemia and associated enhanced ROS production damage cerebral vascular smooth muscle cells (VSMCs) that promote impairment of cerebral autoregulation and contribute to cognitive deficits. The levels of glucose (422 ± 32 vs. 94 ± 3 mg/dL) and glycated hemoglobin (HbA1c, 11.5 ± 0.2 vs. 4.3 ± 0.1%) were higher in old (12-month) T2DN (n=15) in comparison with SD control rats (n=6). We found that the actin cytoskeleton was disorganized in VSMCs freshly isolated from middle cerebral arteries (MCAs) of T2DN compared with SD rats, similar to what was seen in normal primary VSMCs treated with high glucose or H 2 O 2 . Superoxide production was elevated in VSMCs and MCAs freshly isolated from diabetic rats. Elderly diabetic rats exhibit impaired myogenic response of MCA. Cortical blood flow measured by laser Doppler flowmetry rose by 137 ± 15% vs. 36 ± 5% in diabetic (n=6) vs. normal rats (n=6) when MAP was increased from 100 to 180 mmHg. T2DN rats exhibited higher levels of IL-1 β and IL-2, and marked neurodegeneration in the hippocampus and cortex. Elderly T2DN rats showed learning and memory impairments using 8-arm water maze testing. These results indicate that hyperglycemia and elevated ROS production induce VSMCs dysfunction in diabetic rats, which contribute to cerebral vascular dysfunction and promote development of cognitive deficits.

High macrophage activities are associated with advanced periductal fibrosis in chronic Opisthorchis viverrini infection

Liver fluke infection caused by Opisthorchis viverrini induces several hepatobiliary conditions including advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), but >25% of the infected population develops APF and 1% develop CCA. The innate immune response is the first line of defence, and macrophages are critical regulators of fibrosis. We hypothesized that macrophages from infected individuals have different capacities to either promote or suppress periductal fibrosis. We compared phagocytic activities of macrophages of healthy individuals and Oviverrini-infected individuals±APF, and found that macrophages from infected individuals with APF ingested significantly higher numbers of beads compared with healthy controls and Oviverrini-infected individuals without APF. To further investigate proteolytic activity, we monitored real-time phagosomal proteolysis of beads conjugated to DQ-BODIPY-BSA using live cell imaging. We show that macrophages from Oviverrini-infected individuals with APF also have elevated phagosomal proteolysis activity, which is consistent with their increased phagocytic activity. Additionally, stimulated ROS production by blood monocytes was higher in individuals with APF compared with healthy controls and infected individuals without APF. These results suggest that during Oviverrini infection, macrophages with high phagocytic and proteolytic activities together with elevated ROS production are the phenotypes that can promote tissue damage, which results in periductal fibrosis.

RETRACTED ARTICLE: IL-2 augments thesorafenib-induced apoptosis in liver cancer by promoting mitochondrial fission andactivating the JNK/TAZ pathway

BackgroundSorafenib is the standard targeted drug used to treat hepatocellularcarcinoma (HCC), but the therapeutic response between individuals variesmarkedly. Recently, cytokine-based immunotherapy has been a topic of intensediscussion in the fight against cancer. The aim of this study was to explorewhether cytokine IL-2 could augment the anti-tumour effects of sorafenib onHCC.MethodsHepG2 and Huh7 cells were co-treated with sorafenib and IL-2 invitro, and cellular viability and death were analysed through the MTT assay,TUNEL staining, LDH release assay, and western blotting. Mitochondrial functionwas measured via ELISA, immunofluorescence, and western blotting. Pathwayblockers were used to establish the role of the JNK-TAZ pathways in regulatingcancer cell phenotypes.ResultsOur data demonstrated that sorafenib treatment increased the HCCapoptotic rate, repressed cell proliferation, and inhibited migratory responses,and these effects were enhanced by IL-2 supplementation. Mechanistically, thecombination of IL-2 and sorafenib interrupted mitochondrial energy metabolism bydownregulating mitochondrial respiratory proteins. In addition, IL-2 andsorafenib co-treatment promoted mitochondrial dysfunction, as evidenced by thedecreased mitochondrial potential, elevated mitochondrial ROS production,increased leakage of mitochondrial pro-apoptotic factors, and activation of themitochondrial death pathway. A molecular investigation revealed thatmitochondrial fission was required for the IL-2/sorafenib-mediated mitochondrialdysfunction. Mitochondrial fission was triggered by sorafenib and was largelyamplified by IL-2 supplementation. Finally, we found that IL-2/sorafenibregulated mitochondrial fission via the JNK-TAZ pathways; blockade of theJNK-TAZ pathways abrogated the inhibitory effects of L-2/sorafenib on cancersurvival, growth and mobility.ConclusionsAltogether, these data strongly suggest that additionalsupplementation with IL-2 enhances the anti-tumour activity of sorafenib bypromoting the JNK-TAZ-mitochondrial fission axis. This finding will pave the wayfor new treatment modalities to control HCC progression by optimizingsorafenib-based therapy.

Abstract 1980: S116836 overcomes BCR-ABL T315I mutation mediated imatinib resistance in chronic myeloid leukemia

Abstract Chronic Myeloid Leukemia (CML) is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated S116836, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. S116836 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. S116836 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. S116836 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, S116836 increased the cell population in the G0/G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Moreover, S116836 inhibited tumor growth in BaF3/WT and BaF3/T315I tumor xenografts. Taken together, our results indicate that S116836 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. S116836 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients. Keywords: S116836, Chronic myeloid leukemia, BCR-ABL, T315I, Resistance Citation Format: Pranav Gupta, Guannan Zhang, Anna Maria Barbuti, Ke Ding, Jingxuan Pan, Brian J. Druker, Zhe-Sheng Chen. S116836 overcomes BCR-ABL T315I mutation mediated imatinib resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1980.

Pathogen-mimicking nanocomplexes: self-stimulating oxidative stress in tumor microenvironment for chemo-immunotherapy

A novel drug-loaded pathogen-mimicking nanocomplex has been constructed for synergistic chemo-immunotherapy using detoxified lipopolysaccharide coated mesoporous silica nanoparticle. Detoxified lipopolysaccharide behaves as a dual-purpose entity that not only effectively mimics the function of the natural pathogen for triggering immune responses but also acts as a lid for inhibiting premature chemical drug release. In this approach, a knock-on effect would be observed at site of tumor: firstly, pathogen-mimicries elicited the elevated production of ROS; secondly, excessive production of ROS in turn oxidized the arylboronic ester to realize controlled chemotherapy; thirdly, in addition to inducing ROS generation, the nanocomplex would self-stimulate macrophages activation which subsequently activated cytotoxic T cells. Importantly, chemotherapy and immunotherapy were acting in a synergistic manner to inhibit solid tumor growth. Moreover, chemotherapeutic agents could be effectively released upon exposure to self-stimulating oxidative stress in which external addition of ROS was avoided. This proof of concept might open the door to a new generation of carrier materials in the field of cancer therapy.

Abstract 4158: Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor

Abstract Chronic Myeloid Leukemia (CML), a hematological malignancy, is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated PBA2, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. PBA2 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. PBA2 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. PBA2 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, PBA2 increased the cell population in sub G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Taken together, our results indicate that PBA2 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. PBA2 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients. Note: This abstract was not presented at the meeting. Citation Format: Pranav Gupta, Rishil Kathawala, Liuya Wei, Fang Wang, XiaoKun Wang, Brian J. Druker, Li-Wu FU, Zhe-Sheng Chen. Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2017-4158

β-Naphthoflavone-Induced Mitochondrial Respiratory Damage in Cyp1 Knockout Mouse and in Cell Culture Systems: Attenuation by Resveratrol Treatment.

A number of xenobiotic-inducible cytochrome P450s (CYPs) are now known to be localized in the mitochondrial compartment, though their pharmacological or toxicological roles remain unclear. Here, we show that BNF treatment markedly inhibits liver mitochondrial O2 consumption rate (OCR), ADP-dependent OCR, and also reserve OCR, in wild-type mice but not in Cyp1a1/1a2(−/−) double knockout mice. BNF treatment markedly affected mitochondrial complex I and complex IV activities and also attenuated mitochondrial gene expression. Furthermore, under in vitro conditions, BNF treatment induced cellular ROS production, which was inhibited by mitochondria-targeted antioxidant Mito-CP and CYP inhibitor proadefin, suggesting that most of the ROS production was intramitochondrial and probably involved the catalytic activity of mitochondrial CYP1 enzymes. Interestingly, our results also show that the AHR antagonist resveratrol, markedly attenuated BNF-induced liver mitochondrial defects in wild-type mice, confirming the role of AHR and AHR-regulated CYP1 genes in eliciting mitochondrial dysfunction. These results are consistent with reduced BNF-induced mitochondrial toxicity in Cyp1a1/1a2(−/−) mice and elevated ROS production in COS cells stably expressing CYP1A1. We propose that increased mitochondrial ROS production and respiratory dysfunction are part of xenobiotic toxicity. Resveratrol, a chemopreventive agent, renders protection against BNF-induced toxicity.