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At least 829 records · Page 46
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells

Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of more red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-L-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells. - Highlights: • Osimertinib induced the expressions of cytoplasmic vacuoles and autophagic markers in different cancer cells. • Osimertinib induced autophagic flux in NSCLC NCI-H1975 and HCC827 cell lines. • ROS generation contributed to osimertinib-induced cytoplasmic vacuoles, autophagy, cell viability decrease, and apoptosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and downregulation of caveolin-1. • RXM inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. • Our findings expand our knowledge of the role of RXM in airway remodelling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Treatment of cholinergic‐induced status epilepticus with polytherapy targeting GABA and glutamate receptors

Abstract Despite new antiseizure medications, the development of cholinergic‐induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia . 2005;46:142 demonstrated that the initiation and maintenance of cholinergic‐induced RSE are associated with trafficking and inactivation of gamma‐aminobutyric acid A receptors (GABA A R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N‐methyl‐ d ‐aspartate receptors (NMDAR) and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation ( Neurobiol Dis . 2013;54:225; Epilepsia . 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic‐induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic‐induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic‐induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine‐induced seizure in rats, organophosphorus nerve agent (OPNA)‐induced seizure in rats, and OPNA‐induced seizure in two mouse models: (1) carboxylesterase knockout (Es1 −/− ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock‐in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic‐induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic‐induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in human renal proximal tubular epithelial cells

Highlights: •MSP/RON system is activated in rat kidney damaged by gentamicin. •MSP inhibits GM-induced cellular apoptosis and inflammation in HK-2 cells. •MSP attenuates GM-induced activation of MAPKs and NF-κB pathways in HK-2 cells. -- Abstract: The present study aimed to investigate whether macrophage-stimulating protein (MSP) treatment attenuates renal apoptosis and inflammation in gentamicin (GM)-induced tubule injury and its underlying molecular mechanisms. To examine changes in MSP and its receptor, recepteur d’origine nantais (RON) in GM-induced nephropathy, rats were injected with GM for 7 days. Human renal proximal tubular epithelial (HK-2) cells were incubated with GM for 24 h in the presence of different concentrations of MSP and cell viability was measured by MTT assay. Apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. Expression of Bcl-2, Bax, caspase-3, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), IκB-α, and mitogen-activated protein kinases (MAPKs) was analyzed by semiquantitative immunoblotting. MSP and RON expression was significantly greater in GM-treated rats, than in untreated controls. GM-treatment reduced HK-2 cell viability, an effect that was counteracted by MSP. Flow cytometry and DAPI staining revealed GM-induced apoptosis was prevented by MSP. GM reduced expression of anti-apoptotic protein Bcl-2 and induced expression of Bax and cleaved caspase 3; these effects and GM-induced expression of COX-2 and iNOS were also attenuated by MSP. GM caused MSP-reversible induction of phospho-ERK, phospho-JNK, and phospho-p38. GM induced NF-κB activation and degradation of IκB-α; the increase in nuclear NF-κB was blocked by inhibitors of ERK, JNK, p-38, or MSP pretreatment. These findings suggest that MSP attenuates GM-induced inflammation and apoptosis by inhibition of the MAPKs/NF-κB signaling pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Compound 13, an α1-selective small molecule activator of AMPK, inhibits Helicobacter pylori-induced oxidative stresses and gastric epithelial cell apoptosis

Half of the world's population experiences Helicobacter pylori (H. pylori) infection, which is a main cause of gastritis, duodenal and gastric ulcer, and gastric cancers. In the current study, we investigated the potential role of compound 13 (C13), a novel α1-selective small molecule activator of AMP-activated protein kinase (AMPK), against H. pylori-induced cytotoxicity in cultured gastric epithelial cells (GECs). We found that C13 induced significant AMPK activation, evidenced by phosphorylation of AMPKα1 and ACC (acetyl-CoA carboxylase), in both primary and transformed GECs. Treatment of C13 inhibited H. pylori-induced GEC apoptosis. AMPK activation was required for C13-mediated GEC protection. Inhibition of AMPK kinase activity by the AMPK inhibitor Compound C, or silencing AMPKα1 expression by targeted-shRNAs, alleviated C13-induced GEC protective activities against H. pylori. Significantly, C13 inhibited H. pylori-induced reactive oxygen species (ROS) production in GECs. C13 induced AMPK-dependent expression of anti-oxidant gene heme oxygenase (HO-1) in GECs. Zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP), two HO-1 inhibitors, not only suppressed C13-mediated ROS scavenging activity, but also alleviated its activity in GECs against H. pylori. Together, these results indicate that C13 inhibits H. pylori-induced ROS production and GEC apoptosis through activating AMPK–HO–1 signaling. - Highlights: • We synthesized compound 13 (C13), a α1-selective small molecule AMPK activator. • C13-induced AMPK activation requires α1 subunit in gastric epithelial cells (GECs). • C13 enhances Helicobacter pylori-induced pro-survival AMPK activation to inhibit GEC apoptosis. • C13 inhibits H. pylori-induced reactive oxygen species (ROS) production in GECs. • AMPK-heme oxygenase (HO-1) activation is required for C13-mediated anti-oxidant activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer

IL-15 has pivotal roles in the control of CD8{sup +} memory T cells and has been investigated as a therapeutic option in cancer therapy. Although IL-15 and IL-2 share many functions together, including the stimulation of CD8 T cell proliferation and IFN-γ production, the different in vivo roles of IL-15 and IL-2 have been increasingly recognized. Here, we explored the different effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells from resected breast tumors. We found that neither IL-2 nor IL-15 induced intratumoral CD8 T cell proliferation by itself, but after CD3/CD28-stimulation, IL-15 induced significantly higher proliferation than IL-2 during early time points, at day 2, day 3 and day 6. However, the IL-15-induced proliferation leveled off at day 9 and day 12, whereas IL-2 induced lower but progressive proliferation at each time point. Furthermore, IL-15 caused an early and robust increase of IFN-γ in the supernatant of TI cell cultures, which diminished at later time points, while the IL-2-induced IFN-γ production remained constant over time. In addition, the IL-15-costimulated CD8 T cells presented higher frequencies of apoptotic cells. The diminishing IL-15-induced response was possibly due to regulatory and/or exhaustion mechanisms. We did not observe increased IL-10 or PD-1 upregulation, but we have found an increase of Tim-3 upregulation on IL-15-, but not IL-2-stimulated cells. Blocking Tim-3 function using anti-Tim-3 antibodies resulted in increased IL-15-induced proliferation and IFN-γ production for a prolonged period of time, whereas adding Tim-3 ligand galectin 9 led to reduced proliferation and IFN-γ production. Our results suggest that IL-15 in combination of Tim-3 blocking antibodies could potentially act as an IL-2 alternative in tumor CD8 T cell expansion in vitro, a crucial step in adoptive T cell therapy. - Highlights: • We explored the effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells of breast cancer. • IL-15 and IL-2 had different kinetics in inducing TI CD8 T cell responses. • IL-15 induced stronger but shorter-lived TI CD8 T cell responses than IL-2. • IL-15, but not IL-2, caused upregulation of Tim-3 on TI CD8 T cells. • Blocking Tim-3 resulted in increased IL-15-induced proliferation and IFN-γ production in TI CD8 T cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production

LPS stimulation in macrophages/monocytes induces TNFα production. We here tested the potential effect of GSK621, a novel AMP-activated protein kinase (AMPK) activator, against the process. In RAW264.7 macrophages, murine bone marrow-derived macrophages (BMDMs), and chronic obstructive pulmonary disease (COPD) patients' monocytes, GSK621 significantly inhibited LPS-induced TNFα protein secretion and mRNA synthesis. Inhibition of AMPK, through AMPKα shRNA knockdown or dominant negative mutation (T172A), almost abolished GSK621's suppression on TNFα in RAW264.7 cells. Reversely, forced-expression of a constitutively-active AMPKα (T172D) mimicked GSK621 actions and reduced LPS-induced TNFα production. Molecularly, GSK621 suppressed LPS-induced reactive oxygen species (ROS) production and nuclear factor kappa B (NFκB) activation. In vivo, GSK621 oral administration inhibited LPS-induced TNFα production and endotoxin shock in mice. In summary, GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production in macrophages/monocytes. - Highlights: • GSK621 inhibits LPS-induced TNFα production/expression in RAW264.7 cells and BMDMs. • GSK621 inhibits LPS-induced TNFα production/expression in COPD patients' PBMCs. • GSK621's inhibition on TNFα production by LPS requires AMPK activation. • GSK621 inhibits LPS-induced ROS production and NFκB activation, dependent on AMPK. • GSK621 oral administration inhibits LPS-induced TNFα production and endotoxin shock in mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Methionine sulfoxide reductase A deficiency exacerbates acute liver injury induced by acetaminophen

Acetaminophen (APAP) overdose induces acute liver injury via enhanced oxidative stress and glutathione (GSH) depletion. Methionine sulfoxide reductase A (MsrA) acts as a reactive oxygen species scavenger by catalyzing the cyclic reduction of methionine-S-sulfoxide. Herein, we investigated the protective role of MsrA against APAP-induced liver damage using MsrA gene-deleted mice (MsrA{sup −/−}). We found that MsrA{sup −/−} mice were more susceptible to APAP-induced acute liver injury than wild-type mice (MsrA{sup +/+}). The central lobule area of the MsrA{sup −/−} liver was more impaired with necrotic lesions. Serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels were significantly higher in MsrA{sup −/−} than in MsrA{sup +/+} mice after APAP challenge. Deletion of MsrA enhanced APAP-induced hepatic GSH depletion and oxidative stress, leading to increased susceptibility to APAP-induced liver injury in MsrA-deficient mice. APAP challenge increased Nrf2 activation more profoundly in MsrA{sup −/−} than in MsrA{sup +/+} livers. Expression and nuclear accumulation of Nrf2 and its target gene expression were significantly elevated in MsrA{sup −/−} than in MsrA{sup +/+} livers after APAP challenge. Taken together, our results demonstrate that MsrA protects the liver from APAP-induced toxicity. The data provided herein constitute the first in vivo evidence of the involvement of MsrA in hepatic function under APAP challenge. - Highlights: • MsrA deficiency increases APAP-induced liver damage. • MsrA deletion enhances APAP-induced hepatic GSH depletion and oxidative stress. • MsrA deficiency induces more profound activation of Nrf2 in response to APAP. • MsrA protects the liver from APAP-induced toxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

TRPV4 is involved in irisin-induced endothelium-dependent vasodilation

Highlights: • Irisin caused an increase in [Ca{sup 2+}]{sub i} due to extracellular Ca{sup 2+} influx rather than Ca{sup 2+} release in endothelial cells. • Pretreatment with TRPV4 channel inhibitor completely abolished irisin-induced increases in [Ca{sup 2+}]{sub i}. • Irisin induced endothelium-dependent vasodilation, however, it had no effect on endothelium-independent vasodilation. • Pretreatment with TRPV4 channel inhibitor fully abrogated irisin-induced vasodilation. Irisin, an exercise-induced myokine, induces conversion of white into brown adipocytes, promoting mitochondrial biogenesis and energy expenditure. Irisin has a vascular protective effect on endothelial function in animals, including humans. Defects in irisin signaling pathways result in endothelial dysfunction in obesity and diabetes. However, the mechanisms underlying the effects of irisin on endothelial function have not been elucidated. Transient receptor potential vanilloid subtype 4 (TRPV4) channels are one of the most important Ca{sup 2+}-permeable cation channels in vascular endothelial cells. In this study, we hypothesized that irisin may induce endothelium-dependent vasodilation by activating Ca{sup 2+} influx into endothelial cells via TRPV4 channels. In primary cultured rat mesenteric artery endothelial cells, irisin caused an increase in [Ca{sup 2+}]{sub i} due to extracellular Ca{sup 2+} influx rather than release from Ca{sup 2+} stores. Moreover, irisin-induced increases in [Ca{sup 2+}]{sub i} were completely abolished by a TRPV4 inhibitor. In addition, irisin induced endothelium-dependent vasodilation of rat mesenteric arteries. However, irisin had no effect on endothelium-independent vasodilation. Furthermore, irisin-induced vasodilation was fully abolished in the presence of a TRPV4 inhibitor, indicating the involvement of TRPV4 channels in endothelium-dependent vasodilation. This study provides the first evidence that irisin-induced endothelium-dependent vasodilation is related to the stimulation of extracellular Ca{sup 2+} influx via TRPV4 channels in rat mesenteric arteries.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

microRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide

Highlights: • Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation in OB-6 osteoblasts. • miR-29a inhibition upregulates Gab1 to protect OB-6 cells from H{sub 2}O{sub 2}. • Gab1 over-expression promotes Akt activation to inhibit H{sub 2}O{sub 2}-induced OB-6 cell death. • Gab1 is the direct and primary target of antagomiR-29a in mediating its anti-H{sub 2}O{sub 2} actions. The present study determines the role of the Gab1 in hydrogen peroxide (H{sub 2}O{sub 2})-induced death of human osteoblasts. We show that Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation to promote osteoblast survival. In OB-6 human osteoblasts, Gab1 silencing (by targeted-shRNA) or complete knockout (by CRISPR-Cas9 KO plasmid) largely attenuated Akt activation by H{sub 2}O{sub 2}. Gab1-depleted OB-6 cells were more vulnerable to H{sub 2}O{sub 2}. Conversely, forced over-expression of Gab1 by an adenovirus vector increased Akt activation to protect OB-6 cells from H{sub 2}O{sub 2}. Significantly, the anti-sense of microRNA-29a (“antagomiR-29a”) induced Gab1 expression to facilitate H{sub 2}O{sub 2}-induced Akt activation, which protected OB-6 cells from apoptosis. AntagomiR-29a was however ineffective in Gab1-deficient and Akt-inhibited OB-6 cells. Forced over-expression of miR-29a induced Gab1 downregulation to inhibit H{sub 2}O{sub 2}-induced Akt activation, causing enhanced OB-6 cell death. miR-29a-induced actions were abolished by an adenovirus constitutively-active Akt1 (Ad-caAkt1) in OB-6 cells. Together, microRNA-29a inhibition induces Gab1 upregulation and Akt activation to protect OB-6 osteoblasts from H{sub 2}O{sub 2}.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Microstructural characterization of hydrogen induced cracking in TRIP-assisted steel by EBSD

The present work evaluates hydrogen induced cracking by performing an elaborate EBSD (Electron BackScatter Diffraction) study in a steel with transformation induced plasticity (TRIP-assisted steel). This type of steel exhibits a multiphase microstructure which undergoes a deformation induced phase transformation. Additionally, each microstructural constituent displays a different behavior in the presence of hydrogen. The aim of this study is to obtain a better understanding on the mechanisms governing hydrogen induced crack initiation and propagation in the hydrogen saturated multiphase structure. Tensile tests on notched samples combined with in-situ electrochemical hydrogen charging were conducted. The tests were interrupted at stresses just after reaching the tensile strength, i.e. before macroscopic failure of the material. This allowed to study hydrogen induced crack initiation and propagation by SEM (Scanning Electron Microscopy) and EBSD. A correlation was found between the presence of martensite, which is known to be very susceptible to hydrogen embrittlement, and the initiation of hydrogen induced cracks. Initiation seems to occur mostly by martensite decohesion. High strain regions surrounding the hydrogen induced crack tips indicate that further crack propagation may have occurred by the HELP (hydrogen-enhanced localized plasticity) mechanism. Small hydrogen induced cracks located nearby the notch are typically S-shaped and crack propagation was dominantly transgranularly. The second stage of crack propagation consists of stepwise cracking by coalescence of small hydrogen induced cracks. - Highlights: • Hydrogen induced cracking in TRIP-assisted steel is evaluated by EBSD. • Tensile tests were conducted on notched hydrogen saturated samples. • Crack initiation occurs by a H-Enhanced Interface DEcohesion (HEIDE) mechanism. • Crack propagation involves growth and coalescence of small cracks. • Propagation is governed by the characteristics of phases on the crack path.

36 MATERIALS SCIENCE↗
DOE OSTI2013

Mercury induces proliferation and reduces cell size in vascular smooth muscle cells through MAPK, oxidative stress and cyclooxygenase-2 pathways

Mercury exposure is known to increase cardiovascular risk but the underlying cellular mechanisms remain undetermined. We analyzed whether chronic exposure to HgCl{sub 2} affects vascular structure and the functional properties of vascular smooth muscle cells (VSMC) through oxidative stress/cyclooxygenase-2 dependent pathways. Mesenteric resistance arteries and aortas from Wistar rats treated with HgCl{sub 2} (first dose 4.6 mg kg{sup −1}, subsequent doses 0.07 mg kg{sup −1} day{sup −1}, 30 days) and cultured aortic VSMC stimulated with HgCl{sub 2} (0.05–5 μg/ml) were used. Treatment of rats with HgCl{sub 2} decreased wall thickness of the resistance and conductance vasculature, increased the number of SMC within the media and decreased SMC nucleus size. In VSMCs, exposure to HgCl{sub 2}: 1) induced a proliferative response and a reduction in cell size; 2) increased superoxide anion production, NADPH oxidase activity, gene and/or protein levels of the NADPH oxidase subunit NOX-1, the EC- and Mn-superoxide dismutases and cyclooxygenase-2 (COX-2); 3) induced activation of ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized the proliferative response and the altered cell size induced by HgCl{sub 2}. Blockade of ERK1/2 and p38 signaling pathways abolished the HgCl{sub 2}-induced Nox1 and COX-2 expression and normalized the alterations induced by mercury in cell proliferation and size. In conclusion, long exposure of VSMC to low doses of mercury activates MAPK signaling pathways that result in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induce proliferation of VSMC and changes in cell size. These findings offer further evidence that mercury might be considered an environmental risk factor for cardiovascular disease. - Highlights: ► Chronic HgCl{sub 2} exposure induces vascular remodeling. ► HgCl{sub 2} induces proliferation and decreased cell size in vascular smooth muscle cells. ► HgCl{sub 2} induces MAPK activation, oxidative stress and COX-2 expression. ► Inhibition of MAPK reduces HgCl{sub 2}-induced oxidative stress and COX-2 expression. ► Inhibition of MAPK, oxidative stress and COX-2 restores the altered cell proliferation and size.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights: • α5-nAChR and HIF-1α over-expressed in NSCLC and correlated with each other. • Nicotine induces cell proliferation and over-expressions of α5-nAChR, HIF-1α and VEGF in NSCLC. • Silence of α5-nAChR inhibited nicotine-induced A549 cell proliferation. • α5-nAChR involved in nicotine-induced HIF-1α and VEGF expression. • Down-regulation of α5-nAChR decreased the phosphorylation level of ERK1/2 and Akt.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2 prevented methylglyoxal-induced GSH depletion in liver.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5 μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis. - Highlights: • Luteolin inhibited Cr(VI)-induced oxidative stress. • Luteolin inhibited chronic Cr(VI)-induced malignant transformation. • Luteolin inhibited chronic Cr(VI)-induced inflammation. • Luteolin inhibited chronic Cr(VI)-induced angiogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Bleomycin induced epithelial–mesenchymal transition (EMT) in pleural mesothelial cells

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial–mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-β1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis. - Highlights: • Bleomycin induces collagen-I synthesis in pleural mesothelial cells (PMCs). • Bleomycin induces increases in vimentin and α-SMA protein in PMCs. • Bleomycin induces decreases in cytokeratin-8 and E-cadherin protein in PMCs • TGF-β1-Smad2/3 signaling pathway is involved in the PMC EMT induced by bleomycin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. • ABT-751 induces early autophagy and delays apoptosis. • ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways. • ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent. • Inhibition of autophagy enhances ABT-751-induced apoptosis.

60 APPLIED LIFE SCIENCES↗