School Of Basic And Applied Sciences
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Item Gliadin induced oxidative stress and altered cellular responses in human intestinal cells: An in-vitro study to understand the cross-talk between the transcription factor Nrf-2 and multifunctional APE1 enzyme(John Wiley and Sons Inc, 2022-05-09T00:00:00) Gupta, Kunj Bihari; Dhiman, Monisha; Mantha, Anil KumarThe present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H2O2, and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease. � 2022 Wiley Periodicals LLC.Item Organophosphate-pesticides induced survival mechanisms and APE1-mediated Nrf2 regulation in non-small-cell lung cancer cells(John Wiley and Sons Inc, 2020-10-20T00:00:00) Thakur, Shweta; Sarkar, Bibekananda; Dhiman, Monisha; Mantha, Anil K.Epidemiological and molecular studies have indicated that environmental exposure to organophosphate pesticides (OPPs) is associated with increased cancer risk; however, the underlying molecular mechanisms still need to be explained. Increasing cancer incidence is linked�to OPPs-induced oxidative stress (OS). Our study evaluates monocrotophos (MCP) and chlorpyrifos (CP)-induced OS responses and apurinic/apyrimidinic endonuclease 1 (APE1) role in human non-small-cell lung cancer (NSCLC) cells. Our prior study has implicated OPPs-induced base excision repair (BER)-pathway dysregulation and APE1-mediated regulation of transcription factor (TF) c-jun in A549 cells. We further investigated the effects of MCP and CP on apoptosis, proliferation, and APE1's redox-regulation of nuclear factor-like 2 (Nrf2). Data demonstrates that MCP and CP at subtoxic concentrations induced reactive oxygen species generation and oxidative DNA base damage 8-oxo-dG lesions in NCI-H1299 cells. CP moderately upregulated�the apoptosis-inducing factor (AIF) in A549 cells, however, it did not trigger other pro-apoptotic factors viz. caspase-9 and caspase-3, suggesting early caspase-independent apoptosis. However, dose-dependent AIF-downregulation was observed for MCP treatment. Furthermore, CP and MCP treatments upregulated proliferating cell nuclear antigen levels. Immunofluorescent confocal imaging showed the colocalization of APE1 with Nrf2 in 10 �M CP- and MCP-treated NCI-H1299 cells. Immunoprecipitation confirmed that APE1 and Nrf2 physically interacted, indicating the role of APE1-mediated Nrf2 activation following OPPs treatment. This study suggests that low concentration MCP and CP exposure generates OS along with DNA damage, and modulates apoptosis, and APE1-mediated Nrf2 activation, which might be considered as the possible mechanism promoting lung cancer cell survival, suggesting that APE1 may have the potential to become a therapeutic target for the treatment of NSCLC. � 2020 Wiley Periodicals LLCItem Gliadin induced oxidative stress and altered cellular responses in human intestinal cells: An in-vitro study to understand the cross-talk between the transcription factor Nrf-2 and multifunctional APE1 enzyme(John Wiley and Sons Inc, 2022-05-09T00:00:00) Gupta, Kunj Bihari; Dhiman, Monisha; Mantha, Anil KumarThe present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H2O2, and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease. � 2022 Wiley Periodicals LLC.Item Organophosphate-pesticides induced survival mechanisms and APE1-mediated Nrf2 regulation in non-small-cell lung cancer cells(John Wiley and Sons Inc, 2020-10-20T00:00:00) Thakur, Shweta; Sarkar, Bibekananda; Dhiman, Monisha; Mantha, Anil K.Epidemiological and molecular studies have indicated that environmental exposure to organophosphate pesticides (OPPs) is associated with increased cancer risk; however, the underlying molecular mechanisms still need to be explained. Increasing cancer incidence is linked�to OPPs-induced oxidative stress (OS). Our study evaluates monocrotophos (MCP) and chlorpyrifos (CP)-induced OS responses and apurinic/apyrimidinic endonuclease 1 (APE1) role in human non-small-cell lung cancer (NSCLC) cells. Our prior study has implicated OPPs-induced base excision repair (BER)-pathway dysregulation and APE1-mediated regulation of transcription factor (TF) c-jun in A549 cells. We further investigated the effects of MCP and CP on apoptosis, proliferation, and APE1's redox-regulation of nuclear factor-like 2 (Nrf2). Data demonstrates that MCP and CP at subtoxic concentrations induced reactive oxygen species generation and oxidative DNA base damage 8-oxo-dG lesions in NCI-H1299 cells. CP moderately upregulated�the apoptosis-inducing factor (AIF) in A549 cells, however, it did not trigger other pro-apoptotic factors viz. caspase-9 and caspase-3, suggesting early caspase-independent apoptosis. However, dose-dependent AIF-downregulation was observed for MCP treatment. Furthermore, CP and MCP treatments upregulated proliferating cell nuclear antigen levels. Immunofluorescent confocal imaging showed the colocalization of APE1 with Nrf2 in 10 �M CP- and MCP-treated NCI-H1299 cells. Immunoprecipitation confirmed that APE1 and Nrf2 physically interacted, indicating the role of APE1-mediated Nrf2 activation following OPPs treatment. This study suggests that low concentration MCP and CP exposure generates OS along with DNA damage, and modulates apoptosis, and APE1-mediated Nrf2 activation, which might be considered as the possible mechanism promoting lung cancer cell survival, suggesting that APE1 may have the potential to become a therapeutic target for the treatment of NSCLC. � 2020 Wiley Periodicals LLCItem Oxidative stress stimulates invasive potential in rat C6 and human U-87 MG glioblastoma cells via activation and cross-talk between PKM2, ENPP2 and APE1 enzymes.(Springer, 2018) Cholia, Ravi P.; Dhiman, Monisha; Kumar, Raj; Mantha, Anil K.Maintaining genomic integrity is essential for cell survival and viability. Reactive oxygen species (ROS) overproduction results in oxidative stress leading to the genomic instability via generation of small base lesions in DNA and these unrepaired DNA damages lead to various cellular consequences including cancer. Recent data support the concept "oxidative stress is an indispensable participant in fostering proliferation, survival, and migration" in various cancer cell types including glioblastoma cells. In this study we demonstrate that treatment of non-cytotoxic doses of oxidants such as amyloid beta [Aβ(25-35)] peptide, glucose oxidase (GO), and hydrogen peroxide (H2O2) for 24 h and 48 h time points found to increase the expression level and activity of a multifunctional enzyme Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of base excision repair (BER) pathway which takes care of base damages; and also resulted in modulation in the expression levels of downstream BER-pathway enzymes viz. PARP-1, XRCC1, DNA polβ, and ligase IIIα was observed upon oxidative stress in C6 and U-87 MG cells. Oxidants treatment to the C6 and U-87 MG cells also resulted in an elevation in the intracellular expression of glycolytic pathway enzyme Pyruvate kinase M2 (PKM2) and the metastasis inducer protein Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) as analyzed using Western blotting and Immunofluorescence microscopic studies. Our study also reports that oxidative stress induced for 24 h and 48 h in C6 and U-87 MG cells resulted in extracellular secretion of APE1 and ENPP2 as analyzed using Western blotting in conditioned media. However, the biological significance of extracellular secreted APE1 remains elusive. Oxidative stress also elevated the ENPP2's LysoPLD activity in conditioned media of C6 and U-87 MG cells. Our results also demonstrate that oxidative stress affects the expression level and localization of APE1, PKM2, and ENPP2 in C6 and U-87 MG cells. As evidenced by the colocalization pattern at 24 h and 48 h time points, it can be attributed that oxidative stress mediates crosstalk between APE1, PKM2, and ENPP2. In addition, when C6 and U-87 MG cells were treated with lysophosphatidic acid (LPA), a bioactive lipid that negatively regulates ENPP2's LysoPLD activity at 10 μM concentration, demonstrated strong migratory potential in C6 and U-87 MG cells, and also induced migration upon oxidative stress. Altogether, the findings demonstrate the potential of C6 and U-87 MG cells to utilize three proteins viz. APE1, PKM2, and ENPP2 towards migration and survival of gliomas. Thus the knowledge on oxidative stress induced APE1's interaction with PKM2 and ENPP2 opens a new channel for the therapeutic target(s) for gliomas.Item Ginkgolide B Revamps Neuroprotective Role of Apurinic/Apyrimidinic Endonuclease 1 and Mitochondrial Oxidative Phosphorylation Against Ab 25–35 -Induced Neurotoxicity in Human Neuroblastoma Cells(Wiley, 2015) Kaur, Navrattan; Dhiman, Monisha; Perez-Polo, J. Regino; Mantha, Anil K.Accumulating evidence points to roles for oxidative stress, amyloid beta (Aβ), and mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD). In neurons, the base excision repair pathway is the predominant DNA repair (BER) pathway for repairing oxidized base lesions. Apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme with DNA repair and reduction–oxidation activities, has been shown to enhance neuronal survival after oxidative stress. This study seeks to determine 1) the effect of Aβ25–35 on reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels, 2) the activities of respiratory complexes (I, III, and IV), 3) the role of APE1 by ectopic expression, and 4) the neuromodulatory role of ginkgolide B (GB; from the leaves of Ginkgo biloba). The pro-oxidant Aβ25–35 peptide treatment increased the levels of ROS/RNS in human neuroblastoma IMR-32 and SH-SY5Y cells, which were decreased after pretreatment with GB. Furthermore, the mitochondrial APE1 level was found to be decreased after treatment with Aβ25–35 up to 48 hr, and the level was increased significantly in cells pretreated with GB. The oxidative phosphorylation (OXPHOS; activities of complexes I, III, and IV) indicated that Aβ25–35 treatment decreased activities of complexes I and IV, and pretreatment with GB and ectopic APE1 expression enhanced these activities significantly compared with Aβ25–35 treatment. Our results indicate that ectopic expression of APE1 potentiates neuronal cells to overcome the oxidative damage caused by Aβ25–35. In addition, GB has been shown to modulate the mitochondrial OXPHOS against Aβ25–35-induced oxidative stress and also to regulate the levels of ROS/RNS in the presence of ectopic APE1. This study presents findings from a new point of view to improve therapeutic potential for AD via the synergistic neuroprotective role played by APE1 in combination with the phytochemical GB. © 2015 Wiley Periodicals, Inc.