Browsing by Author "Thakur, Shweta"
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Item APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells(Springer New York LLC, 2018) Thakur, Shweta; Dhiman, Monisha; Mantha, Anil K.Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72?h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24?h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol ? and DNA ligase III ? at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6?h time point, whereas it promotes nuclear localization after 24?h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to OPP-induced oxidative stress. Furthermore, nuclear colocalization of APE1 and the TF c-jun was observed in response to the treatment of CP and MCP for different time points in A549 cells. Therefore, in this study we demonstrate that MCP- and CP-induced oxidative stress alters APE1-dependent BER-pathway and also mediates cell survival signalling mechanisms via APE1 regulation, thereby promoting lung cancer cell survival and proliferation. ? 2017, Springer Science+Business Media, LLC.Item APE1/Ref-1 as an emerging therapeutic target for various human diseases: Phytochemical modulation of its functions(Nature Publishing Group, 2014) Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P.; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K.Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1a, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. ? 2014 KSBMB.Item A BIOCHEMICAL STUDY TO EVALUATE THE ROLE OF APURINIC/APYRIMIDINIC ENDONUCLEASE 1 (APE1) IN LUNG CANCER PROGRESSION(Central University of Punjab, 2018) Thakur, Shweta; Mantha, Anil K. and Dhiman, MonishaGlobally, lung cancer has the highest incidence and mortality rate. Environmental factors such as chlorpyrifos (CP) and monocrotophos (MCP), widely used organophosphate pesticides (OPPs), and are reported to be involved in the process of lung carcinogenesis. Present study attempts to investigate the genotoxic potential of CP and MCP, and investigates the oxidative DNA base damage response evoked by CP and MCP in non-small cell lung carcinoma (NSCLC) A549 and NCI-H1299 cells. A549 and NCI-H1299 cells were exposed to a range of concentration of CP and MCP at different time points; cell viability and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether CP and MCP treatment can initiate the DNA repair and cell survival signalling pathways in A549 cells, semi-quantitative RTPCR, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes, transcription factors (TFs) involved in pro-survival mechanisms and apoptosis-related factors. Significant increase in ROS generation was observed when cells were exposed to low and moderate doses of CP and MCP. A549 cells displayed a dose-dependent accumulation of DNA damage apurinic/apyrimidinic (AP) sites after MCP treatment. Cellular responses to CP and MCP-induced oxidative stress resulted in alterations in the mRNA and protein expression of BER-pathway enzymes viz. PARP1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points, which indicates imbalanced BER-pathway. OPPs treatment resulted in alterations in TFs viz. Nrf2, c-jun, phospho-c-jun (p-c-jun); inducible nitric oxide synthase (NOS2) and PCNA, which may lead to cell proliferation and promotion of carcinogenic events. Possible protein-protein interactions of BER-pathway’s key enzyme APE1 were also analyzed using STRING database, and intrinsically disordered (ID) regions of APE1 and TFs AP- v 1, NF-ĸB and Nrf2 were determined using PONDR analysis. PONDR prediction score and presence of ID regions in N-terminal segment of APE1, middle and Cterminal segments of AP-1, NF-ĸB and Nrf2 advocates for the possible direct protein-protein interactions, to be required for redox-regulatory function of APE1. Altered subcellular localization of APE1 and colocalization between APE1 and cjun; APE1 and p-c-jun; and APE1 and NF-ĸB were analyzed in A549 cells and; between APE1 and c-jun and, APE1 and Nrf2 were analyzed in NCI-H1299 cells following CP and MCP exposure using immunofluorescent confocal laser scanning microscopy. The present study results indicate that CP and MCP induces translocation of APE1 within the cytoplasm at an early time point of 6 hr; whereas it promotes nuclear localization at 24 hr, which suggests that APE1 subcellular localization is dynamically regulated in response to the oxidative stress. Furthermore, nuclear colocalization of APE1 and TF c-jun, APE1 and NF-ĸB and, APE1 and Nrf2 was also observed in response to CP and MCP treatment in A549 and NCI-H1299 cells, which indicates for the APE1-mediated redox regulation of TFs c-jun, NF-ĸB and Nrf2. However, no significant nuclear colocalization was observed for APE1 and p-c-jun indicating the role of phosphorylation on c-jun on Ser63 (p-c-jun) to act as a “switch off” form from the redox complex after the regulation. Immunoprecipitation (IP) was also performed to identify the interacting protein partners of APE1 under the influence of CP and MCP exposure; result indicated that MCP could moderately induce the interaction between APE1 and Nrf2, and APE1 and c-jun. Therefore, the present study suggest that CP and MCP-induced oxidative stress alters BER-pathway and mediates cell-survival signalling mechanisms via APE1 regulation thereby promoting lung cancer cell survival and proliferation.Item Exploring the COVID-19 vaccine candidates against SARS-CoV-2 and its variants: where do we stand and where do we go?(Taylor and Francis Ltd., 2021-12-03T00:00:00) Joshi, Gaurav; Borah, Pobitra; Thakur, Shweta; Sharma, Praveen; Mayank; Poduri, RamaraoAs of September 2021, 117 COVID-19 vaccines are in clinical development, and 194 are in preclinical development as per the World Health Organization (WHO) published draft landscape. Among the 117 vaccines undergoing clinical trials, the major platforms include protein subunit; RNA; inactivated virus; viral vector, among others. So far, USFDA recognized to approve the Pfizer-BioNTech (Comirnaty) COVID-19 vaccine for its full use in individuals of 16�years of age and older. Though the approved vaccines are being manufactured at a tremendous pace, the wealthiest countries have about 28% of total vaccines despite possessing only 10.8% of the total world population, suggesting an inequity of vaccine distribution. The review comprehensively summarizes the history of vaccines, mainly focusing on vaccines for SARS-CoV-2. The review also connects relevant topics, including measurement of vaccines efficacy against SARS-CoV-2 and its variants, associated challenges, and limitations, as hurdles in global vaccination are also kept forth. � 2021 Taylor & Francis Group, LLC.Item Histopathological staging in putative prostate cancer tissues and reviewing litearture of correlation between prostate specific antigen levels and prostate cancer inci. 2012.(Central University of Punjab, 2012) Thakur, Shweta; Thakur, SanjeevProstate cancer (PCa) remains the most significant cause of cancer specific mortality in elderly men. Asymptomatic behavior, non-modifiable risk factors and metastatic nature is the main problem of PCa. It remains clinically silent and presents itself only at advanced stage. Thus, diagnosing PCa at an early stage can result in increased chances of better treatment and hence, increased survival rate. An accurate biomarker can detect the cancer at an early stage and hence, at curable stage. Clinical parameters can only suspect prostate cancer. Whereas, histopathological examination can establish definite diagnosis of PCa. Various histological patterns are associated with cancer aggressiveness. Therefore, better understanding of clinical relevance of these histopathological findings can help to evaluate a robust biomarker for early detection of PCa. Present study was divided into two parts. First part was aimed to study the histopathology of putative prostate cancer tissue specimens. In second part, the literature of association of pre-operative serum prostate specific antigen levels with cancer detection and aggressiveness was reviewed. Protocol for histopathology of prostate tissues was established. Results of histopathological findings in putative PCa specimens were evaluated. Prevalence of histological PCa was not found in putative PCa tissues. Image library of results of the study was prepared for future analysis. Review of literature of correlation of serum PSA levels with PCa incidence suggests that PSA screening for PCa is a two-sided debate. No doubt that PSA holds the probability of detecting early PCa before development of symptoms; certain v limitations are associated with it. First, it is not reported to be 100% accurate. Second, it generates false positive and false negative results. A false positive result leads to over-treatment whereas a false negative result generates false sense of security against PCa in patient, both affects quality of life. Another main concern with PSA screening is its inability to differentiate between indolent and aggressive cancer. Therefore; accurate and economical molecular biomarkers for early detection and distinction of indolent versus aggressive cancer are urgently required. Until such biomarkers are developed and more convincing evidences regarding efficacy of PSA screening becomes available, research should focus on improving the diagnostic clinical utility of PSA by utilizing novel PSA isoforms. Identifying and validating correlation of serum PSA with tissue PSA and histological grade would be beneficial in terms of requirement of less invasive diagnostic methods to be used to measure PSA expression level as well as to confirm PCa. Future research may focus on evaluating the histological expression of other putative biomarkers and comparative serum proteomic profiling in different PCa stages.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 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 and inflammation in cardiovascular diseases: Two sides of the same coin(Springer India, 2015) Dhiman, Monisha; Thakur, Shweta; Upadhyay, Shishir; Kaur, Amandeep; Mantha, Anil K.Globally, the major cause of long- term disability and death is an ?epidemiologic transition? from infectious diseases and malnutrition complications to non-communicable chronic diseases like cardiovascular disease (CVD), cancer and diabetes. CVD accounts for major global mortality. Imbalance due to the generation of reactive oxygen species (ROS) levels above normal baseline levels and decreased antioxidant defence reserve makes the cardiovascular system (cardiac and vascular cells) susceptible to oxidative stress and damage. Growing evidences support the notion that oxidative stress plays a crucial role in the development and progression of CVD by altering normal functions such as inactivation of nitric oxide (NO) leading to endothelial dysfunction, intracellular Ca2+overload and others. Oxidative stress also mediates inflammation through various signalling cascades such as the activation of inflammatory transcription factors (TFs) namely NF-?B, AP-1 and Nrf-1. A vicious cycle of oxidative stress-mediated inflammation and inflammation- induced oxidative stress makes the CVD-related complications worse. Therefore, it is also very important to clearly understand the role of enzymatic sources of RO mechanisms underlying pathological conditions and link between oxidative stress and inflammation during each stage of CVD. The present chapter will elucidate the role of oxidative stress and inflammation in CVD development and progression. It is important to find the remedial measures, to develop the efficient biomarkers and to design the therapeutic strategies for CVD in the near future. ? Springer India 2015.