Biochemistry And Microbial Sciences - Research Publications
Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/27
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Item Glycyrrhiza glabra (Licorice) root extract attenuates doxorubicin-induced cardiotoxicity via alleviating oxidative stress and stabilising the cardiac health in H9c2 cardiomyocytes(Elsevier, 2020) Upadhyay, S; Mantha, A.K; Dhiman, MonishaEthnopharmacological relevance: Doxorubicin (DOX) is an effective anti-neoplastic drug, however; it has downside effects on cardiac health and other vital organs. The herbal remedies used in day to day life may have a beneficial effect without disturbing the health of the vital organs. Glycyrrhiza glabra L. is a ligneous perennial shrub belonging to Leguminosae/Fabaceae/Papilionaceae family growing in Mediterranean region and Asia and widespread in Turkey, Italy, Spain, Russia, Syria, Iran, China, India and Israel. Commonly known as mulaithi in north India, G. glabra has glycyrrhizin, glycyrrhetic acid, isoliquiritin, isoflavones, etc., which have been reported for several pharmacological activities such as anti-demulcent, anti-ulcer, anti-cancer, anti-inflammatory and anti-diabetic. Aim of the study: The objective of the present study is to investigate the interaction between the molecular factors like PPAR-?/? and SIRT-1 during cardiac failure arbitrated by DOX under in vitro conditions and role of Glycyrrhiza glabra (Gg) root extract in alleviating these affects. Materials and methods: In the present study, we have examined the DOX induced responses in H9c2 cardiomyocytes and investigated the role of phytochemical Glycyrrhiza glabra in modulating these affects. MTT assay was done to evaluate the cell viability, Reactive Oxygen Species (ROS)/Reactive Nitrogen Species (RNS) levels, mitochondrial ROS, mitochondrial membrane potential was estimated using fluorescent probes. The oxidative stress in terms of protein carbonylation, lipid peroxidation and DNA damage was detected via spectrophotometric methods and immune-fluorescence imaging. The cardiac markers and interaction between SIRT-1 and PPAR-?/? was measured using Real-Time PCR, Western blotting and Co-immunoprecipitation based studies. Results: The Glycyrrhiza glabra (Gg) extracts maintained the membrane integrity and improved the lipid homeostasis and stabilized cytoskeletal element actin. Gg phytoextracts attenuated aggravated ROS level, repaired the antioxidant status and consequently, assisted in repairing the DNA damage and mitochondrial function. Further, the expression of hypertrophic markers in the DOX treated cardiomyocytes reconciled the expression factors both at the transcriptional and translational levels after Gg treatment. SIRT-1 mediated pathway and its downstream activator PPARs are significant in maintaining the cellular functions. It was observed that the Gg extract allows regaining the nuclear SIRT-1 and PPAR-? level which was otherwise reduced with DOX treatment in H9c2 cardiomyocytes. The co-immunoprecipitation (Co-IP) documented that SIRT-1 interacts with PPAR-? in the untreated control H9c2 cardiomyocytes whereas DOX treatment interferes and diminishes this interaction however the Gg treatment maintains this interaction. Knocking down SIRT-1 also downregulated expression of PPAR-? and PPAR-? in DOX treated cells and Gg treatment was able to enhance the expression of PPAR-? and PPAR-? in SIRT-1 knocked down cardiomyocytes. Conclusions: The antioxidant property of Gg defend the cardiac cells against the DOX induced toxicity via; 1) reducing the oxidative stress, 2) maintaining the mitochondrial functions, 3) regulating lipid homeostasis and cardiac metabolism through SIRT-1 pathway, and 4) conserving the cardiac hypertrophy and hence preserving the cardiomyocytes health. Therefore, Gg can be recommended as a healthy supplement with DOX towards cancer therapeutics associated cardiotoxicity. - 2020Item Hydrogen peroxide-induced oxidative stress and its impact on innate immune responses in lung carcinoma A549 cells(Springer, 2019) Upadhyay, S; Vaish, S; Dhiman, MonishaThe immune responses, involved in recognition of cancer-specific antigens, are of particular interest as this may provide major leads towards developing new vaccines and antibody therapies against cancer. An effective treatment for cancer is still a challenge because there are many mechanisms through which the tumor cells can escape the host immune surveillance. Oxidative stress or respiratory burst which is host’s mechanism to kill the foreign particles is used as defense mechanism by the tumor cells. The tumor cells uses this oxidative stress to form neo-antigens which in turn makes them undetectable and can escape the host immune surveillance. The human lung carcinoma (A549) cells were treated using 100 µM H 2 O 2 to induce oxidative stress, and the extent oxidative modifications were detected at the level of membrane and proteins in form of lipid peroxidation and protein carbonyls respectively. Nitric oxide and iNOS levels were estimated by Griess assay and immunostaining, respectively. The oxidized tumor proteins were visualized on one-dimensional SDS–PAGE. The H 2 O 2 -treated (15 min and 24 h post-treatment) A549 cells were co-cultured with THP-1 cells to subsequently visualize the phagocytic activity by Giemsa and CFSE staining to understand the role of neo (oxidized) tumor antigens in eliciting alteration in immune responses. A significant decline in the percent engulfed cells and decrease in the levels of reactive oxygen species was observed. Immunohistostaining for p47 phox , which is an important indicator of the oxygen-dependent phagocytosis, showed a decrease in its levels when cells were treated for only 15 min with 100 µM H 2 O 2 , whereas at 24-h post-treatment there was no change in the p47 phox levels. The study has established oxidative stress as a new pathogenic mechanism of carcinogenesis and will open new avenues for clinical intervention, adjunct therapies for cancer, and its control at the initial stage by targeting these neo-antigens. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.Item Role of Immune System in Tumor Progression and Carcinogenesis.(Wiley, 2018) Upadhyay, S; Sharma N; Gupta, K.B; Dhiman, MonishaTumor micro‐environment has potential to customize the behavior of the immune cell according to their need. In immune‐eliminating phase, immune cells eliminate transformed cells but after tumor establishment innate and adaptive immune cells synergistically provide shelter as well as fulfill their requirement that helps in progression. In between eliminating and establishment phase, equilibrium and escaping phase regulate the immune cells response. During immune‐escaping, (1) the antigenic response generated is either inadequate, or focused entirely on tolerance, and (2) immune response generated is specific and effective, but the tumor skips immune recognition. In this review, we are discussing the critical role of immune cells and their cytokines before and after the establishment of tumor which might play a critical role during immunotherapy.Item Identification of novel indole based heterocycles as selective estrogen receptor modulator.(Elsevier, 2018) Singla, Ramit; Prakash, Kunal; Gupta Kunj Bihari; Upadhyay, Shishir; Dhiman, Monisha; Jaitak, VikasIn the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5cand 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC.Item Inflammasome activation and regulation during Helicobacter pylori pathogenesis(Elsevier, 2018) Kumar, S; Dhiman, MonishaHelicobacter pylori is a leading cause of gastric cancer worldwide, its type four secretary toxin CagA is cited to be primarily responsible for it. Other virulence factors such as urease, VacA, HopQ, BabA and SabA are responsible for bacterial survival in acidic environment, adherence and cellular damage but its molecular mechanism is not completely understood. A number of pathogens including bacteria, fungi and virus are involved in the regulation of cellular machinery of inflammasome. Inflammasomes are multimeric protein complexes formed after external stimuli such as PAMPs/DAMPs or salt crystals and activates cellular caspases causes inflammation via pro-inflammatory cytokines. Virulence factors associated with microbial pathogens causes’ cellular damage through damaging mitochondria, rupturing lysosome, producing endoplasmic stress and dysregulation of cellular ions balance. These cellular dysfunctioning leads to oxidative stress, cathepsin B production, nuclear and mitochondrial DNA damage which activates inflammasome machinery, pro-inflammatory cytokine release and cellular death known as pyroptosis. The mechanism of inflammasome induction by H. pylori is not studied extensively and very few virulence factors such as UreB, CagA, FlaA and VacA and their role in inflammasomes is established. This review elaborates the mechanism of inflammasomes regulation and elucidates the pathways through which H. pylori regulates inflammasome activation.Item 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 Design, synthesis and biological evaluation of novel indole-benzimidazole hybrids targeting estrogen receptor alpha (ER-?)(Elsevier Masson SAS, 2018) Singla R.; Gupta K.B.; Upadhyay S.; Dhiman, Monisha; Jaitak V.In the course of efforts to develop novel selective estrogen receptor modulators (SERMs), indole-benzimidazole hybrids were designed and synthesised by fusing the indole nucleus with benzimidazole. All the compounds were first inspected for anti-proliferative activity using ER-? responsive T47D breast cancer cell lines and ER-? binding assay. From this study, two representative bromo substituted compounds 5f and 8f were found to be most active and thus were escalated for gene expression studies for targeting ER-?. Cell imaging experiment clearly suggest that compounds were able to cross cell membrane and accumulate thus causing cytotoxicity. RT-PCR and Western blotting experiments further supported that both compounds altered the expression of mRNA and receptor protein of ER-?, thereby preventing the further transactivation and signalling pathway in T47D cells lines. Structural investigation from induced fit simulation study suggest that compound 5f and 8f bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these results strongly indicate that compound 5f and 8f represents a novel potent ER-? antagonist properties and will proved promising in the discovery of SERM for the management of breast cancer.Item Inflammatory response of gliadin protein isolated from various wheat varieties on human intestinal cell line(Academic Press, 2018) Gupta, K.B.; Upadhyay, S.; Saini, R.G.; Mantha, Anil K.; Dhiman, MonishaWheat protein contributes a significant part in human diet, apart from its well-known nutritional values, wheat gluten/gliadin proteins are also responsible for the many allergic/inflammatory diseases and chronic inflammation in the small intestine may cause diarrhea and malabsorption, in a specific population of individuals. In the present study, the antigenic characteristics of twelve wheat varieties of diverse origin namely C273, C281, C286, C306, C518, C591, Agra Local, 9D, 8A, Raj4229, HD3027, NP824 released during 1920?2012 were evaluated. Gliadin proteins from these varieties were tested on human colon cancer cell line HCT116 to assess their effect on inflammation, oxidative and nitrosative stress, pro-inflammatory cytokines. The results show that these wheat varieties induced high levels of ROS/RNS and MPO activity which was further supported by the increase in the mRNA levels of a cytokine such as IL-1? and IL-15. It can be concluded that gliadin from these wheat varieties is suggested to act as a potential antigen by enhancing the level of inflammation irrespective of their year of release and origin which if not controlled may lead to the initiation of celiac disease in genetically susceptible individuals or may be responsible for other wheat protein intolerance associated diseases. ? 2018 Elsevier LtdItem Association of elevated levels of C-reactive protein with breast cancer, breast cancer subtypes, and poor outcome(Mosby Inc., 2018) Kaur, R.P.; Rubal, Banipal,; Vashistha, R.; Dhiman, Monisha; Munshi, AnjanaBackground and Purpose: Inflammation and caner are linked in a bidirectional manner. C-reactive protein (CRP) is an important inflammatory marker. The aim of the study was to test whether the inflammatory marker, CRP at the time of diagnosis of breast cancer is associated with metastasis, recurrence, and death in breast cancer patients from Malwa region of Punjab where breast cancer is widely feared. Material and Methods: Two hundred and forty-two breast cancer patients and 242 age and sex matched controls were included in the study. CRP levels were estimated using fully automated bio analyzer Erba200. Follow up interviews were conducted at an interval of 3, 6, 9, 12, 15, 18, 21, 24, and 27 months to determine the outcome among breast cancer patients. Results: Elevated levels of CRP were found among the diseased in comparison with controls (P < 0.0001). Higher CRP levels associated significantly with poor outcome including metastasis and recurrence among breast cancer patients [P = 0.03; 95% confidence interval; odds ratio: 2.954 (0.9125-9.561)]. Conclusion: Elevated levels of CRP associated significantly with increased risk of breast cancer and poor outcome. CRP estimation may be a simple and inexpensive tool for the risk assessment and outcome of the disease in Malwa region of Punjab where incidence of breast cancer is reported to be very high. ? 2018