Department Of Biochemistry And Microbial Sciences
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Item In silico identification of natural anticancer product and their efficacy in breast cancer cells and cancer stem like cells(Central University of Punjab, 2020) Kushwaha, Prem Prakash; Kumar, ShashankBreast cancer is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to breast cancer recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Literature survey and in silico study identified Bulbine frutescens (Asphodelaceae), Kurarinone (KU) and 3-O-(E)-p- coumaroylbetulinic acid (CB) as lead plant product/phytochemicals. Methanolic and hexane extract of B. frutescens (BME and BHE respectively), KU and CB were studied for their anticancer activity and notch signaling pathway inhibitory potential in breast cancer cells. Moreover, KU and CB were also studied for their effect in mammosphere. Literature-based identification of methanol soluble phytochemicals of B. frutescens and in silico docking study revealed Bulbineloneside D as a potent notch signaling inhibitor (ϒ-secretase). In silico docking potential of KU and CB were equal to standard gamma secretase inhibitor DAPT (-8.74 kcal/mol). KU-gamma secretase complex showed lower RMSD value, marginal fluctuation in Radius of gyration (Rg), more number of inter hydrogen bonding, and stable secondary structure of the protein which indicates KU as candidate gamma secretase inhibitor (GSI). B. frutescens extracts (IC50 4.8– 28.4 μg/ml), Kurarinone (IC50 0.43-3.42 µM) and CB (IC50 0.99-5.88 µM) significantly decreased cell viability in MDA-MB-231 and T47D cells in time dependent manner. B. frutescens, KU and CB induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in test extract/phytochemicals treated breast cancer cells. Western Blot analysis showed reduced expression of cyclin D1 and increased procaspase 3 protein expression in extract/phytochemicals treated breast cancer cells in time dependent manner. Fluorescence spectrophotometry and confocal microscopy showed extract/phytochemicals induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells at IC50 and sub IC50 concentration. Flow cytometric apoptosis analysis of extract/phytochemicals treated MDA-MB-231 cells showed significant increase in early apoptotic population in comparison to non-treated cells at IC50 and sub IC50 (half of the IC50) concentration. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens, Kurarinone and CB in HEK293 transfected cells at IC50 concentration. Moreover, RT-PCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract/phytochemical treated breast cancer cells in time dependent manner. Western Blot analysis showed reduced notch responsive protein (Hes1, Hey1 and E-cadherin) expression in extract/phytochemical treated breast cancer cells. KU and CB treatment decreased the mammosphere formation ability in MCF-7 cells at IC50 concentration by lowering the notch signaling target proteins (Hes1, Hey1, and E-cadherin) and proteins involved in cancer cell self-renewal (c-Myc, SOX-2, CD44). In conclusion, extract/phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. KU and CB have the ability to downregulate the notch signaling pathway in breast cancer and cancer stem like cells.Item Anti-cancer drug doxorubicin induced cardiotoxicity: Understanding the mechanisms involved in ros generation resulting in mitochondrial dysfunction(Rasayan Journal of Chemistry, c/o Dr. Pratima Sharma, 2020) Upadhayay, S; Sharma, N; Mantha, A.K; Dhiman, M.Doxorubicin (DOX), despite being an effective anti-cancer drug has offsite targets that affect the vital organs such as heart, brain and kidney. DOX-induced cardiotoxicity is reported as a multi-factorial process that interferes with mitochondrial bioenergetics. These responses increase the threshold of oxidant-mediated injury and redox-mediated apoptosis in the cardiomyocytes. Oxidative stress particularly mitochondrial dysfunction in cardiomyocytes associated with cardiovascular diseases. In the present study we examined the effect of DOX on H9c2 cardiomyocyte where cells were treated with 5 μM DOX. To rule out the source of reactive oxygen species (ROS) during DOX-induced toxicity, the DOX-treated cardiomyocytes were incubated with 100 ?M diphenyleneiodonium (DPI), 50 μM salicyl hydroxamic acid (SHX), 20 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively and 10 μM N-acetyl cysteine (NAC, free radical scavenger) was also used to perceive the role of ROS. H2O2 (100 ?M) treated H9c2 cardiomyocytes were used as positive controls. The cell viability, reactive oxygen species (ROS) level and oxidative stress were determined using MTT assay, NBT assay/Flow-cytometry and Western blotting based assays. The effect of DOX on mitochondria was evaluated using Amplex Red assay; fluorescent probes such as MitoSOX and MitoTracker were used to examine the DOX-induced ROS production from the mitochondrial matrix. The mitochondrial membrane potential was evaluated using JC-1 dye. Western blotting was performed for cytochrome c release and apoptosis was examined with Annexin V-FITC assay. DOX was found to reduce cell viability, increase ROS level followed by enhanced oxidative stress in the form of protein carbonyls. DOX also showed a reduction in the mitochondrial membrane potential and allowed the release of cytochrome c which further leads to apoptosis and cell death. Further to rule out the pathway/mechanism(s) of DOX-mediated cardiac pathologies, the treatment with inhibitors of the classical ROS sources such as NADPH oxidase, Myeloperoxidase, mitochondria and general ROS scavenger (NAC) suggested that ROS via NOX and MPO during DOX-induced toxicity plays a crucial role in cardiomyocytes. The mitochondrial integrity was conserved when the cells were treated with NOX and MPO inhibitors, the cytochrome C release and apoptosis reduced in presence of these inhibitors. Taken together, these results demonstrate that DOX leads to ROS production and oxidative stress in cardiomyocytes which ultimately affects the mitochondrial integrity and functions, most importantly the ROS released via NOX and MPO is critical during DOX-induced cardiotoxicity. - RAS?YAN. All rights reserved.Item Bacteria from native soil in combination with arbuscular mycorrhizal fungi augment wheat yield and biofortification(Elsevier, 2020) Yadav, R; Ror, P; Rathore, P; Ramakrishna, W.Plant growth promoting bacteria (PGPB) have been used to enhance crop productivity. The effect of native PGPB and arbuscular mycorrhizal (AM) fungi in combination on wheat yield, biofortification and soil enzymatic activity is a relatively unexplored area. Twenty seven bacterial isolates from three different soils were characterized for their plant growth promoting traits. A total of three native and five non-native bacteria were used with and without arbuscular mycorrhizal (AM) fungi in an open greenhouse pot experiment with two wheat varieties to evaluate their effect on wheat yield, nutrient uptake, and soil health parameters. Wheat plants subjected to native PGPB (CP4) (Bacillus subtilis) and AM fungi treatment gave the best results with reference to macronutrient (nitrogen and phosphorus), micronutrient (iron and zinc) content in wheat grains and yield-related parameters, including thousand grain weight, number of grains per spike and total tillers per plant in both wheat cultivars. Treatment with CP4 and CP4 plus AM fungi enhanced total chlorophyll in wheat leaves indicating higher photosynthetic activity. Significant improvement in soil health-related parameters, including soil organic matter and dehydrogenase activity, was observed. Significant correlation among grain yield-related parameters, nutrient enhancement, and soil health parameters was observed in PGPB and AM fungi treated plants, especially HD-3086. These results provide a roadmap for utilizing native PGPB and AM fungi for enhancing wheat production in Punjab state of India and exploring their utility in other parts of the country with different soil and environmental conditions. - 2020 Elsevier Masson SASItem Emerging role of ZBTB7A as an oncogenic driver and transcriptional repressor(Elsevier, 2020) Gupta, S; Singh, A.K; Prajapati, K.S; Kushwaha, P.P; Shuaib, M; Kumar, S.ZBTB7A is a member of the POK family of transcription factors that possesses a POZ-domain at the N-terminus and Krüppel-like zinc-finger at the c-terminus. ZBTB7A was initially isolated as a protein that binds to the inducer of the short transcript of HIV-1 virus TAT gene promoter. The protein forms a homodimer through protein-protein interaction via the N-terminus POZ-domains. ZBTB7A typically binds to the DNA elements through its zinc-finger domains and represses transcription both by modification of the chromatin organization and through the direct recruitment of transcription factors to gene regulatory regions. ZBTB7A is involved in several fundamental biological processes including cell proliferation, differentiation, and development. It also participates in hematopoiesis, adipogenesis, chondrogenesis, cellular metabolism and alternative splicing of BCLXL, DNA repair, development of oligodendrocytes, osteoclast and unfolded protein response. Aberrant ZBTB7A expression promotes oncogenic transformation and tumor progression, but also maintains a tumor suppressive role depending on the type and genetic context of cancer. In this comprehensive review we provide information about the structure, function, targets, and regulators of ZBTB7A and its role as an oncogenic driver and transcriptional repressor in various human diseases. - 2020 Elsevier B.V.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 Assessment of risk conferred by coding and regulatory variations of TMPRSS2 and CD26 in susceptibility to SARS-CoV-2 infection in human(Springer, 2020) Senapati, S; Kumar, S; Singh, A.K; Banerjee, P; Bhagavatula, S.At present, more than 200 countries and territories are directly affected by the coronavirus disease-19 (COVID-19) pandemic. Incidence and case fatality rate are significantly higher among elderly individuals (age > 60 years), type 2 diabetes and hypertension patients. Cellular receptor ACE2, serine protease TMPRSS2 and exopeptidase CD26 (also known as DPP4) are the three membrane bound proteins potentially implicated in SARS-CoV-2 infection. We hypothesised that common variants from TMPRSS2 and CD26 may play critical role in infection susceptibility of predisposed population or group of individuals. Coding (missense) and regulatory variants from TMPRSS2 and CD26 were studied across 26 global populations. Two missense and five regulatory SNPs were identified to have differential allelic frequency. Significant linkage disequilibrium (LD) signature was observed in different populations. Modelled protein?protein interaction (PPI) predicted strong molecular interaction between these two receptors and SARS-CoV-2 spike protein (S1 domain). However, two missense SNPs, rs12329760 (TMPRSS2) and rs1129599 (CD26), were not found to be involved physically in the said interaction. Four regulatory variants (rs112657409, rs11910678, rs77675406 and rs713400) from TMPRSS2 were found to influence the expression of TMPRSS2 and pathologically relevant MX1. rs13015258 a 5? UTR variant from CD26 have significant role in regulation of expression of key regulatory genes that could be involved in SARS-CoV-2 internalization. Overexpression of CD26 through epigenetic modification at rs13015258-C allele was found critical and could explain the higher SARS-CoV-2 infected fatality rate among type 2 diabetes. 2020, Indian Academy of Sciences.Item Emerging Role of Migration and Invasion Enhancer 1 (MIEN1) in Cancer Progression and Metastasis(Frontiers Media S.A., 2019) Kushwaha, P.P; Gupta, S; Singh, A.K; Kumar, S.Tumor metastasis is a sequential event accounting for numerous cancer-related fatalities worldwide. The process of metastasis serially involves invasion, intravasation, extravasation, and tumor growth at the secondary site. Migration and invasion enhancer 1 (MIEN1) is a membrane associated protein overexpressed in various human cancers. Biological activity of MIEN1 is driven by geranylgeranyltransferase-I mediated prenylation at CAAX motif and methylation of the prenylated protein that anchors MIEN1 into the cellular membrane. Post-translationally modified MIEN1 interacts with Syk kinase and Annexin A2 protein; polymerizes G-actin and stabilizes F-actin filament; induces focal adhesion kinase phosphorylation and decrease cofilin phosphorylation implicated in both invasion and metastasis of different cancer types. In the present review, we discuss the structure, function, and involvement of MIEN1 in cancer progression. We also highlight the future prospects of MIEN1 as an emerging molecule and novel target in cancer cell invasion and metastasis.Item SNHG12: An LncRNA as a Potential Therapeutic Target and Biomarker for Human Cancer(Frontiers Media S.A., 2019) Tamang, S; Acharya, V; Roy, D; Sharma, R; Aryaa, A; Sharma, U; Khandelwal, A; Prakash, H; Vasquez, K.M; Jain, A.Limitations in current diagnostic procedures warrant identification of new methodologies to improve diagnoses of cancer patients. In this context, long non-coding RNAs (lncRNAs) have emerged as stable biomarkers which are expressed abundantly in tumors. Importantly, these can be detected at all stages of tumor development, and thus may provide potential biomarkers and/or therapeutic targets. Recently, we suggested that aberrant levels of lncRNAs can be used to determine the invasive and metastatic potential of tumor cells. Further, direct correlations of lncRNAs with cancer-derived inflammation, metastasis, epithelial-to-mesenchymal transition, and other hallmarks of cancer indicate their potential as biomarkers and targets for cancer. Thus, in this review we have discussed the importance of small nucleolar RNA host gene 12 (SNHG12), a lncRNA, as a potential biomarker for a variety of cancers. A meta-analysis of a large cohort of cancer patients revealed that SNHG12 may also serve as a potential target for cancer-directed interventions due to its involvement in unfolded protein responses, which many tumor cells exploit to both evade immune-mediated attack and enhance the polarization of effector immune cells (e.g., macrophages and T cells). Thus, we propose that SNHG12 may serve as both a biomarker and a druggable therapeutic target with promising clinical potential.Item Green Silver Nanoparticles for Phytopathogen Control(Springer, 2019) Gautam, N; Salaria, N; Thakur, K; Kukreja, S; Yadav, Neha; Yadav, R; Goutam, U.Plant diseases bring radical problem in the agriculture sector. Phytopathogens mediate diseases that pose considerable loss of yield and quality deterioration which eventually bring down the crop yield and the rural economy. The present study is, thus, focused on developing the optimized protocol for the synthesis of silver nanoparticles (AgNPs) by green chemistry approach and revealing their antimicrobial potential against phytopathogens. The synthesis of AgNPs was carried out by using aqueous plant extracts of three medicinal and aromatic plants, namely Allium cepa (onion), Allium sativum (garlic) and Zingiber officinale (ginger). AgNPs were characterized by various analytical techniques including UV–visible spectra, PSA, FTIR, TEM and XRD analysis. The AgNPs were spherical with size ranging from 1 to 10 nm, crystalline in nature and relatively stable up to 3 months after synthesis. The AgNPs conferred strong antimicrobial activity against selective bacterial and fungal phytopathogens. The antimicrobial activity of the AgNPs was observed against Erwinia sp., Pseudomonas syringe, Bacillus megaterium, Fusarium graminearum, F. avenaceum and F. culmorum. The effective concentration against bacterial pathogens was found to be between 50 µg/ml (garlic/Erwinia sp.) and 130 µg/ml (onion/B. megaterium). In the case of fungal pathogens, the range was 90 µg/ml (garlic/F. avenaceum) to 110 µg/ml (onion/F. graminearum) for an effective dose. © 2019, The National Academy of Sciences, India.Item 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.