School Of Basic And Applied Sciences
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Item Evaluation of amyloid beta (aβ)-induced Mitochondrial dysfunction: Neuroprotective role of Apurinic/apyrimidinic endonuclease (ape1) Via its interaction with cysteamine Dioxygenase (ado)(Central University of Punjab, 2020) Kaur,Navrattan; Mantha, Anil K.Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, ultimately leading to neurodegeneration. Themain pathway for repairing oxidative base lesions is base excision repair (BER), and such repair is crucial for neurons owing to high rate of oxygen metabolism. Apurinic/apyrimidinic endonuclease (APE1) is a protein of this pathway involved in DNA repair and also in the redox co-activating function of different transcription factors. Thus, manipulation of DNA repair mechanisms can be thought of as a putative approach to prevent neuronal loss in neurodegenerative disorders like Alzheimer’s disease (AD). Ginkgo biloba has been studied as a possible treatment for dementia and AD. The ginkgolides present in G. biloba possess antioxidant, neuroprotective and cholinergic activities. The aim of the study was to explore the repair and redox functions of APE1 and a detailed mechanism of association of APE1 with ADO (a thiol dioxygenase) and functional cross-talk between them has been studied. In the present study, we have standardized the differentiation of SH-SY5Y neuroblastoma cells into the cells possessing a mature neuron-like phenotype. The results of cell viability assay showed that differentiated cells are more sensitive/vulnerable to oxidative stress, which is elicited by Aβ. H2DCFDA and DAF- FM-based detection of ROS and RNS strongly advocates that under oxidative stress conditions elicited by Aβ, GB exerts ameliorating effect to render neuroprotection to the SH-SY5Y cells due to its antioxidant nature. Significant decrease in nNOS expression was seen, when cells were pre-treated with GB and then given Aβ treatment in whole cell, cytosol and nucleus. This shows that GB pre-treatment decreases the RNS (NO) levels due to its anti-oxidant property. Determination of DNA damage in terms of measurement of 8-oxo-dG was seen to be more pronounced in mitochondria. In response to DNA damage, pre-treatment with GB decreased the expression of DNA repair enzyme APE1 expression in mitochondria, showing that GB aids in lowering the oxidative stress generated by Aβ in the mitochondria. In the nuclear extracts, upon treatment with GB, there was a significant increase in ADO expression and Aβ treatment also increased the expression of ADO. Whereas, combination treatment of Aβ and GB led to lower expression of ADO. This points towards the possibility that ADO might be translocating to nucleus under oxidative stress and GB might be affecting APE1 – ADO interaction in lowering oxidative stress by the anti-oxidant action of GB, which was clearly observed by immunostaining using confocal microscopy. JC-1 assay points toward GB’s role in restoring the mitochondrial membrane potential against Aβ- challenge. Determination of apoptotic markers (Caspase 9 and AIF) showed that Aβ(25-35) induced oxidative stress caused initiation of apoptosis and GB treatment was able to rescue apoptosis. Our study elucidates activation of synaptic CaMKII and CREB exerting neuroprotective effects; and GB acting to restore the expression and active, phosphorylated state of CaMKII and CREB in presence of Aβ-induced oxidative stress in the SH-SY5Y neuroblastoma cells. This study points towards the use of phytochemicals like GB which will may prove to be beneficial for the enhancement of synaptic functionality and promote neuroprotection.Item OXIDATIVE STRESS INDUCED CELL PROLIFERATION AND DNA REPAIR MECHANISMS IN GLIOBLASTOMA CELLS: ROLE OF ENPP2 AND APE1(Central University of Punjab, 2018) Cholia, Ravi Parkash; Mantha, Anil K. and Kumar, RajGlioblastoma Multiforme (GBM) is a grade IV, most frequent, and invasive devastating brain tumor with poor prognosis, even with the advancement of multimodal therapies, patients have survival period of less than 15 months. GBM is a multifactorial disease with oxidative stress as a key accelerating player. In the present study, rat glioma C6 and human glioblastoma U-87 MG cell lines were exposed to non-cytotoxic concentrations i.e. 10 -35) peptide, 10 U/ml GO, and 50 M H2O2, respectively. Further, the ROS levels were measured via NBT and H2DCFDA assays. Our genome encounters exogenous and intracellular oxidants which result in the DNA damage; small DNA base lesions such as apurinic/apyrimidinic (AP) sites are generated following the oxidants exposure and repairing of these AP sites is the prerequisite to maintaining the genomic integrity. In the present study, it was observed that APE1 being a redox-sensitive protein, with the moderate level of oxidative stress [induced -35) peptide, GO, and H2O2] resulted in the elevation of APE1 expression as measured using Western blotting, RT-PCR, and (repair) activity was boosted after the treatment of oxidants. Oxidative stress also resulted in the secretion of APE1 extracellularly. Additionally, in this study dysregulated expression of BER-pathway enzymes were observed after the treatment of non-cytotoxic concentrations of the oxidants. Cancer shows higher metabolic properties as compared to the normal cells. Pyruvate Kinase M2 (PKM2) one of the isoform of pyruvate kinase (PK), is a key enzyme in the glycolytic pathway, which catalyses the terminal step of the glycolysis, converts phosphoenolpyruvate (PEP) into pyruvate. PKM2 also perform nonglycolytic functions via enhancing the expression of cyclin D, c-myc, and contributing towards the aggressiveness of GBM. In the present study, oxidative stress resulted in up-regulation of PKM2 level, as analyzed using Western blotting and majorly in the cytosolic regions as identified by immunocytochemistry. Ectonucleotide pyrophosphatase/phosphordiestrase2 (ENPP2) is the secretary protein, known to be involved in a variety of processes like embryonic development, blood vessel formation during development, inflammation, favoring PKM2 dimeric form, and progression of cancer through its enzymatic product LPA. ENPP2 is highly expressed in the GBM, and LPA receptors are also predominate in GBM and play a role in its growth and development. In the present study, elevated expression and activity of ENPP2 was observed after the treatment of non-cytotoxic doses of oxidants in C6 and U-87 MG cells as analyzed using Western blotting and immunocytochemistry. In addition, LPA treatment resulted in the induction of migratory potential of C6 and U-87 MG cells. LPA treatment also up-regulated the key transcription factors such as c-jun, p-c-jun, NF- B, and HIF-1- advocating for their involvement in the survival of GBM cells. LPA treatment resulted in the timedependent increase in the PKM2 and ENPP2 expression and subcellular translocation in the C6 and U-87 MG cells. However, LPA treatment resulted in the elevation of nuclear APE1 expression after 48 hr incubation period. Oxidants - 35) peptide, GO, and H2O2 treatment enhanced the secretory levels of ENPP2 in the extracellular media up to 48 hr, suggesting the protective role of ENPP2 against the oxidative stress. Co-localization of APE1, PKM2, and ENPP2 were observed in the C6 and U87 MG cells when treated with -35) peptide, GO, and H2O2 treatment suggesting the role of oxidative stress in the cross talk interaction of three proteins towards the aggressiveness of GBM. In addition, anti-APE1 inhibitors were synthesized activity, and one of the screened molecule GR5G-b showed ani-proliferative property along with dysregulated APE1 level and repair function; and also displayed potential in cell cycle arrest as analyzed by flow cytometry. Taken together, it can be concluded that oxidative stress enhances the aggressiveness of GBM cells via up-regulating the key proteins (APE1, PKM2, and ENPP2) and altering the functions associated as studied in C6 and U-87 MG cell lines. Further studies focusing towards blocking of their activities by designing, help in development of new therapeutic interventions for GBM.Item Phytochemical ginkgolide b protects cultured neuroblastoma SH-SY5Y cells against a?(25-35) induced oxidative stress responses by maintaining the mitochondrial integrity(Rasayan Journal of Chemistry, c/o Dr. Pratima Sharma, 2020) Kaur, N; Kaur, S; Saini, M; Dhiman, M; Mantha, A.K.Alzheimer�s disease is associated with oxidative stress induced by accumulation of A? peptide, by disrupting the mitochondrial function. In this study, the oxidative stress responses induced by A?(25-35) and protective effects of diterpenoid phytochemical Ginkgolide B (GB) were evaluated by the determination of cellular oxidant/antioxidant status, oxidative DNA base damage and repair capacity of cells through evaluation of mitochondrial BER pathway status and the multifunctional enzyme APE1 in human neuroblastoma SH-SY5Y cells, and evaluation of mitochondrial membrane potential and changes in apoptotic pathway. It was found that A?(25-35) treatment increased ROS/RNS production, increased the activities of antioxidant SOD and Catalase enzymes, decreased the expression of mitochondrial SOD (SOD2), induced oxidative DNA base damage, might be altered the repair capacity as analyzed by the transcriptional and translational expression of APE1 and other BER pathway enzymes in the mitochondria, disrupted the mitochondrial membrane potential and induced apoptosis as a result of these responses. Phytochemical modulation by the pre-treatment of GB for 3 hr followed by the treatment of A?(25-35) for a period of 24 hr caused decrease in ROS/RNS, increase in activities of antioxidant enzymes and expression of SOD2, decreased oxidative DNA base damage and increased transcriptional and translational expression of APE1, increased/restored expression of APE1 and polymerase gamma (?) in the mitochondria, restored mitochondrial membrane potential and rescued the SH-SY5Y cells from mitochondrial-mediated apoptosis against A?(25-35) induced oxidative stress responses. Taken together, GB showed neuroprotection by restoring cellular antioxidant defense system, repair capacity of cells and restoring mitochondrial integrity (genome and membrane potential), thus rescuing the SH-SY5Y cells from A?(25-35) induced oxidative stress responses. � RAS?YAN. All rights reserved.Item Investigation of combined genotoxic effect of pesticides on cancer cell lines(Central University of Punjab, 2013) Anju; Bhardwaj, PankajThe use of pesticides in agriculture sector is increasing widely to alleviate crop loss due to pests. To combat problem of pest, various types of pesticides are widely used, out of which insecticide are majorly used. In Malwa region of Punjab lots of insecticides are used. These pesticides when present together they may result in combined response. This combined response can be of three types such as independent, dose addition and interaction (synergism or antagonism). This combined response is very big trouble, such as sometimes even low dose of pesticide which is not toxic individually can result in toxic response in the presence of another pesticide. In the present study, Malathion and Monocrotophos (widely used organophosphorous insecticides) selected for investigation of combined effect. Out of selected pesticide Malathion also detected in water, in present study. Various parameters for genotoxicity evaluation was carried out with exposure of pesticides on HCT-116 WT/ p53 mutant (colon cancer) cell lines. Results of present study suggested that combination of pesticides resulted in more cell death, oxidative stress, membrane instability and imbalanced antioxidative system. It is also evaluated that low dose exposure of pesticides individually and in combination resulted in increased cell survival, may be due to activation of defense mechanism with prior treatment. All parameters evaluated was with short term exposure of 1-2 days, so evaluated long term exposure of ten days and found that this resulted in very less cell number and changed cell morphology. It is observed that both pesticides showed genotoxicity individually as well as in combination. The combined exposure of both pesticides deviate from there expected effect for dose addition towards more toxicity. So it is concluded that there may be some interaction between these pesticides, which may be synergistic in nature, because observed more toxicity.Item Assessment of Extract of Syzygium cumini Against Doxorubicin Induced Cardiotoxicity(Central University of Punjab, 2018) Chayan, Mukherjee; Monisha DhimanFor the past four decades, doxorubicin (DOX) has been used to treat cancer, mainly solid tumours and haematological malignancies. However, clinical community is greatly concerned regarding the administration of this as DOX treatment is commonly associated with dose-dependent cardiotoxicity. Attempts at alleviating drug generated cardiac damage using an extract from different parts of plants with radical scavenging property are a promising area of research. Hydroalcoholic extract derived from fruit pulp of Syzygiumcumini which has a significant antiradical scavenging effect. This study aims to assess the effect of parallel administration of SC fruit pulp extract (SC) on mitigating or preventing DOX induced cardiotoxicity in vitro using H9c2 cardiomyoblast cell lines. Addition of SC fruit pulp extract and DOX were performed for both treatment and control sets on H9c2 cells. SC fruit pulp extract showed strong ABTS cation radical scavenging activity in a dose dependent manner. MTT assay was used to study the cytotoxic effect of SC fruit pulp extract and DOX. ROS levels were estimated using NBT assay and DHE assay. The results showed that DOX has significant cytotoxic effect in a dose dependent manner while SC fruit pulp extract did not display any significant cytotoxicity on H9c2 cells. The DOX induced ROS production was found to be significantly reduced in SC fruit pulp extract treated cells. Results of the current study also suggest that the treatment of SC fruit pulp extract along with DOX, displayed cardioprotective potential in H9c2 cells by: 1) reducing lipid peroxidation; 2) decreasing extracellular nitric oxide (NO); 3) decreasing the expression of the protein p47phox and iNOS/NOS-2. These results clearly suggest that treatment of SC fruit pulp extract along with DOX reduces the DOX induced toxicity and hence can be a promising therapeutic intervention in managing DOX mediated cardiotoxicity.Item Oxidative stress responses to sub-lethal dose of Cry toxin in the larvae of castor semilooper, Achaea janata(Central University of Punjab, 2018) Singh, Kanika; Chaitanya,R.K.Development of synthetic insecticides to reduce the level of infestation led to deleterious effects on environment and human health. This lead to the development of ecofriendly pest management alternatives including Bacillus thuringensis (Bt). Bt produce Crystal (Cry), Cytotoxic (Cyt) and Vegetative (Vip) proteins with insecticidal activity against different orders of lepidoptera. Of late, pest resistance against Bt is reported in countries.The reduced toxicity of Bt formulation from degradation by UV light, wash-off by rain, drying, temperature, and soil acidity as well as its chemistry. Further, insects sense pesticides through odorant receptors and move away quickly, there is always a possibility of a population of larvae to get exposed to sub-lethal doses of toxin which might exhibit variable effects and escape mortality and eventually generate resistance. Sub-lethal dose lead to the generation of oxidative stress in the insect and eventually scavenged by anti-oxidant enzymes. These stress responses would enhance our understanding of adaptations for survival and resistance development. The current study is an attempt to monitor the antioxidative responses at the transcriptional level upon sub-lethal exposure of Cry toxin in the larvae of an polyphagous pest castor semilooper, Achaea janata. prevalent in the Indian subcontinent.Item Role of curcumin on monoamine oxidase(MAO) enzyme expression and activity against Amyloid Beta (A?)-induced oxidative stress in human glioblastoma U-87 MG cell.(Central University of Punjab, 2018) Behera, Nishibala N; Mantha,Anil K.Glioblastoma (GBM) is the most common brain tumor in humans. The major factor responsible for its progression is oxidative stress. Oxidative stress leads to disruption of signaling pathways and damage to cells and tissues. Monoamine oxidase (MAO) is involved in oxidative deamination of endogenous biogenic amine neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine. Therefore, MAO plays a key role in initiation and progression of GBM through oxidative stress. In the present study, A?(25-35) peptide treatment was used to induce oxidative stress in human glioblastoma (U-87 MG) cells. A?(25-35) is known to induce oxidative stress through altering the expression and activity of various antioxidant and mitochondrial enzymes. In this study, the expression and activity of MAO was evaluated through induction of oxidative stress by A?(25-35) and antioxidant treatment of Curcumin. It was found that Curcumin decreases the mRNA expression of MAO but its protein expression increases, whereas A?(25-35) showed little decrease in the mRNA expression of MAO and increase in its protein expression, thus pointing towards differential regulation of translation and transcription. The activity of MAO was found to be increased in A?(25-35), Cur and Cur+A?(25-35) . Therefore, Curcumin has little or no antioxidant effect in altering the expression and activity of MAO and A? showed its oxidative potential by increasing the expression and activity of MAO, although not very significant, possibly because it uses other pathways for inducing oxidative stress.Item Effect of amyloid beta (25-35) peptide on mitochondrial respiratiory function in neuroral cells over expressing ape1(Central University of Punjab, 2014) Kaur, Navrattam; Mantha, Anil K.Alzheimer's disease (AD) is an important public health problem which affects millions of people worldwide. The major pathological hallmarks associated with AD are the accumulation of amyloid beta (A?) in senile plaques and neurofibrillary tangles (NFTs) made up of hyperphosphorylated tau proteins. Accumulating evidences point towards the role of oxidative stress and mitochondrial dysfunction in the pathogenesis of AD. Aging is considered as one of the greatest risk factor for AD. In order to maintain genome integrity, base excision repair (BER) pathway is the predominant pathway for repairing oxidized base lesions in neuronal cells. APE1 is the central enzyme of the BER-pathway, having both repair and redox activities and shown to enhance neuronal survival after oxidative stress. In my study, effect of A?(25-35) on mitochondrial ROS/RNS levels and activities of respiratory complexes (I, III, & IV) in neuronal cells was studied with and without ectopic APE1 expression and the neuro- modulatory role of Ginkgolide B (from leaves of G. biloba) was evaluated. It was seen that A?(25-35) increases the ROS/RNS levels in these cells which was decreased when pre-treated with Ginkgolide B (G.B) before treating with A?(25-35). APE1 levels were found to be decreased on treating with A?(25-35) and were increased on pre- treatment with G.B and subsequent treatment with A?(25-35). These results indicate that ectopic APE1 expression in the mitochondria of the neuronal cells might overcome the oxidative damage caused by A?(25-35). Also, phytochemical G.B has shown to modulate the mitochondrial complex activity upon A?(25-35)-induced oxidative stress and modulate the ROS/RNS levels in the presence of APE1. Further studies are needed to understand the mechanism of action of APE1 in relation to the above results, which will be carried out during my Ph.D. work.Item Oxidative Stress in Invertebrate Systems(InTech, 2016) Chaitanya, R. K.; Shashank, Kumar; Sridevi, P.Invertebrates have been valuable research models in the discovery of many scientific principles owing to the numerous advantages they provide. Throughout the life cycle, many of them thrive in pathogen-rich environments, manage harsh weathers, exposed to a number of allochemicals, and adapt well to both terrestrial and marine ecosystems. Their remarkable ability to cope up with the enormous oxidative stress generated in all these circumstances, make them attractive models in this field of research. Endocrine control of oxidative stress in insects is recently emerging. Adipokinetic hormone, glucagon, ecdysteroids and juvenile hormone have been implicated in antioxidative protective role in insects. Drosophila and Caenorhabditis elegans have provided the largest body of evidence addressing the free radical theory of ageing. Oxidative stress is also induced by pesticides/insecticides. In mollusks, pesticides exert their biological effects via generation of ROS. Oxidative stress has been shown to be associated with exposure to several organophosphorous compounds and different classes of pyrethroids. Malathion is a potential hazard to the environment. Adverse effects induced by malathion in earthworms and insects have been reported. Information is now available in great detail on the role of ROS in modulating insect immunity during parasite invasion and bacterial infection. In Drosophila melanogaster ROS are actively produced in the midgut at a basal level in the presence of commensal microbiota and highly generated upon bacterial challenge. The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. The concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, indicating that oxidative stress may be important for Wolbachia-mediated antiviral protection. In a nutshell, this chapter highlights the current advances of oxidative stress in invertebrate model systems and its implications.Item Ganoderic acid targeting multiple receptors in cancer: in silico and in vitro study(Springer Netherlands, 2016) Gill, Balraj Singh; Navgeet; Kumar, Sanjeev; Gill, B.S.; Navgeet, Kumar, S.Receptor tyrosine kinases (RTKs) are transmembrane high-affinity surface receptors responsible for cell migration, adhesion, apoptosis, metabolism, and cell proliferation activities in various cancers. Minute aberration in the RTK signaling modulates the downstream signaling pathways that results in cancer. Ganoderic acid is a triterpene isolated from Ganoderma lucidum, which is renowned for its therapeutics effect, especially in cancer. The present study discusses receptor-based molecular docking of insulin receptor (IR), insulin-like growth factor receptor 1 (IGFR-1), vascular endothelial growth factor receptor-1 (VEGFR-1), vascular endothelial growth factor receptor-2 (VEGFR-2), and estrogen receptor (ER) with 50 isoforms of ganoderic acid along with natural inhibitors. These receptors were assessed for toxicity (ADMET) by using Maestro 9.6 (Schr?dinger Inc). The calculated docking free energy yielded an excellent dock score for the ganoderic acid when docked with proteins IR, IGFR-1, VEGFR-1, VEGFR-2, and ER, suggesting its potential in combating cancer. Protein?ligand profile highlighted the binding interactions comprising lipophilic, hydrogen bonding, pi-pi stacking interactions, and noncovalent bonding which play a pivotal role in targeting cancer. In silico studies revealed structure of ganoderic acid A as best isoforms among 50 isoforms which exhibits biological activity in liver cancer cells. Ganoderic acids A significantly decrease the viability, proliferation, and oxidative stress in a dose-dependent manner in liver cancer cells. ? 2016, International Society of Oncology and BioMarkers (ISOBM).