Biochemistry And Microbial Sciences - Research Publications

Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/27

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Author: search.filters.author.Kushwaha, Prem PrakashHas files: Yes

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    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, Shashank
    Breast 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.
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    Geminin a multi task protein involved in cancer pathophysiology and developmental process: A review
    (Elsevier B.V., 2016) Kushwaha, Prem Prakash; Rapalli, Krishna Chaitanya; Kumar, Shashank
    DNA replicates in a timely manner with each cell division. Multiple proteins and factors are involved in the initiation of DNA replication including a dynamic interaction between Cdc10-dependent transcript (Cdt1) and Geminin (GMNN). A conformational change between GMNN-Cdt1 heterotrimer and heterohexamer complex is responsible for licensing or inhibition of the DNA replication. This molecular switch ensures a faithful DNA replication during each S phase of cell cycle. GMNN inhibits Cdt1-mediated minichromosome maintenance helicases (MCM) loading onto the chromatin-bound origin recognition complex (ORC) which results in the inhibition of pre-replication complex assembly. GMNN modulates DNA replication by direct binding to Cdt1, and thereby alters its stability and activity. GMNN is involved in various stages of development such as pre-implantation, germ layer formation, cell commitment and specification, maintenance of genome integrity at mid blastula transition, epithelial to mesenchymal transition during gastrulation, neural development, organogenesis and axis patterning. GMNN interacts with different proteins resulting in enhanced hematopoietic stem cell activity thereby activating the development-associated genes' transcription. GMNN expression is also associated with cancer pathophysiology and development. In this review we discussed the structure and function of GMNN in detail. Inhibitors of GMNN and their role in DNA replication, repair, cell cycle and apoptosis are reviewed. Further, we also discussed the role of GMNN in virus infected host cells. ? 2016 Elsevier B.V. and Soci?t? Fran?aise de Biochimie et Biologie Mol?culaire (SFBBM)
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    Recent advancement to target Breast Cancer and Cancer Stem Cells
    (Open Access, 2017) Kushwaha, Prem Prakash; Kumar, Shashank
    Breast cancer is the second most prominent reason of cancer death in women after lung cancer, and it accounts for 25.2% of all cancer in women [1]. In the US, approximately about one in eight women (12%) develop invasive breast cancer [2]. In 2015, approximately 40,290 US women were expected to die from breast cancer. There was an estimation of 231,840 new cases of invasive breast cancer among US women in 2015 [3]. The chance of breast cancer causes woman’s death is about 3%. Various kinds of therapies such as hormonal, immunotherapeutic agents, surgery and cytotoxic currently are being used to target the breast cancer. The response rate from these treatments comprises 60% to 80% for primary breast cancers and about 50% for metastases [4,5]. However, 20%-70% of patients showed reversion of cancer within five year of time [6]. Recurrence development allied with resistance to therapy and augmented death risk. In patients with primary breast cancer, combining cytotoxic and radiation therapy with anastrozole attained four-year survival rate of 91.6 % [7]. Gene mutations and dysregulation has been identified in breast cancers like the enhanced expression of the heparan sulfate interacting protein, p53 mutations (connecting with high histological grade) and mitochondrial D-loop mutation (allied with lymph node-positive breast carcinoma)
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    Novel potent inhibitors of Plasmodium vivax dihydrofolate reductase: An in silico antimalarial drug discovery
    (Association of Pharmaceutical Teachers of India, 2018) Pushpendra, Singh; Kushwaha, Prem Prakash; Shashank, Kumar
    Objectives: In the present study, we targeted the dihydrofolate reductase enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate which is required for the purines and pyrimidine synthesis. Malaria is one of the severe diseases throughout the world caused by blood-borne parasite Plasmodium vivax. Materials and Methods: Eighty-five parthenin analogs were docked against P. vivax and Homo sapiens dihydrofolate reductase proteins (PDB 2BL9 and 1KMS respectively) by using Maestro 9.6 program to evaluate the binding affinities of ligands with the protein. Results and Discussion: Docking analysis revealed some best hit ligands against P. vivax such as CID3467446 and CID56671343 but not inhibited the mammalian dihydrofolate reductase. The Dock score of parthenin analogs ranged from -7.31 to -9.3 while for standard dihydrofolate reductase inhibitors it was -4.78 to -8.04. Structural analysis of docked complexes of selected parthenin like compounds with P. vivax and mammalian dihydrofolate reductase revealed the involvement of Arg 115, Leu 136, Lys 138, Gly 175, Ser 117, Gln 177 and Ile 7, Ala 9, Thr 56, Ile 60, Pro 61 amino acid residues respectively in strong interactions. Absorption, distribution, metabolism, and excretion properties of best-docked compounds were predicted using QikProp application of Maestro 9.6. The results indicated that all the best-docked lead compounds followed Lipinski?s rule of five. Conclusion: Based on the results of the present study it has been concluded that parthenin like compounds may serve as potent dihydrofolate reductase inhibition based anti-malarial drug lead. ? 2018, Association of Pharmaceutical Teachers of India. All rights reserved.