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dc.contributor.advisorJayabaskaran, C
dc.contributor.authorKashyap, Srishti
dc.date.accessioned2018-11-06T10:07:18Z
dc.date.available2018-11-06T10:07:18Z
dc.date.submitted2017
dc.identifier.urihttp://etd.iisc.ac.in/handle/2005/4135
dc.description.abstractCancer has become the leading disease-related cause of death in the human population. For example, in the United States, cancer is the second leading cause of death behind cardiovascular disease, and it is projected that cancer will become the leading cause of death in the coming years. The medical treatment of cancer still has many unmet needs. The main curative therapies for cancer, surgery and radiation, are generally only successful if the cancer is found at an early localized stage. Once cancer has progressed to metastatic stage, these therapies are less successful. Hence, chemotherapeutic drugs are used for the treatment of these advanced tumors, particularly in the case of the common epithelial tumors such as lung, colorectal, breast, prostate, and pancreatic cancers. New chemotherapeutic drugs are necessary since most of the cancers acquire resistance towards existing anticancer drugs. Nature has always been an attractive source of new chemotherapeutic agents, as a tremendous chemical diversity is found in millions of species of plants, animals, and microorganisms.Microorganisms (bacteria, fungi, actinomycetes) serve as readily renewable and inexhaustible source of novel bioactive metabolites.Endophyte, a microorganism that reside in theinternal tissues of living plantswithout causing any immediate overt negative effects, are potential sources of novel natural products for exploitation in medicine. Mappia foetidais distributed in western part of peninsular coastal India from Konkanghatsto northern parts of the Kanara, Nilgiris, Anamalis, and Pullneys hills of India. It is a rich source of alkaloids such as camptothecin, 9-methoxy camptothecin, mappicin,sitosterol and lupeol and other natural products having anticancer and antimicrobial properties. Since, the secondary metabolite production in fungal endophytes is greatly influenced by theircompetitive ecological niche and interaction with host metabolism, Mappia foetida was chosen for endophytic fungal isolation. Sirsi, (North Karnataka, India) because of its rich biodiversity was chosen as a location for collection of the plant samples. The organicsolvent extracts obtained from mycelia and culture filtrates of all the endophytic fungal isolates were screened for their cytotoxic activity against HeLa (human cervical carcinoma) cellline. Organic extracts of 8 fungal isolates exhibited significant cytotoxic activity, among which Phoma tropica(S1/3) culture filtrate extract exhibited most significant cytotoxicactivity againstHeLa cell line with IC50 of 25µg/ml. Dichloromethane extract of the Phoma tropica culture filtrate was subjected to bioassay–guided column chromatographic fractionationwhich resulted in the isolation of purified cytotoxic secondary metabolite. Based on the analysis of variousspectroscopic techniques such as NMR, FTIR, LC-MS/MS, HRMS, CHNOS elemental analysis and X-ray diffraction studies, the purified metabolite was identified as 2-Hydroxy-2,4-dimethyl-5-[-1-propen-1-yl]-3(2H)furanone(Phomafuranone).Phomafuranone exhibited significant cytotoxic activity against various cancer cell lines (HeLa, Jurkat, COLO 205, HT-29, HCT-15, HCT 116, A549, A-431 and OVCAR-3) Further studies were undertaken to elucidate the mechanism of cytotoxicityof the purified metabolite on human cancer cell lines. Phomafuranone contains a conjugated unsaturated α, β, γ, δ carbonyl pharmacophore moiety. Polyunsaturated carbonyl compounds are referred to as “Michael acceptors” and they behave as soft electrophiles. Michael acceptors react with strong biological nucleophiles such as thiols.The reactivity of electrophilic Phomafuranone with thiol containing biomolecules like glutathione, cysteine and N-acetyl cysteine was investigated by spectrophotometric methods. Cell cycle progressionanalyses oftreated HCT 116 (colorectal carcinoma) cell line by flow cytometry revealed that Phomafuranone arrested significant proportion of cells in G2/M phase. HCT 116 cells were arrested specifically in early mitotic phase of cell cycle as indicated by time dependent increase in phospo-histone3 (Ser10) levels and nuclear import of Cyclin B1.On treatment cancer cells with Phomafuranone, time dependent depletion of intracellular reduced glutathione levels was observed. Depletion of reduced glutathione inturn led to elevated intracellular ROS levels.Pre-treatment of HCT 116 cell lines with thiol containing antioxidants like N- acetylcysteine (NAC) and reduced glutathione (GSH) completely abrogated itscytotoxic effect, suggesting Phomafuranone inducedthiol–mediated cytotoxicity. The elevated ROS levels led to mitochondrial membrane depolarization as indicated by cytochrome c release in cytosol from mitochondria in a time dependent manner. Cytochrome c release was followed caspase 9 mediated apoptotic cell death. Thus, our results suggest that Phomafuranone induced redox imbalancemediated apoptosis in HCT 116 cell line by intrinsic pathway.en_US
dc.language.isoen_USen_US
dc.relation.ispartofseriesG28637;
dc.rightsI grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertationen_US
dc.subjectCytotoxic 5-Alkenyl-3 - Elucidationen_US
dc.subjectReactivity of Phomafuranoneen_US
dc.subjectGlutathioneen_US
dc.subjectCysteine, N-acetyl Cysteineen_US
dc.subjectPhomafuranoneen_US
dc.subjectEndophytic Fungien_US
dc.subjectMappia foetidaen_US
dc.subjectHCT 116 Carcinomaen_US
dc.subjectPhoma tropicaen_US
dc.subjectColorectal Carcinomaen_US
dc.subject.classificationBiochemistryen_US
dc.titleIsolation, Structure Elucidation and Functional Characterization of a Novel Cytotoxic Secondary Metabolite Phomafuranone, 2-Hydroxy-2, 4-dimethyl-5-[-1-propen-1-yl]-3(2H)-furanone, from Phoma tropica an Endophytic Fungus Isolated from Mappia foetida.en_US
dc.typeThesisen_US
dc.degree.namePhDen_US
dc.degree.levelDoctoralen_US
dc.degree.grantorIndian Institute of Scienceen_US
dc.degree.disciplineFaculty of Scienceen_US


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