Research Projects

Lichens exemplify the evolution of composite organisms, resulting in a mutualistic interaction between members of distinct phyla and even separate kingdoms. The traditional definition of lichen symbiosis has evolved from a bipartite relationship to a composite organism comprising lichenized fungi, algae, yeast, bacteria, and endolichenic fungi (EF). These supplementary partnerships enhance the symbiotic functioning of the lichens in the Western Ghats (WGs), which has not undergone a comprehensive examination. The emergence of molecular sequencing technologies can unravel the dark taxa and elucidate intricate linkages for bioprospecting purposes. The tropics have one-third to half of the world's lichens. The highest endemism proportion in India is 26.7% for 949 lichen taxa in WGs. The WGs' Gondwanan lichen species have not been evaluated for selectivity and specificity in symbiotic interactions. Endolichenic fungi of tropical and subtropical lichens in WGs have been neglected for a long time, fragmenting research. First objective of this study targets pristine environments in Kerala, Karnataka, Maharashtra, and Tamil Nadu to reflect varied spatiotemporal patterns for lichen collection and isolate EF from these habitats. Endolichenic fungi of India are yet to be studied through a stringent polyphasic taxonomic approach including multigene sequencing, morphology, metabolite profiling and Geneological concordance-based phylogenetic species recognition (GCPSR). Endolichenic fungi represent a valuable reservoir of bioactive chemicals due to their desiccation tolerance nature, extremely slow growth of the lichen thalli, and inter-kingdom interaction within the thallus. Recent studies indicate that natural products, especially those sourced from endophytes and extremophiles, may serve as a viable pathway for the development of anticancer agents. The 2nd objective aims to validate EF from Parmelioid lichens of the Western Ghats and explore their potential as sources of anticancer secondary metabolites. Fungi will be cultured in PD Broth, and metabolites will be extracted using ethyl acetate. The crude extract will be dissolved in DMSO and characterized using UPLC-Q-TOF-MS, LC-MS, and GC-MS to identify bioactive compounds. Preparative HPLC will be used for purification, and HPLC, NMR spectroscopy, and UPLC-Q-TOF-MS/MS will aid in structural elucidation and quantification. Molecular docking studies will predict the anticancer efficacy of the isolated compounds against key breast cancer targets such as ERα, PR, EGFR, mTOR, Cyclin D1, CDK4/6, Bcl-2 family proteins, STAT3, and elements of the PI3K/AKT and MAPK pathways. Pharmacokinetic profiling using Swiss-ADME will assess drug-likeness. The increasing mortality among women due to breast cancer highlights the urgent need for effective, targeted therapies with minimal side effects. In 3rd objective, in vitro cytotoxicity will be evaluated via MTT assays on MCF-7 and MDA-MB-468 breast cancer cell lines using both crude and purified compounds. Fungal metabolites will undergo dose-response testing to measure cell proliferation and viability over 24, 48, and 72 hours, with IC₅₀ values determined. Apoptosis will be assessed through Annexin V-FITC/PI staining and caspase 3 and 9 activation via flow cytometry. Further mechanistic studies will focus on caspase 3/7 activation and apoptotic pathway involvement. In vivo validation of efficacy will be tested on tumor progression in xenograft mouse models. MDA-MB-468 and MCF-7 human cells will be orthotopically implanted into the mammary fat pad of female mice to establish TNBC positive and TNBC negative xenograft models. The proposed research aims to authenticate the endolichenic fungi, identify the key metabolites, investigate their potential as anticancer agents, and contribute to the ongoing search for more effective and targeted breast cancer therapeutics.