Abstract
BackgroundEndophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression.ResultsAmong NCI60 human cancer cell line panel Cladosporol A showed least IC50 value against human breast (MCF-7) cancer cells. The early events of apoptosis were characterized by phosphatidylserine exposure. It disrupts microtubule dynamics and also induces expression of pro-apoptotic protein p21. Moreover treatment of Cladosporol A significantly induced MMP loss, release of cytochrome c, Bcl-2 down regulation, Bax upregulation as well as increased monodansylcadaverine (MDC) staining and leads to LC3-I to LC3-II conversion.ConclusionOur experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death.Graphical abstractThe proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.
Highlights
Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery
Identification, characterization and phylogenetic analysis of endophytic fungus (MRCJ-314) revealed it as Cladosporium cladosporioides The morphological characteristics of isolated endophytic fungus from Datura innoxia Microbial Resource Center Jammu (MRCJ)-314 (DIE-10) supports that it belongs to genus Cladosporium [15]
Flow cytometery and Laser scanning confocal microscopy (LCMS) results suggested that human breast (MCF-7) cancer cells treated with different concentrations of Cladosporol A (Fig. 8 a, b,c) considerable caused significant mitochondrial membrane potential loss
Summary
Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. We report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. We further isolated, purified and characterized a secondary metabolite Cladosporol A from endophytic Cladosporium cladosporioides and investigated the cyotoxic effects of Cladosporol A treatment against various human cancer cell lines. It exhibited promising cytotoxic effect against human breast (MCF-7) cancer cell line having minimum IC50 8.7 μM.
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