Molecular Mechanism Of Angiogenic Inhibitor From Marine Sponge Symbionts On Mammary Carcinogenesis An In Vitro And In Vivo Study

Abstract

The current study focused on the isolation of angiogenic inhibitor newlinemolecule monacolin X from a marine sponge associated fungi and its molecular newlinemechanism of inhibition on mammary carcinogenesis through in vitro and in newlinevivo approaches. The marine sponge Clathria frondifera was collected from newlineGulf of Mannar and the crude extracts from the fungal strain NMKF7 and the newlinebacterial strain NMKB17 were found to inhibit tube formation in HUVEC s newlinecells. The strains were identified as Monascuss ruber NMK7 using 18S rRNA newlinefor fungi and Bacillus subtilis NMK17 by 16S rRNA for bacteria. The extracts newlinewere subjected to column, TLC and HPLC chromatographic purification and newlinescreened all fractions and elutions through MTT to identify the active fractions newlinePPCEF (partially purified crude extract Fungal) and PPCEB (partially purified newlinecrude extract bacteria). PPCEF and PPCEB has showed apoptotic property and newlineantiangiogenic activity in breast cancer and endothelial cells. The presence of newlineactive compound from Monascuss ruber NMK7 was confirmed using Phospho- newlineVEGFR-2 assay. HPLC purification and structural characterization using UV, newlineIR, MS and NMR found out the active molecule monacolin X with molecular newlineweight of 418.5 Da and the formula C24H34O6. newlineMonacolin X was found to induce dose-dependent cytotoxicity in newlinedifferent human breast cancer cell lines MCF-7, T47D, MDA-MB-231, MDAMB- newline468 and MCF-10A normal breast cell. Initially monacolin X apoptotic newlineactivity was confirmed by PI and AO/EB staining in T47D and MDA-MB-468 newlineviii newlinecells. It has also induced the ROS stress (DCFH-DA), which causes the newlinemitochondrial membrane potential difference (Rhodamine-123), the loss of cell newlinemembrane integrity, endoplasmic reticulum stress and eventually cell death. The newlineinitiation of apoptosis was proved by the increased expressions of Bax, cytochrome newlineC, cleaved PARP and cleaved caspase 3 followed by decreased expression of Bcl2 newlinewhich were released due to the ER stress was confirmed by CHOP and ATF4 in in newlinevitro breast cancer cell lines MDA-MB-468 and T47D studies. C