Anti-proliferative and Apoptotic Effect of Compounds Derived from Marine Fungus Aspergillus fumigatus

Abstract

Breast cancer is the most common cancer and principle cause of death from cancer among women globally. Recently, much attention is given towards discovering potential pharmaceutical compounds as anti-cancer candidates from natural resources. In the present study, a marine-derived fungus identified as Aspergillus fumigatus was cultivated and four secondary metabolites, isosclerone (28.6 mg), fumigaclavine C (15.8 mg), bis(methylthio)gliotoxin (19.5 mg) and gliotoxin (11.9 mg) were isolated from the marine fungus strain-150 (MFS-150) culture broth extract (2.3 g) and marine fungus strain-15 (MFS-15) culture broth extract (1.6 g). The structures of four compounds were elucidated though LREIMS, 1D (1H, and 13C) and 2D (DEPT, HMQC, and HMBC) NMR spectra. In order to investigate the impact of secondary metabolites on inhibition of proliferation and induction of apoptosis in breast cancer cells, MCF-7 cells were treated with various concentrations (20 μM, 40 μM, and 60 μM) of isosclerone, fumigaclavine C and bis(methylthio)gliotoxin. Two compounds; isosclerone and fumigaclavine C have shown significant cytotoxicity towards MCF-7 cells. Anti-proliferative effect of the compounds was analyzed via cell mobility and MAPK signaling pathway. Isosclerone and fumigaclavine C have negatively influenced cell mobility in a concentration-dependent manner which was observed through wound healing assay, transwell migration and invasion assay. In addition, Isosclerone and fumigaclavine C inhibited the protein and messenger RNA level expressions of MMP-2, -9 in a dose-dependent manner in MCF-7 cells as per the results of Western blot and RT-PCR. Furthermore, the compounds have significantly inhibited ERK, JNK and P38 phosphorylation which was found as one of the main mechanisms involved in the cell proliferation and migration. The apoptosis induction abilities of the compounds were studied by analyzing the expressions of cytochrome C, Apaf-1, Cdk2, Cdk4, cyclin B1 and cyclin E, PI3K, Akt, caspase-3, -8 and -9, p53, p21, Bax, Bad and Bcl-2, Bcl-xl, cell cycle analysis, Annexin V/PI staining and DNA fragmentation. It was found that both compounds fragmented the MCF-7 cell DNA and arrested the cell cycle. Furthermore, by the treatment of isosclerone and fumigaclavine C; Cytochrome C, Apaf-1, caspase -3, -8 and -9, p53, p21, Bax, and Bad expression levels have been significantly up-regulated, whereas Bcl-2 expression levels have been significantly down-regulated. Moreover, isosclerone and fumigaclavine C have significantly degraded IKK protein, leading to down-regulation of NF-kappa-B P50 and P65 transcription factor activity. Collectively, these results reflect that isosclerone and fumigaclavine C suppress the breast cancer cell proliferation by blocking MAPK and NF-kappa-B activation which leads to induction of apoptosis via regulating anti-apoptotic and pro-apoptotic gene expressions. Therefore, it could be suggested that both isosclerone and fumigaclavine C could be used as potential therapeutic candidates in the treatment of breast cancer.