Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus

Abstract

Background: Mushrooms inspired the cuisines of many cultures and conventional medicaments for cancer. However, a substantial number of mushroom species are yet unexplored, possessing an unknown chemical, biological and pharmacological profiles. Fulviformes fastuosus is a terrestrial mushroom, which is commonly found in Sri Lankan woodlands. The current study was aimed at isolation and characterization of a potent cytotoxic compound from F. fastuosus and investigating the apoptotic effect induced by the active principle against cancer and normal cell lines.

Methods: Bioactivity guided isolation of active principles from the methanol extract of F. fastuosus was performed by a rapid extraction and isolation method using different chromatographic techniques. Potential cytotoxic compound was identified using one and two dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. Isolated compound was screened for in vitro cytotoxicity against Hepatocellular carcinoma (HepG-2), Muscle rhabdomyosarcoma (RD) and Rat Wistar liver normal (CC-1) cell lines using 3 4, 5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide (MTT) cell viability assay. Apoptotic features of cells were observed via microscopic examination and ethidium bromide/acridine orange fluorescent staining.

Results: The interpretation of spectral data resulted in the identification of the chemical structure as ergosta-4,6,8 (14),22-tetraen-3-one (ergone). Ergone exhibited promising cytotoxic properties against RD cells with less cytotoxicity effect on CC-1 cells. In addition, ergone also possesses a strong cytotoxic effect against HepG-2 cells showing low toxic level for CC-1 cells. Apoptotic features of treated cells were detected via morphological characterization and ethidium bromide/acridine orange staining.

Conclusion: The present study elaborates the isolation of a potent cytotoxic compound; ergone, from F. fastuosus via a rapid and efficient isolation method. Importantly, ergone has exhibited greater cytotoxic activity against RD cells with high selectivity index compared to cytotoxicity against HepG-2 cells. Ergone can be used in the development of therapeutic strategies for curbing rhabdomyosarcoma.

Keywords: Cytotoxic activity; Ergone; Fulviformes fastuosus; Hepatocellular carcinoma; Isolation method; Mushrooms; Rhabdomyosarcoma.

Fig. 1

Structural Formula of Ergone

Fig. 2

The percentage inhibition of cell viability against (a) RD (b) Hep-G2 (c) CC-1 cell line as determined by MTT assay, after 24 h treatment with the ergone. The graphical data are represented as mean ± SD of three independent experiments (n = 3)

Fig. 3

Light micrographs of RD cell line after 24 h of incubation with ergone at different concentrations. a- 0.1 μM; (b)- 2 μM; (c)- 50 μM; (d)- Cycloheximide as the positive control (5 mM; 50 μL) (×40)

Fig. 4

Light micrographs of Hep-G2 cell line after 24 h of incubation with ergone at different concentrations. a- 5 mM; (b)- 50 mM; (c)- 80 mM; (d)- Cycloheximide as the positive control (5 mM; 50 μL) (×40)

Fig. 5

Light micrographs of CC-1 cell line after 24 h of incubation with ergone at different concentrations. a- 2 mM; (b)- 10 mM; (c)- 40 mM; (d)- Cycloheximide as the positive control (5 mM; 50 μL) (×40)

Fig. 6

Apoptotic morphology detection by acridine orange-ethidium bromide (AO/EB) fluorescent staining of RD cell line treated with ergone dissolved in methanol: DMSO (1:1). a- Negative control; (b)- 2 μM; (c)- 5 μM; (d)- Cycloheximide as the positive control (5 mM; 50 μL) (×40). Arrows indicate formation of apoptotic bodies

Fig. 7

Apoptotic morphology detection by acridine orange-ethidium bromide (AO/EB) fluorescent staining of HepG-2 cell line treated with ergone dissolved in Methanol: DMSO (1:1). a- Negative control; (b)- 80 μM; (c)- 130 μM; (d)- Cycloheximide as the positive control (5 mM; 50 μL) (×40). Arrows indicate formation of apoptotic bodies

Fig. 8

Apoptotic morphology detection by acridine orange-ethidium bromide (AO/EB) fluorescent staining of CC-1 cell line treated with ergone dissolved in Methanol: DMSO (1:1). a- Negative control; (b)- 6 μM; (c)- 50 μM; (d)- Cycloheximide as the positive control (5 mM; 50 μL). This figure denotes the results of at least 3 independent experiments (Original magnification 40×). Arrows indicate formation of apoptotic bodies