Abstract
Endophytes from marine plants have not been widely explored, and their bioactive compounds have not been well documented. In the present study, 64 endophytic fungi were isolated from 2 Malaysian marine plants (Pandanus odoratissimus and Nypa fruticans) and 6 seaweed species (Turbinaria conoides, Caulerpa lentifera, Caulerpa racemosa, Padina australis, a variant of Caulerpa racemosa, and Sargassum oligocystum). The endophytic extracts were tested for their cytotoxic activity against seven human cancer cell lines. Out of the 64 extracts tested, 49 (77%) of them were able to inhibit cancer cell lines. Eight of the endophytic extracts that were potent (IC50 = ≤0.1 µg ml-1) against colon (DLD-1) and lung (NCI-H1299) cancer cell lines were further tested for their effects on a normal human fibroblast (D551) and liver (WRL-68) cell lines. Cytotoxic effects were not observed in these latter two cell lines.
Among the 8 active extracts, 3 (S1, S2 and P12) with significant traces of metabolites were selected for identification of the bioactive compounds using a dereplication technology. Consequently, 3 known and 12 unknown compounds were determined using the rapid technique. The structures were elucidated by 1H NMR and confirmed by x-ray crystallographic data. Of the known compounds, S1-1 was a monoclinic red crystal, P12-1 was a monoclinic yellow crystal and compound P12-2 was a pale yellow amorphous product. They were identified as bostrycin (S1-1), globosuxanthone A (P12-1) and 1-hydroxy-2 methylanthraquinone (P12-2). Bostrycin and globosuxanthone A exhibited moderate cytotoxicity against colon cancer cell line (IC50 0.9 and 3.25 μg ml-1) and lung cancer cell line(IC50 2.5 and 2.3 μg ml-1 respectively). However, 1-hydroxy-2 methylanthraquinone was not active against the tested cancer lines. This is the first report on globosuxanthone A being isolated from a marine endophytic fungus. The twelve unknown compounds from extract S2 displayed peptidic characteristics such as an α proton signal and a huge molecular weight (1012-1950 Da) but remain unidentified. The structures of the active compounds in S2 extract which are responsible for the bioactivity therefore are not able to be elucidated.
The sequence analysis of internal transcribed spacers ITS1 and ITS4, and phylogenetic analysis identified the three isolates to be ascomycetes designated as Phomopsis sp (S1) and Hypocrea jecorinasp (S2 and P12). Further examination focussed on the molecular mechanism of action of the endophytic extract S2. Extract S2 induced apoptosis in colon cancer cells (DLD-1 and HCT 116) through an extrinsic pathway and was activated by the caspase 3 and 8 cascade. The activation of caspases 3 and 8 were further confirmed with the use of the generic caspase inhibitors Z-VAD-FMK, Z-DEVD-FMK and Z-IETD-FMK. Co-incubation of inhibitors with treated cells significantly (p<0.05) reversed S2 mediated cell death. In contrast, S2 induced apoptosis through cell cycle arrest at S phase and was caspase independent in lung cells (A549). The cDNA microarray identified several genes involved in the apoptosis-regulatory pathway, DNA damage and cell cycle arrest (ATF3, DDIT3, PPPIR15A, CASP4, TP53INPI, TNFSRF10B and BTG1) that significantly (p<0.01) correlated with the response to the mode of action of extract S2 against colon cancer cells (HCT 116).
The safety of the S2 extract was also evaluated. Two doses (100 and 400 mg kg-1) of the extract were administered orally to two groups of rats for acute toxicity testing. The effects of oral administration of the extract for 14 days on body weight, relative weight of six organs, haematological and biochemical and antioxidant properties were investigated. The results showed that oral dose treatment at any two doses did not cause any observed adverse effects in the rats. No significant variation (p>0.05) in the body and organ weights between the control and the treated group was observed. Pathologically, neither gross abnormalities nor histopathological (HE staining) changes were observed in liver. Haematological analysis and clinical blood chemistry revealed no toxic effects of the extract. The level of total cholesterol in treated males with 100 and 400 mg kg-1 significantly (p<0.05) decreased (1.28 and 1.34 mmol L-1) compared to controls (1.55 mmol L-1). This study provides strong evidence of the non-toxic effects of S2 extract. Furthermore the extract exhibited significant (p<0.05) antioxidant activity through increased levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes in serum, liver and kidney.
The research findings from the present study show the potential of natural marine products particularly in Malaysia as a source of new and novel therapeutic entities. Marine endophytic fungi could be a good potential source of anticancer drugs as several can provide extracts that are potent and safe, thus deserving further extensive investigation.