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Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest.

ARTICLE INFO

Keywords:

Apoptosis

Cytotoxicity

Endophytes

Metabolites

ABSTRACT

Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with [IC.sub.50] values less than 17 [micro] g/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Nature has been an attractive source of new therapeutic candidate compounds since a tremendous chemical diversity is found in the multitude of species of plants, animals, marine organisms and microorganisms. Despite the decline of pharmaceutical companies' interest in natural products and the rise in combinatorial chemistry, natural products still have an important role to play, offering an untold diversity of lead compounds. Currently, some interesting natural products have been discovered from microorganisms that reside in the tissues of living plants, known as endophytes. Endophytes grow within their plant hosts without causing apparent disease symptoms (Bacon et al. 2000). The most frequently isolated endophytes are fungi. Tropical rainforests are considered to be a major reservoir of as yet, undescribed fungi. In addition, tropical endophytes are said to provide more active natural products and may produce a larger number of active secondary metabolites than other tropical substrata (Bills et al. 2002).

A large number of secondary metabolites, including some compounds with anticancer activity, have been isolated from endophytic fungi associated with medicinal plants. Taxol and some of its derivatives represent the first major group of cytotoxic agents shown to be produced by endophytes (Pestalotiopsis species) (Wani et al. 1971). This led to worldwide effort by drug companies and research groups to assemble large collections of endophytes in order to discover other anticancer agents. Taxol was subsequently shown to be produced from a number of different fungal endophytes such as Taxodium distichum (Li et al. 1996), Wollemia nobilis (Strobel et al. 1997), Phyllosticta spinarum (Kumaran et al. 2008), Bartalinia robillardoides (Gangadevi and Muthumary 2008), Pestalotiopsis terminaliae (Gangadevi and Muthumary 2009) and Botryodiplodia theobromae (Pandi et al. 2010). Another important anticancer compound, camptothecin-20-O-propionate hydrate (CZ48), a derivative of camptothecin, which is currently in Phase I clinical trials in patients with solid tumors or lymphoma (US National Institute of Health 2010) has also been isolated from an endophytic fungus Fusarium solani.

The mechanisms by which chemotherapeutic agents induce tumor cell death have been widely reported, and there is accumulating evidence that these agents exert their cytotoxic effects mainly by inducing apoptosis. Apoptosis or programmed cell death occurs during cell development and differentiation and is involved in removing damaged cells after cell injury. impairment of apoptosis is known to be related to cell immortality and carcinogenesis (Tan et al. 2005). Therefore, the induction of apoptosis in neoplastic cells is vital in cancer treatment. Bioactive compounds isolated from endophytes that have been observed to induce apoptosis include taxol, hormonemate, camptothecin and brefeldin A (Wani et al. 1971; Wang et al. 2002; Filip et al. 2003; Amna et al. 2006). Mechanism-based studies of active endophytic extracts or compounds hold benefits in the search for drug candidates.

In Malaysia, interest in fungal endophytes as potential producers of novel and biologically active products has increased in the last few years. With over 15,000 plant species, an abundant endophytic fungal diversity in the Malaysian rainforest is expected. To date, only few reports have been documented on the isolation of fungal endophytes from the Malaysian flora for evaluation of their bioactive compounds. In our earlier studies, a number of fungal endophyte isolates from Malaysian rain forest have been shown to he cytotoxic against the murine leukemic (P388) cell line (Hazalin et al. 2009; Ramasamy et al. 2010). This subsequent study focuses on four extracts (KK9L2, KK14L1, KK21FL2 and KK30RH2) with good cytotoxic activity against P388 ([IC.sub.50] < 0.01 [micro] g/ml).

KK9L2 and KK14L1 were isolated from the leaves of Phyllagathis rotundifofia (Jack) Blume and Ampelocissus cinnamomea Planch, respectively. Phyllagathis rotundifolia or commonly known as the 'tapak sulaiman' by the locals, belongs to the botanical family Melastomaceae. This plant is often used by the "Tebuan" tribe of Peninsular Malaysia as contraceptive herbal formulation and also in postpartum therapy (Islam et al. 2007). A decoction of the whole plant is given to women immediately after birth to hasten the process of delivery of the placenta. Ampelocissus cinnamomea is a climber plant that can be found in the lowland forest throughout the Malay Peninsular. Traditionally, Ampelocissus cinnamomea has been used to control asthma, sinusitis and hemorrhoids. KK21FL2 and KK3011112 were isolated from flower and rhizome of Zingiberaceae species, respectively. Zingiberaceae or the ginger family, is a flowering plant commonly used by the local community in Malaysia for its medicinal properties. This plant has been used for many years as spices and traditional forms of medicine to treat a variety of diseases. It has been used as a remedy for diarrhea, thrombosis, sea sickness, migraine and rheumatism (Sirirugsa 1995). The present study assessed the cytotoxic activity of the active endophytic extracts (KK9L2, KK14L1, KK21FL2 and KK30RH2) against human cancer (HCT116, MCF-7 and K562) and normal (WRL-68) cell lines. Mechanistic studies were further carried out to investigate the mode of cell death induced by the cytotoxic effect of the extracts. Chemical characterization of the cytotoxic compounds and molecular characterization of the active fungal endopytes using internal transcribed spacer (ITS1 and ITS4) sequences were also undertaken.

Materials and methods

Selection of endophytic fungi

Plants were collected from Kuala Keniam in National Park, Pahang, Malaysia and treated as reported by Hazalin et al. (2009). Plant samples, which included leaves, stems, roots, rhizomes, flowers, barks and fruits, were thoroughly surface treated with running tap water, 70% EtOH and NaOCl (2.5-5.25%). Each plant sample was cut aseptically into 1 cm long segments and placed on petri dishes containing potato dextrose agar (PDA) (Oxoid, UK) supplemented with chlortetracycline HCL, 50 [micro] g/ml (Sigma, Germany) and streptomycin sulfate, 250 [micro] g/ml (Sigma, Germany). Pure cultures were transferred to PDA plates free of antibiotics and maintained in the culture collection of the Collaborative Drug Discovery Research (CDDR) Group, UiTM, Malaysia.

Growth and semipolar extraction of fungal cultures

Cultures were cultivated for 14 days on PDA plates at 28 [degrees] C. Crude endophytic extracts were prepared as described by Lang et al. (2005) but with slight modification. Cultures (five plates per fungus) were homogenized and transferred to a 500 ml conical flask with 250 ml EtOAc (Merck, Germany) and left to stir overnight at room temperature. The mixture was filtered through Whatman No. 1 filter paper, after which [Na.sub.2][SO.sub.4] (40 [micro] g/ml, Merck, Germany) was added to remove the aqueous layer. The mixture was then transferred to a round bottomed flask and evaporated under reduced pressure at 40 [degress] C. The resultant extract was dissolved in 1 ml of dimethyl-sulfoxide (DMSO) (Sigma, Germany) and kept at 4 [degress] C.

Cytotoxicity evaluation

Human breast adenocarcinoma, MCP-7 (ATCC HTB-22), human colorectal carcinoma, FICT116 (ATCC CCL-247), human chronic myeloid leukemic, K562 (ATCC CCL-243), murine leukemic, P388 (ATCC TIB 63) and normal embryonic liver cells, WRL68 (ATCC CL 48) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. All cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma, Germany) supplemented with 10% heat-activated fetal bovine serum (FAA Laboratories, Austria) and 1% penicillin/streptomycin (FAA Laboratories, Austria). Cultures were maintained in a humidified incubator at 37 [degress] C in an atmosphere of 5% [CO.sub.2].

Cytotoxic activity of extracts at various concentrations (0.01-100 [micro] g/ml) following 72 h of incubation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, Germany) assay, as described previously (Hazalin et al. 2009). Assay plates were read using a spectrophotometer at 520 nm. Data generated was used to plot a dose-response curve from which the concentration of extract required to kill 50% of the cell population ([IC.sub.50]) was determined. Cisplatin (Mayne Pharma, USA) and tamoxifen (Dynapharm, Malaysia), established chemotherapeutics, were used for comparison. Cytotoxic activity was expressed as the mean [IC.sub.50] ([+ or -] standard deviation) of three independent experiments.

Cell death detection by Enzyme-Linked lmmunosorbent Assay (ELISA)

Apoptosis was evaluated using the Cell Death Detection [ELISA.sup.PLUS] assay (Roche Diagnostic, Germany) which is based on the quantitative sandwich-enzyme-immunoassay-principle using mouse monoclonal antibodies.

Cancer cells (HCT116, MCF-7 and K562) were exposed to the endophytic extracts, cisplatin or tamoxifen (positive control) and culture medium (negative control) in microtiter plates for 72 h followed by centrifugation for 10 min at 200 x g. The cell pellet containing the apoptotic bodies was resuspended in lysis buffer. The apoptotic bodies were incubated before centrifugation to obtain the supernatant containing the cytoplasmic fraction which was subsequently analyzed for apoptosis. The amount of antibody that bind to the DNA components of the nucleosomes was determined using ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. To quantify the extent of apoptosis, enrichment factor was calculated using the formula as provided by the manufacturer.

Enrichment factor = (mU of the sample (treated cells))/(mU of the corresponding control (untreated cells))

where mU = absorbance at 450 nm.

Data are expressed as the mean values of the enrichment factor [+ or -] standard deviation of three independent experiments.

DNA extraction, PCR amplification and sequencing

A single mycelial agar block (2 [cm.sup.2]) from each original culture was placed in 200 ml Potato Dextrose Broth (PDB). Cultures were incubated without shaking at 28 [degress] C for 3-5 days. DNA was then extracted from the mycelium using the MasterPure[TM] Yeast DNA Purification Kit according to the manufacturer's instructions (Epicentre Biotechnologies, USA). The target rDNA region including the internal transcribed spacer (ITS1, ITS2) regions and 5.8S gene, was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), a specific primer for fungi and ITS4 (51-TCCTCCGCTTATTGATATGC-31), a universal primer (White et al. 1990). The internal transcribed spacers (ITS) rDNA were used because the ITS regions evolve much more rapidly than other conserved regions of the DNA. Amplifications were performed in a total reaction volume of 100 [micro]l containing 50 [micro]l of GoTaq Green Master Mix (Promega, USA), 4 [micro]1 of each of the primer, 6 [micro]l of DNA template and 36 [micro]l of nuclease free water. PCR was performed in a thermal cycler (Biorad, USA) with an initial denaturing step of 94 [degress] C for 1 min, followed by 40 cycles of amplification at 95 [degress] C for 30 s, annealing at 50 [degress] C for 1 min and primer extension at 72 [degress] C for 30 s, followed by final extension step for 7 min at 72 [degress] C. The amplified PCR products were separated by electrophoresis in 1.5% (w/v) agarose gel at 90 V for 40 min in 1 x Tris acetate EDTA (TAE) buffer, stained with ethidium bromide and visualized under UV light and photographed. A 100-bp size marker (Promega, USA) was used as reference.

The amplified PCR products were further purified using Wizard[R] SV Gel and PCR Clean-Up System (Promega, USA). Purified PCR products with ITS primers were sent for sequencing. Sequences were manually edited and aligned using BioEdit v 7.0.5, and matched with DNA sequences from GenBank using the BLAST software (BLASTN) at the National Center of Biotechnology Information, NCBI (http://www.ncbi.nlm.nih.gov). The sequences were deposited in the GenBank for accession numbers.

Molecular phylogenetic analysis

The ITS sequences of the four different fungi were aligned to each other as well as the sequences retrieved from NCBI databases, using multiple sequence alignment software, CLUSTAL W program with default settings (Thompson et al. 1994). Phylogenetic analyses were performed by the neighbor-joining (NJ) method using Molecular Evolutionary Genetic Analysis 4 (MEGA4) software (Tamura et al. 2007). Parsimony trees were obtained using the Close-Neighbor-Interchange algorithm with search level 3, in which the initial trees were obtained with the ten random addition replicates of the sequences (Nei and Kumar 2000). All positions containing gaps and missing data were eliminated from dataset (complete deletion option). The tree stability was evaluated by 1000 parsimony bootstrap replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. Phylogenetic trees were constructed from distance matrix values by neighbor-joining (NJ) method. NJ trees were constructed using p-distance parameter model to estimate evolutionary distance. A bootstrap analysis was performed using 1000 resamples of the data. Saccharomyces cerevisiae clone FLH143771 was used as an out-group.

Chemical characterization of extracts

The four extracts were fractionated by HPLC (Dionex, USA) equipped with an ISCO Foxy Jr (Teledyne ISCO, USA) sample collector using a reversed-phase analytical column (Phenomenex Luna C18, 10 mm x 250 mm, 5 p.m) with diode array detector (DAD) and evaporative light scattering detector (ELSD) (AllTech, USA). An aliquot of the extracts (500 [micro]g) was analyzed by HPLC [solvents: (A) [H.sub.2]O + 0.05% trifluoroacetic acid, TFA, (B) ACN; gradient: 0 min 10% B, 2 min 10% B, 14 min 75% B, 24 min 75% B, 26 min 100% B; flow: 1 ml/min; 40 [degress] C]. The eluent from the DAD was split in a 1:10 ratio between the ELSD and the fraction collector configured to collect into a 96-well microtiter plate (15 s/well). A total of 88 wells were collected (2.5-24.5 min). The plate was dried using a centrifugal evaporator. After complete evaporation of the solvent, the wells in the plate were analyzed for cytotoxic activity against a murine leukemic (P388) cell line using the MTT assay (Hazalin et al. 2009). Peaks that exhibited active cytotoxic activity were identified using a dereplication technique.

The dereplication of fungal metabolites was carried out using the in-house library database by establishing whether any of the significant peaks showed matches (both UV chromophore and Rt) with known compounds already present within the database. The HPLC-UV data were compared with the in-house HPLC-UV/[R.sub.t] library database for known fungal and bacterial metabolites using the Chromeleon software on Dionex analytical HPLC. The database had been established by the Marine Chemistry Group, University of Canterbury. Particular attention has been paid to ensure that all HPLC parameters have been maintained constant over the years so that it is possible to realistically include the retention time along with a direct comparison of the UV data (Lang et al. 2008). If a match of data with a known compound was found, then no further effort was expanded for the identification of the compounds.

Results and discussion

Cytotoxic and cell death assay

A total of 300 endophytic fungi were isolated from 43 plants of the National Park, Pahang (Hazalin et ai. 2009). Four isolates, KK9L2 (isolated from leaf of Phyllagathis rotundifolia), KK14L1 (isolated from leaf of Ampelocissus cinnamomea), KK21FL2 (isolated from flower of Zingiberaceae sp.) and KK30RH2 (isolated from rhizome of Zingiberaceae sp.) were found to show potent ([IC.sub.50] < 0.01 [micro] g/ml) cytotoxic activity against P388 cell line (Hazalin et al. 2009). In the present study, the four active extracts were further tested against three common cancer cell lines (HCT116, MCF-7 and K562) and a normal cell line (WRL-68). These extracts showed activity that was within the cutoff point set by the National Cancer Institute for cytotoxicity ([IC.sub.50] < 20 [micro] g/ml) (Lee and Houghton 2005) and in certain cases the value was approximately 500 times lower than the cutoff point (Table 1). Among the extracts, KK14L1, showed remarkable cytotoxic activity against the tested cancer cell lines but exhibited an equally strong cytotoxic effect against a normal cell line, WRL-68 ([IC.sub.50] = 0.12 [micro] g/ml). Extract KK14L1 was found to be as effective as "gleevec" (IC50 = 0.107 [micro] g/ml), a targeted chemotherapeutic used in leukemia cancer management when tested against K562 (Niwa et al. 2007).

Table 1
Cytotoxic activity of endophytic extracts against cancer and
normal cell lines. (e)

Extract    [IC.sub.50]
           ([mu] g/ml)
           [+ or -] SD

             HCT116         MCF7           K562           WRL68

KK30RH2      10.06 [+ or  5.83 [+ or -]  4.81 [+ or -]  1.30 [+ or -]
             -] 2.24 (a)       3.92 (a)       4.17 (a)       0.53 (d)

KK9L2      3.67 [+ or -]  9.44 [+ or -]  7.33 [+ or -]  0.16 [+ or -]
            2.40 (a) (b)   6.58 (a) (b)       0.88 (b)       0.13 (d)

KK14L1     3.72 [+ or -]  4.78 [+ or -]  0.03 [+ or -]  0.12 [+ or -]
                1.00 (a)       3.34 (a)       0.01 (a)       0.16 (d)

KK21FL2      16.22 [+ or    15.89 [+ or    13.56 [+ or    17.66 [+ or
             -] 4.74 (a)    -] 5.23 (a)    -] 3.87 (a)    -] 6.90 (d)

Cisplatin  0.60 [+ or -]    57.78 [+ or  2.00 [+ or -]  0.30 [+ or -]
(c)             0.15 (d)       -] 18.36       0.00 (b)       0.26 (a)
                                (a) (b)
Tamoxifen  6.33 [+ or -]  0.04 [+ or -]     4.17 [+ or    17.22 [+ or
(c)         1.53 (d) (b)       0.01 (d)    -] 0.29 (b)       -] 11.10
                                                              (a) (b)

Gleevec               ND             ND          0.107             ND
(c), (d)

(a-b.) Means within a row with no common superscripts differ
significantly (p < 0.05).
(c.) Standard chemotherapeutic used in cancer treatment as positive
control in the study.
(d.) Data adapted from Niwa et al. (2007).
(c.) Data represents mean values of three replicates (n = 3).


The potent anti-proliferative activity of the endophytic extracts may be associated with the ability of these extracts in inducing apoptosis against the tested cells. Induction of apoptosis in cancer cells is an important property in anticancer drug development (Frankfurt and Krishan 2003). The level of apoptosis was measured using the enrichment factor. A high enrichment factor indicates high expression of apoptosis. The ability of the endophytic extracts to induce apoptosis is shown in Fig. 1. There was a significant (p < 0.05) increase in the level of apoptosis observed in HCT116 cells treated with extracts KK9L2, KK14L1 and KK30RH2 when compared to the untreated control cells. The highest level of apoptosis was observed in HCT116 treated with extract KK9L2 which gave increment to an enrichment factor of 5.0. However, a significant (p < 0.05) increase in the level of apoptosis was only observed in the MCF-7 cells treated with KK30RH2. The measurement for apoptosis in K562 cells revealed that all the four endophytic extracts and cisplatin (positive drug) caused a statistically significant (p < 0.05) higher level of apoptosis when compared to the untreated control cells. Extract KK30RH2 may have a great potential as an apoptotic agent due to its ability to cause significant (p < 0.05) increment in the level of apoptosis in all the three tested cell lines.

The present study shows that a few of the endophytic extracts as well as cisplatin and tamoxifen failed to significantly induce apoptosis in FICT116 andjor MCF-7 cells. The failure of tamoxifen to induce apoptosis is surprising since it has been reported to cause apoptosis through a p53 independent pathway in human breast adenocarcinoma cells (Fattman et al. 1998). Even cisplatin has been widely reported as a potential apoptotic agent in colon cancer (Dietze et al. 2001). The lack of apoptosis in cisplatin treated FICT116 cells could be due to the simultaneous necrotic and apoptotic cell death. Pestell et al. (2000) reported the occurrence of both apoptosis and necrosis in the same population of cisplatin-treated cells. The failure to induce apoptosis may also be due to the ability to induce cell death through other pathways including non-apoptotic cell death (Type II non-apoptotic cell death) (Tan et al. 2005). The cells that undergo non-apoptotic cell death have been found to produce apoptotic features such as chromatin changes, convolution and apoptotic bodies but absence of DNA fragmentation (Bowen et al. 1998). In addition, Tan et al. (2005) reported that type II non-apoptotic cell death might also behave like necrotic cells since the cells have the ability to take up propidium iodide although the plasma membrane remained intact. However, the appearance of Type II non-apoptotic cell death in the present study remains unclear since ultrastructural analysis was not performed for both FICT116 and MCF-7 cells.

It is also important to note that treatment period or incubation time may also affect the occurrence of apoptosis in treated cells. The time point used in the present study may not be sufficient to induce full events of apoptosis in the cancer cells. Apart from that, cells that undergo initial apoptosis may cause secondary necrosis when they are left for a long period of time (Er et al. 2007) or if inappropriate doses of drugs are used (Perez et al. 1999). An incomplete apoptotic process may also have caused some cells to die and be detected in the measurement of necrosis (Gonzalez et al. 2001). Necrotic cell death may also occur due to irreversible injury to the cells which then further continue to become necrotic. Compared to apoptosis, which is widely regarded as an active or programmed cell death, the importance of necrotic cell death in cancer therapy remains controversial.

Molecular identification of the endophytic fungal strains

The morphological features of the four active isolates after two weeks of incubation on FDA were observed macroscopically and microscopically. In the present study, only one isolate, KK21FL2 was shown to sporulate. Cultural characteristics and spore morphology was consistent with that of Fusarium sp. The remaining three were sterile even after prolonged incubation. In order to achieve a more definitive identification, all four isolates were subjected to molecular analysis. Using ITS1 and ITS4 primer pair, rDNA containing ITS1, ITS2 and the intervening 5.85 rDNA gene region was amplified. The length of the amplified rDNA fragment ranged from 450 to 700 bp. The amplified products of the ITS regions were sequenced and analyzed. Sequence strains with the accession numbers including their host plant and the result of the BLAST searches are listed in Table 2.

Table 2

Molecular identification of four ascomycetes
(KK9L2, KK14L1, KK21FL2 and KK30RH2).

Isolate  Host plant               Accession  Closest GenBank
                                  number     match

KK9L2    PhyUagathisrotundifoiia  HQ223035   Corynesporacasiicola
                                             isolate GD

KK14L1   Ampelocissuscinnamomea   HQ232850   Phomopsis sp. Mfer 5

KK21FL2  Zingiberaceae sp.        HQ413706   Fungal sp. 143C3

KK30RH2  Zingiberaceae sp.        HQ413707   Fungal endophyte
                                             isolate 515

Isolate  Accession  Max
         number     identity
         (GenBank   (%)
         database)

KK9L2    FJ196284         98

KK14L1   AY907347         98

KK21FL2  EU563516         99

KK30RH2  EU687127         99


Phylogenetic analysis

A further phylogenetic analysis based on ITS sequences was conducted to compare the species sequenced in this study with those in GenBank to determine their relationships and to authenticate the fungi. Maximum parsimony analysis of the rDNA sequences gave 98 parsimonious trees. Of 436 characters, 167 were parsimony informative. The most parsimony rooted tree (total length = 490 steps, CI = 0.825, RI =0.937) is shown in Fig. 2. Molecular analysis indicates all four isolates to be ascomycetes. The Ascomycota is the largest phylum in the Kingdom Fungi, with approximately 32,000 species (Hawksworth et al. 1995). In this study, isolate KK21FL2 showed typical Fusarium features. Fusarium is the asexual stage of several genera within the Hypocreales and many are reported as plant pathogens and endophytes (Feldman et al. 2008). Recently, several Fusarium species isolated as endophytes have been reported to produce metabolites with antimicrobial and cytotoxic activity (Deng et al. 2009). These include F. solani isolated from Taxus chinensis (Deng et al. 2009) and F. oxysporum isolated from Palicourea tetraphylla (Rosa et al. 2010).

Isolates KK14L1 and KK3ORH2 identified as Phomopsis spp. and isolate 515 respectively were grouped in the order Diaporthales. Phomopsis is a large genus that includes over 1000 species which have been described primarily on the basis of plant host (Farr et al. 2002). Recently, the genus Phomopsis yielded numerous biologically active metabolites including the antimicrotubule phomopsidin (Kobayashi et al. 2003), antimalarial phomoxanthones Osaka et al. 2001), antifungal phomoxanthone A (Elsaesser et al. 2005), antimicrobial phomopsichalasin (Horn et al. 1995) and the plant growth regulator cytochalasin H (Wells et al. 1976).

The result of the sequence analysis suggests that isolate KK9L2 may be related to Corynespora casiicola a member of the order Pleosporales. Corynespora casiicola has been recorded in over 70 countries from more than 280 plants including fruits, vegetables, grains, perennial crops, forest trees and ornamental plants (Nghia et al. 2008).

Identification of bioactive metabolites

The cytotoxic compounds isolated are shown in Table 3. Six compounds were identified from the rapid dereplication of fungal metabolites using an in-house UV library without having to put in effort for total purification of metabolites (Fig. 3). Chemical analysis of the secondary metabolites obtained from 14-day-old culture of the Colynespora casiicola (KK9L2) strain led to the isolation of two-compounds (dechlorogriseofulvin and griseofulvin) that possessed good cytotoxic activity against P388 (Fig. 4). Meanwhile, bioassay guided separation of EtAOc extract from the mycelium of Fusarium sp. (KK21FL2) resulted in the isolation of known compounds, demethylharzianic acid and harzianic acid (Fig. 5) which eluted at 17.738 and 19.426 min respectively. HPLC screening of isolate 515 (KK30RH2) showed two significant peaks eluting at 9.10 min and 14.70 min (Fig. 6). However, the results from the P388 assay indicated the activity to be related to a compound eluted over the period 14.70 min. A search using the in-house HPLC/ [R.sub.t] library showed that this peak matched with the known compound 2-hexylidene-3-methyl-succinic acid. The HPLC analysis of Phomopsis sp. (KK14L1) revealed seven main peaks appearing at [R.sub.t] 10-17 min (Fig. 7). The extract was further assayed to determine the peak responsible for cytotoxic activity. The results from the P388 quick screen assay indicated that the activity was associated only with peak eluted at 14.32 min. This peak matched with a known compound, cytochalasin J in the HPLC/[R.sub.t] library.

Table 3

Identification of the active metabolites from endophytic
fungal extracts.

Extract                       Code       Metabolites

Corynesporacasiicola          KK9L2-A    Dechlorogriseofulvin

                              KK9L2-B    Griseofulvin

Phomopsissp. Mfer 5           KK14L1-A   Cytochalasin J

Fusahum sp.                   KK21FL2-A  Demethylharzianic-acid

                              KK21FL2-B  Harzianic acid

Fungal endophyte isolate 515  KK30RH2-A  2-Hexylidene-3methyl
                                         -succinic
                                         acid


All compounds have been previously reported from a variety of fungi. Cytochalasin J, dechlorogriseofulvin, griseofulvin, harzianic acid and succinic acid have been reported from extracts of endophytic fungi. Cytochalasin J and H with five novel nonenolides have been isolated from an endophytic strain of Phomopsis (Li et al. 2010). This strain was isolated from leaves of the mangrove species, Kandelia candel, collected in China. The cytochalasins are a group of toxic fungal metabolites which exhibited a broad spectrum of biological activity including antibiotic and antitumor activity (Katagiri and Matsura 1971), inhibition of HIV-1 protease (Wells et al. 1976), and phytotoxic activity (Alvi et al. 1997). Griseofulvin isolated from Penicillium griseofulvum in the year 1939, is an interesting heptaketide molecule, which has been commercially used as an antifungal agent (Park et al. 2005; Rahman 2005). The isolation of griseofulvin has been reported from various organisms which include Aspergillus sp. and Streptomyces sp. (Rahman 2005). Recent study reported the isolation of heptaketides from a Corynespora sp. which includes 9-O-methylscytalol A, 7-desmethylherbarin and 8-hydroxyherbarin (Wijeratne et al. 2010). However, the present study is the first to report the production of the heptaketide compounds dechiorogriseofulvin and griseofulvin from Corynespora sp. Meanwhile, harzianic acid is a compound produced by a Trichoderma harzianum which has been widely reported as an antifungal and antimicrobial agent (Vinale et al. 2009). Isolation of harzianic acid from T. harzianum has been recently investigated for plant growth promoting activity. Succinic acid has been reported to be isolated from various filamentous fungi (Ling et al. 1978). In the past, succinic acid or its derivatives showed appreciable cytotoxic activity which supported the current findings (Nakamura et al. 2004). To date, demethylharzianic acid and harzianic acid have not been isolated from endophytic Fusarium sp. This is also the first report of 2-hexylidene-3-methyl-succinic acid from Phomopsis spp. In this study, succinic acid isolated from the EtAO of fungal endophyte isolate 515 (KK30RH2) was observed to be the same compound that was isolated from Zingiberaceae sp. (Qiao et al. 2002). This finding indicates that some endophytes may produce similar or the same metabolites as their hosts. Meanwhile, to the best of our knowledge, no reports have been documented on the isolation of cytochalasin J, dechlorogriseofulvin, griseofulvin and harzianic acid from their host plants, Phyllagathis rotundifolia or Ampelocissus cinnamomea.

In conclusion, it is evident that the endophytic extracts generally have excellent in vitro cytotoxic activities. It is noteworthy that extract KK30RH2 from the present study possess the ability to induce apoptosis in all the tested cell lines. In addition, isolation of cytochalasin J, dechlorogriseofulvin, griseofulvin, demethylharzianicacid, harzianic acid and 2-hexylidene-3methyl-succinic acid from the present study shows that plants from Malaysia rainforest could be hosts to a great diversity of endophytic fungi that serve as a remarkable source of bioactive compounds. Further studies are in progress to realize the potential of the Malaysian tropical rainforest for biologically active compounds and elucidate the detailed molecular pathway(s) of these bioactive compounds for future development of chemotherapeutics for cancer.

Funding

Ministry of Science, Technology and Innovation (MOSTI), Malaysia and Ministry of Higher Education (MOHE), Malaysia under the R & D IPHARM Initiative Grant [100-IRDC/BIOTEK 16/6/2 B (1/2008)] and Fundamental Research Grant Scheme (Num 5.3.1) respectively are thanked for the financial support.

Acknowledgments

We thank Professor M. Munro, Professor J. Blunt, Dr. L. Sun and Ms. G. Ellis of the Chemistry Department, University of Canterbury, New Zealand for their invaluable help and advice. Pharmacology and Toxicology Research Laboratory, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) is thanked for the cell lines provided.

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Nurul Aqmar Mohamad Nor Hazalin (a), Kalavathy Ramasarny (a), *, Siong Meng Lim (a), Anthony L.J. Cole (b), Abu Bakar Abdul Majeed (c)

(a.) Collaborative Drug Discovery Research (CDDR) Group, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

(b.) School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand

(c.) Brain Research Laboratory, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alum, Selangor Darul Ehsan, Malaysia

* Corresponding author. Fax: +60 3 32584692.

E-mail address: kalav922@salam.uitritedu.my (K. Ramasamy).

0944-7113/$ - see front matter [c] 2012 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2012.01.007
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Author:Hazalin, Nurul Aqmar Mohamad Nor; Ramasamy, Kalavathy; Lim, Siong Meng; Cole, Anthony L.J.; Majeed,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9MALA
Date:May 15, 2012
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