Inhibition of Aspergillus parasiticus and Cancer Cells by Marine Actinomycete Strains

LI Ping, and YAN Peisheng,

1) School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150001, P. R. China

2) School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai 264209, P. R. China

Inhibition of Aspergillus parasiticus and Cancer Cells by Marine Actinomycete Strains

LI Ping1), and YAN Peisheng1),2),*

1) School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150001, P. R. China

2) School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai 264209, P. R. China

Ten actinomycete strains isolated from the Yellow Sea off China’s coasts were identified as belonging to two genera by 16S rDNA phylogenetic analysis: Streptomyces and Nocardiopsis. Six Streptomyces strains (MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1) and one Nocardiopsis strain (MA03) were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSα (ketoacyl-synthase) gene in the type II PKS (polyketides synthase) gene cluster. Four strains (MA03, MA01, MA10 and MA05-2) exhibited significant inhibitory effects on mycelia growth (inhibition rate >50%) and subsequent aflatoxin production (inhibition rate >75%) of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated (MA03: 0.275 mg mL-1, MA01: 0.106 mg mL-1, MA10: 1.345 mg mL-1and MA05-2: 1.362 mg mL-1). Five strains (2SHXF01-3, MA03, MA05-2, MA01 and MA08-1) were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix (HeLa), lung (A549), kidney (Caki-1) and liver (HepG2) (IC50: 2.890, 1.981, 3.032 and 2.603 μg mL-1, respectively). The extract also remarkably inhibited colony formation of HeLa cells at an extremely low concentration (0.5 μg mL-1). This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.

actinomycetes; antifungal; antitumor; tip culture method; MTT; colony formation assay

1 Introduction

Actinomycetes are particularly important for the discovery of valuable antibiotics and metabolites (Bode HB et al., 2002; Bérdy, 2005; Kurtb?ke, 2012; Charoensopharat, 2008) that have been widely applied in the development of antimicrobial (e.g., amphotericin B, erythromycin and vancomycin), antitumor (e.g., daunorubicin, bleomycin and mitomycin) and immunosuppressive (e.g., rapamycin) drugs (Watve et al., 2001; Zotchev, 2012). Most of the known microbial compounds are derived from terrestrial actinomycetes (Bérdy, 2005). Recently, researchers have switched to marine actinomycetes in the search for novel bioactive products because of the belief that terrestrial microorganism resources have been exhaustively studied (Zotchev, 2012). It appears that marine actinomycetes have different characteristics from terrestrial actinomycetes and may yield novel bioactive secondary metabolites because of their extremely different growing environment (Kijjoa et al., 2004; Ramesh et al., 2009). In fact, novel compounds with various biological activities have been purified from several new marine actinomycete taxa (Prudhomme et al., 2008; Olano et al., 2009; Asolkar et al., 2010; Rahman et al., 2010) in recent years. These reports clearly indicate that marine actinomycetes are an important source of novel secondary metabolites.

However, valuable compounds with biological activities (e.g., antitumor, antimicrobial and anti-inflammatory) obtained from marine actinomycetes are only a small portion of the marine-derived natural products (Mayer et al., 2011). El-Shatoury et al. discovered that metabolic extracts from several actinomycetes inhabiting marine shellfish displayed obvious antimicrobial, antitumor and in vivo cytotoxicity (El-Shatoury et al., 2009). Gao and colleagues reported that a novel phenazine derivative from a marine actinomycete BM-17 repressed a diploid fungus, Candida albicans, and displayed extreme induction of cancer cell death in vitro and in vivo (Gao et al., 2012). Bahamaolide A, isolated from a marine Streptomyces sp., also demonstrated significant inhibitory activity against various pathogenic fungi, such as Candida albicans (Kim et al., 2012). Carlson et al. identified a new macrolide antibiotic from a marine Micromonospora sp. that mediated cancer chemopreventive activity (Carlson et al., 2013). These valuable compounds indicate thetremendous potential of marine actinomycetes, and the screening and isolation of marine actinomycetes with various bioactivities is an urgent need in the exploration of the novel compounds produced from these microorganisms.

In this paper, ten new actinomycete strains isolated from the Yellow Sea off China’s coasts were identified by molecular methods. With the aim of predicting the potential in producing bioactive secondary metabolites, we assayed a functional gene, type II polyketide synthase (PKS), by PCR. Then, the potential antifungal and antitumor activities of the strains were evaluated.

2 Materials and Methods

2.1 Microorganisms and Media

Ten actinomycete strains (Yan et al., 2011) isolated from the sea sediments of the Yellow Sea (China) were used in the study. The mutant aflatoxigenic A. parasiticus NFRI-95, in which the synthesis pathway of aflatoxin is blocked, can accumulate an orange-red precursor, norsolorinic acid (NA) (Yan et al., 2004), and was used to detect the antifungal activity.

Marine actinomycetes were grown in Gause’s Synthetic (GS) medium (2% starch, 0.05% K2HPO4, 0.05% MgSO4·7H2O, 0.05% NaCl, 0.001% FeSO4·7H2O, 0.1% KNO3, 2% agar, pH 7.2-7.4, wt/v). GY medium contained 2% glucose and 0.5% yeast extract (wt/v).

2.2 Genomic DNA Extraction

Approximately 50 mg of the mycelia pellet was ground completely with liquid nitrogen before incubation in a tube containing 500 μL of lysozyme solution (5 mg mL-1) at 37℃ for 1 h. Then, the tube received 250 μL of 2% SDS and 10 μL of proteinase k (20 mg mL-1) followed by incubation at 37℃ overnight. Thereafter, the tube was inverted for 30 min after the addition of 500 μL of carbolic acid/trichloromethane (1:1). The pellet was removed by centrifugation (12 000 r min-1for 5 min), and the supernatant solution was transferred to a new microcentrifuge tube and extracted with carbolic acid/trichloromethane (1:1) 3 times to remove proteins and lipids. Genomic DNA was precipitated after inversion with 0.1 volume of NaAC (3 mol L-1) and an equal volume of isopropanol. Genomic DNA was obtained by centrifugation and washed with 70% ethanol twice and was then redissolved in 200 μL of TE for immediate use or storage at -20℃.

2.3 PCR Amplification

PCR amplification of the 16S rRNA gene was conducted using the general primers MaF8 (5’-GAGAGT TTGATCCTGGCTCAG-3’) and MaR1492 (5’-CGGCTA CCTTGTTACGAC-3’) (Woese et al., 1983). The reaction conditions were as follows: initial denaturation at 95℃for 5 min, 35 cycles of 95℃ for 30 s, 55℃ for 45 s, and 72℃ for 1 min 30 s, and a final extension of 7 min at 72℃.

The degenerate primers IIPF6 (5’-TSGCSTGCTTCG AYGCSATC-3’) and IIPR6 (5’-TGGAANCCGCCG AABCCGCT-3’) were used to amplify the KSα gene of PKS II (Mets?-Ketel? et al., 1999). PCR was performed as previously described (Mets?-Ketel? et al., 1999).

2.4 Fermentation and Organic Metabolite Extraction

All of the actinomycete strains were incubated in GS liquid medium at 28℃ and 160 r min-1for 9 d. The broth was centrifuged at 8000 r min-1for 20 min. The supernatant was extracted three times using an equal volume of ethyl acetate by vigorous shaking for 30 min. Ethyl acetate fractions were concentrated at 40℃ in a rotary vacuum distillation apparatus and then dissolved in methanol (100 mg mL-1) and DMSO (1 mg mL-1) for the investigation of antifungal and antitumor activities, respectively.

2.5 Tip Culture Assay

The tip culture method (Yan et al., 2004) was used to assess the antifungal activity. Initially, the supernatants of these 10 actinomycete strains supplemented with nutritional components of GY medium were filtered using 0.22 μm pore size filters. Then, 700 μL of the filtered supernatant was added to a 5 mL tip as a culture vessel, and two tips were used as negative controls as follows: GY medium without any filtered supernatant and GS medium without any filtered supernatant. Next, 10 μL of A. parasiticus NFRI-95 spores was seeded into each tip followed by incubation at 28℃ for 6 d. Thereafter, the mycelia were obtained and weighed following centrifugation at 96 r min-1. To detect the precursors of aflatoxin, the mycelia were extracted with a reagent containing 1 mol L-1NaOH and methanol (9:1) for 2 h. The extracts were obtained after the centrifugation at 4000 r min-1for 5 min, and the absorbance was detected at a wavelength of 560 nm.

2.6 Agar Diffusion Assay

A ten microliter spore suspension of each actinomycete strain was seeded at a point approximately 2.5 cm away from the center on the GS agar plate prepared with fresh water uniformly, and then, the plates were incubated at 28℃ for 4 d. Thereafter, 10 μL of a spore suspension of A. parasiticus NFRI-95 was swabbed at the center of each plate. The plates were photographed after the incubation for another 6 days.

2.7 MTT Cell Proliferation Assay

All of the cell lines used in this study were obtained from the American Type Culture Collection. HeLa (Cervix carcinoma) and HepG2 (liver carcinoma) were grown in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). A549 (lung carcinoma) and Caki-1 (renal cell cancer) were cultured in RPM1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Then all of the cells were maintained at 37℃ in a 5% CO2incubator.

Approximately 1×104cells were seeded into 96-wellculture plates for 24, 48, 72, 96 or 120 h and were then incubated with 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) solution (final concentration 0.5 mg mL-1) at 37℃ for 4 h, followed by 150 μL of DMSO to solubilize the crystals after the supernatant was discarded. The absorbance at a wavelength of 570 nm was measured.

2.8 Cell Colony Formation Assay

Cells were seeded at a density of 3×102per dish (35-mm diameter) and incubated at 37℃ overnight. The cells were then treated with crude extracts or cisplatin (DDP), which was used as a positive control, for 1 h. After treatment, the cells were washed with a phosphate buffer solution (PBS) and allowed to grow for 10-14 d. The cultured colonies were fixed with methanol for 5 min and then stained with 2.5% Crystal Violet (Sigma).

3 Results

3.1 Molecular Taxonomy of Ten Marine Actinomycete Strains

Fig.1 Neighbor-joining phylogenetic tree based on the 16S rDNA sequence of actinomycetes from the Yellow Sea off China’s coasts. The sequences obtained in this study are marked by black dots. The numbers are percentages, which represent the level of boot strap support. The scale bar represents 0.02 nucleotide substitutions per site.

Based on the BLAST analysis of 16S rRNA gene sequences, the phylogenetic tree in Fig.1 was derived from the distance matrices using the neighbor-joining method, and these 10 marine actinomycete strains were assigned to the actinobacteria in the GenBank database, including 2 genera: Streptomyces (8 isolates) and Nocardiopsis (2isolates) (Fig.1 and Table 1). Five strains (MA08-1, 2SHXF01-3, MA05-2, MA35 and MA05-2-1) displayed over 98% similarity to Streptomyces variabilis strain NRRL B-3984Tand were clustered in close clades. The 16S rDNA sequences of strain MA04 and MA10 shared high similarity (99%) with the 16S rDNA sequences of Streptomyces sp. VTT E-99-1336 (B329). In addition, they were clustered in close clades. However, the 16S rDNA sequence of strain MA01 displayed over 99% and 97% similarity with the 16S rDNA sequence of Streptomyces sp.LS-A24 and of Streptomyces fradiae strain NRRL B-1195T, respectively. Thus, MA01 belongs to a distinct clade, whereas strains MA03 and MA09 are diversely distributed in the genus Nocardiopsis.

3.2 The Potential for Producing Bioactive Metabolites Based on Functional Gene Analysis

Polyketide synthases (PKS, e.g., type I, type II and type III) catalyze the synthesis of a large number of important bioactive compounds in microorganisms. In addition, many aromatic metabolites with unique structures, including many important antimicrobials and anticancer agents, arise from type II PKS (Hertweck et al., 2007). To predict the production of the bioactive metabolites of these actinomycete strains, the KSα gene of PKS II was analyzed by PCR using genomic DNA. Based on BLAST analyses, the presence of KSα gene was detected in 7 isolates (Table 1 and Table 2), i.e., MA03 (Nocardiopsis),MA10 (Streptomyces), 2SHXF01-3 (Streptomyces), MA35 (Streptomyces), MA05-2 (Streptomyces), MA05- 2-1 (Streptomyces) and MA08-1 (Streptomyces), and the KSα domain amino acid sequences displayed high (97-100%) sequence similarity to their BLAST matches. Fig.2 shows that KSα amino acid sequences from 5 Streptomyces strains (MA35, MA08-1, 2SHXF01-3, MA05-2-1 and MA05-2) were classified into one phylogenetic division. In addition, based on the prediction of the production of secondary metabolites with pharmacologically important activities using the DoBISCUIT database (Ichikawa et al., 2013), these 5 strains all displayed over 72% sequence similarity to 3 polyketide synthases from the genus Streptomyces (AF098965, AY196994 and X63449) that produce naphthocyclinone (antibacterial), hedamycin (antitumor) and actinorhodin (antibacterial). Notably, the KSα domain amino acid sequences of MA03 (Nocardiopsis) and MA10 (Streptomyces) shared over 97% similarity with putative ketosynthase alpha EncA (Streptomyces maritimus, AF254925), which can synthesize enterocin with antibacterial activity. In addition, the KSα amino acid sequences of MA03 and MA10 both exhibited over 72% similarity with the synthases of alnumycin (antitumor, gyrase inhibitory and topoisomerase inhibitory), aranciamycin (antibacterial and collagenase inhibitor), naphthocyclinone (antibacterial), chromomycin (antibacterial, antitumor and antiviral), granaticin (antibacterial) and chartreusin (antibacterial and antitumor). Thus, these 7 strains, particularly MA03 and MA10, were the focus of the next part of the study.

Table 1 Ten actinomycete strains from the marine sediments of Yellow Sea (China)

Fig.2 Neighbor-joining tree constructed using the aligned KSα amino acid sequence of type II PKS. The sequences obtained in this study are marked by black dots. The scale bar represents 0.01 substitutions per amino acid position.

Table 2 BLAST analysis based on KSα domain amino acid sequence

3.3 Inhibitory Effect of Actinomycete Metabolites on Aspergillus parasiticus

A previous study indicated that these 10 marine actinomycete strains grew equally well in medium prepared with fresh water (FW) and sea water (SW). However, it was not clear whether the replacement of SW by FW affected the production of bioactive metabolites. To investigate this issue, the equivalent spores of each actinomycete strain were cultured in GS medium prepared with FW and SW, respectively. Simultaneously, to verify the prediction based on functional gene analysis, we assessed the antifungal activity using the initial sterile supernatants of the actinomycete broths that were supplemented with the nutritional components of GY medium by the tip culture assay. As shown in Fig.3, strain MA01 produced a remarkable inhibitory effect on mycelia growth (ratio > 90%) (Figs.3A and B) and aflatoxin production (ratio > 90%) (Figs.3A and C), whereas the replacement of SW by FW decreased the inhibitory effect of MA03 on mycelia growth (approximately 40%) (Figs.3A and B) but did not affect the antiaflatoxigenic activity (Figs.3A and C), and similar results were observed for the strain MA10. In addition, MA35, MA05-2 and MA09 grown in FW medium also repressed mycelia growth (approximately 50%). In regard to the subsequent aflatoxin production, as shown in Figs.3A and C, MA09 and MA05-2 displayed significant inhibition of NA production by A. parasiticus NFRI-95 (antiaflatoxigenic rate >60%), despite the fact that SW could decrease this effect (antiaflatoxigenic rate <10%). In conclusion, MA03, MA10 and MA01 prevented NFRI-95 production of NA both in FW and SW GS medium, whereas MA05-2 suppressed NA accumula-tion only in FW GS medium but not in SW GS medium. To confirm the results of the tip culture method, an agar diffusion assay was performed. As shown in Fig.S1, the results were consistent with the tip culture assay in that MA03 and MA01 displayed significantly inhibitory effects on the growth of NFRI-95, but MA08-1 did not affect the growth of NFRI-95 and neither did the negative control, MA08-2, which is an actinomycete strain with a known lack of inhibitory effect on NFRI-95 growth.

Fig.3 Inhibitory effect of 10 actinomycetes strains on Aspergillus parasiticus. A, Every strain was cultured in FW and SW. The inhibitory effect of the filtered supernatants of actinomycete broth was determined using the tip culture method. CK1 indicates GY medium without actinomycetes. CK2 indicates GS medium without actinomycetes. B, Ratio of 10 strains inhibiting A. parasiticus NFRI-95 mycelia growth. C, Inhibitory ratio of 10 strains against accumulation of NA.

According to the results of the tip culture method and agar diffusion assay, the ethyl acetate extracts of the broth of the four strains with high inhibitory effects on both mycelia growth and NA production of NFRI-95, including MA03, MA01, MA10 and MA05-2, were selected for the evaluation of the IC50 value against NFRI-95 growth by the tip culture method. As shown in Fig.4, their inhibitory effect on both mycelia growth and NA production was dose-dependent. Thus, we calculated the IC50 value, and the crude extracts of MA03 (IC50: 0.275 mg mL-1) and MA01 (IC50: 0.106 mg mL-1) displayed remarkable inhibitory activity against NFRI-95 growth, whereas the IC50 values of MA10 (IC50: 1.345 mg mL-1) and MA05-2 (IC50: 1.362 mg mL-1) were much larger than those of MA03 and MA01. Therefore, these four actionmycete strains, especially MA03 and MA01, deserve in-depth studies regarding biocontrol.

Fig.4 Inhibitory effect of the crude extracts of 4 actinomycete strains on Aspergillus parasiticus. A, MA03; B, MA01; C, MA10; D, MA05-2. The IC50 value of the crude extract of each actinomycete strain was calculated.

3.4 Antitumor Activities of Actinomycete Metabolites on Human Cell Lines

The antitumor activities of these 10 actinomycete strains were investigated by the MTT cell proliferation assay while the antifungal abilities were being determined. Initially, the sterile supernatant of each actinomycete broth (final concentration 1%) was used in the MTT assay. As shown in Fig.5, three types of cancer cells, i.e. HeLa (cervix carcinoma), A549 (lung carcinoma) and Caki-1 (renal cell cancer), were cultured for 48 h after treatment with sterile supernatants. Six strains (2SHXF01-3, MA03, MA35, MA05-2, MA01 and MA08-1) exhibited stable antitumor activities against these 3 types of cells (inhibition rate > 60%). Interestingly, MA03, MA05-2, MA01 and MA08-1, which were cultured in FW GS medium, all displayed much greater inhibitory effects than the strains cultured in SW GS medium. Based on these observations, five strains (2SH XF01-3, MA03, MA05-2, MA01 and MA08-1) were selected for the subsequent assays, except for MA35, which was used in another study.

To screen the strains with highest antitumor activity, crude extracts of the fermentation broths of these five strains were extracted using ethyl acetate. The survival fractions of each cell line were plotted against the different concentrations (1-20μg mL-1) of each extract (Fig.6A), and the IC50 values were calculated (Table 3). Compared with the crude extracts of the other 3 strains as well as the positive control DDP, MA03 displayed the highest inhibitory effect on all the four tested cell lines. The con-centrations of the MA03 crude extract that reduced survival of cell lines of cervical (Hela), lung (A549), kidney (Caki-1) and liver (HepG2) carcinoma to 50% were 2.890, 1.981, 3.032 and 2.603 μg mL-1, respectively. Nevertheless, even 20 μg mL-1of the crude extract of 2SHXF01-3 could not reduce A549 and Caki-1 to 50% survival, and neither did MA05-2 and MA08-1 against A549, nor did MA01 against A549.

Table 3 IC50 value of antitumor activity (μg mL-1)

Fig.5 Antitumor activities of 10 actinomycete strains. The inhibitory effect of the sterile supernatant of each actinomycete broth when added to the medium for cell culture (1%) was assessed using human cancer cell lines of the cervix (HeLa), lung (A549) and kidney (Caki-1) by the MTT assay.

To further confirm our observations, a colony formation assay was performed to examine long-time cell survival. HeLa cells were treated with a low concentration (0.5 or 1 μg mL-1) of the indicated crude extracts or DDP for 1 h and were then assayed after 10 days of treatment. As shown in Fig.6B and C, 2SHXF01-3 and MA03 inhibited the colony formation of HeLa cells significantly at a very low concentration (0.5 μg mL-1) (P<0.01) compared with the control group, but MA01 and DDP could not. However, 2SHXF01-3, MA03 and MA01, as well as DDP, all reduced the number of colonies significantly (P<0.01) at 1μg mL-1.

According to the above results, the crude extract of MA03 fermentation broth displayed the highest and the most stable killing efficiency against cancer cells compared with other strains as well as commercial DDP. These findings imply that MA03 might produce some types of bioactive compounds that possess significantly high antifungal and antitumor activities, and MA03 deserves an in-depth study into its secondary metabolite components in the future.

4 Discussion

Since the 2000s, many novel bioactive natural products from marine-associated organisms, especially actinomycetes, have been discovered (Zheng et al., 2000; Fenical and Jensen, 2006; Bull et al., 2007; Leary et al., 2009; Sun et al., 2012). The ten strains of actinomycetes used in this study were isolated from the sea sediments of the Yellow Sea (China) and classified as falling into two genera, Streptomyces and Nocardiopsis, by phylogenetic tree analysis using 16S rRNA gene sequences (Fig.1 and Table 1). Most of our isolates (8 strains) belong to the genus Streptomyces, which is an economically important and dominant group of actinomycetes, and they are the pivotal source for compounds with multiple biological activities (Bérdy, 2005). Approximately 75% of the commercially and medicinally useful antibiotics (Kieser et al., 2000) and some agriculturally important agents (Okami et al., 1988) have been derived from streptomycetes. Genus Nocardiopsis are a group of rare actinomycetes with a rapidly increasing number of species (Bérdy, 2005). In addition, the number of newly identified bioactive compounds from Nocardiopsis (Engelhardt et al., 2010; Manivasagan et al., 2013) is increasing because of recently developed genetic and isolation techniques. The strains of the two genera above mentioned would yield some compounds with potential activities. Actinomycetes with the PKS gene have produced many novel compounds (Schneemann et al., 2010). To predict the antifungal and antitumor activities and verify our hypothesis, we detected the functional gene, the KSα domain of type II PKS, by PCR using the genomic DNA of the 10 isolates mentioned earlier. Among the 10 strains of actinomycetes, 6 strains from the genus Streptomyces yielded positive results. Streptomyces species are well-known polyketide producers, so it is quite expected that the KSα gene was detected in most of the 8 Streptomyces strains (Fig.2 and Table 1). As Nocardiopsis is a rare genus of actinomycetes featured in a few reports, the presence in MA03 of the KSα gene should be further examine in future studies (Fig.2 and Table 1).

Based on the molecular analysis, we further performed antifungal and antitumor assays to confirm the above results. Previously, these 10 strains of actinomycetes have been proven to grow well in medium prepared with fresh water. Although there are reports indicating that many strains from marine sediments require sea water for growth (Jensen et al., 2005; Bredholt et al., 2008), the Yellow Sea lies between mainland China and the Korean peninsula with terrestrial environments. Thus, it is possible that there are also a large number of actinomycete strains of terrestrial origin that grow well in fresh water environment as well. However, it has rarely been examined whether sea water is required for the production of bioactive metabolites. Here, we compared the antifungal and antitumor activities of these 10 actinomycete strains cultured with fresh water or sea water (Fig.3 and Fig.5), and four strains (MA03, MA10, MA01 and MA05-2) with high inhibitory effect on both fungal growth and NA accumulation and five strains (2SHXF01-3, MA03, MA01, MA05-2, and MA08-1) with high antitumor activity were screened out. MA03 and MA01 displayed significantly high antifungal and antitumor activities simultaneously; sea water, however, decreased the production of the potential compounds with antitumor activity (Fig.5) but did not affect the antifungal activity (Fig.3). There might be several types of compounds with multiple and valuable biological activities, such as antifungal, antitumor and antibacterial activities, in the secondary metabolites of the strains MA03 and MA01. It is interesting that MA01, without a type II PKS gene, NRPS or type I PKS (data not shown), exhibited a very high inhibitory effect on fungal and cancer cells, whereas MA05-2-1 with the KSα gene did not show an obviously inhibitory effect on fungus or tumor cells. In addition, every biosynthetic pathway (e.g., NRPS, type I PKS and type II PKS) is a complicated enzyme system under strict regulation by internal and external signals, including some negative regulators, such as the phosphorylated form of AbsA2 (Sheeler et al., 2005), and many biosynthetic enzymes also undergo some important post-translational protein modifications before participating in the synthesis of bioactive compounds. For example, the first 31 or 34 amino acid residues of the dehydratase ActIV-ORF3 need to be cut off before translocating to the functional foci (Hesketh et al., 2002). These results imply that although functional gene-based analysis provides a powerful method to predict valuable microorganisms, culture-dependent as-says are also needed to confirm related hypotheses.

Based on the results of initial antifungal and antitumor assays using the sterile supernatants of the broths, we obtained crude extracts using ethyl acetate to prepare for the separation of the bioactive compounds. A high inhibitory effect of the crude extracts confirmed the observations using the sterile supernatants of the broths (Fig.4 and Fig.6A). In particular, the antitumor activities of the crude extracts of MA03 and MA01 are both higher than the antitumor activity of commercial DDP. In addition, the findings are consistent with the above results that a tiny amount of the crude exacts of MA03 and MA01 could inhibit cancer cell colony formation (Fig.6B). Thus, we infer that the cytotoxicity of bioactive compounds produced by these two strains is extremely high or that these two strains achieve significantly high yields under the condition of this study, and ethyl acetate is a suitable extraction reagent. Therefore, our future studies will focus on the separation and purification of these potentially valuable compounds.

5 Conclusion

Screening and isolation are the key steps of the exploitation of valuable actinomycete strains producing potential bioactive compounds. In this study, four isolates with high antifungal activities and three strains with significant inhibitory activities against tumor cells were screened out. Notably, one of these selected strains, MA03, produced some secondary metabolites that inhibited both the growth and production of aflatoxin of A. parasiticus in addition to the proliferation of cancer cells. The present data suggest that compared with the other isolates, MA03 is a much more valuable Nocardiopsis strain and deserves more attention in future studies.

Acknowledgements

This work was supported by the COMRA project (No. DY125-15-R-01).

Supplementary Material

Fig.S1 Inhibitory effects on Aspergillus parasiticus of actinomycete strains were assessed by an agar diffusion assay. A, Four strains (MA08-2, MA08-1, MA03 and MA01) were seeded at the points on the GS agar plate around the center, where A. parasiticus NFRI-95 was uniformly cultured four days later. B, A. parasiticus NFRI-95 was cultured on the GS agar plate without the actinomycete strain. This plate was used as a negative control.

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(Edited by Ji Dechun)

(Received February 16, 2014; revised July 6, 2014; accepted August 4, 2014)

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-631-5687230

E-mail: psyan6@hotmail.com