Introduction

Cancer has now emerged as a major public health threat worldwide. World population growth and aging imply a progressive increase in the cancer burden in the future (Parkin 2001). In the USA, cancer has become the number one killer in age under 85 since 1999 (Twombly 2005). Although many anticancer drugs have been developed, most are also toxic to normal cells and tissues. Therefore, only a few of them have found a certain place in the treatment of cancer (Waksman 1966; Lokich 1980). Furthermore, intrinsic and/or acquired (multi-)drug resistance is often a major impediment to successful cancer chemotherapy (Hait 1996). To overcome these limitations and improve the effectiveness of chemotherapy for cancer, it is necessary to isolate new anticancer drugs. Fortunately, numerous anti-tumor metabolites with a variety of structures are produced by actinomycetes (Waksman 1966). These compounds are unrivaled and unmatched in medical significance.

In this work, we describe a new soil-inhabiting actinomycete strain, L033, which produces anticancer agent and exhibits potent anti-tumor activity against human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The isolate was identified as a strain of Streptomyces avermitilis based on taxonomic experiments. One anti-tumor component was purified, and its structure was determined to be the same as those of oligomycin A by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Strain L033 was distinguished from other wild-type strains of S. avermitilis reported (Miller et al. 1979; Ikeda et al. 1993) by high yield of oligomycin A among its fermentation products.

Materials and methods

Microbial strains

An actinomycete strain, termed L033, was isolated from a soil sample collected in Guangzhou City, China and grown on yeast extract–malt extract–soluble starch medium (YMS) agar (Ikeda et al. 1988) at 28°C. L033 was deposited at the China General Microbiological Culture Collection, as CGMCC4.5508. S. avermitilis ATCC 31267T (MA-4680T = NCIMB 12804T = NRRL 8165T) was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The above strains were maintained on YMS agar at 4°C and as spore suspension in 20% (v/v) glycerol at −70°C.

Human tumor cell lines

Human hepatoma cell line Bel-7402 was obtained from the China Center for Type Culture Collection (Wuhan City). Human chronic myelogenous leukemia cell line K-562 and human colonic carcinoma cell line HCT-8 were from the Cell Culture Centre, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing City).

Cultural and morphological properties of strain L033

Strain L033 was inoculated onto peptone yeast extract iron agar (ISP-6) medium in order to determine melanin-producing ability. Its colony-forming and pigmentation properties were examined on inorganic salt-starch agar (ISP-4) and oatmeal agar (ISP-3) media after 14 days at 28°C. Spore chain morphology and spore surface features were examined by scanning electron microscopy of 7-day cultures grown on Bennett’s agar. Using the cover technique described previously (Zhou et al. 1998; Kawato and Shinobu 1959), samples were observed with a Hitachi S-3400N scanning electron microscope, with secondary mode operating at 20 kV.

Carbon source utilization tests

Utilization of substrates as sole carbon and energy sources was tested as described by Kämpfer et al. (1991). Carbon sources were filter-sterilized, with a final concentration of 0.2% (w/v).

16S rDNA sequence and phylogenetic analysis

Genomic DNA was isolated from cells as described by Hopwood et al. (1985). The 16S rRNA gene of strain L033 was amplified by polymerase chain reaction, using two universal bacterial primers, 1492R (5′-GGTTACCTTGTTACGACTT-3′) and Eubac27F (5′-AGAGTTTGATCCTGGCTCAG-3′; Jiang et al. 2006). The amplified products were purified using TIANgel mini purification kit (TianGen Biotech Beijing), ligated to pMD18-T simple vector (TaKaRa), and transformed into competent cells of Escherichia coli DH5α. 16S rRNA gene fragment was sequenced using forward primer M13F (−47) and reverse primer M13R (−48). The derived 16S rRNA gene sequence was compared to the GenBank database (NCBI), to search for similar sequences using the basic local alignment search tool algorithm. Similarity analysis was performed using ClustalW program (Thompson et al. 1994). A phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei 1987) and the Molecular Evolutionary Genetics Analysis (MEGA 3.1) software program (Kumar et al. 2004). Tree topology was evaluated by bootstrap analysis (Felsenstein 1985) based on 1,000 replicates.

DNA–DNA relatedness

Level of DNA–DNA relatedness between strain L033 and the type strain S. avermitilis ATCC 31267T was determined by thermal denaturation method (De Ley et al. 1970), using a lambda 35 UV/Vis spectrophotometer (PerkinElmer) fitted with a PTP1 temperature programmer (PerkinElmer) and standard software. Results were expressed as the mean of three determinations.

Fermentation in shaken flasks

Spores of strain L033 stored in 20% glycerol at −70°C were inoculated on YMS agar plates and cultured at 28°C for 12 days. Five-hundred-milliliter Erlenmeyer flasks were filled with seed medium consisting of soluble starch (30 g), malt extract (2 g), soy peptone (2 g), and CoCl2.6H2O (5 mg) per liter deionized water, which was inoculated with strain L033 by addition of small areas of growth cut from the agar plate. pH was adjusted to 7.0–7.2 before sterilization. Flasks were incubated at 28°C for 48 h on a rotating shaker (170 rpm).

Production medium G, also placed in 500-ml flasks, consisted of soluble starch (70 g), dried yeast (16 g), MgSO4.7H2O (0.5 g), K2HPO4.3H2O (0.5 g), KCl (4 g), CoCl2.6H2O (5 mg), and CaCO3 (2 g) per liter deionized water, and was inoculated with 4-ml seed solution. pH was adjusted to 7.0–7.2 before sterilization, and flasks were incubated at 28°C on a rotating shaker at 170 rpm. After 10 days, broth from 156 flasks was combined and centrifuged, and the cell pellet was washed with deionized water and centrifuged again for isolation of substances showing anti-tumor activity.

Purification and structure determination of compound 1

The strain L033 cell mass (4,937 g) was mixed with acetone (3,800 ml), stood for 2 days, and filtered. The residue was added twice with acetone (2,000 ml, 1,500 ml), stood for 1 day, and filtered after each addition. The combined filtrates of three marinations were evaporated (55–65°C) to remove acetone. The aqueous residue was kept at 18°C overnight. A white solid appeared, which was collected, washed, with distilled water, and filtered. The resulting white solid was mixed with n-hexane/ethanol (96:4), stirred, and centrifuged. The clear supernatant was moved to a different container, dried (Na2SO4), decolored (activated carbon), and centrifuged. The resulting supernatant was kept at 18°C overnight. A cream-colored crystalline precipitate appeared, which was collected, dissolved with n-hexane/ethanol (96:4), and separated by preparative positive-phase high-performance liquid chromatography (HPLC) using SiO2 column (250 × 20 mm, 10 μm) [Chuang Xin Tong Heng (Beijing City)], with n-hexane/ethanol mixture (95:5) as eluent, at a flow of 25 ml/min. Solutions of the same peak were combined and evaporated to remove n-hexane/ethanol. n-Hexane/ethanol (95:5) was added to the residue of fraction containing compound 1, and the mixture was stirred until pure white crystal precipitated. The crystal and supernatant were divided by centrifugation.

Structure of compound 1 was determined by 1H- and 13C-NMR spectroscopy, 2D NMR spectroscopy, and fast atom bombardment mass spectrometry (FAB-MS), performed at the National Center of Biomedical Analysis (Beijing City). 1H- and 13C-NMR, and 2D NMR spectra were recorded at ambient temperature or 300 K on a Varian Inova 600 (600 M) spectrometer. The solvent was CDCl3.

HPLC analysis

Secondary metabolites were analyzed and quantified using a Waters 600 reversed-phase HPLC. Methanol (1 ml) was added to broth (1 ml), stirred for 30 min, and stood for 12 h. Cell residue was removed by centrifugation, and clear supernatant (20 μl) was injected onto a YMG-C18 column (250 × 4.6 mm, 10 μm). The solvent system was methanol/water (9:1) at a flow rate of 1 ml/min. Products were monitored by a Waters 2487 dual lambda absorbance detector at 246 nm.

Anti-tumor effect of compound 1 in vitro

3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Hansen et al. 1989) was employed to evaluate the anti-tumor effect of compound 1 on Bel-7402 hepatoma, K-562 leukemia and HCT-8 colonic carcinoma cells in vitro. Cells in a volume of 100 μl (5 × 103 cells/ml) were seeded onto 96-well plates and incubated at 37°C for 24 h in 5% CO2 atmosphere. Various concentrations of compound 1 (each 10 μl) plus 90 μl RPMI-1640 medium were added; 100 μl RPMI-1640 medium was added to control wells. Plates were incubated at 37°C in 5% CO2 atmosphere for 3 days. Supernatant was removed, 100 μl freshly prepared 0.5 mg/ml MTT per well was added to form formazan salt, and incubation continued at 37°C for 4 h. Supernatant was carefully removed and 200 μl/well dimethyl sulphoxide was added to dissolve the MTT formazan salt. The solution was mixed, absorbance was read on an enzyme-linked immunosorbent assay (ELISA) reader at 544 nm, and growth-inhibitory ratio was calculated using the formula (1 − A/B) × 100%, where A and B are mean absorbance of the treated and control wells, respectively. 5-Fluorouracil (5-FU) was used as reference compound for positive control.

Nucleotide sequence accession number

The nucleotide sequence of 16S rRNA gene reported in this article was assigned to the GenBank accession no. EU621830.

Results

Characterization and identification of isolated strain L033

A bacterial isolate from a soil sample collected in Guangzhou City, China displayed strong anti-tumor activity against human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The cultural and morphological properties suggested that the isolate, termed L033, was a strain of actinomycete. Sporulation occurred on standard media such as inorganic salt-starch agar and oatmeal agar. Aerial spore masses of light gray and gray color, respectively, were formed on these media. Melanin pigments were produced on peptone yeast extract iron agar. L033 formed an extensively branched substrate mycelium and aerial hyphae that differentiated into long, compact spiral chains, which became looser as the culture aged. The spore chains were composed of spherical to oval-shaped spores, with smooth surfaces (Fig. 1). L033 was able to use glucose, d-fructose, inositol, mannitol, l(+)-arabinose, d-xylose, L(+)-rhamnose, sodium citrate, d-galactose, glycerol, maltose, lactose, or d-mannose as sole carbon source, but was not able to use melezitose, l(−)-sorbone, inulin, or cellulose. Nearly complete 16S rRNA gene sequence (1487 bp) of L033 was obtained and was found to be most similar to those of S. avermitilis ATCC 31267T, S. cellostaticus NBRC 12849T, and S. griseochromogenes NBRC 13413T, with sequence identities of 99.9%, 98.9%, and 98.9%, respectively. S. avermitilis ATCC 31267T appeared to be the closest relative. A phylogenetic tree was constructed, using the neighbor-joining method based on similarity of a 1,461-bp consensus length of 16S rRNA gene sequence (Fig. 2), and confirmed that L033 grouped most closely with S. avermitilis ATCC 31267T. DNA–DNA relatedness studies were conducted between L033 and S. avermitilis ATCC 31267T. The mean DNA–DNA hybridization value of three determinations was 100%. The properties of culture, morphology, and carbon source utilization of strain L033 were consistent with those of S. avermitilis ATCC 31267T as described by Kim and Goodfellow (2002). The 16S rRNA gene sequence identity and DNA–DNA relatedness data confirmed that strain L033 belonged to S. avermitilis.

Fig. 1
figure 1

Scanning electron micrograph of Streptomyces avermitilis L033 grown on Bennett’s agar at 28°C for 7 days. The spiral spore chains consist of spores with smooth surfaces

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Fig. 2
figure 2

Neighbor-joining tree based on nearly complete 16S rRNA gene sequences, showing phylogenetic relationships between Streptomyces avermitilis L033 and related Streptomyces species. Numbers at nodes indicate bootstrap values from 1,000 replicates. GenBank accession numbers are given in parentheses. Bar, 0.2% sequence divergence

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Fermentation

Production of compound 1 by strain L033 in medium G at 28°C started after 1 day of fermentation. The maximal concentration of compound 1 was observed after 9 days of fermentation. A 4,937-g cell pellet was obtained from combined broth of 156 flasks for isolation of substances displaying anti-tumor activity. When cottonseed protein flour (16 g/l) was substituted for dried yeast as the nitrogen source in production medium G, production of compound 1 was 1,461 μg/ml.

Isolation and in vitro anti-tumor activity of compound 1

In tests against human tumor cells, six fractions prepared from crude extract by preparative positive-phase HPLC were determined. Of these, fraction 3 (containing compound 1, retention time = 5.867′; Fig. 3) was found to have anti-tumor activity; 191.4 mg of compound 1 were isolated from the 4,937-g cell pellet as above and purified to homogeneity by preparative positive-phase HPLC and crystallization.

Fig. 3
figure 3

High-performance liquid chromatography profile of extract from broth of Streptomyces avermitilis L033 at 246 nm. The solvent system was methanol/ water (9:1). Compound 1 displayed potent anti-tumor activity against human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines

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The ability of compound 1 to inhibit human tumor cell proliferation was investigated in vitro using the MTT assay. 5-FU, a commonly used agent for treatment of malignant tumors of the digestive tract, was used as the reference compound for positive control. Compound 1 inhibited cell proliferation of Bel-7402, K-562, and HCT-8 in a dose-dependent manner (Table 1). For compound 1 at a concentration of 5.0 × 10−5 μg/ml (i.e., 6.3 × 10−5 μM), rates of inhibition against Bel-7402, K-562, and HCT-8 were 29.9%, 43.7%, and 53.5%, respectively. In contrast, for 5-FU at a concentration of 5.0 × 10−2 μg/ml (i.e., 3.8 × 10−1 μM), rates of inhibition against these three cell lines were near or below zero. Thus, compound 1 was much more potent than 5-FU at inhibiting proliferation of these human tumor cell lines.

Table 1 In vitro sensitivity of cancer cell lines to compound 1, detected by MTT assay
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Structural determination of compound 1

The structure of compound 1 was determined by 1H-NMR, 13C-NMR, and MS analysis and compared with those of oligomycin A. Signals corresponding to 26-membered macrolide structures were found in the 1H- and 13C-NMR spectra of compound 1 (see Supplemental material). 13C-NMR chemical shifts of compound 1 were identical to those of oligomycin A (Carter 1986). The molecular ion of compound 1 (M + H, m/z 791.3) indicated a molecular weight (MW) of 790.3 (Fig. 4), consistent with the MW of oligomycin A. On the basis of the above data in combination with 2D NMR studies (data not shown), we concluded that compound 1 is oligomycin A (Fig. 5).

Fig. 4
figure 4

Determination of molecular weight of compound 1 by FAB-MS in positive-ion mode. Spectral data showed that the molecular weight of compound 1 was 790.3

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Fig. 5
figure 5

Structure of compound 1 isolated from Streptomyces avermitilis L033

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Discussion

A new actinomycete strain, S. avermitilis L033, was isolated from a soil sample during a screening program and displayed strong anti-tumor activity against human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines in vitro. Strain L033 was identified on the basis of cultural property, morphology, carbon source utilization, 16S rRNA gene sequence, and DNA–DNA relatedness. Compound 1, with anticancer activity, was isolated from the cells and identified as oligomycin A. L033 is the first reported naturally occurring strain of S. avermitilis producing a high yield of oligomycin A (maximal 1,461 μg/ml). In contrast, other reported wild-type strains of S. avermitilis produced low oligomycin (Miller et al. 1979; Ikeda et al. 1993).

Oligomycin and its analogues are a series of 26-membered macrocyclic lactones. Many of them display strong anti-tumor activity (Kobayashi et al. 1987; Yamazaki et al. 1992; Kim et al. 1997). For example, oligomycin SC-1 and SC-2 exhibit antiproliferative activity against mouse P388 lympholeukemia cells (IC50 = 0.0013 and 0.00033 μg/ml, respectively; Daisuke et al. 1997). The antibiotic NK86-0279, a structural analogue, displays growth-inhibitory effect on a variety of mouse and human cancer cells (IC50 = 0.0027~2.13 μg/ml; Nishikiori et al. 1991). In a study of 37,000 molecules tested against 60 human cancer cell lines as mitochondrial targeting agents, oligomycin was among the top 0.1% most cell line selective agents. In a study using R-HepG2 cells, oligomycin was able to bypass doxorubicin (Dox) resistance and trigger apoptosis (Li et al. 2004). Oligomycin ABC mixture reduced survival of P388 lympholeukemia cells to 54% at a concentration of 30 pg/ml, which is >103 times lower than the level required to inhibit respiration. Oligomycin A was more efficient than oligomycin ABC mixture in inhibiting P388 growth. The authors hypothesized that oligomycin at low concentration interferes with signaling events of apoptosis, which differ in tumor vs. normal cells (Korystov et al. 2003). Thus, oligomycin A has potential application as an anti-tumor agent. However, little is known about the effect of oligomycin A on other cancer cell lines.

When anti-tumor activity against human tumor cell lines Bel-7402, K-562, and HCT-8 in vitro was examined by MTT assay in this study, oligomycin A was found to inhibit proliferation of these cell lines much more strongly than 5-FU. Inhibition rates of oligomycin A at a concentration of 5.0 × 10−5 μg/ml against Bel-7402, K-562, and HCT-8 cells were 29.9%, 43.7%, and 53.5%, respectively. This concentration was thousands of times lower than that required to inhibit respiration (Currie and Gregg 1965). These results were similar to those reported by Korystov et al. (2003).

During procedures for isolation of compound 1, the aqueous residue of the marinated solution formed aqueous, oily, and solid layers (from bottom to top) when left at 18°C overnight. The solid layer, which contained most of the desired antibiotic compound, was recovered for further purification; the other two layers, which contained small quantity of the compound, were discarded. An immiscible organic solvent for second extraction (Albers-Schönberg et al. 1982; Visser et al. 1960) was therefore not required in the present study but could provide an economical method for additional isolation if applied industrially.

In summary, oligomycin A, a useful anti-tumor agent, was isolated with high yield and high purity from strain L033 cells by a relatively simple procedure. The yield can be further improved through induced breeding, optimized composition of culture medium, and production in tanks. S. avermitilis strain L033 should be great potential for industrial production of oligomycin A.