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Aureolic acids from a marine-derived Streptomyces sp. WBF16
Microbiological Research 167 (2012) 590–595
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Microbiological Research journal homepage: www.elsevier.de/micres
Aureolic acids from a marine-derived Streptomyces sp. WBF16 Jiansheng Lu a,b , Yihua Ma b , Jianjia Liang b , Yingying Xing b , Tao Xi b,∗ , Yuanyuan Lu a,b,∗∗ a b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China Department of Marine Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
a r t i c l e
i n f o
Article history: Received 1 March 2012 Received in revised form 31 May 2012 Accepted 5 June 2012 Keywords: Marine Streptomyces Phylogenetics analysis Secondary metabolites Aureolic acid Chromomycin
a b s t r a c t A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A2 (2) and Chromomycin A3 (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1–3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The aureolic acids are a family of related antitumor antibiotics that include olivomycin A, Chromomycin A2 or A3 , mithramycin, chromocyclomycin and so on (Remers 1979a; Skarbek and Speedie 1981; Remers and Iyengar 1995; Wohlert et al. 2001; Ogawa et al. 1998; Katahira et al. 1998; Lombó et al. 2006). All aureolic acid type compounds (with the exception of chromocyclomycin) contain a tricyclic aglycon with two aliphatic side chains attached at C-3 and C-7. Now, Chromomycin have been used clinically in the treatment of some tumor diseases (Jia et al. 2007; Remers 1979b) such as disseminated embryonal cell carcinoma (Skarbek and Speedie 1981; Kennedy et al. 1968; Elias and Evans 1972), hyperglycaemia (Dewick 1997), -thalassemia and sickle cell anemia (Fibach et al. 2003). As suggested mechanism of action, MTM binds to the DNA, as a Mg2+ dimer, which cross-links the two strands (Sastry and Patel 1993; Sastry et al. 1995; Huang et al. 1995), so this action block RNA synthesis, rendering the drug potent anti-tumor activity. However, clinical use of Chromomycin was limited by the side effects (Calabresi and Chabner 1991). Therefore, we paid more attention to investigate new aureolic acids from marine-derived Streptomyces sp.
∗ Corresponding author. Tel.: +86 25 83271022; fax: +86 25 83271249. ∗∗ Corresponding author at: Department of Marine Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271022; fax: +86 25 83271249. E-mail addresses: [email protected] (T. Xi), [email protected] (Y. Lu). 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2012.06.001
In this paper, a marine-derived actinomycete was identified as Streptomyces sp. WBF16 by morphological, biochemical, genetic techniques and 16S rRNA analysis. And its secondary metabolites including one new Chromomycin derivatives (1) (Fig. 3), together with the known Chromomycin A2 (2) and A3 (3) were elucidated by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. 2. Material and methods 2.1. Experiment materials NMR, Bruker DRX-500; HREIMS, Mariner ESI spectrometer. Sephadex LH-20 (Amersham Pharmacia Biotech Co., Ltd, America). HPLC was performed using a JASCO2000 series instrument equipped with an UV-2075 detector and an YMC-Pack ODS-A (250 mm × 20 mm) column. 2.2. Sample collection Samples were collected from sea sediments located in Bijiatuan (37◦ 32 N, 122◦ 03 E), in the city of Weihai, China. They were collected and kept in the box with ice until transported to laboratory for further isolation. 2.3. Biochemical and physiological characterization The morphological characteristics of the isolate were examined according to the methods reported by Shirling for the International Streptomycete Project (Shirling and Gottlieb 1966). Cultural
J. Lu et al. / Microbiological Research 167 (2012) 590–595
characters of the strain were studied on International Streptomyces Project (ISP) media including YMD agar (ISP-2), oat meal agar (ISP3), starch inorganic salt agar (ISP-4), glycerol aspar agine salts agar (ISP-5), peptone yeast extract iron agar (ISP-6), tyrosine agar (ISP-7) and non-ISP media like Gause-agar and Waksman No. 2 (Waksman 1961). The utilization of carbon sources was determined by referring the method of Gottlieb (1961). The strain’s tolerance of NaCl, the ability to produce H2 S and different enzymes were obtained by standard protocols (Holding and Collee 1971). Cell wall composition was analyzed by the method of Lechevalier and Lechevalier (1970), using thin layer chromatography plates as described by Staneck and Roberts (1974).
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Table 1 Cultural characteristics of actinobaterium strain WBF-16. Culture media
Growth
Pigment production
a
Good Good Good Good Good Good Good Good
–b – – – Light yellow Dark yellow Dark yellow –
ISP-2 ISP-3 ISP-4 ISP-5 ISP-6 ISP-7 Gause-agar Waksman No. 2 a b
International Streptomyces Project Media. No production.
2.7. Cytotoxicity assays 2.4. Phylogenetic analysis of the strain Genomic DNA of the strain was extracted by the method according to Weisburg et al. (1991) and Chun and Goodfellow (1995). 16S rRNA gene was amplified with primers forward (5 -CGGAGAGTTTGATCCTGGCTCAG-3 ) and reverse (5 AAAGGAGGTGATCCA GCCGCA-3 ). The conditions for thermal cycling were as follows: denaturation of the target DNA at 94 ◦ C for 5 min followed by 30 cycles at 94 ◦ C for 45 s, primer annealing at 55 ◦ C for 45 s, and primer extension at 72 ◦ C for 10 min. The fragments were separated on 1% agarose gel, eluted and purified by the Clean-up gene kit (U-gene, China). The obtained PCR product was sequenced by the ABI 3730XL Genetic Analyzer (Applied Biosystems, USA). The sequence was compared for similarity with the reference species of bacteria contained in genomic database banks, using the NCBI BLAST program. The sequences were aligned via Clustal X 2.1 software (Thompson et al. 1997). The phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei 1987). The statistical significance of the tree was obtained by bootstrap analysis of sequence data with help of MEGA version 4.1 (Kumar et al. 2001). 2.5. Fermentation, extraction, purification and structural elucidation of bioactive metabolites The marine actinomycete WBF16 was cultivated in 0.5 L conical flasks containing 0.3 L of soy medium (20 g soluble starch, 20 g soybean powder, 1.0 g KNO3 , 25 g sea salt) for 7 days and shaken at 200 rpm at 28 ◦ C. After cultivation, fermentation broth was concentrated and added to macroporous resin to adsorb the organic products. The resin was eluted with deionized water–30% ethanol/water–100% ethanol/water in turn. The ethanol soluble fraction was dried in vacuo to yield 10 g of extract. The crude extract was dissolved in methanol and chromatographed on a silica gel column using CHCl3 /MeOH (gradient 20:1, 10:1, and 2:1) to deliver 3 fractions Fr. I–Fr. III. Fr. II was purified on Sephadex LH-20 using MeOH to afford two sub-fractions (Fr. IIa and Fr. IIb). Fr. IIa was purified by reversed phase HPLC (250 mm × 20.0 mm, 8 mL/min) using 48% CH3 CN (0.02% Formic acid) to afford Chromomycin A2 (2, 200 mg, tR = 47 min) and Chromomycin A3 (3, 90 mg, tR = 24 min). Fr. IIb was purified with the HPLC using 90% methanol (0.02% Formic acid) to afford compound 1 (12 mg, 40 min).
Human gastric cancer cell line (SGC7901), human liver hepatocellular carcinoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549), human colon cancer cell line (HCT116), Human ovarian cancer cell line (COC1) and Human Umbilical Vein Endothelial Cells (HUVEC) were maintained in RPMI-1640 medium with 5% (v/v) fetal bovine serum and cultured in 96 well microtiter plates. Appropriate dilutions of 1 were added into wells and the cells cultured in a humidified environment in the presence of 5% CO2 at 37 ◦ C for 72 h. The CCK-8 method was used to evaluate the survival rates of the cancer cell (Ma et al. 2007). The final concentration of compounds 1–3 that gave 50% inhibition of cell growth was expressed as the IC50 values. Results were expressed as the mean value of triplicate determinations. 3. Results 3.1. Taxonomy of the strain Cultural, morphological and physiological characteristics of WBF16 from marine sediments were studied according to the standard procedures. Table 1 depicts the cultural characteristics of strain WBF16. It exhibited good growth on ISP-2, ISP-3, ISP-4, ISP5, ISP-6, ISP-7, Gause-agar and Waksman No. 2 media. The color of the aerial mycelium appeared light yellow (see Supplementary Data File I), while that of substrate mycelium went from light yellow to dark gray (see Supplementary Data File II). The strain produced reddish dark yellow pigment on ISP-7 and Gause-agar and exhibited light yellow pigmentation on ISP-6. Fig. 1 shows the sporophor morphology of the strain was a spiral type, and it may
2.6. Chromomycin B (1) Yellow amorphous powder; 1 H NMR (500 MHz, CDCl3 ) see Table 3, 13 C NMR (125 MHz, CDCl3 ) see Table 3; ESI-MS [M−H]− m/z 1152, HRESIMS [M−H]− m/z 1151.5239 (calcd for [C57 H84 O24 −H]− , 1151.5274)
Fig. 1. Scanning electron micrograph of sporotrichial and spore of Streptomyces sp. WBF16.
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J. Lu et al. / Microbiological Research 167 (2012) 590–595
Fig. 2. Neighbor-joining tree based on the nearly complete sequences of WBF-16 (in bold) observed in this study and closely related sequences obtained from an NCBI BLAST search. GenBank accession numbers are given in parentheses. Bootstrap values calculated from 5000 resamplings using the neighbor-joining method are shown at the nodes for values of ≥50%. The scale bar corresponds to 0.02 substitutions per nucleotide position. The genetic distance was calculated with Kimura’s two-parameter model.
J. Lu et al. / Microbiological Research 167 (2012) 590–595
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Fig. 3. Structures of compounds 1–3.
Table 2 Physiological and biochemical characteristics of Streptomyces sp. WBF-16. Utilization of carbon sources
Production of P W P W P P P Up to 8%
d-Lacrose d-Xylose d-Glucose d-Fructose Maltose Sucrose Mannose Tolerance to NaCl
Melanoid pigments Amylase Catalase Nitrate reductase Protease H2 S
+ − + − + +
P, positive; W, weak; +, positive result; −, negative result.
be placed in the genus Streptomyces. The physiological and biochemical characteristics of the strain were presented in Table 2. d-Lacrose, d-glucose, Maltose, Sucrose and Mannose can be utilized by the strain as carbon sources. It was noticed that production of melanoid pigments on ISP-7 medium and enzymes such as Catalase and Protease. It also can produce H2 S. Furthermore it exhibited salt
tolerance up to 8% that indicated it may be placed in the intermediate group of salt tolerance (Tresner et al. 1968). The phylogenetic position of Strptomyces sp. WBF16 was established by amplifying the 16S rRNA region and the sequence of the strain (1522 bases) was analyses by BlAST analysis. The 16S rRNA genome sequence of the strain showed 99% identity with that of S. coelicolor A3 (2) (Fig. 2); hence, the strain was assigned to the S. coelicolor A3 (2) cluster and the 16S rRNA sequence was submitted to the NCBI Genbank with accession number HQ848084.
3.2. Structure elucidation of compounds 1–3 Compound 1 was obtained as yellow amorphous powder. Its molecular formula was assigned as C57 H84 O24 on the basis of HR-ESIMS data [found m/z 1151.5239, calcd for [C57 H84 O24 −H]− 1151.5274]. 1 H NMR spectrum 1 resembled that of Chromomycin compounds, with two distinctive aromatic protons at ı 6.76 (1H, s) and 6.66 (1H, s) and five anomeric protons from ı 4.59
Table 3 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data of compound 1. Position
ıC
1 2 3 4 5 6 7 8 9 10 4a 8a 9a 10a 7-CH3 1 2 3 1 -OCH3 Sugar A A1 A2 A3 A4 A5 A6 CH3 –CO CH3 –CO Sugar B B1 B2
202.3 76.1 42.4 27.4 100.8 159.7 111.8 165.3 156.2 116.8 134.7 108.2 108.2 138.3 8.2 80.1 70.1 20.5 59.4
o, overlap; m, multiple.
ıH 4.71, d (6.1) 2.59, m 3.00, d (14.6); 2.53 m 6.66, s
6.76, s
2.19, s 5.00 br d (9.2) 4.01 o 1.28 d (6.7) 3.41, s
97.5 32.9 70.0 67.3 69.8 16.8 20.8 170.8
5.21, dd (9.7, 2.2) 2.23, m; 2.08, m 4.04, m 5.23, d (2.6) 3.82, br q (6.0) 1.29, d (6.0) 2.17, s
95.3 33.5
5.12, br s 1.76, m; 1.64, m
Position
ıC
ıH
B3 B4 B5 B6 B4-OCH3 Sugar C C1 C2 C3 C4 C5 C6 Sugar D D1 D2 D3 D4 D5 D6 Sugar E E1 E2 E3 E4 E5 E6 E3-CH3 (CH3 )2 CH–CO (CH3 )2 CH–CO (CH3 )2 CH–CO
65.9 82.3 67.0 17.8 62.3
3.97, m 3.61, d (2.4) 3.99, br q (6.2) 1.36, d (6.2) 3.60, s
100.3 37.3 81.5 75.3 72.1 18.0
5.07, dd (10.3, 2.0) 2.48, m; 1.60, m 3.22, m 3.12, o 3.39, m 1.23, d (6.2)
99.7 37.1 80.8 75.1 72.2 17.8
4.59, dd (9.2, 1.6) 2.39, o; 1.70, o 3.51, m 3.12, o 3.31, m 1.38, d (6.3)
97.1 43.8 70.6 79.5 66.6 17.2 23.01 177.5 34.3 18.9
5.03, br s 1.91, m – 4.61, d (10.3) 3.88, m 1.30, d (6.3) 1.35, s – 2.64, m 1.21, d (2.0)
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Fig. 4. Key HMBC correlations of compounds 1.
Table 4 IC50 values (M) of compounds 1–3 tested in vitro for cytotoxicity. Compound
SGC7901
1 2 3 Cisplatin Doxorubicin
0.002 0.004 0.004 0.004 0.076
± ± ± ± ±
0.0001 0.0002 0.0002 0.0007 0.0074
HepG2 0.007 0.0005 0.1 0.003 0.084
A549 ± ± ± ± ±
0.0004 0.00003 0.006 0.0004 0.004
0.03 0.003 0.003 0.03 0.020
HCT116 ± ± ± ± ±
0.002 0.0002 0.0002 0.003 0.001
0.007 0.005 0.02 0.01 0.067
± ± ± ± ±
COC1 0.0004 0.0003 0.001 0.002 0.003
0.008 0.005 0.002 0.02 0.01
HUVEC ± ± ± ± ±
0.0004 0.0002 0.0001 0.001 0.001
0.01 0.008 0.005 0.02 0.01
± ± ± ± ±
0.001 0.0002 0.0001 0.001 0.002
IC50: 50% inhibitory concentration; the data show represent means ± S.E.M. of three replicates.
(1H, dd, J1 = 9.2 Hz, J2 = 1.6 Hz), 5.03 (1H, br s), 5.07 (1H, dd, J1 = 10.3 Hz, J2 = 2.0 Hz), 5.12 (1H, br s), and 5.21 (1H, dd, J1 = 9.2 Hz, J2 = 1.6 Hz) (Table 3). In the 13 C NMR spectrum, totally 57 signals were observed. Comparison of the 1 H and 13 C NMR spectra of 1 with those of Chromomycin A2 (2), showed considerable structural similarity and indicated that there have a different side-chain substituted on C-3 of aglycon. Based on HSQC, COSY relations and the difference in molecular formula, we deduced that the pentyl sidechain of the Chromomycin A2 had been truncated to three carbons. In the HMBC spectrum (Fig. 4), the side-chain were further evidenced by the correlations from H-3 (ıH 1.28) to C-2 (ıC 70.1), from H-2 (ıH 4.01) to C-3 (ıC 42.4), respectively. In addition, its sugar residues attachments were unambiguously assigned by the HMBC experiment (Fig. 4). The known compounds Chromomycin A2 and Chromomycin A3 were isolated from fermentation extracts and identified by comparison of NMR data with those in the literatures (Miyamoto et al. 1967; Tsuyoshi et al. 1990). 3.3. Cytotoxicity assays Three isolated compounds Chromomycin B (1), Chromomycin A2 (2) and Chromomycin A3 (3) were tested for cytotoxic activity against SGC7901, HepG2, A549, HCT116, COC1 and HUVEC. Based on IC50 values from Table 4, we can see that compounds 1–3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1. Meanwhile, Chromomycin B against non-tumor cell HUVEC also showed significant cytotoxicity. The results of cisplatin and doxorubicin as controls are also listed in Table 4.
And a new aureolic acid Chromomycin B (a new aureolic acid) with strong anti-tumor activity was isolated from marine-derived Streptomyces sp. in this paper. According to previously report (Lo Giudice et al. 2007; Hu et al. 2011), a series of Chromomycin analogs such as Chromomycin SA3 , Chromomycin SA3 , Chromomycin SA and aburamycin which show strong anti-tumor activities were isolated from marine-derived Streptomyces sp. (Hu et al. 2011). Therefore, the marine-derived actinomyces acts as a prominent reservoir for aureolic acid family and continuing to be a source of new aureolic acids. And the new Chromomycins might be synthesized by different biosynthetic pathway or ketoreductase process, which will be great interest for future attempts to generate new biosynthetic pathway or enzymatic process. Acknowledgments This study was supported by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGQ201102) and National Nature Science Foundation of China for Young Scientists (No. 81001395). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2012.06.001. References
4. Discussion Aureolic acids are one of the most important bioactive metabolites. According to earlier reports, most of the aureolic acid antibiotics were produced by terrestrial actinomyces (Watve et al. 2001).
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J. Lu et al. / Microbiological Research 167 (2012) 590–595 Dewick PM. Medicinal natural products: a biosynthetic approach. New York: John Wiley and Sons, Inc; 1997. p. 123–4. Elias EG, Evans JT. Mithramycin in the treatment of Paget’s disease of bone. Bone Joint Surg 1972;54:1730–6. Fibach E, Bianchi N, Borgatti M, Prus E, Gambari R. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood 2003;102:1276–81. Gottlieb D. An evolution of criteria and procedures used in the description and characterization of Streptomyces, a co-operative study. Appl Microbiol 1961;9:55–60. Holding AJ, Collee JG. Routine biochemical tests. Methods Microbiol 1971;6A:1–31. Huang HW, Li D, Cowan JA. Biostructural chemistry of magnesium regulation of mithramycin-DNA interactions by Mg2+ coordination. Biochimie 1995;77:729–38. Hu YC, Ewpindola PDM, Stewart NA, Wei SG, Posner BA, Macmillan JB. Chromomycin SA analogs from marine-derived Streptomyces sp. Bioorg Med Chem 2011;19:5183–9. Jia Z, Zhang J, Wei D, Wang L, Yuan P, Le X, et al. Molecular basis of the synergistic antiangiogenic activity of bevacizumab and mithramycin A. Cancer Res 2007;67:4878–85. Katahira R, Uosaki Y, Ogawa H, Yamashita Y, Nakano H, Yoshida M. UCH9, a new antitumor antibiotic produced by Streptomyces. II. Structure elucidation of UCH9 by mass and NMR spectroscopy. J Antibiot 1998;51:267–74. Kennedy BJ, Yarbro JW, Kickertz V, Sandberg-Wollheim M. Effect of mithramycin on a mouse glioma. Cancer Res 1968;28:91–7. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA 2: molecular evolutionary genetics analysis software. Bioinformatics 2001;17:1244–5. Lechevalier HA, Lechevalier MP. A critical evaluation of the genera of aerobic actinomycetes. In: Prauser H, editor. The actinomycetales. Jena: Gustav Fischer Verlag; 1970. p. 393–405. Lo Giudice A, Bruni V, Michaud L. Characterization of Antarctic psychrotrophic bacteria with antibacterial activities against terrestrial microorganisms. J Basic Microbiol 2007;47:496–505. Lombó F, Menéndez N, Salas JA, Méndez C. The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis, and novel derivatives. Appl Microbiol Biotechnol 2006;73:1–14. Miyamoto M, Kawamatsu Y, Kawashima K, Shinohara M, Tanaka K, Tatsuoka S. Chromomycin A2 , A3 and A4 . Tetrabedron 1967;23:421–37. Ma XC, Cui J, Zheng J, Guo DA. Microbial transformation of three bufadienolides by Penicillum aurentigriseum and its application for metabolite identification in rats. J Mol Catal B: Enzym 2007;48:42–50.
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Ogawa H, Yamashita Y, Katahira R, Chiba S, Iwasaki T, Ashizawa T, et al. UCH9, a new antitumor antibiotic produced by Streptomyces: I. Producing organism, fermentation, isolation and biological activities. J Antibiot 1998;51:261–6. Remers WA. In the chemistry of antitumor antibiotics, vol. 2. Wiley-Interscience; 1979a. p. 133–75. Remers WA, Iyengar BS. In cancer chemotherapeutic agents. Am Chem Soc 1995:577–679. Remers WA. The chemistry of antitumor antibiotics, vol. 1. Wiley-Interscience; 1979b, 1133–41. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–25. Sastry M, Fiala R, Patel D. Solution structure of mithramycin dimers bound to partially overlapping sites on DNA. J Mol Biol 1995;251:674–89. Sastry M, Patel D. Solution structure of the mithramycin dimer–DNA complex. Biochemistry 1993;32:6588–604. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacteriol 1966;16:313–40. Skarbek JD, Speedie MK. In antitumor compounds of natural origin. Chem Biochem 1981;1:191–235. Staneck JL, Roberts GD. Simplified approach to identification of aerobic actinomycetes by thinlayer chromatography. Appl Microbiol 1974;28:226–31. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876–82. Tresner HD, Hayes JA, Backus EJ. Differential tolerance of streptomycetes to sodium chloride as a taxonomic aid. Appl Microbiol 1968;16:1134–6. Tsuyoshi K, Tomomi H, Kazuo F, Mochizuki J, Hiroshi N, Yoichi H, et al. Isolation and structures of mono- and di-deacetyl chromomycin antibiotics 02-3D and 02-3G from Streptomyces avellaneus. J Antibiot 1990;8:110–3. Waksman SA. The Actinomycetes, Classification, Identification and Description of Genera and Species, vol. 2. Baltimore: The Williams and Wilkins Company; 1961. pp. 61–292. Watve MG, Tickoo R, Jog MM, Bhole BD. How many antibiotics are produced by the genus Streptomyces. Arch Microbiol 2001;176:386–90. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697–703. ˝ Wohlert SE, Kunzel ES, Chatterjee K, Zaman H, Ryu A, Conforto A, et al. Sequenceselective DNA binding drugs mithramycin A and Chromomycin A3 are potent inhibitors of neuronal apoptosis induced by oxidative stress and DNA damage in cortical neurons. Ann Neurol 2001;49:345–54.
Contents lists available at SciVerse ScienceDirect
Microbiological Research journal homepage: www.elsevier.de/micres
Aureolic acids from a marine-derived Streptomyces sp. WBF16 Jiansheng Lu a,b , Yihua Ma b , Jianjia Liang b , Yingying Xing b , Tao Xi b,∗ , Yuanyuan Lu a,b,∗∗ a b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China Department of Marine Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
a r t i c l e
i n f o
Article history: Received 1 March 2012 Received in revised form 31 May 2012 Accepted 5 June 2012 Keywords: Marine Streptomyces Phylogenetics analysis Secondary metabolites Aureolic acid Chromomycin
a b s t r a c t A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A2 (2) and Chromomycin A3 (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1–3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The aureolic acids are a family of related antitumor antibiotics that include olivomycin A, Chromomycin A2 or A3 , mithramycin, chromocyclomycin and so on (Remers 1979a; Skarbek and Speedie 1981; Remers and Iyengar 1995; Wohlert et al. 2001; Ogawa et al. 1998; Katahira et al. 1998; Lombó et al. 2006). All aureolic acid type compounds (with the exception of chromocyclomycin) contain a tricyclic aglycon with two aliphatic side chains attached at C-3 and C-7. Now, Chromomycin have been used clinically in the treatment of some tumor diseases (Jia et al. 2007; Remers 1979b) such as disseminated embryonal cell carcinoma (Skarbek and Speedie 1981; Kennedy et al. 1968; Elias and Evans 1972), hyperglycaemia (Dewick 1997), -thalassemia and sickle cell anemia (Fibach et al. 2003). As suggested mechanism of action, MTM binds to the DNA, as a Mg2+ dimer, which cross-links the two strands (Sastry and Patel 1993; Sastry et al. 1995; Huang et al. 1995), so this action block RNA synthesis, rendering the drug potent anti-tumor activity. However, clinical use of Chromomycin was limited by the side effects (Calabresi and Chabner 1991). Therefore, we paid more attention to investigate new aureolic acids from marine-derived Streptomyces sp.
∗ Corresponding author. Tel.: +86 25 83271022; fax: +86 25 83271249. ∗∗ Corresponding author at: Department of Marine Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China. Tel.: +86 25 83271022; fax: +86 25 83271249. E-mail addresses: [email protected] (T. Xi), [email protected] (Y. Lu). 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2012.06.001
In this paper, a marine-derived actinomycete was identified as Streptomyces sp. WBF16 by morphological, biochemical, genetic techniques and 16S rRNA analysis. And its secondary metabolites including one new Chromomycin derivatives (1) (Fig. 3), together with the known Chromomycin A2 (2) and A3 (3) were elucidated by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. 2. Material and methods 2.1. Experiment materials NMR, Bruker DRX-500; HREIMS, Mariner ESI spectrometer. Sephadex LH-20 (Amersham Pharmacia Biotech Co., Ltd, America). HPLC was performed using a JASCO2000 series instrument equipped with an UV-2075 detector and an YMC-Pack ODS-A (250 mm × 20 mm) column. 2.2. Sample collection Samples were collected from sea sediments located in Bijiatuan (37◦ 32 N, 122◦ 03 E), in the city of Weihai, China. They were collected and kept in the box with ice until transported to laboratory for further isolation. 2.3. Biochemical and physiological characterization The morphological characteristics of the isolate were examined according to the methods reported by Shirling for the International Streptomycete Project (Shirling and Gottlieb 1966). Cultural
J. Lu et al. / Microbiological Research 167 (2012) 590–595
characters of the strain were studied on International Streptomyces Project (ISP) media including YMD agar (ISP-2), oat meal agar (ISP3), starch inorganic salt agar (ISP-4), glycerol aspar agine salts agar (ISP-5), peptone yeast extract iron agar (ISP-6), tyrosine agar (ISP-7) and non-ISP media like Gause-agar and Waksman No. 2 (Waksman 1961). The utilization of carbon sources was determined by referring the method of Gottlieb (1961). The strain’s tolerance of NaCl, the ability to produce H2 S and different enzymes were obtained by standard protocols (Holding and Collee 1971). Cell wall composition was analyzed by the method of Lechevalier and Lechevalier (1970), using thin layer chromatography plates as described by Staneck and Roberts (1974).
591
Table 1 Cultural characteristics of actinobaterium strain WBF-16. Culture media
Growth
Pigment production
a
Good Good Good Good Good Good Good Good
–b – – – Light yellow Dark yellow Dark yellow –
ISP-2 ISP-3 ISP-4 ISP-5 ISP-6 ISP-7 Gause-agar Waksman No. 2 a b
International Streptomyces Project Media. No production.
2.7. Cytotoxicity assays 2.4. Phylogenetic analysis of the strain Genomic DNA of the strain was extracted by the method according to Weisburg et al. (1991) and Chun and Goodfellow (1995). 16S rRNA gene was amplified with primers forward (5 -CGGAGAGTTTGATCCTGGCTCAG-3 ) and reverse (5 AAAGGAGGTGATCCA GCCGCA-3 ). The conditions for thermal cycling were as follows: denaturation of the target DNA at 94 ◦ C for 5 min followed by 30 cycles at 94 ◦ C for 45 s, primer annealing at 55 ◦ C for 45 s, and primer extension at 72 ◦ C for 10 min. The fragments were separated on 1% agarose gel, eluted and purified by the Clean-up gene kit (U-gene, China). The obtained PCR product was sequenced by the ABI 3730XL Genetic Analyzer (Applied Biosystems, USA). The sequence was compared for similarity with the reference species of bacteria contained in genomic database banks, using the NCBI BLAST program. The sequences were aligned via Clustal X 2.1 software (Thompson et al. 1997). The phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei 1987). The statistical significance of the tree was obtained by bootstrap analysis of sequence data with help of MEGA version 4.1 (Kumar et al. 2001). 2.5. Fermentation, extraction, purification and structural elucidation of bioactive metabolites The marine actinomycete WBF16 was cultivated in 0.5 L conical flasks containing 0.3 L of soy medium (20 g soluble starch, 20 g soybean powder, 1.0 g KNO3 , 25 g sea salt) for 7 days and shaken at 200 rpm at 28 ◦ C. After cultivation, fermentation broth was concentrated and added to macroporous resin to adsorb the organic products. The resin was eluted with deionized water–30% ethanol/water–100% ethanol/water in turn. The ethanol soluble fraction was dried in vacuo to yield 10 g of extract. The crude extract was dissolved in methanol and chromatographed on a silica gel column using CHCl3 /MeOH (gradient 20:1, 10:1, and 2:1) to deliver 3 fractions Fr. I–Fr. III. Fr. II was purified on Sephadex LH-20 using MeOH to afford two sub-fractions (Fr. IIa and Fr. IIb). Fr. IIa was purified by reversed phase HPLC (250 mm × 20.0 mm, 8 mL/min) using 48% CH3 CN (0.02% Formic acid) to afford Chromomycin A2 (2, 200 mg, tR = 47 min) and Chromomycin A3 (3, 90 mg, tR = 24 min). Fr. IIb was purified with the HPLC using 90% methanol (0.02% Formic acid) to afford compound 1 (12 mg, 40 min).
Human gastric cancer cell line (SGC7901), human liver hepatocellular carcinoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549), human colon cancer cell line (HCT116), Human ovarian cancer cell line (COC1) and Human Umbilical Vein Endothelial Cells (HUVEC) were maintained in RPMI-1640 medium with 5% (v/v) fetal bovine serum and cultured in 96 well microtiter plates. Appropriate dilutions of 1 were added into wells and the cells cultured in a humidified environment in the presence of 5% CO2 at 37 ◦ C for 72 h. The CCK-8 method was used to evaluate the survival rates of the cancer cell (Ma et al. 2007). The final concentration of compounds 1–3 that gave 50% inhibition of cell growth was expressed as the IC50 values. Results were expressed as the mean value of triplicate determinations. 3. Results 3.1. Taxonomy of the strain Cultural, morphological and physiological characteristics of WBF16 from marine sediments were studied according to the standard procedures. Table 1 depicts the cultural characteristics of strain WBF16. It exhibited good growth on ISP-2, ISP-3, ISP-4, ISP5, ISP-6, ISP-7, Gause-agar and Waksman No. 2 media. The color of the aerial mycelium appeared light yellow (see Supplementary Data File I), while that of substrate mycelium went from light yellow to dark gray (see Supplementary Data File II). The strain produced reddish dark yellow pigment on ISP-7 and Gause-agar and exhibited light yellow pigmentation on ISP-6. Fig. 1 shows the sporophor morphology of the strain was a spiral type, and it may
2.6. Chromomycin B (1) Yellow amorphous powder; 1 H NMR (500 MHz, CDCl3 ) see Table 3, 13 C NMR (125 MHz, CDCl3 ) see Table 3; ESI-MS [M−H]− m/z 1152, HRESIMS [M−H]− m/z 1151.5239 (calcd for [C57 H84 O24 −H]− , 1151.5274)
Fig. 1. Scanning electron micrograph of sporotrichial and spore of Streptomyces sp. WBF16.
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Fig. 2. Neighbor-joining tree based on the nearly complete sequences of WBF-16 (in bold) observed in this study and closely related sequences obtained from an NCBI BLAST search. GenBank accession numbers are given in parentheses. Bootstrap values calculated from 5000 resamplings using the neighbor-joining method are shown at the nodes for values of ≥50%. The scale bar corresponds to 0.02 substitutions per nucleotide position. The genetic distance was calculated with Kimura’s two-parameter model.
J. Lu et al. / Microbiological Research 167 (2012) 590–595
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Fig. 3. Structures of compounds 1–3.
Table 2 Physiological and biochemical characteristics of Streptomyces sp. WBF-16. Utilization of carbon sources
Production of P W P W P P P Up to 8%
d-Lacrose d-Xylose d-Glucose d-Fructose Maltose Sucrose Mannose Tolerance to NaCl
Melanoid pigments Amylase Catalase Nitrate reductase Protease H2 S
+ − + − + +
P, positive; W, weak; +, positive result; −, negative result.
be placed in the genus Streptomyces. The physiological and biochemical characteristics of the strain were presented in Table 2. d-Lacrose, d-glucose, Maltose, Sucrose and Mannose can be utilized by the strain as carbon sources. It was noticed that production of melanoid pigments on ISP-7 medium and enzymes such as Catalase and Protease. It also can produce H2 S. Furthermore it exhibited salt
tolerance up to 8% that indicated it may be placed in the intermediate group of salt tolerance (Tresner et al. 1968). The phylogenetic position of Strptomyces sp. WBF16 was established by amplifying the 16S rRNA region and the sequence of the strain (1522 bases) was analyses by BlAST analysis. The 16S rRNA genome sequence of the strain showed 99% identity with that of S. coelicolor A3 (2) (Fig. 2); hence, the strain was assigned to the S. coelicolor A3 (2) cluster and the 16S rRNA sequence was submitted to the NCBI Genbank with accession number HQ848084.
3.2. Structure elucidation of compounds 1–3 Compound 1 was obtained as yellow amorphous powder. Its molecular formula was assigned as C57 H84 O24 on the basis of HR-ESIMS data [found m/z 1151.5239, calcd for [C57 H84 O24 −H]− 1151.5274]. 1 H NMR spectrum 1 resembled that of Chromomycin compounds, with two distinctive aromatic protons at ı 6.76 (1H, s) and 6.66 (1H, s) and five anomeric protons from ı 4.59
Table 3 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data of compound 1. Position
ıC
1 2 3 4 5 6 7 8 9 10 4a 8a 9a 10a 7-CH3 1 2 3 1 -OCH3 Sugar A A1 A2 A3 A4 A5 A6 CH3 –CO CH3 –CO Sugar B B1 B2
202.3 76.1 42.4 27.4 100.8 159.7 111.8 165.3 156.2 116.8 134.7 108.2 108.2 138.3 8.2 80.1 70.1 20.5 59.4
o, overlap; m, multiple.
ıH 4.71, d (6.1) 2.59, m 3.00, d (14.6); 2.53 m 6.66, s
6.76, s
2.19, s 5.00 br d (9.2) 4.01 o 1.28 d (6.7) 3.41, s
97.5 32.9 70.0 67.3 69.8 16.8 20.8 170.8
5.21, dd (9.7, 2.2) 2.23, m; 2.08, m 4.04, m 5.23, d (2.6) 3.82, br q (6.0) 1.29, d (6.0) 2.17, s
95.3 33.5
5.12, br s 1.76, m; 1.64, m
Position
ıC
ıH
B3 B4 B5 B6 B4-OCH3 Sugar C C1 C2 C3 C4 C5 C6 Sugar D D1 D2 D3 D4 D5 D6 Sugar E E1 E2 E3 E4 E5 E6 E3-CH3 (CH3 )2 CH–CO (CH3 )2 CH–CO (CH3 )2 CH–CO
65.9 82.3 67.0 17.8 62.3
3.97, m 3.61, d (2.4) 3.99, br q (6.2) 1.36, d (6.2) 3.60, s
100.3 37.3 81.5 75.3 72.1 18.0
5.07, dd (10.3, 2.0) 2.48, m; 1.60, m 3.22, m 3.12, o 3.39, m 1.23, d (6.2)
99.7 37.1 80.8 75.1 72.2 17.8
4.59, dd (9.2, 1.6) 2.39, o; 1.70, o 3.51, m 3.12, o 3.31, m 1.38, d (6.3)
97.1 43.8 70.6 79.5 66.6 17.2 23.01 177.5 34.3 18.9
5.03, br s 1.91, m – 4.61, d (10.3) 3.88, m 1.30, d (6.3) 1.35, s – 2.64, m 1.21, d (2.0)
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Fig. 4. Key HMBC correlations of compounds 1.
Table 4 IC50 values (M) of compounds 1–3 tested in vitro for cytotoxicity. Compound
SGC7901
1 2 3 Cisplatin Doxorubicin
0.002 0.004 0.004 0.004 0.076
± ± ± ± ±
0.0001 0.0002 0.0002 0.0007 0.0074
HepG2 0.007 0.0005 0.1 0.003 0.084
A549 ± ± ± ± ±
0.0004 0.00003 0.006 0.0004 0.004
0.03 0.003 0.003 0.03 0.020
HCT116 ± ± ± ± ±
0.002 0.0002 0.0002 0.003 0.001
0.007 0.005 0.02 0.01 0.067
± ± ± ± ±
COC1 0.0004 0.0003 0.001 0.002 0.003
0.008 0.005 0.002 0.02 0.01
HUVEC ± ± ± ± ±
0.0004 0.0002 0.0001 0.001 0.001
0.01 0.008 0.005 0.02 0.01
± ± ± ± ±
0.001 0.0002 0.0001 0.001 0.002
IC50: 50% inhibitory concentration; the data show represent means ± S.E.M. of three replicates.
(1H, dd, J1 = 9.2 Hz, J2 = 1.6 Hz), 5.03 (1H, br s), 5.07 (1H, dd, J1 = 10.3 Hz, J2 = 2.0 Hz), 5.12 (1H, br s), and 5.21 (1H, dd, J1 = 9.2 Hz, J2 = 1.6 Hz) (Table 3). In the 13 C NMR spectrum, totally 57 signals were observed. Comparison of the 1 H and 13 C NMR spectra of 1 with those of Chromomycin A2 (2), showed considerable structural similarity and indicated that there have a different side-chain substituted on C-3 of aglycon. Based on HSQC, COSY relations and the difference in molecular formula, we deduced that the pentyl sidechain of the Chromomycin A2 had been truncated to three carbons. In the HMBC spectrum (Fig. 4), the side-chain were further evidenced by the correlations from H-3 (ıH 1.28) to C-2 (ıC 70.1), from H-2 (ıH 4.01) to C-3 (ıC 42.4), respectively. In addition, its sugar residues attachments were unambiguously assigned by the HMBC experiment (Fig. 4). The known compounds Chromomycin A2 and Chromomycin A3 were isolated from fermentation extracts and identified by comparison of NMR data with those in the literatures (Miyamoto et al. 1967; Tsuyoshi et al. 1990). 3.3. Cytotoxicity assays Three isolated compounds Chromomycin B (1), Chromomycin A2 (2) and Chromomycin A3 (3) were tested for cytotoxic activity against SGC7901, HepG2, A549, HCT116, COC1 and HUVEC. Based on IC50 values from Table 4, we can see that compounds 1–3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1. Meanwhile, Chromomycin B against non-tumor cell HUVEC also showed significant cytotoxicity. The results of cisplatin and doxorubicin as controls are also listed in Table 4.
And a new aureolic acid Chromomycin B (a new aureolic acid) with strong anti-tumor activity was isolated from marine-derived Streptomyces sp. in this paper. According to previously report (Lo Giudice et al. 2007; Hu et al. 2011), a series of Chromomycin analogs such as Chromomycin SA3 , Chromomycin SA3 , Chromomycin SA and aburamycin which show strong anti-tumor activities were isolated from marine-derived Streptomyces sp. (Hu et al. 2011). Therefore, the marine-derived actinomyces acts as a prominent reservoir for aureolic acid family and continuing to be a source of new aureolic acids. And the new Chromomycins might be synthesized by different biosynthetic pathway or ketoreductase process, which will be great interest for future attempts to generate new biosynthetic pathway or enzymatic process. Acknowledgments This study was supported by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGQ201102) and National Nature Science Foundation of China for Young Scientists (No. 81001395). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2012.06.001. References
4. Discussion Aureolic acids are one of the most important bioactive metabolites. According to earlier reports, most of the aureolic acid antibiotics were produced by terrestrial actinomyces (Watve et al. 2001).
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