Activity of p53, a known tumor suppressor gene, is regulated by Mdm2, an E3 ubiquitin ligase.1 Mdm2 binds to the N-terminal transactivation domain of p53, and then Mdm2 mediates the ubiquitination of p53. Finally, ubiquitinated p53 is degraded by the proteasome. Inhibitors of the p53–Mdm2 interaction suppress p53 degradation and potentiate the transcription of p53. Furthermore, these inhibitors induce p53-dependent cell death and suppress osteosarcoma tumor growth in a xenograft model.2, 3, 4, 5, 6 Therefore, inducers of p53-dependent cell death may provide a promising strategy for anticancer therapies.

To discover new compounds that induce p53-dependent cell death, we screened microbial metabolites using glioblastoma LNZTA3 cells in which the expression levels of wild-type p53 can be regulated by a tetracycline-inducible system.7 In our previous report, we isolated five novel compounds that induce p53-dependent cell death, quinofuracins A–E, from fungus Staphylotrichum boninense PF1444.8 Further investigation of microbial metabolites led to the discovery of new anthraquinones, named coccoquinones A (1) and B (2) from another fungus Staphylotrichum coccosporum PF1460 (Figures 1a and b). Here we describe the isolation, structural elucidation and biological properties of 1 and 2, as well as their ability to suppress p53-dependent growth in LNZTA3 cells.

Figure 1
figure 1

Structures and biological activities of coccoquinones A (1) and B (2). (a) Structure of 1. (b) Structure of 2. (c) Key correlations of 1 obtained by 1H-1H COSY and HMBC spectroscopy. (d) Key correlations of 2 obtained by 1H-1H COSY and HMBC spectroscopy. (e) p53-dependent growth suppression of 1. (f) p53-dependent growth suppression of 2.

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The fungal strain PF1460 was isolated from a soil sample collected in Ishigaki Island, Okinawa prefecture, Japan. This strain was deposited as NITE P-02037 at the NITE Patent Microorganisms Depository, Japan. The following media were used to identify strain PF1460: potato dextrose agar, 2% malt agar, oatmeal agar and corn meal agar. The formed colonies were observed during the 1- to 2-week incubation period at 25 °C. The 28S rRNA-D1/D2 and ITS-5.8S rRNA sequences of strain PF1460 were identical to those of Staphylotrichum coccosporum NBRC33272 (100%). Therefore, this strain was identified as Staphylotrichum coccosporum PF1460. The sequence data of S. coccosporum PF1460 were deposited in GenBank as LC061579 and LC061580.

The strain S. coccosporum PF1460 was grown in 100-ml Erlenmeyer flasks, each containing 20 ml of a seed medium comprising 2% soluble starch, 1% glucose, 0.5% polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2% soybean meal and 0.2% CaCO3 in deionized water adjusted to pH 7.0 with NaOH solution before sterilization. The flasks were incubated at 25 °C for 4 days on a rotary shaker at 220 r.p.m. Aliquots of 3 ml of this seed culture were inoculated by spot inoculation into 500-ml Erlenmeyer flasks each containing a solid production medium, which consisted of 2 g of oatmeal and 80 g of water-absorbed brown rice. The flasks were gently shaken and incubated statically at 25 °C for 14 days.

Four-hundred-gram grown solid medium of S. coccosporum PF1460 was extracted with 800 ml of 67% aqueous acetone. The filtrate of the extracts was concentrated under reduced pressure, and the remaining residue was partitioned between EtOAc (400 ml) and H2O (400 ml). The organic layer was concentrated under reduced pressure, and the remaining residue was chromatographed using a silica gel column (Merck, Darmstadt, Germany) with a CHCl3–MeOH solvent system. The active fraction 1 (eluent CHCl3–MeOH=25:1) was purified by reverse-phase HPLC (CapcellPak UG120, φ 20 mm × 250 mm, Shiseido, Tokyo, Japan; 40% aqueous CH3CN containing 0.1% AcOH) to give 1 (6.3 mg). The active fraction 2 (eluent CHCl3–MeOH=10:1) was purified using same method as 1 to give 2 (1.5 mg).

Compound 1 was isolated as a red amorphous solid. The UV spectrum of 1 exhibited absorption maxima at 223, 265, 293, 316 and 455 nm in MeOH, closely resembling that of quinofuracins8 and averantin.9 The IR spectrum of 1 showed absorption maxima at 3200, 2920, 1710, 1620, 1440, 1400, 1320, 1260, 1170, 1095, 1030 and 770 cm−1. The molecular formula of 1 was determined to be C22H20O9 with high resolution ESI MS (HRESI-MS); (found, m/z 429.1182 (M+H)+, calcd for C22H21O9, 429.1180). The 1H and 13C NMR data for 1 are shown in Table 1. Analysis of 13C NMR and DEPT, along with HMQC spectra, revealed the presence of 22 carbons in 1, which were categorized as 2 methyl, 2 methylene, 1 oxymethylene, 1 sp3 methine, 3 sp2 methine, 9 sp2 quaternary, and 4 carbonyl carbons. The structure of 1 was elucidated primarily using 2D NMR experiments (1H-1H COSY and HMBC) as illustrated in Figure 1c. In the HMBC spectrum, correlations between aromatic protons at H-2, H-4, H-6 and a methine proton at H-2′ and relevant carbons in the anthraquinone skeleton suggested that 1 was a penta-substituted anthraquinone moiety. In the 1H-1H COSY spectrum, a spin network was observed from the oxymethylene protons at H-1′ to the methylene protons at H-4′ through one methine proton at H-2′ and one methylene proton at H-3′. In the HMBC spectrum, methyl protons at H-6′ correlated with the methylene carbon at C-4′ and the carbonyl carbon at C-5′, and two methylene protons at H-3′ and H-4′ correlated with the carbonyl carbon at C-5′. Furthermore, acetyl methyl protons (δH 1.99) correlated with the acetyl carbonyl carbon (δC 173.1), and the oxymethylene protons at H-1′ correlated with the acetyl carbonyl carbon, suggesting that an acetyl group bound to the 1′-oxygen. On the basis of these findings, the structure of 1 was closely related to versicolorone10 and paeciloquinones,11 and was determined to be 1′-O-acetylversicolorone (Figure 1a).

Table 1 13C (150 MHz) and 1H (600 MHz) NMR spectroscopic data for 1 and 2 in CD3OD
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Compound 2 was isolated as a red amorphous solid. The UV spectrum of 2 exhibited absorption maxima at 224, 263, 294, 314 and 469 nm in MeOH. The IR spectrum of 2 showed absorption maxima at 3200, 2920, 1715, 1625, 1415, 1400, 1310, 1260, 1170, 1125, 1030 and 770 cm−1. The 1H and 13C NMR data for 2 are shown in Table 1. These spectroscopic data of 2 were similar to those of 1. The molecular formula of 2 was determined to be C22H22O9 by HRESI-MS (found m/z 431.1338 (M+H)+, calcd for C22H23O9, 431.1337), indicating that 2 had two additional hydrogen atoms in comparison with 1. The absence of a carbonyl carbon signal (δC 211.7) and the presence of an oxymethine signal (δC 68.3, δH 3.73) were observed in the NMR spectra of 2, suggesting reduction of a carbonyl carbon at C-5′. Indeed, a spin network was observed from the oxymethylene protons at H-1′ to the methyl protons at H-6′ in the 1H-1H COSY spectrum (Figure 1d). Furthermore, in the HMBC spectrum, methyl protons at H-6′ correlated with the methylene carbon at C-4′ and oxymethine at C-5′, and the methylene protons at H-4′ correlated with the oxymethine carbon at C-5′. Thus, the structure of 2 was determined to be a dihydro derivative of 1 (Figure 1b).

The p53-dependent growth suppression of 1 and 2 was determined using human glioblastoma LNZTA3 cells. These cells contain a wild-type p53-encoding sequence controlled by tetracycline. Wild-type p53 is produced in the absence of tetracycline, whereas, p53 is not produced in the presence of tetracycline. LNZTA3 cells were treated with 1 or 2 in the presence or absence of tetracycline for 72 h, and then the cell viability was determined by MTT assay (Figures 1e and f). Compounds 1 and 2 showed preferential cytotoxicity to LNZTA3 cells expressing p53 compared with LNZTA3 cells not expressing p53, indicating p53-dependent growth suppression in LNZTA3 cells. Further studies of the biological activities of 1 and 2, and the molecular mechanisms of p53 dependency are currently underway.