Anti-glioma Natural Products Downregulating Tumor Glycolytic Enzymes from Marine Actinomycete Streptomyces sp. ZZ406

Marine natural products are important resources for discovering novel anticancer drugs. In this study, an extract prepared from the culture of a sea anemone-derived actinomycete Streptomyces sp. ZZ406 in soluble starch and casein-related liquid medium was found to have activity in inhibiting the proliferation of glioma cells and reducing the production of lactate in glioma cells. Chemical investigation of this active crude extract resulted in the isolation of four new compounds and seven known ones. Structures of the new compounds were determined by a combination of extensive NMR analyses, HRESIMS and MS-MS data, electronic circular dichroism calculation, chemical degradation, and Marfey’s method. New compound 1 showed potent activity against the proliferation of different glioma cells with IC50 values of 4.7 to 8.1 μM, high selectivity index (>12.3 to 21.3), and good stability in human liver microsomes. Western blot analysis revealed that compound 1 remarkably downregulated the expressions of several important glioma glycolytic enzymes. The data from this study suggested that compound 1 might have potential as a novel anti-glioma agent to be further investigated.


Results and Discussion
The strain Streptomyces sp. ZZ406 was a sea anemone-associated actinomycete. Its 16S rDNA gene sequence (Fig. S1, supporting information) completely matched (99% identity for a 1390 bp stretch of sequence) those of several Streptomyces strains including S. fulvissimus DSM 40593, S. pratensis ATCC 33331, S. griseus subsp. griseus NBRC 13350, and S. halstedii NRRL ISP-5068 (Table S1). A large culture (70 L) of strain ZZ406 was conducted in the SC liquid medium. The crude extract prepared from the culture was separated by column chromatography, following by HPLC purification to give compounds 1-11 (Fig. 1).
Compound 1 has a molecular formula C 16 H 10 O 6 deduced from its negative HRESIMS [M − H] − and 13 C-NMR data. Its UV absorption and NMR data suggested that 1 is an anthraquinone derivative 29,30 . The substituted pattern In the HSQC spectrum of 1, a 1 H NMR signal at δ H 5.02 (2 H) was corrected to a 13 C NMR signal at δ C 62.0, suggesting the presence of an oxymethylene group. In addition, in the 1 H NMR spectrum of 1, a low field singlet at δ H 12.89 confirmed the presence of a chelated hydroxy group. The 13 C NMR spectrum of 1 displayed 16 carbon signals, of which 14 were assigned to the anthraquinone backbone, one to the oxymethylene group, and the remaining one to a carboxylic group. As depicted in Fig. 2, HMBC correlations determined the oxymethylene group at C-1 and the carboxylic group at C-3. The 13 C and 1 H assignments of 1 was made based on the 1 H-1 H COSY, HSQC, and HMBC correlations. The structure of 1 was elucidated as 1-hydroxymethyl-8-hydroxy-anthraquinone-3-carboxylic acid, a new anthraquinone. The molecular formula C 16 H 16 O 6 of 2 was determined according to its positive HRESIMS and 13 C NMR data. The 13 C NMR spectrum showed 16 signals for three carbonyls, eight aromatic carbons, one oxymethine, three methylenes, and one methyl. Further HSQC and HMBC spectroscopic analyses indicated that the structural backbone of 2 is 2-methylchromone 33 , which resonated at δ C 165.4 (C, C-2), 110.8 (CH, C-3), 178.4 (C, C-4), 121.0 (C, C-4a), 136.6 (C, C-5), 128.9 (CH, C-6), 132.9 (CH, C-7), 117.1 (CH, C-8), 157.2 (C, C-8a), 19.6 (CH 3 , C-9) and δ H 6.11 ( (3 H, s, H-9). In addition, the 13 C NMR signals at δ C 48.8 (C-1′), 205.7 (C-2′), 50.6 (C-3′), 64.7 (C-4′), 43.3 (C-5′), 175.6 (C-6′) and the 1  were also observed. These NMR signals were contributed to the side chain of 2′-oxo-4′-hydroxy-hexanoic acid, which was confirmed by HSQC, 1 H-1 H COSY, and HMBC correlations (Fig. 2). The absolute configuration of C-4′ was established by theoretical electronic circular dichroism (ECD) calculation. Conformational analyses were carried out by random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 2.5 kcal/mol 36 . The results showed six lowest energy conformers (2-1 C to 2-6 C, Fig. S60) for R-2 whose relative energy is within 2.5 kcal/mol. Subsequently, the conformers were re-optimized using the DFT method at the B3LYP/6-31 + G(d) level in the gas phase by the GAUSSIAN 09 program (Table S6) 37 . The energies, oscillator strengths, and rotational strengths (velocity) (Tables S7-S12) of the first 60 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311++ G(d,p) level in vacuum. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, σ = 0.25 eV) 38 . To obtain the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). Through comparison of the calculated ECD spectra with the experimental spectrum (Fig. 3), the absolute configuration at C-4′ of 2 was assigned to be R. The 13 C and 1 H NMR signals were assigned using 1 H-1 H COSY, HSQC, and HMBC correlations (Fig. 2). Based on the foregoing evidence, the structure of 2 was established as 5-(2′-oxo-4′R-hydroxy-hexanoic acid)-2-methyl-chromone, named as phaeochromycin I. The planer structure of compound 2 with CAS registry number 1796973-58-4 was recorded in the SciFinder database. However, no NMR data, stereochemistry assignment, physical and chemical properties, or even reference was provided for this synthesized compound. Therefore, compound 2 was designated as a new compound, at least a new natural product.
Compound 3 was obtained as a colorless amorphous powder. Its molecular formula of C 14 (Fig. 2) demonstrated that compound 3 composed of three amino acids of N-acetyl-leucine, serine, and alanine. The sequence of the three amino acids was established to be N-acetyl-leucine-serine-alanine based on the HMBC correlations as depicted in Fig. 2. In order to assign the absolute configuration, compound 3 was hydrolyzed by hydrochloric acid to release three free amino acids of l-leucine, l-serine, and l-alanine, which were confirmed by Marfey's method using standard amino acids as references. The retention times were found to be 7.67 min for l-serine-FDAA, 10.87 min for l-alanine-FDAA, and 17.69 min for l-leucine-FDAA (Fig. S47). Therefore, the structure of 3 was determined as N-acetyl-l-leucine-l-serine-l-alanine, a new linear peptide.
Compound 4 was isolated as a colorless amorphous powder and has a molecular formula of C 10 H 16 N 2 O 4 deduced from its HRESIMS [M + H] + and [M + Na] + ions and 13 C NMR data. Analyses of 1 H, 13 C, and HSQC NMR spectra indicated that 4 contained three carbonyls, two methines, two methylenes, and three methyls. An acetyl in 4 was easily recognized by a HMBC correlation of H-2′ (δ H 2.18, 3 H, s) with C-1′ (δ C 158.3) and located at N-1 position as H-5 (δ H 3.73, 2 H, br s) had a HMBC correlation with C-1′. The presence of a 2-methyl-1-oxopropyl group at N-2 was indicated by its NMR signals at δ C 175.3 (C-1″), 33.2 (C-2″), 18.6  . In order to assign the absolute configuration of C-4, theoretical ECD calculation was conducted for 4. The results showed that the different orientations of the two substitutes at N-1 and N-2 had significant effects on the calculated ECD spectra of 4. In the case where the chiral center at C-4 is constant, the calculated ECD spectra of 4 were completely opposite when the two substitutes at N-1 and N-2 positioned at up and down or down and up. Therefore, the configuration of C-4 in 4 was not able to be determined in this study. Based on the above NMR spectroscopic analyses, in combination with the HRESIMS and MS-MS data, the structure of 4 was identified as a new pyrazolidine derivative, 1-acetyl-2-isobutyrylpyrazolidine-4-carboxylic acid. Although pyrazolidine derivatives such as 4a 39 and 4b 40 were previously reported by synthesis, this type of pyrazolidines just like 4 was found from natural resources for the first time.
New compounds 1-4 were tested for their activity in inhibiting the proliferation of human glioma U87MG, U251 and SHG44 cells by SRB assay. Doxorubicin (DOX, a chemotherapeutic drug) was used as a positive control. It has been found that 1 had potent activity against different glioma cells with IC 50 values in a range of 4.7 to   Table 1). The control DOX had antiproliferative activity with IC 50 values of 1.9-9.6 μM. Unfortunately, new compounds 3 and 4 were inactive. Because new compound 1 is an anthraquinone with good activity in suppressing the proliferation of glioma cells, its analogues 5 and 6 were also assayed for their activity against glioma. The results showed that both known anthraquinones 5 and 6 also had anti-glioma activity with IC 50 values of 0.5 to 3.0 μM for 5 and 10.4 to 36.3 μM for 6. A marine anthraquinone SZ-685C 41 and herbal anthraquinones aloe emodin 42 and rhein 43 were reported to have activity against glioma, suggesting that these simple anthraquinones are sensitive to glioma cells. The cytotoxicity (CC 50 ) of the two active compounds 1 and 2 towards normal human astrocytes (HA) was also assayed. The results (Table 1) indicated that 1 had much higher selectivity index (CC 50 /IC 50 , >12.3 to 21.3) than 2 (>3.8 to 4.6) and DOX (0.9 to 4.6).
The active compounds 1 and 2 were further assayed for their effects on the expression levels of important tumor glycolytic enzymes (regulators) of HK2, PFKFB3, PKM2, and LDH5. Previous study demonstrated that these four tumor metabolic regulators were highly expressed in U87MG cells 27 . Therefore, the effects of 1 and 2 on the expressions of these regulators in U87MG cells were evaluated. 2-Deoxyglucose (2-DG, a hexokinase inhibitor) 8 , was used as a positive control. The U87MG cells were treated by 1 (30.0 μM), 2 (60.0 μM), or 2-DG (6.0 mM) for 48 h. Protein prepared from the compound-treated U87MG cells was subjected to western blot analysis. As shown in Fig. 5, both 1 and 2 clearly reduced the expression levels of HK2, PFKFB3, PKM2, and LDH5. The full-length blots of Fig. 5 were provided as supplementary information (Fig. S61).
Because compound 1 showed potent activity against glioma cells with very high selectivity index and unique anti-glioma mechanism, suggesting 1 might have potential as an anti-glioma agent. Therefore, it is interesting to understand the stability of compound 1 in human liver microsomes. Human liver microsomes were incubated with compound 1 at 37 °C in a water bath and the concentration of 1 at designed time points of 0, 15, 30, 60, 90, 120, 180, 240, and 360 min was determined by HPLC analysis. The results ( Table 2    It is well known that many microbial secondary metabolic biosynthetic gene cluster are silent under common cultural conditions and different methods are used to activate the cryptic gene cluster to obtain novel secondary metabolites 44,45 . The easiest and most common method is OSMAC (One Strain, Many Compounds), which uses different cultural media to induce different silent gene cluster to express and then produce different novel bioactive natural products 45 . In this study, ten different media were applied to culture strain ZZ406. A crude extract prepared from the culture of strain ZZ406 in the SC liquid medium showed more secondary metabolites by HPLC analysis and had the strongest activity in suppressing the proliferation of human glioma cells and in reducing the production of lactate. Chemical investigation of this crude active extract led to the isolation and structural elucidation of four new compounds (1-4) with different structural classes. The results from this study further supported the OSMAC strategy for the discovery of novel microbial natural products.
New compounds 1 showed potent activity in inhibiting the proliferation of human U251, U87MG, and SHG44 glioma cells with less cytotoxicity towards normal HA and good stability in human liver microsomes. Compound 1 also remarkably downregulated the expressions of several important tumor glycolytic enzymes. The data suggested that targeting multiple glioma metabolic enzymes might be one of the anti-glioma mechanisms of 1. Full understanding of this mechanism of action and the anti-glioma effect of 1 in animal models need to be further explored.
Our previous studies 23,27,28 proposed that the in vitro SRB screening assay combined with the detection of lactate production might be a new way to discover novel anti-glioma compounds from marine actinomycetes with unique mechanism by regulating multiple tumor metabolic enzymes. The data from this study further support this proposal.

Methods
General experimental procedures. UV spectra were recorded on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China). Optical rotation and ECD spectra were measured on a JASCO DIP-370 digital polarimeter and a JASCO J715 spectropolarimeter (JASCO, Japan), respectively. NMR spectra were acquired on a Bruker 500 spectrometer using standard pulse programs and acquisition parameters and chemical shifts were expressed in δ (ppm). HRESIMS data were obtained from an Agilent 6230 TOF LC/ MS spectrometer. Diaion HP-20 (Mitsubishi Chemical, Japan), silica gel (100-200 mesh, Qingdao Marine Chemical Co. Ltd., China), and octadecyl-functionalized silica gel (ODS, Cosmosil 75C 18 Prep, Nacalai Tesque Inc., Japan) were used for column chromatography. HPLC separation was performed on a CXTH LC-3000 prepared HPLC system (Beijing Chuangxintongheng Science & Technology Co. Ltd., China) using a Fuji-C 18 CT-30 column (280 × 30 mm, 10 μm) or an Agilent 1260 HPLC system using an Agilent Zorbax SB-C 18 column (250 × 9.4 mm, 5 μm). All solvents used in this study were purchased from the Sinopharm Chemical Reagent Co.   Isolation of strain ZZ406. Fresh sea anemone H. lineata (5.0 g) was washed with sterile sea water three times and ground into homogenate as 10 −1 g/mL suspension in sterile sea water. The 10 −1 g/mL suspension was diluted to be 10 −2 , 10 −3 , 10 −4 g/mL stepwisely. Each diluted suspension (200 μL) was covered on the surface of Gauze's solid medium in petri dish and incubated at 28 °C for five days. The single colony was picked with sterile needles and transferred to a Gauze's agar plate. After another five days of growth at 28 °C, the single colony (strain ZZ406, Fig. S62) from 10 −2 g/mL suspension that grew well was transferred onto Gauze's agar slants, which were stored at 4 °C until use. Preparation of crude extract for bioactive assay. The procedure of the preparation of crude extract for bioactive assay was described in the previous publication 26 . The crude extract was made to a concentration of 1.0 mg/mL. Sulforhodamine B (SRB) assay was used to evaluate the activity of the crude extract against the proliferation of glioma U87MG cells.

Lactate measurement.
The lactate level in the extracted-treated U87MG cells was measured using a commercial lactate assay kit according to the manufacturer's protocols. Briefly, 100 μL U87MG cells (3000/well) were treated with 100 μL extract solution (1 mg/mL) for 72 h. The extract-treated U87-MG cells (2.5 μL) in each well of the 96-well plate were taken into the corresponding well of a new 96-well plate. The enzyme working solution (50 μL) and chromogenic agent (10 μL) from the lactate assay kit were added into each well. The reaction mixture was incubated at 37 °C for 10 min, added 100 μL stop buffer to terminate the reaction, and then measured at 550 nm on a microplate reader. The lactate content was calculated based on the regression equation (standard curve). The alternation (100%) of lactate in the extract-treated U87MG cells was compared to that in the U87MG cells by the treatment of DMSO (CON), which was 100%.
Large culture of strain ZZ406. Strain ZZ406 from the Gauze's agar slant was refreshed on the plates of Gauze's agar at 28 °C for six days. The pure colonies of ZZ406 were inoculated in eight Erlenmeyer flasks (500 mL) each containing 250 mL Gauze's liquid medium. The flask cultures were incubated at 28 °C on a rotary shaker at 180 rpm for five days to produce seed broth. The spore seed broth (5 mL) was inoculated into a Erlenmeyer flask (500 mL), containing 250 mL of SC liquid medium (soluble starch 10 g, casein 0.3 g, KNO 3 2 g, MgSO 4 •7H 2 O 0.5 g, K 2 HPO 4 2 g, CaCO 3 0.02 g, FeSO 4 •7H 2 O 0.01 g, sea water 1.0 L, pH 7.2). All flasks were incubated at 28 °C for 13 days on a rotary shaker at 180 rpm. A total of 70 L culture was prepared for this study.
Extraction and isolation of compounds 1-11. The 70 L culture of strain ZZ406 was filtered into filtrate and mycelia. The filtrate was applied to a HP-20 column eluting with water and then 100% MeOH to obtain MeOH fraction. The mycelia were extracted with MeOH three times to give MeOH extract. The mixture of MeOH fraction and MeOH extract was concentrated under the reduced pressure to afford part A. The part A was successively partitioned with EtOAc and n-BuOH to give part B and part C after removal of the organic solvent. Part B was fractioned by a silica gel column with gradient elution of cyclohexane and EtOAc to give fractions B 1 -B 9 based on the results of TLC analysis. Fraction B 2 was separated by preparative HPLC using a CT-30 column (Fuji-C 18 , 280 × 30 mm, 10 μm, mobile phase: MeOH/H 2 O, 82/18; flow rate: 10 mL/min) to give 5 (50 mg, t R 50 min) and 11 (267 mg, t R 90 min); while 1 (9.3 mg, t R 31 min) was obtained from fraction B 4 by preparative HPLC purification using the same CT-30 column (mobile phase: MeOH/H 2 O, 75/25; flow rate: 10 mL/min). Similarly, by preparative HPLC purification using the same CT-30 column and the same flow rate, compound 8 (2.6 mg, t R  Part C was fractioned on a Sephadex LH-20 column eluting with MeOH to give fractions C 1 -C 6 . By HPLC purification using an Agilent Zorbax column (SB-C 18 , 250 × 9.2 mm, 5 μm) and a flow rate of 0.8 mL/min, 3 (11.3 mg, t R 4.6 min, MeOH/H 2 O: 20/80) was obtained from fraction C 2 and 4 (14.7 mg, t R 5.7 min, MeOH/H 2 O, 25/75) from C 3 . (1). Yellowish amorphous powder; molecular formula C 16  Marfey's analysis. The hydrolysate was dissolved in 120 μL water and then NaHCO 3 (1 M, 20 μL) and 1% FDAA in acetone (400 μL) were added to the solution of the hydrolysate. The mixture was stirred at 43 °C for 2 h and the reaction was terminated by an addition of 20 μL of 1 N HCl. The reaction mixture was diluted with 500 μL acetone to give amino acid-FDAA derivatives for HPLC analysis. Standard amino acids (each 1 mg) of l-serine, d-serine, l-leucine, d-leucine, l-alanine, and d-alanine were also converted to corresponding FDAA derivatives. Each of the amino acid-FDAA derivatives (0.8 μL) was analyzed by HPLC (Agilent SB-C 18 column: 250 × 4.6 mm, 5 μm; flow rate: 1.0 mL/min; detection wavelength: 340 nm). Water containing 0.1% HOAc was employed as mobile phases A and 100% acetonitrile as phase B. The binary gradient program was 0.00-30.00 min with 20-70% B, 30.01-36.00 min with 100% B, and 36.01-43.00 min with 20% B. The amino acid-FDAA derivatives prepared from the hydrolysate of 3 were found to be l-serine-FDAA (t R 7.71 min), l-alanine-FDAA (t R 10.84 min), and l-leucine-FDAA (t R 17.79 min) by comparison with the retention times of authentic l-serine-FDAA (t R 7.67 min), d-serine-FDAA (t R 8.28 min), l-alanine-FDAA (t R 10.87 min), d-alanine-FDAA (t R 12.98 min), l-leucine-FDAA (t R 17.69 min), and d-leucine-FDAA (t R 20.57 min).
Western blot analysis. Western blot analysis was used to detect the expression levels of tumor glycolytic regulators HK2, PFKFB3, PKM2, and LDH5. The detailed procedure, including protein sample preparation, determination of protein concentration, and western blot analysis, was described in the previous publication 21 .
Determination of the stability of compound 1 in human liver microsomes. Compound 1 was dissolved in 100 mM NaPO 4 solution (pH 7.4) to make a sample solution with a final concentration of 125 μg/mL. Human liver microsomes were diluted by 100 mM NaPO 4 solution (pH 7.4) to make liver microsomal solution with a concentration of 1.25 mg/mL. A mixture of the sample solution (90 μL) and the liver microsomal solution (720 μL) was mixed well and then pre-incubated at 37 °C for 7 min. The pre-incubated mixture was initiated by adding 90 μL 10 mM NADPH solution, mixed well, and then incubated at 37 °C in a water bath. At each designed time point of 0, 15, 30, 60, 90, 120, 180, 240 and 360 min, aliquot 80 μL of the incubated mixture was pipette by micropipet and then quenched by the addition of 320 μL methanol. The reaction mixture was centrifuged at 12000 rpm for 10 min at 5 °C to give the supernatant, which was submitted to HPLC analysis.