Angucycline Glycosides from an Intertidal Sediments Strain Streptomyces sp. and Their Cytotoxic Activity against Hepatoma Carcinoma Cells

Four angucycline glycosides including three new compounds landomycin N (1), galtamycin C (2) and vineomycin D (3), and a known homologue saquayamycin B (4), along with two alkaloids 1-acetyl-β-carboline (5) and indole-3-acetic acid (6), were isolated from the fermentation broth of an intertidal sediments-derived Streptomyces sp. Their structures were established by IR, HR-ESI-MS, 1D and 2D NMR techniques. Among the isolated angucyclines, saquayamycin B (4) displayed potent cytotoxic activity against hepatoma carcinoma cells HepG-2, SMMC-7721 and plc-prf-5, with IC50 values 0.135, 0.033 and 0.244 μM respectively, superior to doxorubicin. Saquayamycin B (4) also induced apoptosis in SMMC-7721 cells as detected by its morphological characteristics in 4′,6-diamidino-2-phenylindole (DAPI) staining experiment.


Introduction
Angucycline is a group of aromatic polyketides containing a benz[a]anthraquinone framework of the aglycone which is mostly attached with C-glycosidic moiety [1]. Naturally occurring angucyclines are exclusively produced by terrestrial and marine actinomycetes, especially Streptomycetes species, in which a decaketide initially derived from acetyl-CoA is catalytically cyclized to four-ring core of angucycline by polyketide cyclase [2]. The structures of angucycline glycosides always vary in the oxidation degree of aglycones along with the number and position of diverse deoxy sugars [1][2][3][4]. In some cases, e.g., galtamycin B [5], grincamycin B [6], and vineomycin B 2 [7], the angular four-ring of typical angucycline is rearranged to linear tetracyclic or tricyclic system by enzymatic or non-enzymatic modification. Although firstly discovered half a century ago and possessing potent antibacterial, antiproliferative, and cytotoxic activities [6][7][8][9][10][11], so far, none of angucycline compounds has been successfully developed into clinical drug due to toxicity or solubility issues, which is unlike their biogenetic relatives tetracycline and anthracycline antibiotics [2]. Recent researches on angucyclines mainly concentrated on the understanding of their biosynthetic pathways in order to obtain modified analogues with medicinal potentiality through genetic manipulation [12][13][14].
Intertidal ecosystems are significantly different from those of seafloor. Regular tide immersion and emersion result in the dissolution of more organic carbon as well as oxygen and sulfate into intertidal sediment, which is beneficial to microbes' survival, particularly to aerobic actinomycetes. Both metagenomes and culture-dependent isolation have verified the abundance and diversity of intertidal sediment, which is beneficial to microbes' survival, particularly to aerobic actinomycetes. Both metagenomes and culture-dependent isolation have verified the abundance and diversity of Actinobacteria in intertidal sediment [15]. Thus, we exploited the Actinobacteria resources from the intertidal sediment of Xiaoshi Island in Weihai, China, to screen for new antitumor agents. As a result, a Streptomyces sp., designated OC1610.4, was obtained, and its 16S rRNA nucleotide sequence (Accession no. MK045847) shared only 81.8% and 81.6% similarity, respectively, with those of Streptomyces chromofuscus (FJ486284) and Streptomyces lannensis (KM370050) in GenBank. The thin layer chromatography (TLC) analysis of its EtOAc extract of liquid culture medium displayed several yellow and brown spots, presumably due to aromatic polyketides. Subsequent large-scale fermentation and chromatographic isolation led to the identification of four angucycline glycosides including three new compounds, namely landomycin N (1), galtamycin C (2) and vineomycin D (3), and the previously reported saquayamycin B (4) (Figure 1), along with two alkaloids 1-acetyl-βcarboline (5) and indole-3-acetic acid (6) [16,17]. Saquayamycin B (4) displayed potent cytotoxic activity against hepatoma carcinoma HepG-2, SMMC-7721 and plc-prf-5 cell lines, and it caused apoptosis in SMMC-7721 cells.

Results and Discussion
From 30 L liquid fermentation broth of the strain Streptomyces sp. OC1610.4, cultured for 9 days, 4.6 g of EtOAc extract was obtained. After fractionation by column chromatography and preparative HPLC purification, six yellow or brown amorphous powdered-compounds were isolated from the crude EtOAc extract. The major constituent in the extract was firstly purified and whose molecular formula C43H48O16 was established by the HR-ESI-MS m/z 838.3298 ([M + NH4] + , calcd for C43H52NO16, 838.3286) and m/z 843.2842 ([M + Na] + , calcd for C43H48NaO16, 843.2840) ( Figure S1). Its 1 H NMR spectrum displayed complex signals including three pairs of aromatic or olefinic protons from δH 6.06 to 7.91, more than a dozen methylene and methine protons from δH 1.40 to 5.39 and five methyl groups ( Figure S2). The four oxygenated methine proton signals between δH 5.01 and 5.40 which,

Results and Discussion
From 30 L liquid fermentation broth of the strain Streptomyces sp. OC1610.4, cultured for 9 days, 4.6 g of EtOAc extract was obtained. After fractionation by column chromatography and preparative HPLC purification, six yellow or brown amorphous powdered-compounds were isolated from the crude EtOAc extract. The major constituent in the extract was firstly purified and whose molecular formula C 43 H 48 O 16 Figure S1). Its 1 H NMR spectrum displayed complex signals including three pairs of aromatic or olefinic protons from δ H 6.06 to 7.91, more than a dozen methylene and methine protons from δ H 1.40 to 5.39 and five methyl groups ( Figure S2). The four oxygenated methine proton signals between δ H 5.01 and 5.40 which, through HSQC spectrum, directly attached to the carbons signals at δ C 96.0, 92.8, 92.1 and 72.0 ( Figure S3), along with four doublets of methyl groups are the characteristic of four deoxy sugar molecules, one of which probably formed a C-glycoside since its anomeric carbon appeared at δ C 72.0 [18,19]. These data, especially the signals of the deoxy sugar C-glycosidic moiety suggested the structure of angucycline glycoside [1]. Detailed comparison of its 1 H and 13 C NMR data with those previously reported in the literature and analysis of the 2D NMR sprectra ( Figures S5-S8), led to the identification of this compound as saquayamycin B (4) [3,18].
Galtamycin C (2) is an isomer of 1, due to its HRESIMS data m/z 561.1752 [M + H] + (calcd for C31H29O10, 561.1761). The 1 H and 13 C NMR spectra showed that its aliphatic proton and carbon signals were similar to those of 1, suggesting the presence of the disaccharide α-L-cinerulose B-(1→4, 2→3)β-D-olivosyl moiety ( Table 1). The 1 H NMR of 2 also showed five aromatic proton signals at δH 8.39 (s), 7.87 (d, J = 7.8 Hz), 7.73 (d, J = 7.8 Hz), 7.52 (brs) and 6.95 (brs), where the singlet at δH 8.39 (s) has higher frequency than the corresponding singlet of 1. The 13 C NMR spectrum (Table 1) dispalyed sixteen aromatic carbons with chemical shifts ranging from δC 108.8 to 162.1 and two quinone carbonyl carbons at δC 187.3 and 186.3 were similar to those of rearranged linear angucycline glycosides, galtamycinone, grincamycin and grincamycin H [7,21]. Hence, 2 was suggested to possess a linear tetracyclic system. The structure of the compound and the relative configurations of the two deoxysugars were confirmed by COSY, HMBC and NOESY correlations (Figures 2 and 4). Therefore, 2 was named galtamycin C ( Figure 1).  Table 1). The 1 H and 13 C NMR  CH-1A). The most obvious difference in 13 C NMR spectra of 3 and 4 is the absence of a signal above δ C 200 in 3, and the presence of a signal at δ C 172.2, characteristic of a carboxylic acid or ester group. Accordingly, 3 was suggested to have a tricyclic system with a side chain, probaly due to the opening of the cyclohexanone ring of saquayamycin B (4) [6,7,22].
A few anguclines, such as saquayamycin B, landomycin E, vineomycin A 1 etc., have been reported to exhibit remarkable antitumor activity against a series of tumor cell lines [3,7,10]. Though, the distinct in vivo toxicity restricted the further development of these compounds to be clinical drugs. Recently, an atypical angucycline, lomaiviticin A, was reported to be under preclinical evaluation for antitumor treatment due to its prominent cytotoxicity and effects of inducing double-strand breaks in DNA [14,23]. In present work, 1-4 were assayed for their cytotoxic activity against normal liver cell LO 2 , hepatoma carcinoma HepG-2, SMMC-7721 and plc-prf-5 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Table 2). At the concentrations of 40 µM, 1-3 displayed no cytotoxicity against any of the tested cell lines. Saquayamycin B (4) displyed potent cytotoxic activity against HepG-2, SMMC-7721 and plc-prf-5 cells, with IC 50 values 0.135, 0.033 and 0.244 µM, respectively, which are less than the IC 50 of doxorubicin. Treatment of SMMC-7721 cells with saquayamycin B at concentrations ranging from 0.025 to 0.100 µM for 24 h, SMMC-7721 cells resulted in chromatin dispersion and formation of apoptotic body in DAPI staining test (Figure 5a). The apoptotic ratio of SMMC-7721 cells was dependent on the concentrations of saquayamycin B (Figure 5b).

Actinomycetes Strain
The intertidal sediment was collected after the tide has ebbed in Xiaoshi Island, Weihai, China in September 2016. The strain OC1610.4 was isolated from this sediment using Gause's synthetic medium (20 g/L amylogen, 1 g/L KNO3, 0.5 g/L NaCl, 0.5 g/L K2HPO4·H2O, 0.5 g/L MgSO4·H2O, 0.01 g/L FeSO4·H2O, and 3.0% sea salt) containing potassium dichromate (6 μg/mL) and nalidixic acid (20 μg/mL) as antifungal and antibacterial agents. The procedures of DNA extraction and PCR amplification of 16S rRNA were same as described in reference [24]. The nucleotide sequence of the OC1610.4 strain was sequenced at the Shanghai Sangon Biotech Co., China, and deposited at GenBank (Accession no. MK045847). Voucher strain (No. OC1610.4) was deposited at Laboratory of Natural Products Chemistry, Department of Pharmacy, Shandong University at Weihai.

Actinomycetes Strain
The intertidal sediment was collected after the tide has ebbed in Xiaoshi Island, Weihai, China in September 2016. The strain OC1610.4 was isolated from this sediment using Gause's synthetic medium (20 g/L amylogen, 1 g/L KNO 3 , 0.5 g/L NaCl, 0.5 g/L K 2 HPO 4 ·H 2 O, 0.5 g/L MgSO 4 ·H 2 O, 0.01 g/L FeSO 4 ·H 2 O, and 3.0% sea salt) containing potassium dichromate (6 µg/mL) and nalidixic acid (20 µg/mL) as antifungal and antibacterial agents. The procedures of DNA extraction and PCR amplification of 16S rRNA were same as described in reference [24]. The nucleotide sequence of the OC1610.4 strain was sequenced at the Shanghai Sangon Biotech Co., China, and deposited at GenBank (Accession no. MK045847). Voucher strain (No. OC1610.4) was deposited at Laboratory of Natural Products Chemistry, Department of Pharmacy, Shandong University at Weihai.

Cytotoxicity Assays, DAPI Staining Test and Flow Cytometric Analysis
The cytotoxicity evaluations of 1-4 against normal liver cell and hepatoma carcinoma cells were carried out using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as positive control drug and deionized H 2 O with the same DMSO concentration was used as parallel control. DAPI staining test was employed to qualitatively observe apoptosis, and the apoptotic ratio was measured by flow cytometric analysis (Becton Dickinson FACScan, San Jose, CA, USA). These tests were conducted using the methods as previously described [25,26].