Cytotoxic, Anti-Migration, and Anti-Invasion Activities on Breast Cancer Cells of Angucycline Glycosides Isolated from a Marine-Derived Streptomyces sp.

Four angucycline glycosides were previously characterized from marine-derived Streptomyces sp. OC1610.4. Further investigation of this strain cultured on different fermentation media from that used previously resulted in the isolation of two new angucycline glycosides, vineomycins E and F (1–2), and five known homologues, grincamycin L (3), vineomycinone B2 (4), fridamycin D (5), moromycin B (7), and saquayamycin B1 (8). Vineomycin F (2) contains an unusual ring-cleavage deoxy sugar. All the angucycline glycosides isolated from Streptomyces sp. OC1610.4 were evaluated for their cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231, and BT-474. Moromycin B (7), saquayamycin B1 (8), and saquayamycin B (9) displayed potent anti-proliferation against the tested cell lines, with IC50 values ranging from 0.16 to 0.67 μM. Saquayamycin B (9) inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner, as detected by Transwell and wound-healing assays.


Introduction
Angucyclines and their aglucones (angucyclinones) are a class of natural products containing an angularly assembled tetracyclic scaffold or a corresponding rearranged frame in the structure.They are the largest family of polycyclic aromatic polyketides produced by Gram-positive actinomycetes [1].Since the first member of the class, tetrangomycin, was identified from Streptomyces rimosus in 1965 [1], dozens of angucyclines and angucyclinones have been found to display prominent cytotoxic and anti-proliferative properties [2][3][4].Although their severe in vivo toxicity and limited water-solubility restricted their clinical application, this class of natural products still continuously draws attention due to their structural diversity and anti-neoplastic potential [5][6][7][8].To date, several subclasses of angucycline derivatives have been extensively investigated regarding their in vitro and in vivo anti-tumor effects.For instance, landomycin E displayed promising anti-cancer activity against multidrug-resistant cancer cells and induced apoptotic cell death as a consequence of rapid mitochondrial damage [9].
Further investigation indicated that rapid H 2 O 2 generation and complex caspase activation contribute to its anti-neoplastic effects [10].Jadomycins are atypical angucyclinones containing nitrogenous heterocycles in their structure, and jadomycins B and F were reported to induce DNA cleavage through the generation of cytosolic superoxide or the inhibition of type II topoisomerases, leading to the death of multidrug-resistant breast cancer cells [11,12].Lomaiviticins were first isolated from Micromonospora lomaivitiensis as dimers of kinamycin angucyclines with two diazofluorene functional groups [3].One of them, lomaiviticin A, exhibited remarkable cytotoxicity against a panel of human cancer cells at nanomolar-picomolar concentrations by inducing double-strand breaks in DNA, and it is currently under preclinical evaluation [13][14][15].Accordingly, angucyclines are still considered as promising candidates for anti-tumor drug development.
Angucyclines were mainly separated from terrestrial actinomycetes, though in recent years an increasing number of them have been identified from marine actinomycetes associated with seafloor sediments [4,8,[16][17][18][19], sponges [7], and mangrove forests [20].We also initiated a screening for angucycline-producing strains employing PCR amplification of the β-ketoacyl synthase (KSα) gene from marine actinomycetes, and a Streptomyces sp.designated as OC1610.4 was obtained from the intertidal sediments.Its 16S rRNA nucleotide sequence (GenBank number: MK045847) is similar to those of Streptomyces chromofuscus (FJ486284) and Streptomyces lannensis (KM370050), with 81.8% and 81.6% similarity, respectively (Supplementary Figure S1).After this strain was cultured in Morel & Wetmore Modification medium (S-medium), four angucycline glycosides were identified, and one of the isolates, saquayamycin B, displayed potent cytotoxicity on human hepatoma carcinoma cells [21].For the purpose of discovering more diversified analogues with cytotoxicity, three kinds of medium-Gauze's synthetic solid medium (GAU), yeast extract-malt extract starch medium (YMS), and yeast extract-malt extract agar medium (YMEA)-were further employed to reculture this strain.Thin-layer chromatography (TLC) profiles showed that more yellow spots displaying orange fluorescence under UV 365 nm light were observed in the EtOAc extract of GAU.Large-scale fermentation using GAU and isolation resulted in the identification of another seven angucyclines including two new derivatives.This paper reports their structure identification and cytotoxic, anti-migration, and anti-invasion activities on breast cancer cells of angucyclines obtained from Streptomyces sp.OC1610.4.

Actinomycetes Strain
The strain OC1610.4 was obtained from intertidal sediment using the method reported in our previous research [19].DNA extraction and PCR amplification of 16S rRNA were conducted according to the instructions of the DNA isolation kit and PCR kit supplied by Shanghai Sangon Biotech Co., China, and 16S rRNA was sequenced by the same company.The 16S rRNA sequence was deposited at GenBank (accession no.MK045847), and the closely related taxa were retrieved from GenBank using BLAST software.The voucher strain (no.OC1610.4) was deposited at the Laboratory of Natural Products Chemistry, College of Marine Science, Shandong University at Weihai.

Cell Culture
Human breast cancer cells MCF-7, MDA-MB-231, and BT-474 were bought from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, China.The cell lines were cultured in RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum (FBS) supplemented with 100 units/mL of penicillin and 100 µg/mL of streptomycin.All cells were incubated in 5% CO 2 at 37 • C.

MTT Assay
The cytotoxic activity of compounds 1-11 against breast cancer cells MCF-7, MDA-MB-231, and BT-474 was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in our previous research [30].Doxorubicin was used as a positive control drug and deionized H 2 O with the same DMSO concentration was used as a parallel control.

Wound-Healing Assay
The anti-migration effect of saquayamycin B (9) against breast cancer cells MDA-MB-231 was evaluated using a wound-healing assay [31].Briefly, MDA-MB-231 cells were cultured in a 24-well plate at a concentration of 5 × 10 5 cells/mL with RPMI-1640 (10% FBS).When the cell density grew to 90%, linear gaps were scratched by micropipette tips, and the suspension cells were flushed out using phosphate-buffered saline (PBS).Then, the cells in the plate were starved for 12 h to eliminate the interference of proliferation.Wound healing rate (%) = [1 -(scratch width of saquayamycin B-treated group/scratch width of control group)] × 100%.

Transwell Assay
A Transwell assay was employed to evaluate the invasion capacity of MDA-MB-231 cells [29].A mixture of Matrigel and serum-free RPMI-1640 (1:8) was precoated on the upper chamber membrane of the filter insert.Then, 200 µL cell suspension (5 × 10 5 cells/mL) in serum-free RPMI-1640 was poured into the upper chamber and treated with 25 and 50 nM saquayamycin B or DMSO for 12 h.RPMI-1640 medium containing 20% FBS was filled in the lower chamber as a chemoattractant.The invading cells in the lower chamber were fixed with methanol for 15 min and stained by 0.5% cresyl violet, followed by washing 3 times with double-distilled water and air drying.Three random views were photographed, and the invasive cells were counted under a Nikon TE2000 microscope with NIS elements viewer 4.2.0 software (Nikon Instech Co., Ltd., Tokyo, Japan).

Figure 2 .
Figure 2. COSY, HMBC, and key NOESY correlations for 1. Vineomycin F (2) was isolated as a yellow solid and has the molecular formula C 31 H 32 O 14 , determined by the m/z 629.1867 ([M + H] + , calc.d for C 31 H 33 O 14 , 629.1870) from HR-ESI-MS.The 1 H NMR spectrum of 2 displayed the resonances of four aromatic protons and a set of aliphatic protons, similar to those of 1.The prominent difference is that the anomeric proton signal of the terminal deoxy sugar in 2, whose corresponding carbon resonated at δ C 90.5 assigned by the HMQC spectrum, shifted to downfield δ H 6.06 (H-1B).The 13 C NMR spectrum of 2 showed 31 carbon signals in which δ C 169.8, 171.4,and 173.8 were assigned to three carboxyl or ester carbonyl carbons.The chemical shift of the methyl group at δ H 2.17 (s, H-6B) and the HMBC correlation from this signal to carbon at δ H 169.8 (C-5B) supported the presence of an acetyl group (Figure3).After the signals of protons and carbons in 2 were completely assigned by HMQC and COSY spectra (Table1), the structure of aglycone was established to be identical to that of 1 by the HMBC correlations associated with the four aromatic protons (δ H 7.78, 7.84, 7.85, and 7.96) and the methane protons (δ H 2.56, 2.59, and 3.10) (Figure3).The presence of the d-olivose moiety, including its configurations, was confirmed by the COSY correlations from H-1A (δ H 5.03, d, J = 9.1 Hz) through H-6A (δ H 1.26, d J = 5.6 Hz) and the NOESY correlations H-1/H-3A,5A and H4A/H-6A.The HMBC correlations from H-1A (δ H 5.03) to C-8 (δ C 159.6), C-9 (δ C 138.8), and C-10 (δ C 134.2) indicated the attachment of β-d-olivose at C-8 to form a C-glycoside (Figure3).The COSY correlations from H-1B (δ H 6.06) through H-3B (δ H 2.54), together with the HMBC correlations from H-2B (δ H 4.34) to the carboxyl carbon at δ H 171.4 (C-4B), suggested that the terminal sugar is a butyrate acid derivative.In addition, it linked with β-d-olivose through two ether bonds, which were deduced by the HMBC correlations from H-1B (δ H 6.06) to C-4A (δ C 75.3) and H-2B (δ H 4.34) to C-3A (δ C 74.6).The downfield shift of H-1B to δ H 6.06 and the HMBC correlation from H-1B to the carbonyl carbon at δ C 169.8 (C-5B) implied that the acetyl group attached at C-1B.The acetyl group and the butyrate acid derivative were probably the results of an oxidative break of the C-C bond between C-4 and C-5 in l-cinerulose B (Figure3).

Figure 4 .
Figure 4. Saquayamycin B (9) treatment dose-dependently inhibited invasion and migration in the breast cancer cell line MDA-MB-231.Cell invasion and migration were observed with incubation for 12 h by Transwell and wound-healing assays.A: cresyl violet staining in the Transwell assay, captured by a microscope (100× magnification).B: quantification by counting the number of cells in the Transwell assay.C: effects of wound-healing captured by a microscope (100× magnification).D: analysis of the woundhealing rate.Results are presented as mean ± SD. ** p < 0.01, *** p < 0.001 compared to the control group.
a Residual signals of the solvent as a reference.b Measured in CD 3 OD.c Measured in acetone-d 6 .