Effects of Myxococcus Fulvus KYC4048 Metabolites on Breast Cancer Cell Death

J. Microbiol. Biotechnol. (2018), 28(5), 765–775 https://doi.org/10.4014/jmb.1711.11003 Research Article Review jmb Effects of Myxococcus fulvus KYC4048 Metabolites on Breast Cancer Cell Death S Chayul Lee1†, Sanghyun Park1†, Ikhbayar Ayush2, Kyungyun Cho3, Sung Soo Kim1,4, Insug Kang1,4, Wonchae Choe1,4, Yoon-Seong Kim1,4,5, and Kyung-Sik Yoon1,4* 1Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea 2Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea 3Myxobacteria Bank, Department of Biotechnology, Hoseo University, Asan 31399, Republic of Korea 4Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea 5Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, USA Received: November 2, 2017 Revised: February 7, 2018 Using MCF7 breast cancer cells, we tested the anticancer activity of metabolites from 130 Accepted: March 3, 2018 strains of myxobacteria newly isolated in South Korea. Of these, three strains whose First published online metabolites had high anticancer activity and low cell toxicity were selected and identified by March 16, 2018 their fruiting body morphology, cell morphology, and 16S rRNA sequence. Strains KYC4030 *Corresponding author and KYC4048 were determined to be Myxococcus fulvus, whereas strain KYC4081 was Phone: +82-2-961-0388; identified as Corallococcus coralloides. We found that metabolites of M. fulvus KYC4048 Fax: +82-2-965-6349; E-mail: [email protected] demonstrated no toxicity in normal cells but specifically induced cancer cell death by suppressing the expression of WNT2B. This discovery highlights the value of assessing the † These authors contributed metabolic and biomedical potential of myxobacteria, even those that are already known but equally to this work. were isolated from new areas, and the possible use of metabolites from M. fulvus KYC4048 in S upplementary data for this cancer treatment. paper are available on-line only at http://jmb.or.kr. Keywords: Myxobacteria, WNT2B, anticancer, breast cancer pISSN 1017-7825, eISSN 1738-8872 Copyright© 2018 by The Korean Society for Microbiology and Biotechnology Introduction epothilone B analog, ixabepilone, was approved in October 2007 by the United States Food and Drug Administration Myxobacteria, a group of gram-negative soil bacteria as a treatment for breast cancer resistant or refractory to belonging to the class Deltaproteobacteria, occur in a variety currently available chemotherapies [8, 10, 11]. Because of of habitats such as soil, decaying plant material, rotting the success of epothilone, research on myxobacterial wood, and the dung of herbivorous animals. Myxobacteria metabolites has focused mainly on those produced by are classified into 7 families, 22 genera, and 47 species [1, S. cellulosum. Notably, 48.4% of myxobacterial species that 2], and produce a variety of bioactive secondary metabolites produce secondary metabolites belong to the genus with unique characteristics and cellular effects. More than Sorangium [3]. However, this finding might be due to the 500 bioactive substances produced from myxobacteria have enrichment in Sorangium (23.2%) during the isolation of been identified, many of which are novel [3-5]. For strains from the wild. instance, Sorangium cellulosum produces epothilone [6], a The capacity to produce bioactive metabolites in various novel anticancer agent that stabilizes microtubules via a myxobacteria was recently confirmed by genomic analysis. taxol-like mechanism of action [6-9]. Notably, a semisynthetic For example, gene clusters for the synthesis of 11 bioactive May 2018 ⎪ Vol. 28⎪ No. 5 766 Lee et al. polyketide and hybrid polyketide-nonribosomal peptides and 100% acetone. After the acetone was evaporated from the have been identified in S. cellulosum So ce56 [12, 13], a strain extract, the resulting residue was dissolved in dimethyl sulfoxide that produces the antibiotics chivosazol and etnangien. (DMSO, D4540; Sigma, USA) at a concentration of 0.4 g/ml. Notably, biosynthetic enzyme gene clusters for 18 polyketide and nonribosomal peptides were found in Myxococcus Human Cell Culture and Treatments MCF-7 human breast cancer cells were obtained from Seoul xanthus DK1622 [13, 14], a strain that had been presumed to National University (Seoul, Korea), whereas MCF10A normal be unable to produce bioactive substances and had only breast cells were purchased from the American Type Culture been used as a model strain to study fruiting body formation Collection (USA). MCF-7 cells were grown in RPMI-1640 medium and gliding motility. Subsequently, five bioactive molecules (HyClone, UK) containing 10% fetal bovine serum (HyClone, UK). were isolated from this strain, using new methods of MCF10A cells were grown in Dulbecco’s modified Eagle’s medium/ extraction and culture [13]. Taken together, these data Ham’s F-12 Nutrient Mixture (Gibco, USA) containing 5% horse suggest that new bioactive substances can be isolated from serum (Gibco, USA), 2 mM L-glutamine, 10 μg/ml insulin, 0.1 μg/ml myxobacteria, depending on the strain, geographic origin, cholera toxin, 0.5 μg/ml hydrocortisone, and 20 ng/ml recombinant extraction, and culture conditions. human epidermal growth factor. Cells were cultured at 37°C in a In this study, we tested the anticancer activity of humidified incubator with 5% (v/v) CO2. metabolites from various myxobacterial strains newly Cell viability was determined based on the conversion of 3-(4,5- isolated from soil in different environmental conditions dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystals by mitochondrial enzymes. Cells were seeded throughout South Korea. In addition, the effects of these in 12-well plates at 2×105 cells/well in growth medium and metabolites on genes and proteins associated with cell cultured to approximately 60–70% confluency. The cells were then death were analyzed by RNA sequencing and a Phospho treated with metabolites at concentrations of 0.5× (102 μg/ml), 1× Antibody Explorer Array. (2 × 102 μg/ml), 2× (4 × 102 μg/ml), and 5× (103 μg/ml). Subsequently, the media were carefully removed and DMSO (300 μl) was added Materials and Methods to solubilize the blue formazan crystals in the living cells. Absorbance was measured at 540 nm using an ELISA reader Myxobacterial Culture (Multiskan EX; Thermo Lab Systems, USA). VY/3 and ST21CX agar media were used to isolate myxobacteria [15, 16] and isolates were cultured in CYS liquid media (Table S1) Flow Cytometry and Annexin V-Fluorescein Isothiocyanate to induce metabolite production as described below. All (FITC)/Propidium Iodide (PI) Double Staining myxobacterial strains were cultured at 32°C. To measure apoptosis, human cells were seeded at 2 × 105 cells/ml in 100-mm dishes, cultured for 1 day, and treated for 72 h with Isolation and Identification of Myxobacteria and Preparation of 2×102 μg/ml myxobacterial metabolite. Cells were then harvested, Metabolites washed with phosphate-buffered saline (PBS), fixed at 4°C for Myxobacteria were isolated as previously described [16, 17]. 24 h with ice-cold 75% ethanol, and pelleted by centrifugation. Briefly, soil samples collected throughout South Korea were placed Subsequently, the cells were washed with PBS, treated for 30 min on ST21CX agar plates and incubated at 32°C for 2–4 weeks. with 0.5 μg/ml RNase A in PI buffer, and stained for 30 min in the Myxobacterial 16S ribosomal RNA (rRNA) genes were amplified dark with 20 μg/ml PI. Cell cycle distribution was then analyzed using the 5’-GAGTTTGATCCTGGCTGAG-3’ (27f) and 5’-AGA on the basis of the DNA content using Kaluza flow cytometry AAGGAGGTGATCCAGCC-3’ (1525r) primers, as previously software (Beckman Coulter, USA). Apoptosis was also quantified reported [18, 19]. Phylogenetic analysis of the isolates was carried by flow cytometry on the basis of annexin V–FITC and PI double out using ClustalW [20]. staining. Briefly, human cells exposed to myxobacterial metabolites For morphologic characterization and identification, the vegetative were trypsinized, collected by centrifugation, resuspended in cells of the myxobacteria grown on VY/3 agar media were annexin V-FITC binding buffer, and incubated for 15min at room observed using a CKX41 phase-contrast microscope (Olympus, temperature (20-25°C) in the dark with 1 μg/ml annexin V-FITC Japan) and the fruiting bodies developed on the VY/3 agar media and 10 μg/ml PI. Samples were analyzed as described above, were observed using an Olympus SZ61 stereomicroscope. The using Kaluza flow cytometry software. microscopic images were then photographed using an Olympus C-5060 digital camera. RNA Sequencing and Transcript Analysis For metabolite preparation, the myxobacteria cells cultured on Total RNA was prepared, using QIAzol Lysis Reagent (Qiagen, VY/3 agar medium and 2% Amberlite XAD-16 resin (Sigma, USA) The Netherlands), from MCF-7 and MCF10A cells grown and were cultured in CYS liquid medium for 7 days at 32°C and treated as described above. 180 rpm. The resin and cells were extracted twice each with 50% To construct cDNA libraries, 1 μg of total RNA was used. The J. Microbiol. Biotechnol. M. fulvus Metabolite Effect on Breast Cancer Cells 767 Table 1. Strains of myxobacteria producing anticancer metabolites and their activities. MCF7a cell MCF10A cell Strain

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