Abstract Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1α, VEGF, BrK, and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90α, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis. Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.

Citation: Lim EJ, Hong DY, Park JH, Joung YH, Darvin P, Kim SY, et al. (2012) Methylsulfonylmethane Suppresses Breast Cancer Growth by Down-Regulating STAT3 and STAT5b Pathways. PLoS ONE 7(4): e33361. https://doi.org/10.1371/journal.pone.0033361 Editor: Jun Li, Sun Yat-sen University Medical School, China Received: October 20, 2011; Accepted: February 7, 2012; Published: April 2, 2012 Copyright: © 2012 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the Next generation BioGreen 21 Program (No. PJ0081062011), by the Rural Development Administration, and partially supported by the Ministry of Commerce, Industry and Energy through the Bio-Food and Drug Research Center at Konkuk University, Glocal Campus, South Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Breast cancer (BC) is the major cancer affecting females in the United States. Additionally, more than 1 million women worldwide are diagnosed with this disease per year. BC is the second most common cause of cancer-related deaths with ∼400,000 patients dying due to this disease every year [1], [2]. This disease is the major cause of death in women between the ages 45 and 55 y [3]. Approximately, 15% of BCs are triple-negative breast cancer, a type that is more prevalent among young African, African-American, and Latino women [4]. This type of aggressive breast cancer has unique molecular profiles. This subtype is clinically negative about the expression of estrogen receptor (ER) and progesterone receptor (PR), and does not over-express human epidermal growth factor receptor-2 (Her-2) protein. No targeted therapies exist for treating TNBC, and this disease frequently displays distinct patterns of metastasis [3]. Human BC frequently expresses the epidermal growth factor (EGF) receptor. Human epidermal growth factor-2 (Her-2), -3, and -4, orphan receptors of the EGF receptor family, that are co-expressed with other EGF receptors. The proto-oncogene Her-2 is located on chromosome 17. In case of 25 – 30% breast cancers, Her-2 is over-expressed. Apart from this, over-expression of Her-2 has been reported in many other aggressive breast cancers [5]. Ligand binding activates these receptors so that they form homo/heterodimers and stimulate downstream signalling pathways. The Ras/Raf/MAPK and PI3-K/Akt pathways involved in cell proliferation, and survival are major targets of activated EGF receptors [6]. Her-2 over-expression has been shown to result in increased transformation, tumorigenicity, proliferation, and invasiveness [7]. Approximately one-half of primary breast tumors are ER+/PR+, whereas less than 5% are ER−/PR+ [8]. PR is a specific receptor that belongs to the superfamily of ligand-activated nuclear receptors [9]. PR exists in two isoforms, PR-A and PR-B; both are expressed in humans [10]. Both receptors bind progestins and promote epithelial cell proliferation as well as lobulo-alveolar development [11]. The binding of progesterone to PRs induces the formation of receptor homo- or heterodimers. This conformational change leads to increased receptor phosphorylation, and interaction with target gene promoters, specific co-activators, and general transcription factors [12]. PRs have some prognostic and predictive implications [13], [14]. Together with ERs, PRs make cells sensitive or resistance to different therapies [15]. Based on the expression pattern, PR breast cancer may be ER+/PR+ or ER+/PR−, and PR+ breast cancers have been found to be more differentiated than PR- breast cancers [8]. High levels of estrogen receptor-α (ER-α) promote hormone-dependent tumor growth by converting the receptor as a ligand-dependent transcription factor. ER-α-dependent processes require different concentrations of receptors and is not always the factor limiting hormone responsiveness. In breast tumors, increased proliferation rates have been observed with high ER-α expression [16] and thymidine kinase activity [17]. The ER-α receptor and steroid hormones regulate vascular endothelial growth factor (VEGF) in breast cancer in vivo [8]. Vascular endothelial growth factor-A (VEGF-A) is considered to be the most important and potent pro-angiogenic factor involved in tumor growth [19]. The binding of VEGF to VEGFR induces conformational changes in the receptor followed by auto-phosphorylation of the receptor [20]. VEGF expression is regulated by hypoxia, steroid hormones, nitric oxide, and cytokines [21], [22]. Signal transducer and activator of transcription 3 (STAT3) is important for breast involution after weaning [23] and a prognostic factor for breast cancer [24]. We have reported that STAT3 modulates VEGF through HIF-1α [25]. Tumor angiogenesis is enhanced as VEGF expression is up-regulated by increased STAT3 activity. In addition, decreased Src-induced VEGF expression is observed when Stat3 signaling is blocked [26]. This indicates that Stat3 represents a common anti-angiogenesis target for blocking multiple signaling pathways in human cancers. Another important member of the STAT family, STAT5b, regulates growth, differentiation, and survival of mammary and solid tumors. Recently, we reported that STAT5b regulates the transactivation of cyclin D1 and IGF-1 upon hypoxia stimulation in breast cancer cells [27], [28], [29]. Methylsulfonylmethane (MSM) is a very simple organic sulfur-containing compound with a molar mass of 94.13 g/mol. MSM contains only eleven atoms and is found in foods, including fruits, vegetables, grains, and beverages [30]. It is a symmetric molecule with no isomeric forms. Dimethyl sulfone has also been detected in the human brain [31], blood plasma, and cerebrospinal fluid [32] by proton magnetic resonance spectroscopy. MSM is volatile, easily lost during cooking, and is believed to be non-toxic [33], [34]. MSM decreases arthritis pain and improves physical function of osteoarthritis human knees without major adverse events [35]. This compound has also been found to be effective for treating allergies [36], osteoarthritis pain [35], inflammation [37], repetitive stress injuries [38], and bladder disorders like intestinal cystitis [39]. MSM can induce wound healing, contact inhibition, and can block the ability of cells to migrate through the extra-cellular matrix. Furthermore, it can restore anchorage-dependent growth and irreversible senescence followed by arborization with melanosomes in arbors seen in murine melanoma cell lines [40]. In this study, we proposed that MSM suppresses tumor growth via inhibition of the STAT3 and STAT5b pathways. To test this hypothesis, we investigated the effects of MSM on human breast cancer cells and in the experimental animal model. The effects of MSM on the expression of STAT3, STAT5b, and their downstream targets were analysed. From the results obtained, we found that MSM down-regulates triple-negative hormone receptor expression in hormone-responsive cell lines and suppresses the growth of breast cancer xenografts through its multi-targeted action.

Materials and Methods Ethics Statement All procedures for animal experiment were approved by the Committee on the Use and Care on Animals (Certificate No: KUB00313, Institutional Animal Care and Use Committee, Seoul, Korea) and performed in accordance with the institution guidelines. Materials Methylsulfonemethane (MSM) was purchased from Fluka/Sigma Co. (St. Louis, MI). Antibodies and Reagents Dulbecco’s modified eagle’s medium (DMEM), DMEM/F-12, RPMI 1640, 10% fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco-BRL (GrandIsland, NY). L-15 medium, anti-actin antibody, insulin and EGF were obtained from Sigma Chemical (St. Louis, MO). Anti- STAT5b antibodies, secondary antibodies (goat anti-mouse IgG-horseradish peroxidase) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-STAT3(Tyr705) and Anti-phospho-IGF-1R(Tyr1131) antibodies were obtained from cell signaling (Beverly, MA). Anti-phospho-STAT5b(Tyr699) antibodies was obtained from upstate (Lake Placid, NY). The secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit IgG), the enhanced chemiluminescence (ECL plus) detection kit was purchased from Amersham Pharmacia Biotech. (Piscataway, NJ). RestoreTM Western Blot Stripping Buffer and NE-PER kit were purchased from Pierce (Rockford, IL). The luciferase assay substrates, reporter lysis buffer, and electrophoretic mobility shift assay (EMSA) kit were purchased from Promega Corp. (Madison, WI). FuGene 6 transfection reagent was from Roche (Basel, Switzerland), RNeasy mini kit and Qiaprep spin miniprep kits were purchased from Qiagen (Germany). Cell Culture MCF-10A (kind gift from Dr. Ssang-Goo Cho, Konkuk University, Korea), immortalized normal human breast epithelial cells were grown to confluency in phenol red free DMEM/F2 medium supplemented with cholera toxin (20 µg/mL), insulin (10 mg/mL), EGF (250 µg/mL), 1% penicillin/streptomycin, and horse serum (5%). MCF-7 (No: 30022, KCLB, Korea), T-47D (No: 30133, KCLB, Korea) and SK-BR3 (No: 30030, KCLB, Korea), human breast cancer cells, were grown to confluency in RPMI 1640 medium containing 10% FBS, insulin (5 • g/ml), and EGF (10 ng/ml). COS-7 (No: 21651, KCLB, Korea), monkey kidney cells, and MDA-MB 231 (No: 30026, KCLB, Korea), human breast cancer cells, were cultured in DMEM containing 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin at 37°C in 5% CO 2 . At the start of each experiment, the cells were resuspended in the medium at a density of 2.5×105 cells/ml. MTT assay Cell viability was assayed by measuring blue formazan that was metabolized from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenase, which is active only in live cells. One day before drug application, cells were seeded in 96-well flat-bottomed microtiter plates (3,000–5,000 cells/well). Cells were incubated for 24 h with various concentrations of MSM. MTT (5 mg/ml) was added to each well and incubated for 4 h at 37°C. The formazan product was dissolved by adding 200 μl dimethylsulfoxide (DMSO) to each well, and the plates were read at 550 nm. All measurements were performed in triplicate, and each experiment was repeated at least three times. Apoptosis Analysis Fluorescein-conjugated Annexin V (Annexin V-FITC) was used to quantitatively determine the percentage of cells undergoing apoptosis. Treated cells were washed twice with cold PBS and then resuspended in binding buffer at a concentration of 1x106 cells/ml. Five microliters of Annexin V-FITC and 10 µl of propidium iodide were added to suspended cells. After incubation for 15 min at room temperature in the dark, the percentage of apoptotic cells was analyzed by flow cytometry (Becton-Dickinson FACScan, San Jose, CA). For positive controls 10 µM camptothecin and 23 µM actinomycin D were used. Total Cell Lysis Breast cancer cells were treated with MSM for determined times. Cells were lysed on ice for 10 min in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors. Cells were disrupted by aspiration through a 23-gauge needle, and centrifuged at 15,000 rpm for 10 min at 4°C to remove cellular debris. Protein concentations were measured using the Bradford method. Western Blot Whole cell extracts (WCE) from breast cancer cells were prepared by described previously and quantified using Bradford’s method. Equal amounts of protein obtained by total lysis were subjected to 10% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The blots were blocked with 5% skim milk or BSA in TBS-T buffer. It was then incubated overnight with primary antibody followed by washing with TBS-T and incubation with secondary antibody (anti-mouse or anti rabbit IgG HRP conjugate, 1∶1,000 dilutions with skim milk or BSA). Detection was done by using enhanced chemiluminescence (ECL plus) detection kit. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from the cells by using Tri reagent (Sigma Chemical Co., St. Louis, MO) and quantitated spectrophotometrically at 260 nm. RT-PCR analysis for VEGF, IGF-1R and 18s RNA was performed (Table S1). Briefly, 1 µg of RNA was reverse transcribed, and nested PCR was carried out by using 2 µl of cDNA. The PCR condition consisted of denaturation for 1 min at 94°C, annealing for 1 min at 58°C, and extension for 1 min at 72°C. RT-PCR products were analyzed on 1% agarose gel stained with ethidium bromide. Electrophoretic Mobility Shift Assay (EMSA) STAT5 and STAT3 DNA binding activity was detected using an electrophoretic mobility shift assay (EMSA), in which a labeled double-stranded DNA sequence was used as a DNA probe to bind active STAT5b and STAT3 protein in nuclear extracts. Nuclear protein extracts were prepared with the Nuclear Extract Kit (Panomics, AY2002). EMSA experiment is performed by incubating a biotin-labeled transcription factor (TF-STAT5 and STAT3) probe with treated and untreated nuclear extracts. Co-transfection and Luciferase Assay The expression vectors for mouse STAT5b (pMX/STAT5b; kindly provided by Dr. Koichi Ikuta, Kyoto University, Japan) were constructed as previously described. cDNA for STAT5b was inserted into the EcoR?and Sal?sites of the pMX vector. IGF-1R (kindly provided by Dr. Haim Werner, Tel Aviv University, Israel) genomic DNA fragments, including nucleotides -2350 to +640 (nucleotide 1 corresponds to the transcription start site of the rat IGF-1R gene), was sub-cloned upstream of a promoterless firefly luciferase reporter in the pGL2P vector (Promega, Madison, WI). For reporter gene assays, COS-7 cells were transiently co-transfected with the plasmid pGL2P, IGF-1R or HSP90α (kindly provided by Dr. Carrie Shemanko, Calgary University, Canada) construct and the STAT5b expression vector. Cells were co-transfected with various combinations the following constructs; wild-STAT3 (gifts from Dr. Shong, Chungnam National University, Korea); the VEGF reporter construct containing 2.7 kb of the VEGF promoter region. Transfected cells were washed with ice-cold PBS, lysed, and lysates were used directly to measure luciferase activity. The luciferase activity of each sample was determined by measuring luminescence for 10 s on a Lumat LB 9507 luminometer (EG&G Berthold, Oak Ridge, TN). The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments. Live Cell Microscopy Cells were plate on 6 well culture dishes and incubated at 37°C, 5% CO 2. Time series (10 min) of phase contrast images were acquired at a video rate of 1 frame/5s with a Real-time cell observer (Carl Zeiss). Time series of cells with and without methylsulfonylmethane were obtained at 0-120 min after adding the compound and every 24 h for up to four days. Tumorigenicity All procedures for animal experiment were approved by the Committee on the Use and Care on Animals (Institutional Animal Care and Use Committee, Seoul, Korea) and performed in accordance with the institution guidelines. MDA-MB 231 tumor xenograft were established by subcutaneously inoculating 1x107 cells into the right flanks of 5-week-old Balb/c nude mice (Orient Bio, Seongnam-Si, Korea). When tumors reached between 6 to 8 mm in diameter, mice were randomly assigned to control group, MSM 3%-treated group and MSM 5%- treated group respectively with 6 mice in each group. The drug was administered as intragastric injections of 100 μl, containing 3% MSM or 5% MSM in triple distilled water. The injections were repeated one time every other day. Tumor growth was monitored by periodic measurements with calipers. Tumor volume was calculated using the formula: tumor volume (mm3) = maximal length (mm) × (perpendicular width) (mm2)/2. Animals were sacrificed when the diameter of tumors reached 2 cm or after 30 days of treatment. In our experiments, no mice were observed to be died of tumor loading. All available human breast cancer xenograft collected from mice were reviewed and included in the study. Real-time Polymerase Chain Reaction Total RNA was isolated from tumor xenograft and quantified by a spectrophotometric analysis at 260 nm. The cDNA synthesis and the probe used for the detection of IGF-1 and β-actin from a TaqMan gene expression assay kit (Applied Biosystems Inc.). PCR was monitored in real time using the ABI Prism 7900 HT Real time PCR System (Applied Biosystems Inc., CA). Immunohistochemistry Formalin-fixed paraffin-embedded breast tumor xenografts were sliced into 5 μm thick section. These sections were deparaffinized with 100% xylene, rehydrated with decreasing concentration of ethyl alcohol, permeabilised with 0.1% triton X-100 and blocked with 10% NGS (Nomal Goat Serum in PBS). These were then incubated with the STAT5b, IGF-1R, STAT3 and VEGF antibody followed by incubation with the secondary antibody, Alexa Fluor 488 (rabbit) and Alexa Fluor 594 (mouse) (Invitrogen). For the detection of nuclear level, tissue sections were incubated on DAPI for one minute and rinsed with PBS. Samples were observed and photographed under the fluorescent microscope. Data analysis and Statistics The results of the experiments are expressed as mean ± SEM. Statistical analysis was done by t-tests or ANOVA-tests using the SAS program.

Author Contributions Conceived and designed the experiments: YY. Performed the experiments: EL DH JP YN. Analyzed the data: YJ SK TP. Contributed reagents/materials/analysis tools: TH SY EM BC KP HL. Wrote the paper: PD.