The following were purchased from the indicated sources: penicillin-streptomycin solution and fetal bovine serum (FBS) from Hyclone (South Logan, UT, USA); RPMI-1640 from Sigma Chemical Co. (St. Louis, MO, USA); trypsin-EDTA (0.05%) from Gibco-BRL (Grand Island, NY, USA); STAT5b, HER2 antibodies, and secondary antibody (goat anti-mouse and rabbit IgG-horseradish peroxidase) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); phosphorylated STAT5 from Upstate Biotechnology (Lake Placid, NY, USA); β-actin from Sigma Chemical Co.; the enhanced chemiluminescence (ECL) detection kit from Amersham Pharmacia Biotech (Piscataway, NJ, USA); Restore™ Western Blot Stripping Buffer and NE-PER kit from Pierce (Rockford, IL, USA); the electrophoretic mobility shift assay (EMSA) kit, oligonucleotide probes (STAT5b), luciferase assay substrates, and reporter lysis buffer from Promega Corp. (Madison, WI, USA); FuGENE6 transfection reagent from Roche (Basel, Switzerland); RNeasy mini kit and Qiaprep spin miniprep kit from Qiagen (Hilden, Germany); the RT-PCR Premix kit and VEGF, IGF-1R, 18s primer for RT-PCR were synthesized by Bioneer (Dajeon, Korea); imprint chromatin immunoprecipitation assay kit from Sigma Chemical Co.; and MSM from Fluka/Sigma Co. (St. Louis, MO, USA).

The human breast adenocarcinoma cell lines, SK-BR3 and MCF-7, were maintained in RPMI-1640 medium containing 10% FBS and 100 U/ml penicillin and streptomycin at 37°C in 5% CO₂. The cells were placed in airtight chambers (Nu Aire, Plymouth, MN, USA). At the beginning of each experiment, the cells were resuspended in the medium at a density of 2.5×10⁵ cells/ml. Cells were treated with 300 mM MSM.

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 only active in live cells. The cells were resuspended in the medium one day before drug treatment, at a density of 3×10³ cells per well in 96-well culture plates. Liquid medium was replaced with fresh medium containing dimethyl sulfoxide (DMSO) as a control (vehicle). Cells were incubated with various concentrations of MSM. Then, MTT (5 mg/ml) was added to each well and incubated for 4 h at 37°C. The formazan product formed was dissolved by adding 200 μl DMSO to each well, and the absorbance was measured at 550 nm on an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC, USA). All measurements were performed in triplicate, and were repeated at least three times.

The SK-BR3 and MCF-7 cell lines were treated with MSM. Whole cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer, containing phosphatase and protease 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 concentrations were measured using the Bradford method. Equal amounts of proteins were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk. Membranes were probed overnight at 4°C with a primary antibody followed by horseradish peroxidase-conjugated secondary antibodies. Detection was performed using the ECL Plus detection kit and a LAS-4000 imaging device (Fujifilm, Japan).

Total RNA was extracted using the RNeasy Mini kit (Qiagen) and quantified spectrometrically at 260 nm. Then, RT-PCR analysis for HER2 and 18s RNA was performed. The cDNA was synthesized from total RNA by RT at 42°C for 1 h and 95°C for 5 min using first strand cDNA synthesis kits (Bioneer, Korea). The cDNA was used in PCR with the following primers: HER2 sense, 5′-TGCGGCTCGTACACAGGGACTT-3′ and HER2 antisense, 5′-TGCGGAGAATTCAGACACCAACT-3′, with a 420-bp amplified HER2 mRNA fragment; 18s sense, 5′-CGGCTACCACATCCAAGGAA-3′ and 18s antisense, 5′-CCGGCGTCCCTCTTAATC-3′, with a 489-bp amplified 18s mRNA fragment. The PCR conditions consisted of denaturation for 1 min at 95°C, annealing for 1 min at 58°C, and extension for 1 min at 72°C. The PCR products were analyzed on a 1% agarose gel stained with ethidium bromide.

The DNA binding activity of STAT5b was assessed using EMSA, in which labeled double-stranded DNA was used as a DNA probe to bind active STAT5b proteins in nuclear extracts. Nuclear protein extracts were prepared with a nuclear extract kit (Panomics, AY2002). The EMSA experiment was performed by incubating a biotin-labeled transcription factor-STAT5b probe with treated and untreated nuclear extracts. Proteins were resolved on a non-denaturing 6% polyacrylamide gel (Bio-Rad, Korea). The proteins in the gel were transferred to a nylon membrane and detected using streptavidin-horseradish peroxidase and a chemiluminescent substrate.

The ChIP assay was performed using the Imprint chromatin immunoprecipitation kit (Sigma) according to the manufacturer's protocol. Briefly, SK-BR3 cells were fixed with 1% formaldehyde and quenched with 1.25 M glycine. After washing with PBS, the cells were suspended in nuclei preparation buffer and shearing buffer and sonicated under optimized conditions. This sheared DNA was then centrifuged and the cleared supernatant used for protein/DNA immunoprecipitation. The clarified supernatant was diluted with buffer (1:1 ratio) and 5 μl of the diluted samples was removed as an internal control. The diluted supernatant was incubated with antibody (STAT5b) in pre-coated wells for 90 min. The negative and positive controls were normal goat IgG and anti-RNA polymerase II, respectively. The unbound DNA was washed off with IP wash buffer and the bound DNA was collected by cross link reversal using DNA release buffer containing proteinase K. The released DNA and DNA from the internal control were purified with the GenElute Binding Column G. The DNA was then quantified using conventional PCR.

Cells were co-transfected with various combinations of the following constructs: wild-STAT5b (pMX/STAT5b; kindly provided by Dr Koichi Ikuta, Kyoto University, Japan), constructed as previously described (38), and the HER2 reporter construct containing 5.6 kb of the HER2 promoter region. Transfected cells were washed with ice-cold PBS and lysed. Lysates were used directly to measure luciferase activity. The luciferase activity of each sample was determined by measuring luminescence for 10 sec on a Lumat LB 9507 luminometer (EG&G Berthold, TN, USA). The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments.

SK-BR3 cells (1×10⁵) were cultured on 6-well plates and grown to 50% confluence. The cells were then transfected with On-Target plus SMARTpool siRNA targeting STAT5b or On-Target plus non-targeting siRNA (Dharmacon, Chicago, IL, USA) using FuGENE6 (Roche, IN, USA), according to the manufacturer's instructions. Following transfection with this mixture for 48 h, invasion assays were conducted without adding drugs for an additional 24 h. Different areas were captured and the cells were counted.

The effect of MSM on the viability of the human breast cancer cell line SK-BR3 was examined using the MTT assay. The SK-BR3 cells were exposed to increasing concentrations of MSM (100, 200, 300 and 400 mM) for a period of 24 h. Following this, the metabolically viable cells were quantified based on the amount of formazan crystals formed. Treatment with MSM substantially decreased the viability of SK-BR3 cells in a dose-dependent manner. It was observed that 100 mM MSM inhibited SK-BR3 cell growth by 32%, 200 mM MSM by 45%, and 300 mM MSM by 51% (Fig. 1). Therefore, the 300-mM concentration of MSM is considered the IC₅₀ dosage and was used in the further experiments.

To study the effect of MSM on STAT5b and HER2, SK-BR3 and MCF-7 cells were treated with 300 mM MSM for 24 h. Whole cell lysates were prepared in 1X RIPA lysis buffer containing 1X protease and 1X phosphatase inhibitor. Western blotting of the whole cell lysates prepared from MSM-treated and non-treated cells showed a decrease in the expression and phosphorylation of STAT5 in MSM-treated cells. Concurrently, the expression of HER2 also decreased in both SK-BR3 and MCF-7 cells (Fig. 2A). Compared with the control group, the MSM-treated group showed a 30% decrease in STAT5 phosphorylation (Fig. 2B); while, STAT5b and HER2 expression levels were inhibited ~50 and 40%, respectively (Fig. 2B).

We analyzed the expression of the STAT5b target gene, HER2. The RT-PCR analysis showed a decrease in the transcription of HER2 mRNA in MSM-treated cells (Fig. 2C) when amplifying HER2 with gene-specific primers and using 18S RNA as a loading control. Treated cells had ~25 and 40% inhibition of HER2 expression when compared with non-treated control SK-BR3 and MCF-7 cells, respectively (Fig. 2D). The ability of MSM to suppress HER2 expression was confirmed at the translational level.

A previous study showed STAT5 was a transcription factor for HER2. We then analyzed the nuclear level expressions of STAT5b and HER2 after MSM treatment. The nuclear extracts of the MSM-treated groups showed decreased levels of p-STAT5 and HER2 (Fig. 3A). Results from the electrophoretic mobility shift assay indicated that MSM suppressed the STAT5b-DNA binding activity in SK-BR3 cells (Fig. 3B). Therefore, it was necessary to determine the DNA-binding site of STAT5b, which we found corresponds to the interaction of STAT5b with the GAS element (TTCagcGAA) of the HER2 gene (Fig. 3C). There results proved that MSM inhibited the binding of p-STAT5 to the HER2 gene promoter site.

To perform transcriptional functions, phosphorylated STAT5b should translocate to the nucleus from the cytosol. Thus, these nuclear translocations were analyzed using the ChIP assay. We found that MSM treatment led to a decrease in the STAT5b binding to the HER2 promoter in SK-BR3 cells. The quantitative analysis by qPCR showed that MSM treatment inhibited binding of STAT5b to the HER2 promoter region in SK-BR3 cells (Fig. 3D). There was significant downregulation of DNA binding of STAT5b in MSM-treated cells. These results indicate MSM plays an important role in the suppression of binding.

The transcription activation functions of STAT5b after MSM treatment were studied using a luciferase assay. After treatment with MSM for 24 h, relative luciferase activity was decreased significantly for STAT5b/HER2 (Fig. 4A, ^***P<0.001). This finding confirms the critical role of MSM in inhibiting the transcription promoter activities of STAT5b.

To analyze the correlation between STAT5b and HER2, we overexpressed the STAT5b protein in MCF-7 cells. After transfection with STAT5b for overexpression, we analyzed the expression of STAT5b and HER2 using western blotting (Fig. 4B). Treatment with MSM led to ~15% inhibition of STAT5b and HER2 compared with the control in MCF-7 cells. In cells overexpressing STAT5b, the MSM treatment led to ~60% inhibition of STAT5b and HER2 compared with non-transfected MSM-treated MCF-7 cells (Fig. 4C). Expression levels of STAT5b and HER2 after MSM treatment showed similar expression patterns in cell lysates, demonstrating the ability of MSM to suppress STAT5b and HER2 expression.

To explore whether STAT5b is involved in HER2 expression, we used the siRNA strategy. Before MSM treatment, STAT5b in SK-BR3 cells was knocked down with a specific STAT5b siRNA. Interestingly, the results showed a similar pattern for STAT5b and HER2, with the knockdown of STAT5b leading to decreased HER2 protein expression. After MSM treatment, the HER2 expression was less in cells targeted with siSTAT5b than in cells treated with non-targeting siRNA (Fig. 5A). The relative expression of proteins, with respect to actin, gave a clear view of the effect of regulating STAT5b-related HER2 expression with MSM (Fig. 5B). From there results, we concluded that STAT5b plays an essential role in HER2 activation.