Stathmin is involved in the cooperative effect of Zoledronic acid and gefitinib on bone homing breast cancer cells in vitro

2.1. Cell cultures and drug treatment

The human breast cancer cell line MDA-MB-231 and its bone-seeking clone were studied. The bone-seeking clone had been established by repeated cardiac inoculation of the MDA-MB-231 parental cells into nude mice [16]. These cells were cultured in RPMI 1640 medium (Life Technologies Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum (GE Healthcare Japan, Hino, Japan) and 1% penicillin (GE Healthcare Japan) at 37 °C under 5% CO₂.

Zol was provided by Novartis Pharma (Basel, Switzerland). Gefitinib (Iressa^®) was purchased from Tocris Bioscience (St. Louis, MO, USA). Zol powder was dissolved in 0.1 N NaOH, and gefitinib powder was dissolved in dimethyl sulfoxide (DMSO). Cells were treated with 10 μM Zol and/or 1 μM gefitinib for 24 h or 72 h. The final concentration of NaOH or DMSO was less than 0.01% in all experiments.

2.2. Proliferation assay

Effects of Zol or gefitinib on cell proliferation were determined using the WST-8 assay (Dojindo, Kumamoto, Japan). This cell counting assay is sensitive for the determination of cell viability in cell proliferation assay. WST-8 is reduced by dehydrogenase activities in cells to give a yellow color formazan dye. The amount of formazan dye is directly proportional to the number of living cells. Brief protocol is as follows. Cells were seeded at a concentration of 1×10⁴ cells per well in triplicate in a 96-well plate (Asahi Glass, Tokyo, Japan), and pre-incubated at 37 °C for 24 h to allow cell attachment. At 72 h after drug treatment, 10 μl of WST-8 solution was added to each well and incubated at 37 °C for 1 h. The assay is based on the extracellular reduction of WST-8 to a water-soluble formazan dye formed by NADH produced in mitochondria. The augmentation of enzyme activity leads to an increase in the amount of formazan dye, which was quantified using a microplate reader (BIOTEC, Tokyo, Japan) by measuring the absorbance at 450–550 nm. The above procedure was repeated at least three times.

2.3. Invasion assay

Invasion was estimated using a 24-well Boyden chamber (BD BioCoat^TM Matrigel^TM Invasion Chamber, BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer's recommendations. After hydration of the matrigel inserts for 2 h, 5.0×10⁴ cells in 500 μl RPMI medium containing 10 μM Zol, 1 μM gefitinib, or Zol plus gefitinib were added to the upper chambers. The lower chambers were filled with 750 μl RPMI medium containing drugs at the same concentrations as those in the upper chambers. After 24 h of incubation at 37 °C, non-invasive cells were removed from the membranes, and the membranes were fixed with methanol and stained with crystal violet. Only cells that had penetrated the membranes were counted in 10 randomly chosen high-power fields (HPFs) (×400), and the mean number per HPF was adopted as the number of cells that had invaded through the membrane. The above experiments were performed in triplicate and repeated at least three times.

2.4. Protein extraction

The bone-seeking clone was cultured to 70% confluency and treated with each drug (Zol, gefitinib, or Zol plus gefitinib) for 24 h. The cells were then washed twice with ice-cold PBS, followed by fixation with 10% trichloroacetic acid, and scraped off into a tube and centrifuged at 3000 rpm for 10 min. The cell pellet was treated with lysis buffer (30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, dilute HCl, and dH₂O), and the resulting cell lysate was separated from debris by centrifugation and sonicated for 1.5 min at 250 W (Bioruptor, COSMO BIO, Yokohama, Japan). Protein concentration was measured with a 2-D Quant kit (GE Healthcare, Milwaukee, WI, USA).

2.5. 2-D DIGE (two-dimensional fluorescence difference gel electrophoresis)

Proteins were labeled with fluorescent cyanine dyes (GE Healthcare) in accordance with the manufacturer's instructions. In brief, 50 μg of a pair of samples extracted from the bone-seeking clone treated or untreated with an anti-cancer drug was adjusted to pH 8.5 with 50 mM NaOH and labeled with 400 pmol Cy3 or Cy5, while another pair of samples was labeled inversely with Cy5 or Cy3. The fluorescence labeling was performed on ice in the dark for 30 min, and then quenched with 1 μl of 10 mM lysine (Sigma–Aldrich, St. Louis, USA) for 10 min. Each preparation was treated with 2×sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS,1% immobilized pH gradient (IPG)-buffer, pH range 4–7, and 2% dithiothreitol (DTT), and the final volume was adjusted to 260 μl with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG-buffer, pH range 4–7, 0.2% DTT). The proteins were applied to Immobiline DryStrips (pH 4–7, 13 cm) and focused on an Ettan IPGphor II (GE Healthcare).

Four focused IPG strips were equilibrated, and then loaded onto 12.5% SDS-polyacrylamide gels (30% acrylamide, 1.5 M Tris–HCl pH 8.8, 10% SDS, 10% APS, 10% TEMED) using low-fluorescence glass plates on an Ettan DALT II system (GE Healthcare). All electrophoresis procedures were performed in the dark. After SDS-PAGE, a pair of gels were scanned with a Typhoon 9410 scanner (GE Healthcare) with appropriate excitation/emission wavelengths specific for Cy3 (532/580 nm) and Cy5 (633/670 nm) to generate protein spot maps. The above procedure was repeated three times.

DeCyder 7.0 software (GE Healthcare) was used for 2-D DIGE analysis in accordance with the manufacturer's recommendation. The DeCyder differential in-gel analysis (DIA) module was used for pairwise comparisons of each sample. Differential protein spots (|ratio|>2, P<0.05) that showed consistent alteration were selected.

2.6. In-gel digestion and mass spectrometric analysis

Peptide mass fingerprinting (PMF) was performed for mass spectrometric analysis. To extract the differential protein spots, 500 μg of unlabeled sample was loaded onto SDS gel as described above. The gel was then stained with Coomassie blue for 1 h, and destained for 1 h in 10% acetic acid and 40% methanol. After washing with distilled water overnight, each spot was excised from the gel using a 1-ml pipet and kept in a PCR plate with water. In-gel digestion was performed as described by Kondo et al. [17]. Briefly, reduction was done using 45 mg DTT in 10 mM ammonium bicarbonate (ABM) and alkylation using 40 mg iodoacetamide in 50 mM ABM. After addition of 50% methanol, the gel plugs were washed in an ultrasonic water bath with ice for 10 min, then dehydrated with 50–100% acetonitrile (ACN) and incubated in vacuo for 10 min. Each procedure was performed 4–6 times. After drying for 1 h, the gel plugs were digested overnight in 0.5% trypsin in 50 mM ABM. The trypsin digests were recovered with 45% ACN and 0.1% trifluoroacetic acid (TFA), and the gel plugs were subjected to speedvac at room temp for 20 min. Finally, 15 μl of 0.1% TFA was added.

The trypsin digests were subjected to spiral mass spectrometry (JMS-S3000 MALDI-spiral TOF-TOF, Japan Electron Optics Laboratory, Akishima, Japan) and a database search was done using the Mascot search engine at http://www.matrix-science.com. Proteins that scored greater than 62 were judged to be significant (P<0.05).

2.7. Western blot analysis

We further validated the identification of proteins using Western blot analysis. Samples from MDA-MB-231 cells and the derivative bone-seeking clone treated with 1 μM gefitinib and/or 100 μM Zol were extracted with cell lysis buffer (1 M Tris pH 7.5, NP-40, 5 M-NaCl, 0.5 M EDTA pH 7.5–8.0, 2 mM Na₃VO₄, 100 mM NaF, 10 mM NaP₂O₇). Fifty micrograms of each sample was separated using 12% SDS-PAGE gels, and transferred to a membrane using a Trans-Blot^® Turbo^TM Blotting System (Bio-Rad, Hercules, CA USA). After blocking with 5% non-fat milk powder in PBS overnight at 4 °C, the membranes were incubated with mouse monoclonal antibody against stathmin diluted 1:500 (BD Biosciences, San Jose, CA) or an anti-β-actin antibody (dilution 1:2000) (Sigma) for 1 h at room temperature. After washing with PBS containing 0.1% Tween 20, the membranes were incubated with the secondary antibody, anti-mouse IgG diluted at 1:2000 (GE Healthcare), for 30 min at room temperature and then washed for 30 min 4 times. The bands were reacted with chemiluminescence reagent (SuperSignal^®, Thermo, Rockford, IL, USA) and visualized using a luminescence image analyzer (LAS-3000, Fuji Film, Kanagawa, Japan). The level of protein expression was evaluated using Multi Gauge ver. 3.0 (Fuji Film).

2.8. Statistical analysis

The significance of differences between the mean values of triplicate experiments in the proliferation and invasion assays was calculated using one-way analysis of variance (ANOVA) test. Differences were considered to be statistically significant at P<0.05.

3.1. Cell proliferation

Dose–response studies of Zol and gefitinib for both cell lines were shown in Fig. 1, Fig. 2, respectively. Treatment with 10 μM Zol significantly reduced the absorbance of the bone-seeking clone from 0.77 nm (without any treatment) to 0.28 nm (reduction rate: 64%; 0.77–0.28/0.77; P<0.001 indicated by ⁎), and also that of MDA-MB-231 from 0.61 nm (without any treatment) to 0.17 nm (reduction rate: 72%; P<0.001 indicated by **) (Fig. 3). In the bone-seeking clone, combined treatment with 10 μM Zol and 1 μM gefitinib reduced the absorbance by 50% to 0.14 nm, in comparison with a reduction to 0.28 nm by treatment with Zol alone (P<0.01 indicated by ***). In contrast, the reduction rate was 18% in MDA-MB-231, and no significant cooperative effect of gefitinib with Zol was detected.

3.2. Invasion assay

The effects of Zol and/or gefitinib on invasion by the bone-seeking clone and MDA-MB-231 are shown in Fig. 4. The median number of cells that invaded through the membrane was significantly reduced by treatment with 10 μM Zol from 113.7 (without any treatment) to 35.0 (69%) for the bone-seeking clone (P<0.001 indicated by *) and from 80.0 (without any treatment) to 36.7 (54%) for MDA-MB-231cells (P<0.05 indicated by **). Combined treatment with 10 μM Zol plus 1 μM gefitinib markedly reduced the number of invading cells from 35.0 (treatment with Zol alone) to 4.3 (88%) for the bone-seeking clone (P<0.01 indicated by ***), but no such significant effect was detected for MDA-MB-231 cells.

3.3. 2-D DIGE

2-D DIGE identified four spots whose expression levels were significantly changed by treatment with Zol (Fig. 5a). All four spots were up-regulated more than 3-fold. Treatment with 1.0 μM gefitinib alone induced differential up-regulation of the expression of one spot (indicated by the arrowhead in Fig. 5c and d), but the other spots showed no significant change in expression though many spots were differentially expressed within 2-folds up- or down-regulation.

When the bone-seeking clone was treated with both Zol and gefitinib, these four spots were confirmed to show a change in protein level. Moreover, three new spots, numbered ①∼③, appeared and showed significant down-regulation of their expression (Fig. 5d). These three spots, whose expression levels were not changed by treatment with either Zol or Gef alone (Fig. 5b and c, respectively), were located within a pH range of 5–6 and had molecular masses of 17–20 kDa. They were then subjected to identification by mass spectrometry.

3.4. Mass spectrometric analysis

We focused on the three protein spots (Fig. 5b–d ①∼③) whose expression was significantly down-regulated by combined treatment with the two drugs. Fig. 6 shows the peak flow of the digested peptide derived from spot ③, which was identified as stathmin by a Mascot search. Among the three spots, ② and ③ shared a common sequence of 16 peptides (P=0.005). There was no evident difference in the amino acid sequences that could explain their different electrophoretic mobilities. Spot ① could not be identified in this study.

3.5. Western blot analysis

Expression of stathmin in the bone-seeking clone was increased 6-fold in comparison with that in the parental line, MDA-MB-231 (Fig. 7a). In the bone-seeking clone, it was confirmed that the expression level of stathmin was halved by combined treatment with Zol and gefitinib, in comparison with Zol alone (Fig. 7b). This kind of down-regulation was not clearly detected in MDA-MB-231 because MDA-MB-231 expressed stathmin lower than bone-seeking clone without the treatment of Zol or gefitinib. (data not shown).