Clinical Significance of Galectin-7 in Epithelial Ovarian Cancer

Materials and Methods

Tumor samples. In this study, a total of 68 paraffin-embedded tissues were used. These included 63 specimens of EOC. As controls, we also obtained normal ovarian tissues (n=5) from patients who underwent hysterectomies for benign disease. All operations were performed at the Department of Obstetrics and Gynecology at Samsung Medical Center in Seoul, Korea between 1997 and 2005. All of the patients were treated with maximal debulking surgery, which was followed by administration of intravenous paclitaxel (175 mg/m²) or docetaxel (75 mg/m²) plus carboplatin [area under the curve (AUC) of 5] combination chemotherapy every three weeks for 6-8 cycles. We divided patients into two groups according to their sensitivity to the first-line platinum-based combination chemotherapy: i) platinum-resistant, as defined by a platinum-free interval of less six months, and ii) platinum-sensitive, as defined by platinum-free interval greater than or equal six months. Surgical staging was established according to the International Federation of Gynecology and Obstetrics (FIGO) system (19). Optimal cytoreduction was defined as no grossly visible tumor at the completion of the surgical procedure. Sub-optimal or non-complete cytoreduction was defined as residual tumor measuring >0 cm at the completion of the surgical procedure. All samples were collected according to the Institutional Review Board of the Samsung Medical Center (IRB number: 2009-09-002-002).

Immunohistochemistry. Immunohistochemical studies were carried out on formalin-fixed, paraffin-embedded 4-μm-thick tissue sections. The primary antibodies used were rabbit polyclonal antibodies against GAL-7 (a gift from Dr. Sabine Andre, Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Munich, Germany). Tissue sections were de-paraffinized three times in xylene for a total of 15 min and subsequently rehydrated. Antigen retrieval was carried out at 97°C, with PTLink (DAKO, Glostrup, Denmark) for 20 min in citrate buffer (pH 6.0). After blocking endogenous peroxidase activity with 3% hydrogen peroxidase for 10 min, the primary antibody incubation for GAL-7 was carried out for 120 min at room temperature, with a dilution of 3 μl/ml. The antigen–antibody reaction was detected using the DAKO REAL TM EnvisionTM Detection system, Peroxidase/DAB K5007 (DAKO). Counterstaining was performed with Mayer's hematoxylin. Staining for GAL-7 was considered positive when tumor cells exhibited nuclear and/or cytoplasmic reactivity. Negative controls [substituting Tris-buffered saline (TBS) for primary antibody] were run simultaneously. Two pathologists (Dr.C.O.Sung and Dr.I.G.Do) without previous knowledge of the clinical outcomes assessed each slide. The intensity of staining was graded on a semi quantitative scale from 0 to 3, where 0=no staining, 1=weak staining, 2=moderate staining, and 3=strong staining. The percentage of positive cells was stratified from 0 to 3, where 0%=0, 1-20%=1, 21-50%=2, 51-100%=3 (20). The total score was calculated by multiplying the intensity score and the stratified score for the percentage of positive cells and this ranged from 0 to 9. Scores from 0 to 4 were considered low and scores from 5 to 9 were considered high.

Cell lines. Human EOC cells (HeyA8, SKOV3ip1 and A2780-PAR) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The taxol-resistant EOC cells (HeyA8-MDR, SKOV3-TR) and the cisplatin-resistant EOC cells (A2780-CP20) were a gift from Dr. Anil K. Sood, Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA. Human EOC cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA, USA) in 5% CO₂ at 37°C.

Transfection of GAL-7 siRNA. GAL-7 siRNA and negative control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A2780-PAR cells were seeded at 3x10³ cells per well in a 96-well microplate in culture media with 10% FBS. siRNA was transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Cells were then incubated at 37°C for 72 h.

Western blot analysis. Cells were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein lysates were separated on 15% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-enhanced chemiluminescence (ECL) nitrocellulose filter paper (Amersham, Little Chalfont, Buckinghamshire, UK). Membranes were blocked with 5% skimmed milk in Tris-buffered saline, containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with an antibody against GAL-7 (Abcam, Cambridge, UK), and α-tubulin (Epitomics, Burlingame, CA, USA), and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (Amersham, Piscataway, NJ, USA). Bands were visualized using an ECL kit (Amersham), according to the manufacturer's protocol.

Isolation of total RNA, cDNA synthesis, and RT-PCR. Total RNA from each cell line was extracted using an Ambion mirVana miRNA isolation Kit (Ambion, Austin, TX, USA). First-stranded cDNA was synthesized by reverse transcriptase using the High Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. The cDNA was amplified by PCR using the following primer sequences for GAL-7: 5’-ACCAACCCGGTCCCAG-3’ (forward) and 5’-GCGGGCTAACG CTTTATTTGC-3’ (reverse) (13). An endogenous control cDNA was formed using β-actin primers: 5’-GATGCAGAAGGAGATCACTG-3’ (forward) and 5’-AGTCATAGTCCGCCTAGAAG-3’ (reverse). PCR was carried out by initial denaturation at 95°C for 5 min, followed by either 35 cycles or 28 cycles of denaturation (95°C, 40 s), annealing (68°C for GAL-7, 57°C for β-actin, 40 s), and extension (72°C, 1 min) for GAL-7 and β-actin, respectively. This was followed by a final extension step at 72°C for 10 min. Amplification products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining under ultraviolet transillumination.

Proliferation assay. For the proliferation assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) solution (Amresco, Solon, OH, USA) was subsequently added to each well. After an additional 4 h of incubation, the medium was discarded, 100 μl of acidic isopropanol (0.1 N HCl in absolute isopropanol) was added, and the plate was shaken gently. Absorbance was measured on an enzyme-linked immunosorbent assay (ELISA) reader at a test wavelength of 540 nm.

Statistical analysis. Fisher's exact probability test or the chi-square test was used to assess for statistical significance between GAL-7 expression and clinicopathological parameters. The Fisher's exact test was used if the expected frequency was less than five. Kaplan-Meier curves were plotted to assess the effects of GAL-7 expression on survival. These survival curves were compared using the log-rank test. Variables shown to be significant or borderline-significant (p<0.25) in the univariate analysis were selected for the Cox model. p-Values less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS (version 10.0; SPSS, Inc, Chicago, IL, USA).