Hypoxia-induced Maspin Expression Affects the Prognosis of Ovarian Clear Cell Carcinoma

Materials and Methods

Reagents, cell lines, and tissue samples. Cisplatin and N-acetylcysteine (NAC) were acquired from Sigma-Aldrich (St. Louis, MO, USA), and paclitaxel was donated by Samyang Biopharmaceuticals Corp. (Seongnam-si, Republic of Korea). For western blot and immunohistochemistry, anti-HIF-1α was obtained from Abcam (Cambridge, UK). Anti-CBP, anti-FIH, an-ti-HIF-1β, and anti-VHL antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Moreover, anti-P300, anti-VEGF, and anti-maspin antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

ES-2, a clear cell carcinoma cell line of human ovarian origin (12), and five frozen tissues, each from either a normal ovary or OCCC, were purchased from the Cancer Tissue Bank of Seoul National University (Seoul, Republic of Korea).

Cell culture. ES-2 cells were cultured in a 1:1 mixture of MCDB 105/Medium 199 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. ES-2 cells were grown in the culture media until reaching up to 70% confluency in monolayers in 100-mm tissue culture dishes; subsequently, cells were serum-starved for 24 h before the assays.

Incubation under normoxic and hypoxic conditions. For normoxic conditions, ES-2 cells were grown in a standard humidified incubator containing 5% CO₂ and 21% O₂ at 37°C. For hypoxic conditions, cells were incubated in a commercially available hypoxia incubator chamber (STEMCELL Technologies Inc., Vancouver, Canada) containing 90% N2; 5% CO₂; 5% O₂, 92% N₂; 5% CO₂; 3% O₂, and 94% N₂; 5% CO₂; 1% O₂.

Cytotoxicity and cell viability assays. The cultured ES-2 cells were treated with gradually escalated doses of paclitaxel, and cisplatin from 0 to 100 μmol/l was applied to cultured ES-2 cells for determining the half-maximal inhibitory concentration (IC₅₀) under normoxic conditions. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] colorimetric assay was performed for assessing cell viability. Following serum starvation for 24 h, MTT solution at 5 mg/ml was applied to ES-2 cells for 4 h at 37°C. After that, the medium was substituted with 100 μl of 0.04 N HCl in isopropanol to dissolve the formazan crystals. Cell viability was quantified with a microplate reader (Bio-rad, Seoul, Republic of Korea) at 595 nm and 655 nm as a background. The percentage of cell viability was calculated according to the following equation:

Cell viability percentage (%)=(absorbance of hypoxia or chemical compound-treated cells divided by absorbance of control cells)×100.

Quantitative real time polymerase chain reaction assay. Total cellular RNA of ES-2 cells and each frozen tissue derived from either normal ovary or OCCC were extracted by utilizing Trizol reagent (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s guidelines. Complementary DNA was synthesized using total RNA extracted from ES-2 cells with the AccuPower RT PreMix (Bioneer, Daejeon, Republic of Korea). Gene expression was measured using SYBR Green (Sigma) and a StepOnePlus qRT-PCR system (Applied Biosystems, Foster City, CA, USA). For the control and normalization of variation, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was analyzed simultaneously in each experiment. The sequences of the forward and reverse primers of the eight hypoxia-related genes are shown in Supplementary Table I. The forward primer (5’-ACA CAG AAG ACG GTG GAT GG-3’) and the reverse primer (5’-GGC AGG TCA GGT CAA CAA CA-3’) for the GAPDH gene were constructed for the amplification of GAPDH, a 193 bp product. Subsequently, expression levels of the eight hypoxia-related genes were determined using the cycle threshold (CT) values and the standard curve method. The values were normalized based on the expression of the GAPDH gene.

The conditions of polymerase chain reaction (PCR) were the following: denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 30 s, annealing at 60°C for 30 s, and DNA synthesis at 72°C for 30 s. PCR was performed using a melting curve program, which increased the temperature at a rate of 0.5°C every 10 s, ranging from 55°C to 95°C with a continuous fluorescence measurement. ROX reference dye (Invitrogen, Carlsbad, CA, USA) was used as a negative control for the fluorescence measurements. Sequence-specific products were identified by generating a melting curve, where CT value represented the cycle number at which a fluorescent signal was statistically more significant than the background. Relative gene expression levels were quantified using the 2-ΔΔCTmethod (13).

Determination of the levels of intracellular reactive oxygen species. Intracellular reactive oxygen species (ROS) production was evaluated by using 2’,7’-dichlorofluorescein diacetate (DCFH-DA, Sigma), which was converted to fluorescent 2’,7’-dichlorofluorescein (DCF) in the presence of peroxides. ES-2 cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA), collected by centrifugation, and washed with phosphate-buffered saline (PBS). The cells were treated with 10 μM DCFH-DA for 30 min at 37°C. Then, the cells were washed with PBS twice and treated with NAC in a dose-dependent manner (0, 1, 2, 5, and 10 mM) for 1 h at 37°C in a CO₂ incubator, with a combination of either paclitaxel or cisplatin. The treated cells were re-washed with PBS. Fluorescent DCF intensity was analyzed using a flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).

Western blot. Protein concentration of CBP, P300, HIF-1α, HIF-1β, FIH, VHL, VEGF, and maspin in whole-cell extracts was estimated using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. Protein extract was denatured, separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and developed through enhanced chemiluminescence detection (Super-Signal, West Pico, IL, USA). The protein amount was quantified by measuring light intensity from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). Detection of immunore-active proteins was done using goat anti-rabbit polyclonal antibodies against phospho-proteins and total-proteins at a 1:1,000 dilution and 10% SDS/PAGE gel. Total protein levels were used as a loading control for the normalization of results from protein detection by western blotting. Multiple exposures of each western blot were performed to ensure the linearity of chemiluminescent signals.

Human population. The clinicopathological data of 61 patients with OCCC between July 2001 and November 2012 were gathered. The inclusion criteria were as follows: diagnosis of OCCC; primary cytoreductive surgery followed by adjuvant chemotherapy using paclitaxel (175 mg/m²) and cisplatin (50 mg/m²); Eastern Cooperative Oncology Group (ECOG) performance status ranging from 0-2; and no underlying diseases which may affect survival.

Data on the patients’ stage based on the International Federation of Gynecology and Obstetrics (FIGO) classification, age, the extent of cytoreductive surgery, platinum-resistance, progression-free survival (PFS), and overall survival (OS) were extracted. Optimal cytoreduction was defined as cytoreductive surgery with a maximal diameter of residual tumors measured ≤1 cm. Platinum resistance was defined as a treatment-free interval less than six months following completion of adjuvant chemotherapy. PFS was defined as the period between the date of surgery and the date of disease recurrence, and OS was defined as the period from the date of surgery to the date of death or end of this study.

Immunohistochemistry. We conducted immunohistochemistry (IHC) to evaluate the expression of the eight hypoxia-related genes in patients with OCCC. For the negative control, purified non-immune mouse IgG as primary antibody substitution was utilized at the same concentration. Representative sections of core tissue (2 mm diameter) were sliced from paraffin blocks and arranged in new tissue microarray blocks using the trephine apparatus (SuperBioChips Laboratories, Seoul, Republic of Korea). Subsequently, a pathologist who was unaware of the patients’ clinicopathological characteristics interpreted and scored the eight hypoxia-related genes’ expression levels semi-quantitatively. The scoring system was based on two parameters - the product of staining intensity, read as I score (1, weak; 2, moderate; and 3, strong) and the percentage of positive cells, read as P score (1, <25%; 2, 25-50%; 3, 51-75%; and 4, >75%). The immunoreactivity score was defined as I score multiplied by P score, ranging from 1 to 12. Protein expression was considered high when the score was six or more because the median score was six.

Statistical analysis. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Seoul National University Hospital (No. 1311-050-533). Informed consent was waived due to the retrospective design of the study and there were no ethical risks.

All in vitro experiments were replicated three times for obtaining consistent results, and cell viability assays were analyzed by the analysis of variance (ANOVA) method based on the general linear model (PROC-GLM) of the SAS program (SAS Institute Inc., Cary, NC, USA). For identifying the roles of the eight hypoxia-related genes in influencing platinum resistance and PFS, the Chi-square test and Student’s t-test were performed. Kaplan-Meier analysis with the log-rank test, Cox’s proportional hazard, and logistic regression analyses were done to determine the hazard ratio (HR), odds ratio (OR), and 95% confidence interval (CI). Statistical analysis was performed using SPSS software version 21.0 (SPSS Inc., Chicago, IL, USA). A p-value <0.05 was considered statistically significant.