The present study included 54 patients with ovarian serous carcinoma (FIGO stages III and IV). All patients were treated at Osaka City University Hospital (Osaka, Japan) from January 2005 to December 2012. Patients were divided into two groups based on recurrence of cancer within 6 months from the last platinum administration. Both groups underwent maximum debulking surgery followed by platinum-based chemotherapy. In the first group (platinum-sensitive group), disease did not recur within 6 months from the last platinum administration whereas in the second group (platinum resistant group), disease recurred within 6 months. Written informed consent was obtained from each patients prior to surgery. The study proposal was approved by the Institutional Review Board (IRB) of Osaka City University Hospital (IRB no. 3525).

Immunohistochemical analysis was performed to examine UCP2 expression in paraffin-embedded sections using an anti-UCP2 antibody (cat. no. ab116263; Abcam, Cambridge, UK) and a Dako LSAB2 Peroxidase kit (cat. no. K0675; Agilent Technologies, Inc., Santa Clara, CA, USA). The paraffin-embedded sections (4 µm-thick) were de-paraffinized, hydrated, and immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. For antigen retrieval, sections were immersed in 10 mM citrate buffer (pH 6.0) and heated in an autoclave at 110°C for 20 min. Then tissue sections were washed in PBS and incubated overnight at 4°C with a 1:100 dilution of the aforementioned rabbit polyclonal antibody for UCP2. Next, sections were washed in PBS for 15 min and incubated with biotinylated goat anti-rabbit immunoglobulin G (Dako; Agilent Technologies, Inc.) for 10 min. After washed with PBS, sections were incubated with a streptavidin-peroxidase solution and 3,3′-diaminobenzidine was used as the chromogenic reagent. Finally, sections were counterstained with hematoxylin. The specificity of the immunohistochemical reactions was confirmed by omitting the primary antibody.

The immunohistochemical expression of UCP2 were assessed quantitatively according to the weighted score method of Sinicrope et al (24). Staining intensity was categorized into three classes: 1+, weak; 2+, moderate; and 3+, intense. The mean percentage of stained tumor cells was classified as follows: 0, ≤5%; 1, 5< and <25%; 2, 25< and <50%; 3, 50< and <75%; 4, >75%. The weighted score was determined by multiplying the score of staining intensity for each tissue specimen by that of percentage of stained tumor cells.

The human ovarian serous carcinoma cell line OVSAHO (no. JCRB1046; National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan) was cultured in RPMI medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured in a humidified 5% CO² atomosphere and at 37°C.

The sensitivity of cells to carboplatin was examined using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were collected and seeded into a 96-well tissue culture plate at a density of approximately 2×10³ cells/ml. After 24 h the culture medium was replaced with 100 µl of fresh medium per well and 10 µl dimethyl sulfoxide (DMSO) alone or containing 50 µM genipin (cat. no. G-4796; Sigma Aldrich, Missouri, USA) was added to each well. Cells were then treated with carboplatin (10–1,000 µM) for 24 h. At the end of treatment 10 µl CCK-8 was added and the plates were incubated for 2 h before measurement of the absorbance at 450 nm with a microplate reader (Corona Electric Co., Ltd., Ibaraki, Japan). Dose-response graphs were constructed as the percentage of viable cells compared with the control cells.

Total RNA was extracted from the human ovarian serous carcinoma cell line OVSAHO using a RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) and reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Gene expression of UCP2 was determined using a TaqMan Gene Expression Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) in the Applied Biosystems 7500 Fast Real-Time PCR System. For quantification, gene expression was normalized to that of GAPDH.

Statistical analyses were performed using SPSS software version 21.0 (IBM SPSS, Armonk, NY, USA). Data are presented as the mean ± standard error in Figures and as the mean ± standard deviation in Tables. Kaplan-Meier and log-rank analyses were performed to assess prognosis. Mann-Whitney U test was performed to compare the weighted scores. The differences between the means of two groups were assessed using Student's t-test, and associations of the categorical variables in two groups were assessed using χ² tests. P<0.05 was considered to indicate a statistically significant difference.

A total of 54 patients with ovarian serous carcinoma were divided into the platinum-sensitive group (n=27) and the platinum-resistant group (n=27). Table I shows age, FIGO stage, tumor marker, and post-surgery observations for the study patients. There were no significant differences in these parameters between the two groups other than postoperative residual disease.

Cytoplasmic expression of UCP2 was observed in tumor cells (Fig. 1). Table II shows the UCP2 weighted scores in tissues of the two patient groups. The mean weighted score for UCP2 expression was significantly lower in the platinum-sensitive group compared with the platinum-resistant group (5.1 and 7.9, respectively, P=0.005; Table II and Fig. 2).

In continuation, cases were classified into two groups according to their UCP2 expression levels: the low UCP2 expression group (weighted score, 0–6) and the high UCP2 expression group (weighted score, 8–12). Table III shows the characteristics of the high and low expression groups, with analyses revealing no significant differences between the two groups other than postoperative residual disease.

Within the low UCP2 expression group, 19 cases (73.1%) belonged to the platinum-sensitive group while 7 (26.9%) belonged to the platinum-resistant group. In the high UCP2 expression group, 8 cases (28.6%) belonged to the platinum-sensitive group and 20 (71.4%) belonged to the platinum-resistant group. The low UCP2 expression group was significantly more sensitive to platinum-based chemotherapy than the high UCP2 expression group (P=0.001; Table IV).

The low UCP2 expression group showed significantly better overall survival compared with the high UCP2 expression group (P=0.006; Fig. 3).

Expression of UCP2 mRNA in the ovarian serous carcinoma cell line OVSAHO was confirmed by real-time PCR. UCP2 expression in OVSAHO cells was suppressed following 24 h of incubation with 10 or 50 µM genipin (Fig. 4). Then, we examined whether the sensitivity of ovarian serous carcinoma cells to carboplatin was affected by treatment with genipin. Genipin-mediated inhibition of UCP2 expression in OVSAHO cells significantly enhanced their sensitivity to carboplatin (Fig. 5).