All investigations described in this study were carried out after obtaining informed consent and in accordance with an Institutional Review Board protocol approved by the Partners Human Research Committee at the Affiliated Hospital of Nantong University, Nantong, China. A total of 119 human EOC tissue specimens (45 papillary serous adenocarcinoma, 16 papillary mucinous carcinoma, 14 endometrioid adenocarcinoma and 14 clear cell carcinoma specimens, as well as 30 specimens classified as ‘other’) were provided by the Department of Pathology, the Affiliated Hospital of Nantong University from 2004 to 2009. None of the patients had received chemotherapy or radiotherapy prior to surgery. A total of 10 tissue samples were obtained at the time of surgery and were immediately stored in liquid nitrogen and maintained at −80°C until used in western blot analysis, including 1 normal tissue sample from a woman who underwent hysterectomy for benign disease. The clinicopathological characteristics of all the participants are shown in Table I. According to the WHO system, the histological classification of tumors was graded as follows: well differentiated [grade 1 (G1); n=15], moderately differentiated [grade 2 (G2); n=33] and poorly differentiated [grade 3 (G3); n=71].

The antibodies used for western blot analysis and immunohistochemistry were as follows: mouse anti-CAP1 monoclonal antibody (SC-376286), mouse anti-human Ki-67 monoclonal antibody (SC-101861), mouse anti-human proliferating cell nuclear antigen (PCNA) monoclonal antibody (sc-56), rabbit anti-human cyclin A polyclonal antibody (sc-751) and rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (sc-25778; all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Total protein extracts (100 mg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) filter membranes (Millipore, Bedford, MA, USA). After the membranes were blocked in 5% non-fat milk in TBST (150 mM NaCl, 20 mM Tris, 0.05% Tween-20) for 2 h, they were incubated with the primary antibodies overnight at 4°C. After washing the membranes with TBST 3 times for 5 min each, horseradish peroxidase-linked IgG secondary antibodies (donkey anti-mouse IgG-HRP, sc-2314; Santa Cruz Biotechnology, Inc.) were added followed by incubation for 2 h at room temperature. The membranes were developed using the ECL detection system.

Serial sections (5-μm-thick) were mounted on glass slides coated with 10% polylysine and were dewaxed in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by soaking in 0.3% hydrogen peroxide. The sections were then processed in 10 mmol/l citrate buffer (pH 6.0) and heated to 121°C in an autoclave for 20 min to retrieve the antigen. After rinsing in phosphate-buffered saline (PBS, pH 7.2), the sections were incubated with mouse anti-human CAP1 antibody (diluted 1:10,000) and mouse anti-human Ki-67 antibody (diluted 1:600) for 2 h at room temperature. Negative control slides were also processed in parallel using a non-specific immunoglobulin IgG (Santa Cruz Biotechnology, Inc.) at the same concentration as the primary antibody. All slides were processed using the peroxidase-anti-peroxidase method (Dako, Hamburg, Germany). Following rinsing with PBS, the peroxidase reaction was visualized by incubating the sections with the liquid mixture, DAB (0.1% phosphate buffer solution, 0.02% diaminobenzidine tetrahydrochloride and 3% H₂O₂). After rinsing in water, the sections were counterstained with hematoxylin, dehydrated and cover-slipped. All the immunostained sections were observed under a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

For the assessment of CAP1 and Ki-67, at least 10 high-power fields in each specimen were randomly selected, and the percentage of cells with nuclear and cytoplasmic staining was examined with a total number of >500 cells counted to determine the labeling index in a single section. Tissues with no staining were rated as 0, with a faint staining or moderate to strong staining in ≤25% of cells as 1, with moderate staining or strong staining in 25–50% of cells as 2, and strong staining in ≥50% of cells as 3. For statistical analysis, a score of <2 was counted as low expression, while a score of ≥2 was counted as overexpression, as previously described (24). When evaluating Ki-67 protein immunoreaction, staining was scored in a semi-quantitative manner. A cut-off value of 50.7% or more positively stained nuclei in 5 high-power fields were used to identify Ki-67 staining as follows: the high expression group (≥50.7%) and low expression group (<50.7%). All the immunostained sections were evaluated in a blinded manner without knowledge of the clinicopathological variables of the patients. In half of the samples, staining was repeated twice to avoid technical errors, but similar results were obtained in these samples.

The human EOC cells, HO-8910, were purchased from the Shanghai Institute of Cell Biology and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum and 100 U/ml penicillin-streptomycin mixture (both from Gibco-BRL, Grand Island, NY, USA) at 37°C and 5% CO₂.

Cells were harvested at the proper time and fixed in 70% ethanol overnight at 4°C and then incubated with 1 mg/ml RNase A for 30 min at 37°C. Subsequently, the cells were stained with propidium iodide (PI, 50 mg/ml; Becton-Dickinson, San Jose, CA, USA) in PBS, 0.5% Tween-20, and analyzed using a flow cytometer BD FACScan (Becton-Dickinson, San Jose, CA, USA) as well as CellQuest acquisition and analysis programs. As regards cell synchronization, serum deprivation was used for cell cycle G1-S phase arrest.

Duplex siRNAs were synthesized as follows by Biomics Biotechnologies Co., Ltd. (Nantong, China): The sequences were as follows CAP1-specific, 5′-GAAAUGAA UGAUGCCAdTdT-3′ and control, 5′-UGGCGGCAUUCAUU UCdTdT-3′. The HO-8910 cells were seeded the day prior to transfection at a confluence of 50–70%. In addition, a Mock group with no transfection was also used as a control. The transient transfection of CAP1 and non-specific siRNA oligos was carried out using Lipofectamine 2000 (Invitrogen, St. Louis, MO, USA) in accordance with the manufacturer’s instructions. The cells were harvested 48 h after transfection and used for the experiment. The experiments were repeated at least 3 times.

The cell counting kit-8 (CCK-8) was used to measure cell proliferation according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). The cells were seeded into a 96-well cell culture cluster plates (Corning, Inc., Corning, NY, USA) at a concentration of 2×10⁴ cells/well in a volume of 100 μl culture medium and grown overnight. CCK-8 reagents (Dojindo) were then added to each well followed by incubation for an additional 2 h at 37°C. The absorbency was measured at a test wavelength of 490 nm and a reference wavelength of 630 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The experiments were repeated at least 3 times.

All statistical analyses were performed using SPSS 17.0 statistical software. The association between CAP1 and Ki-67 expression and clinicopathological characteristics was analyzed using Pearson’s χ² test. Multivariate analysis was performed using Cox’s proportional hazards model. CAP1 and Ki-67 expression was quantified using Pearson’s correlation co-efficient. Kaplan-Meier analysis was used to evaluate the survival curve, and the log-rank test was performed for analysis.

To confirm the role of CAP1 in EOC, western blot analysis for CAP1 was performed using surgical specimens. We found that CAP1 protein expression gradually increased in 9 EOC tissues from 3 tumors classified as G1 to G3 (3 tissues from each tumor) in comparison with 1 normal ovarian tissue sample in which CAP1 expression was barely detected (Fig. 1). In order to determine the role of CAP1 in the progression and development of EOC, we examined the intracellular expression of CAP1 and Ki-67 in 119 specimens of EOC by immunohistochemical analysis. We found that CAP1 was mainly located in the cytoplasm of the EOC cells (Fig. 2). The high expression of CAP1 was accompanied by the high expression of Ki-67 localized in the nucleus. In addition, CAP1 was highly expressed in the poorly differentiated tumor specimens compared to the well differentiated ones; similar results were obtained for Ki-67 expression.

To clarify the clinicopathological significance of CAP1, we analyzed the correlation of CAP1 expression with the clinicopathologic characteristics of patients with EOC (Table I). We divided the tumor specimens into 2 groups according to the expression of CAP1. CAP1 expression was significantly associated with the histological grade (P=0.010) and Ki-67 expression (P<0.001), whereas there was no correlation observed with the FIGO stage (P=0.477), menopause (P=0.395), ascites (P=0.666), lymph node status (P=0.209), malignant tumor cells (P=0.472), metastases to other organs (P=0.666), age (P=0.775) and histologic subtype (P=0.071). In addition, Pearson’s correlation co-efficient revealed that there was a positive correlation between CAP1 expression and Ki-67 expression (r=0.722, P<0.001) (Fig. 3).

Kaplan-Meier analysis revealed that out of the 119 clinical cases examined, the patients with a high expression of CAP1 had a poorer overall survival than those with a lower expression (P<0.001; Fig. 4). Pearson’s χ² test indicated that the histological grade (P=0.047), CAP1 (P<0.001) and Ki-67 (P<0.001) were independent prognostic indicators of EOC (Table II). Moreover, multivariate analysis using Cox’s proportional hazards model showed that the histological grade (P<0.001), CAP1 (P= 0.029) and Ki-67 (P<0.001) were independent prognostic indicators for patient overall survival (Table III). The survival curve indicated that the median survival for patients with a high CAP1 expression was significantly shorter than the median survival for those patients with a low CAP1 expression (Fig. 4).

It has been reported that the knockdown of the expression of CAP1 affects the breast cancer cell cycle, inhibiting the growth of breast cancer cancer (25). In this study, we found that a high CAP1 expression associated with a poor prognosis of patients with EOC. Thus, we hypothesized that CAP1 participates in the cell cycle of HO-8910 cells. We subjected the cells to serum starvation and serum re-feeding and found that the expression of CAP1 gradually increased, as well as that of the proliferation markers, PCNA and cyclin A, during cell progression. Flow cytometric analysis revealed that the HO-8910 cells subjected to serum deprivation for 48 h were arrested in the G1 phase, and following serum re-feeding, the population of HO-8910 cells in the S phase increased from 18.08 to 61.12% (Fig. 5C). To further validate these results, we collected HO-8910 cellular protein at different time points, and western blot analysis was performed to determine whether CAP1 expression is cell cycle-dependent in HO-8910 cells. We found that CAP1 expression was significantly increased as early as 4 h following serum re-feeding in the HO-8910 cells. The expression of the cell proliferation markers, PCNA and cyclin A, showed a similar tendency (Fig. 5A and B).

To further determine the effect of CAP1 on cell proliferation, we knocked down endogenous CAP1 in HO-8910 cells using siRNA targeting CAP1 and the effects to the cells transfected with control siRNA and mock siRNA (Fig. 6A and B). The knockdown of CAP1 using siRNA inhibited the accumulation of cyclin A and PCNA compared with control- and mock-transfected cells (Fig. 6A and B). Additionally, we found that the proliferation rate of the HO-8910 cells transfected with siRNA exhibited a decrease compared with the cells transfected with the control siRNA and the mock-transfected cells (Fig. 6D). From these results, we ascertained that CAP1 plays a positive role in the regulation of cell proliferation.