Effects of Sodium Valproate on the Growth of Human Ovarian Cancer Cell Line HO8910

Sodium valproate, as a member of the short chain fatty acids family, is a broad-spectrum anti-epileptic drug that has been extensively used in clinical practices. It is also a histone deacetylase inhibitor, which suppresses the activity of histone deacetylating (Tryfon et al., 2009). Histone deacetylase inhibitors are promising new class of anticancerous agents that affect proliferation, differentiation, and apop¬tosis in both solid and hematologic tumors (Blaheta et al., 2002). In the nucleus, increased levels of histone acety¬lation lead to relaxation of the chromatin structure, allowing access of transcription factors and increased transcription, while decreased levels of acetylation are associated with repressed tran¬scription. Therefore, inhibition of the function of histone acetylation affects the regulation of gene expression, cell growth, differentiation, and apoptosis (Duenas-Gonzalez et al., 2008). Sodium valproate has been shown to reduce cancer proliferation to some extent by inducing cancer cell apoptosis, differentiation and cycle arrest (Davies, 2002; Michaelis et al., 2007; Shi et al., 2007; Dutertre et al., 2010; Li et al., 2012). Therefore, we hypothesized that sodium valproate is a remarkable drug and could be used as a new treatment for tumors. In this present study, we selected human ovarian cancer cell line HO8910 as


Introduction
Sodium valproate, as a member of the short chain fatty acids family, is a broad-spectrum anti-epileptic drug that has been extensively used in clinical practices. It is also a histone deacetylase inhibitor, which suppresses the activity of histone deacetylating (Tryfon et al., 2009). Histone deacetylase inhibitors are promising new class of anticancerous agents that affect proliferation, differentiation, and apop¬tosis in both solid and hematologic tumors (Blaheta et al., 2002). In the nucleus, increased levels of histone acety¬lation lead to relaxation of the chromatin structure, allowing access of transcription factors and increased transcription, while decreased levels of acetylation are associated with repressed tran¬scription. Therefore, inhibition of the function of histone acetylation affects the regulation of gene expression, cell growth, differentiation, and apoptosis (Duenas-Gonzalez et al., 2008).
Sodium valproate has been shown to reduce cancer proliferation to some extent by inducing cancer cell apoptosis, differentiation and cycle arrest (Davies, 2002;Michaelis et al., 2007;Shi et al., 2007;Dutertre et al., 2010;Li et al., 2012). Therefore, we hypothesized that sodium valproate is a remarkable drug and could be used as a new treatment for tumors. In this present study, we selected human ovarian cancer cell line HO8910 as Department of Obstetrics and Gynecology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu, China *For correspondence: hongchaoyan@hotmail.com and plated in triplicate into 96-well plates (200μl each well) and then incubated for 24h to allow cell attachment. In the experiment groups, cells were treated with 1.0, 2.0, 3.0, and 4.0mmol/L concentrations of sodium valproate, while normal culture medium was used in the control group. After 48h, the MTT assay was performed by using 20μl of serum-free medium containing MTT (0.5 g/l) and incubating at 37C for 4 h. Then, 150μL DMSO was added to each well, and all the plates were shaken at room temperature for 10min. Optical density (OD) was measured at 490nm using a spectrophotometer microplate reader, and the cellular proliferation graphs were plotted.

Cellular morphological changes observed under microscope
HO8910 cells were trypsinized, added into 24-wells plates (1ml each well), and then incubated for 24h in the serum-free medium. In the experiment groups, cells were treated with 1.0, 2.0, 3.0, 4.0 mmol/L concentrations of sodium valproate, while normal culture medium was used in the control group. After 48h, cells were examined under microscope for cell growth and morphology.

Flow Cytometric analysis for Cell Cycle and apoptosis
Cell cycle assay: This assay was performed by cell cycle detection kit (Beijing Biosea Biotechnology Co., Ltd., China). Briefly, HO8910 cells were plated into 6-wells plates (3×10 4 /ml, 2ml each well) and then incubated for 24h in serum-free medium. The cells were treated with normal culture medium for control groups and different concentrations of sodium valproate (1.0, 2.0, 3.0, and 4.0mmol/L respectively) for experimental groups. After 48h treatment, the cells were harvested, fixed with 70% ethanol, and then adjusted to the concentration of 1×10 6 /ml. According to the manufacturer's instruction, the cells were incubated in 0.5ml propidium iodide with RnaseA for 30 min at 4℃ in the dark. Results were analyzed by Flow Cytometry (6 color FACSCalibur, Promega, CellQuest was analysis software).
Cell apoptosis assay: This assay was performed by Annexin V-FITC/PI Apoptosis Detection Kit purchased from Beijing Biosea Biotechnology Co., Ltd., China. In short, HO8910 cells were plated into 6-wells plates (6×10 4 cells/well) and then incubated for 24h in the serum-free medium. In the experiment groups, cells were treated with above mentioned concentrations of sodium valproate; normal culture medium was used in the control group. After 48h, the percentage of apoptotic cells was measured according to manufacturer's instructions.

Western blot analysis
HO8910 cells (3×10 4 /ml) were trypsinized and harvested as the concentration of 2×10 6 in 100ml medium. In the experiment groups, cells were treated with above mentioned concentrations of sodium valproate, while the cells in the control group were treated with normal culture medium. The cells were maintained in a humidified atmosphere of 5% CO 2 in air at 37°C for 24h. After cell attachment, the mediums were replaced with the fresh RPMI1640 medium, in which different doses of sodium valproate were added for the experiment and control groups. After 48h, the cells were harvested and protein concentration was determined by a BCA protein assay kit (Sigma aldrich, USA) 30μg proteins were subjected to SDS-PAGE and electro-blotted on a PVDF membrane. The membranes were blocked with 5% fat-free milk solution at room temperature for 4h. After that, the anti-WWOX and anti-P27 antibodies at 1:250 ratios (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., China) were incubated for 12h at 4°C. The secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., China) conjugated with horseradish peroxidase with dilution ratio 1:1000 was incubated for 3h incubation at room temperature. Bands were visualized by enhanced chemiluminescence (ECL) kit (Xuzhou College Biochemistry Laboratory, China). The same nitrocellulose membrane was stripped and incubated with β-actin monoclonal antibody (Xuzhou College Biochemistry Laboratory, China) at a 1:2000 dilution for 2 h, which acted as a control for loading and blotting.

Xenograft Studies
20 Female BALB/c nude mice (Shanghai Experiment Animal Center, Chinese Academy of Medical Sciences, China), 4-6weeks old, were used. Trypsinized HO8910 cells suspension (2×10 6 /ml) was subcutaneously injected per mouse at right forelimb. After one week, the existence of tumor had been confirmed at subcutaneous tissue of the nude mice. Then after, mice were equally divided into two groups: the experiment group and the control group. They were maintained at 25-27℃ , 45%-50% humidity, fresh air, and pathogen-free conditions. In the control group, the mice were allowed to free access for food and water, while, in the experimental group, the water was substituted by 0.4% sodium valproate solution. The nude mice were observed daily and the death date had been recorded. After the mouse death, it was anatomized immediately and then the tumor sizes were measured. The tissues from the control and experimental groups were collected. The expressions of WWOX and P27 were detected by western blot as described earlier.

Statistical analysis
Statistical and graphic analyses were performed with the SPSS 13.0 software (Chicago, USA). All results were expressed in mean ± SD. Comparisons between two groups were made using Student's t test; multiple-group analyses were made by one-way ANOVA test and two groups comparisons in multiple-groups were made by q test. P <0.05 was considered significantly different.

Results
The effect of sodium valproate on the proliferation of HO8910 cells MTT cell proliferation assay results showed that there was a significant decrease (P <0.05) in cell proliferation (ODs) in the experimental groups than the control group (p=0.032). Moreover, a gradual decrease in cell proliferation was also observed with the gradual increase of sodium valproate concentration within the experimental groups. Importantly, these differences were significant (P <0.05) at each level among different experimental groups (Figure 1). These results suggested that ovarian cancer HO8910 cells treated with sodium valproate had a profound dose-dependent inhibition of growth.

The effect of sodium valproate on the morphology of HO8910 cells
After 48h incubation of cells with sodium valproate, the morphology of HO8910 cells was observed under microscope. Cells cultured in control media grew well, attached in clumps and sheets with the increased diopter and the smooth margins. The numerous cells at fissiparous stage had been observed (Figure 2A). In the experimental group of 1mmol/L sodium valproate, few apoptotic cells at early stage were observed. Moreover, cells turned to small and spherical with concentrated cytoplasm with decreased diopter and coarse margins ( Figure 2B). Cells treated with increasing concentrations of sodium valproate showed markedly morphological changes. More apoptotic cells were found and the cells became more spherical with aggravated concentration of cytoplasm and with an increase in the floating cells ( Figure 2CDE).

The effect of sodium valproate on the cell cycle of HO8910 cells
Flow cytometric analysis of cell cycle assay indicated that a significant higher number of cells in the experimental groups with various doses (1.0, 2.0, 3.0 and 4.0mmol/L respectively) of sodium valproate were blocked at G 0 / G 1 phase compared with the control group (P <0.05). In addition, with the increased dose of sodium valproate, the percentages of cells that blocked at S phase obviously decreased ( Table 1).

The effect of sodium valproate on the apoptosis rate of HO8910 cells
Flow cytometric analysis for the percentage apoptosis rates of the experimental groups with 1.0, 2.0, 3.0 and 4.0 mmol/L doses of sodium valproate were 10.39±1.1, 29.31±1.19, 38.7±1.23 and 46.96±1.37. Our results indicated that all apoptotic rates in the experimental groups from low to high doses of sodium valproate were statistically and gradually higher (P <0.05) as compared to the control group (4.4±1.16) (Figure 3). Thus, sodium valproate induces apoptosis in HO8910 cells in dose-dependent manner.

The effect of sodium valproate on the expression levels of WWOX and P27
Western blots studies showed that the expression levels of WWOX in the experimental groups was significantly and gradually increased (P <0.05) compared with control group. The increase of the protein was in a dose-dependent manner. Similarly, the expression levels of P27 was also significantly and gradually increased (P <0.05) with the increase concentrations of sodium valproate (Figure 4). In addition, correlation analysis indicated that sodium valproate doses had a positive correlation with the expression levels of P27 (p=0.038, r=0.801). These results suggested that sodium valproate induce expression of tumor suppressor genes in a dose-dependent manner.

The effect of sodium valproate on tumor burden induced by HO9810 cells in vivo
Tumors were detected after one week in all the nude mice injected with the HO8910 cells. The average tumor size of the experimental group (fed on sodium valproate solution) was significantly lesser (1264.59±24.83 vs. 2155.62±73.38mm3, P <0.05) than control group. The average survival time was significantly higher in the experimental group (47.12±0.73 vs. 34.58±0.46 days, P <0.05) compared to the control group. These results indicate that sodium valproate reduce tumor burden and increase survival time.

Tumoral proteins and mRNA expression of WWOX and P27 in nude mice
Western blot results showed that the expression levels of WWOX and P27 proteins were significantly higher in the experimental group (fed on sodium valproate solution) than in the control group (0.46±0.065 & 0.39±0.018 vs. 0.15±0.025 & 0.20±0.067, P <0.05 respectively) ( Figure 5A). Similarly, RT-PCR results indicated that the expression levels of WWOX and P27 mRNA were also significantly higher in the experimental group than in the control group (0.305±0.015 & 0.926±0.034 vs. 0.193±0.056 & 0.702±0.047, P <0.05 respectively) ( Figure  5B). These results demonstrated that sodium valproate induce in vivo expression of tumor suppressor genes (WWOX and P27) both at the levels of mRNA and protein.

Discussion
There are several kinds of ovarian cancers, however, epithelial ovarian carcinomas account for 85 to 90 percent of all cancers of the ovaries. It is a leading gynecologic cancer with high mortality rate and poses a great threat to female health for its failure in early diagnosis and treatment (Jemal et al., 2011). Therefore, the search for effective and successful therapies for this cancer is mandatory.
Sodium valproate, sodium salt of valproic acid, is a histone deacetylase inhibitor. Acetylation of histones is regulated by the opposing activities of histone acetyltransferase or histone deacetylase. Chromatin structure and gene expression and regulation are controlled under the dynamic balance of histone acetylation and deacetylation. It is becoming a hot topic that histone deacetylase inhibitors (HDIs) are promising anticancer agents. It is generally believed that HDIs  induce acetylation of histones, modify the expression of oncogenes and tumor suppressor genes, rescue normal cell growth and differentiation, and thus elicit anticancer effect (Balakin et al., 2007). Sodium valproate is a traditional anti-epilepsy drug with few side effects and excellent tolerability for long-term use. Therefore, sodium valproate as a strong and safe HDI which has a selective cytotoxic effect on cancer cells but no serious influence on normal cells.
Recent evidences suggested that sodium valproate participates in multiple processes to treat cancer i.e. blocking tumor cells signaling, down-regulating the proliferation of tumor cell and the production of vascular growth factor, and inducing cell cycle arrest (Venugopal et al., 2011). Some researches revealed up-regulation of Bak protein expression, degradation of cyclin D1 and an increase in cyclin-dependent kinases p21 and p27 after sodium valproate treatment. In addition, sodium valproate also increased the levels of full-length Notch-1 and active Notch-1 intracellular domain, which intern inhibit proliferation and induced apoptosis of neoplasm (Greenblatt et al., 2007;Fortunati et al., 2008;Platta et al., 2008). Sami et al. in cervical cancer found that sodium valproate can increase the expression of histone H3 acetylation and up-regulate p21 expression (Sami et al., 2008). Moreover, long-term administration of sodium valproate had a cytostatic effect resulting in a significant reduction of tumor growth. Another study revealed that sodium valproate suppress in vitro proliferation of endometrial cancer cells in nude mice and increase the proportion of cells at G 0 /G 1 phases of the cell cycle by possible increase of p21, p27, and E-cadherin (Takai et al., 2004).
To further evaluate whether sodium valproate can inhibit the proliferation of ovarian cancer cells, we investigated the effect of different doses of sodium valproate on HO8910 cells in vitro, and in BALB/c nude mice in vivo. Our result demonstrated that sodium valproate inhibit the growth of HO8910 cells in a dose-dependent manner. Moreover, flow cytometric and microscopic examination data indicated that sodium valproate not only has cytostatic effect and block the cell cycle at G 0 /G 1 or S phase, but also promoted the apoptosis of HO8910 cells. In addition, the effect of sodium valproate has an obvious association with its concentrations. Based upon these facts, we extend our study on the possible molecular mechanisms of sodium valproate on HO8910 cells. Western blot analysis indicated that WWOX and P27 expression levels were increased in HO8910 cells treated with various doses of sodium valproate. These result suggested that sodium valproate up-regulate WWOX and P27 expression levels and then inhibit growth and induce apoptosis in HO8910 cells. Our in vivo experiments on HO8910 cell induced tumor in nude mice also revealed that sodium valproate treated mice showed decrease tumor burden, and up-regulate tumor suppressors WWOX and P27, thus confirming the inhibitive effect of sodium valproate on HO8910 cells.
In summary, we showed that sodium valproate inhibits HO8910 cells growth in a dose-dependent manner. We also present evidences that sodium valproate up-regulate tumor suppressor genes WWOX and P27 in vitro as well as in vivo in these cancerous cells. Taken together, sodium valproate has a potential role in inhibition and control of ovarian cancer cell line HO8910, and could be the basis of future ovarian cancer treatments.