Adiponectin exhibits proliferative and anti-apoptotic effects on ovarian cancer cells via PI3K/Akt and Raf/MEK/ERK pathways

Purpose: To elucidate the effects and the underlying mechanism of adiponectin on human ovarian cancer cells. Methods: The level of adiponectin, adiponectin receptor-1, caspase-3 and bcl-2 in the serum and ascites of the patients were measured with enzyme-linked immunosorbent assay (ELISA), qPCR and Western blotting. The human ovarian cancer cell lines (Caov3 and SKOV3) were enumerated using 3(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide (MTT) assay. Western blotting was also used to determine the levels of p-Akt, p-ERK and cyclin B. Results: Serum and ascite levels of adiponectin were significantly higher in ovarian cancer patients than in healthy patients (p < 0.05). Expression of adiponectin in the serum and ascites of patients in FIGO stage IV was remarkably higher than in earlier stages (p < 0.05). The proliferative effect of adiponectin on ovarian cells was dose-dependent. Adiponectin treatment significantly increased the expression of cyclin B in Caov3 and SKOV3, and reduced the levels of caspase-3 and bcl-2. Inhibitors of PI3K and MEK pathways significantly reduced the proliferation of attenuated Caov3 and SKOV3 by up-regulating cyclin B upon adiponectin treatment (p < 0.05), and thus alleviated the inhibitory effect of adiponectin on the expressions of caspase-3 and bcl-2. Conclusion: The findings demonstrate that adiponectin promotes proliferation of the cells via the PI3K/Akt and Raf/MEK/ERK pathways, and also provide new insights into ovarian cancer treatmment.


INTRODUCTION
Adiponectin, one of the cytokines secreted by adipose tissue [1,2], is a lipid-like and collagenlike protein encoded by apM1 gene [3]. The role of adiponectin in the occurrence and development of tumors is multiple and complex [4,5]. For example, it inhibits the proliferation and migration of tumor cells while promoting their apoptosis via activation of the cAMP/PKA and AMPK pathways [6,7]. It also down-regulates the expression of vascular endothelial growth factor while suppressing the blood vessel formation [8,9].
Recently, increasing literatures have indicated that adiponectin may act as a tumor suppressor gene. Adiponectin suppresses the proliferation and migration of hepatocellular carcinoma cells [10], gastric cancer cells [11], breast cancer cells [13], prostate cancer cells [14] and endometrial cancer cells [15]. Adiponectin can activate different pathways by binding to its receptors; it regulates the activity of some growth factors and regulates the expression of oncogenes or tumor suppressor genes. Adiponectin stimulates the expression of nicotinamide adenine dinucleotidedependent histone deacetylase (Sirt1) via the promotion of the phosphorylation of AMPactivated protein kinase (AMPK), and inhibition of apoptosis in pancreatic cancer cells [17]. Adiponectin also promotes the migration and invasion of breast cancer cells via activating MAPK and PI3K pathways [7,18], and promote the phosphorylation of signaling molecules such as STAT3, ERK1/2 and Akt in 786-O renal carcinoma cells.
Ovarian cancer is one of the most common gynecological malignancies since patients do not present obvious early symptoms and its diagnosis is usually poor. More than 70 % of ovarian cancer patients are identified and confirmed at the terminal stage of the disease [19]. In a previous study, the expression ratio of adiponectin receptor 1/2 in follicular cells of polycystic ovary syndrome patients was significantly higher compared with that in normal ovaries [20], but there are few studies on the role of adiponectin in ovarian cancer's development and progression. This study aims to investigate the effects and underlying mechanism of adiponectin on ovarian cancer cells.

Materials and reagents
Adiponectin ELISA kit and recombinant protein were produced by R&D, USA; rabbit anti-human adiponectin receptor 1 and bcl-2 antibodies were purchased from Abcam, USA; HRP labeled goat anti-mouse IgG and goat anti-rabbit IgG antibodies were obtained from ZSGB-BIO, China. Polymerase chain reaction (PCR) primers and MTT assay kit were purchased from Sangon Biotech, China. Rabbit anti-human Akt, p-AKt, ERK, p-ERK primary antibodies, and rabbit antihuman caspase-3 were products of Cell Signaling Technology, USA. Mouse anti-human GAPDH antibody was obtained from Biotime, China, while rabbit anti-human cylcin antibody was produced by Santa Cruze, USA. Trizol reagent was obtained from Sigma Aldrich Co., Germany.

Patients and clinical data
Sixty three patients in total were included for this one year study. All the patients were divided into two groups: an observation group consists of 46 patients with ovarian cancer, and a control consists of 17 healthy patients. Patients in the observation group were aged between 40 and 59 years (mean age = 53.00 ± 9.50 years), while those in control were aged between 30 and 55 years (mean age = 43 ± 12.50 years). Patients in the observation group were diagnosed pathologically and met the following conditions: (1) [21]. The patients and their family members signed written informed consent.

Enzyme-linked immunosorbent assay (ELISA)
Serum and ascites samples obtained from the patients were incubated overnight at 4 o C after the addition of enzyme-labeled primary antibody. The blocking condition was placing in room temperature for one hour. The mixture was further added with enzyme-labeled secondary antibody and kept at room temperature for one hour. Positive and negative controls were routinely employed.

Cell culture
Human ovarian cancer cell lines (Caov3 and SKOV3) were cultured with complete culture medium (FCS-RPMI 1640 medium) , spiked with 10 % fetal bovine serum and kept at 37 o C in a humidified incubator in an atmosphere of 5 % CO 2 . The entire operation was carried out in 10,000-grade purification room in sterile conditions.

MTT assay
The cells at the concentration of 1 × 10 4 cells/well were inoculated into a 96-well plate in DMEM, and were incubated for 24 h. Different concentrations of adiponectin (0 -200 ng/mL) was spiked to the cells and the mixtures were further incubated for 24, 48, and 72 h, respectively. After 72 h, 20 µL of MTT solution (5 mg/mL) was supplemented to each well, and then incubated for another 4 h. The medium was finally replaced with dimethyl sulfoxide (DMSO) solution and agitated (50 oscillations/min, 10 min). A microplate reader were used to determine the absorbances of the mixtures at 570 nm. MTT assays of cells in each group were conducted in quintuplicate.

Real-time quantitative PCR
Trizol reagent was used to extract the total RNA from the cells. The RNA was then reversetranscribed to cDNA and subjected to real-time quantitative PCR (qPCR). The reactions were performed in a final 25-µL reaction mixture volume. Reaction conditions were listed as follow: initial denaturation at 95 o C for 5 min, followed by 40 cycles of 94 o C for 20 sec, 61 o C for 20 sec, 72 o C for 20 sec, and a final elongation at 72 o C for 5 min. The following protocol of melting curve was used: 10 seconds at 56 o C, and then 10 seconds at a 0.5 o C increment to a final temperature of 95°C. The resultant PCR products were confirmed by subjecting them to gene sequencing. The examination of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The primer sequences were listed in Table 1.

Western blotting
We used the phosphate-buffered saline (PBS) to wash the cells treated with varied concentrations of adiponectin. After washing, the clean cells were lysed with ice-cold radioimmunoprecipitation assay buffer (RIPA). The BCA protein assay kit was used to determine the protein content. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the protein. The protein bands were transferred to PVDF membranes and incubated with primary antibodies (Akt, p-Akt, ERK, p-ERK, cyclin B and caspase-3). The membranes were then washed and subsequently incubated with horseradish peroxidaseconjugated secondary antibodies. We attenuated the primary and secondary antibodies w at a ratio of 1 to 500 overnight at 4 o C. The densities of the bands were estimated using Chemi-doc XRS imaging system.

Statistical analysis
All statistical analyses were performed with SPSS (13.0). Significant differences between the observation group and the control group were estimated using t-test. The data are presented as mean ± standard devistion (SD). Statistically significant were accepted when p < 0.05.

Expression of adiponectin
The levels of serum and ascites adiponectin expression were significantly higher in the patients belonging to the observation group than the control group (p < 0.05). The expression of adiponectin in the serum of patients in FIGO stage IV was significantly higher than that in stage I (p < 0.05), but no significant difference was observed in the corresponding expressions between the other stages (p > 0.05: Figure 1 A). The expressions of adiponectin in ascites of patients in stages III and IV were significantly higher than the corresponding expressions in the other stages (p < 0.05; Figure 1 B).

Expressions of adiponectin receptor-1 in human ovarian cancer cell lines
The expression of adiponectin receptor-1 was significantly higher in SKOV3 than in Caov3 (p < 0.05; Figure 2 A and B).

Effect of exogenous adiponectin on proliferation of Caov3 and SKOV3
The proliferation of Caov3 and SKOV3 cells was dose-dependent and significantly higher than the corresponding proliferation in the control group (p < 0.05 Figures 3 A and B).

Expression of cyclin B in Caov3 and SKOV3 48 h after treatment with adiponectin
After 48 h adiponectin treatment, the expressions of cyclin B in Caov3 and SKOV3 were significantly increased (p < 0.01; Figure 4 A and B).

Expression of apoptosis-related proteins after treatment with adiponectin
The expressions of caspase-3 and bcl-2 in Caov3 and SKOV3 were determined before and after treatment with adiponectin. The expressions of caspase-3 and bcl-2 were up-regulated in Caov3 and SKOV3 treated with H 2 O 2 (50 μM). However, after treatment with both H 2 O 2 (50 μM) and adiponectin, there were significant reductions in the expressions of caspase-3 and bcl-2 ( Figure 5).

Effect of adiponectin on phosphorylation of Akt and ERK in ovarian cancer cells
A significant increase was observed in the expression of p-Akt after the adiponectin treatment. The expression peaked 10 and 30 min after treatments of Caov3 and SKOV3, respectively, with adiponectin. The expression of p-ERK also increased after adiponectin treatment and peaked 60 min and 30 min after the treatment of Caov3 and SKOV3, respectively ( Figure 6).

Effect of inhibitors of PI3K and MEK pathways on adiponectin-induced inhibition of apoptosis
The inhibitors of PI3K and MEK pathways significantly attenuated the up-regulation of cyclin B upon adiponectin treatment (p < 0.05). After treatment with inhibitors of PI3K and MEK pathways, the inhibitory effect of adiponectin on the expressions of caspase-3 and bcl-2 was also attenuated (Figure 8).

DISCUSSION
Adiponectin is a fat factor that is relatively abundant in plasma [22] and mediates a variety of biological effects through its receptors expressed in cells of different tissues. Several studies have shown that there is a degree of correlation between its expression in the systemic circulation and the incidence of malignant tumors [23,24].
In the present study, the expression of serum and ascites adiponectin was significantly higher in the observation group patients than the control group patients. The expression of adiponectin in the serum of patients in FIGO stage IV was significantly higher than that of stage I, but there were no significant differences of the corresponding expressions among other stages. The expressions of adiponectin in ascites of patients in stages III and IV were significantly higher than the corresponding expressions in the other stages. These results suggest that adiponectin may act as an oncogene in ovarian cancer. Our result is inconsistence with findings of Chen et al [25] and Arano et al [26], indicating that adiponectin may increase the risk of hepatocellular carcinoma.
It has also been reported that an increase in the serum level of adiponectin is associated with the late stage and poor differentiation of GC. Some authors have reported a negative correlation between the expression of adiponectin in the serum and the incidence of malignant tumors such as GC, breast cancer, endometrial cancer and prostate cancer [27,28]. High level of expression of adiponectin is an indication of better outcomes for patients with tumor metastasis [29]. Studies have shown that adiponectin can act as an oncogene or tumor suppressor gene in different tumors. However, some researchers have speculated that it plays contradictory roles in the development of tumors, regulation of metabolism, changes in cell microenvironment and direct influence on tumor cells [4]. A meta-analysis of patients with breast cancer revealed inconsistent results. So far, it is difficult to determine whether adiponectin acts as an oncogene or not [30].
In this study, the expression of adiponectin receptor-1 was significantly higher in SKOV3 than in Caov3. These results suggest that adiponectin may exert its biological effects by interacting with adipoR1 on the surface of ovarian cancer cells. In a previous study it was reported that 87.30 % of patients with epithelial ovarian cancer expressed adipoR1, an indication that adipoR1 is highly expressed in obesityrelated cancers [31]. Some authors have reported that the ratio of adipoR1 to R2 in follicular cells of patients with polycystic ovary was significantly higher than that in normal tissues [32]. In their studies, adiponectin inhibited the synthesis of androstenedione in follicular cells, luteinizing hormone (LH) receptors and the key enzymes of androgen synthesis. In this study, the effect of adiponectin on proliferations of Caov3 and SKOV3 cells were in a dosedependent manure and the level of proliferations in the observation group was significantly higher than that of control.
The expressions of cyclin B in Caov3 and SKOV3 were significantly increased after treatment with adiponectin. These results suggest that adiponectin up-regulated the expression of cyclin B. Apoptosis is the orderly programmed death of cells regulated by genes and enzymes so as to maintain the stability of the cell inner microenvironment. Cytochrome C released into the cytosol binds to apoptosisrelated factor 1 (Apaf-1) and promotes the formation of apoptotic bodies by recruiting caspase-9, which is followed by the activation of other caspases such as caspase-3, resulting in initiation of apoptosis.
It is believed that activated casepase-3 initiates caspase-chain reaction in the apoptotic pathway.
In the present study, the expressions of caspase-3 and bcl-2 were up-regulated in Caov3 and SKOV3 treated with H 2 O 2 . However, after treatment with H 2 O 2 and adiponectin, significant reductions in their expressions were detected. These results are consistent with a previous study which showed that increased serum level of adiponectin significantly inhibited apoptosis in pancreatic cancer cells [33]. Recent studies have also shown that AMPK may play an important role in the suppression of adiponectin-induced proliferation of tumor cells [34].
The results of this study showed that adiponectin promote the phosphorylation of Akt and ERK. After treatment with inhibitors of PI3K and MEK pathways, the proliferations of Caov3 and SKOV3 at 48 or 72 h after the treatment were significantly reduced, an indication that adiponectin inhibit apoptosis in ovarian cancer cells and attenuate the down-regulation of casepase-3 and bcl-2. These results suggest that adiponectin may promote the proliferation of ovarian cancer cells by inducing apoptosis via the PI3K/Akt and the Raf/MEK/ERK pathways. In recent years, the role of PI3K/Akt pathway in tumorigenesis has attracted huge attention, probably because it plays an important role in cell proliferation, apoptosis, migration, adhesion and degradation of extracellular matrix.
The molecule PI3K is a key in many physiological processes, having both serine/threonine protein kinase and phosphatidylinositol kinase activities. Activation of PI3K by cytokines promotes the binding of Akt to phosphatidylinositol to the serosal membrane through its PH domain, thereby activating it. The activated Akt is released into the cytosol where it activates downstream factors causing them to regulate cell proliferation, differentiation, growth and migration. The Ras/Raf/MEK/ERK pathway is considered to be a classical MAPK pathway. The MAPK pathway is at the center of several signaling pathways, and plays an important role in receiving signals from the membrane and transferring them to the cell nucleus. It also plays the leading role in many pathways related to cell proliferation.
MAPK family includes three major members of ERK, C-JUN amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK). ERK pathway can be activated by receptor tyrosine kinase, G protein-coupled receptors and partial cytokine receptors. Studies have shown that MAPK pathway mediates signal transduction of several cytokines, thus acting on transcription factors to regulate cell proliferation, differentiation and apoptosis [39]. This is important in malignant transformation of cells.

CONCLUSION
The findings of this study demonstrate that adiponectin promotes proliferation of the cells via PI3K/Akt and Raf/MEK/ERK pathways, and thus provide new insights into ovarian cancer treatment.

Conflict of Interest
No conflict of interest associated with this work.

Contribution of Authors
This work was done by the authors named in this article and the authors accept all liability resulting from claims which relate to this article and its contents. The study was conceived and designed by Xuemei Wang; Fengjin Hao and Weina Wan collected and analysed the data. Yueqin Feng wrote the text. All authors have read and approved the text prior to publication.