The Role Of Eicosapentaenoic Acid (EPA) In Ovarian Clear Cell Carcinoma

Ovarian cancer is the most lethal gynecologic malignancy worldwide. It is generally accepted that epithelial ovarian cancers (EOCs) is not a single disease but is composed of heterogeneous group of tumors with distinctive morphologic and molecular genetic features. Ovarian clear cell carcinomas (OCCCs), one type of EOCs, are more common in women of oriental descent. Compared with other type of EOCs, OCCCs carries a poorer prognosis and are more frequently platinum resistant. Obviously, there is a need to optimise or develop new therapeutic strategies in the management of this disease.EPA (eicosapentaenoic acid), one kind of n-3 polyunsaturated fatty acids, has been observed significantly lower in plasma of patients with several cancers than that of healthy individuals, implying its considerable role in cancer development. At present, EPA has been demonstrated to preserve or gain lean body mass in patients with cancer cachexia, and have the potential to sensitize tissues to chemotherapy. However, there is still no experiment to illustrate the relationship between EPA and OCCCs. In our experiment, we provide evidence that EPA could influence the proliferation and apoptosis of ES2 cell (ovarian clear cell line). Compelling evidence shows that fatty acids (FAs) could realize its biological effect through governing fluidity and configuration of membrane receptors, cell signaling pathways, transcription of genes and inflammatory response. Recently, a series of G protein-coupled receptors (GPRs) for FAs has been described and characterized. These receptors have been reported as critical component of sensing apparatus in human body and shown differing specificities for FAs of differing saturation degree and chain length. FFAR1 (GPR40) and GPR120, whose Ga subunit are both Gaq/11, are ligands for long-chain unsaturated FAs. We firstly hypothesized that EPA could influence the ovarian cancer through these GPRs. But finally we found very low expression of GPR40 and GPR120 in ES2 cells, so we search for other G protein-coupled receptors that probably mediate the role of EPA in ES2 cells.G protein-coupled receptor 30 (GPR30), which is a 7-transmembrane estrogen receptor of Gs family members predominantly localized on endoplasmic reticulum, functions alongside traditional estrogen receptors to modulates both rapid non-genomic events and genomic transcriptional events of estrogen. An affinity of GPR30 for 17β-E2 is about 3～6nM, but little for the non-physiological estrogen stereoisomer 17a-estradiol or for other physiological estrogen agonist such as extrone and estriol. Other steroids, including progesterone, cortisol and testosterone exhibit almost no binding to GPR30 at μM concentration. Besides, some synthetic estrogen-receptor ligands, such as tamoxifen and ICI 182 780, and phytoestrogens or xenoestrogens, such as soy products, are also ligands for GPR30. When ligands bind to GPR30, the activated heterotrimeric G proteins then activate Src and adenylyl cyclase (AC) resulting in intracellular cAMP production, triggering cleavage of membrane-tethered heparin-bound epidermal growth factor (EGF) by matrix metalloproteinases (MMP) that resulting transactivation of epidermal growth factor receptors (EGFRs). According to recent literatures, GPR30 is not only expressed in normal tissues, including the uterus, ovaries, and mammary glands, but also in several cancers, including breast cancer, ovarian cancer, endometrial cancer, thyroid cancer, prostate cancer and lung cancer. As for ovarian cancer, GPR30 is demonstrated to be highly expressed and related to poor prognosis. However, our understanding of GPR30 at both the molecular and physiological level is very much in its infancy. Here, we show for the first time that GPR30 is also a receptor of EPA and mediate the biological functions of EPA in ES2 ovarian cancer cells.Part1 EPA influences the growth of ES2 in vitro Objective To evaluate the influences of EPA on ES2 cells in vitro, including cell proliferation and apoptosis.Methods ES2 cells were first treated with different type (palmitic acid, palmitoleic acid, oleic acid, linoleic acid, arnchidnic acid, linolenic acid and EPA) and concentration of fatty acids for 24h(0-300uM), the proliferation of ES2 was evaluated by the CellTiter 96(?) Aqueous Non-Radioactive kit. We also analyzed toxicity of various fatty acids on ES2 cells using CCK-8 assay and selected an appropriate concentration to further subsequent experiments. In the appropriate concentration, we observed the impact of proliferation of different FFAs on ES2 in different time. The mRNA expression of pro-apoptotic genes (bax、bim、puma) and anti-apoptotic genes(bcl-2, bcl-xl, survivin) were analyzed by RT-PCR.Results We found that cell toxicity increased after 300uM concentration. The inhibitory effect of EPA on ES2 was dose-dependent and time-dependent. mRNA expression analysis confirmed that EPA leads to activation of pro-apoptotic genes (bax, bim, and puma) with inhibition of anti-apoptotic genes (bcl-2, bcl-xL, and survivin) in ES2 cells. Conclusion EPA inhibits cell growth and induces apoptosis in ES2 cells.Part 2 EPA influences the growth of ES2 via GPR30 Objective To find out the receptor on ES2 that mediates the effects of EPA.Methods We measured changes of [Ca2+]I in fura-2/AM-loaded cells following palmitic acid or EPA treatments in the absence and presence of Ym25486, an inhibitor of Gq protein. Next, we studied the effect of Ym25486 on EPA anti-proliferative and pro-apoptotic action in ES2 cells. Analyze the expression of GPR30 and the reported FFARs by quantitative PCR analysis. We used Protein Lipid Overlay Assay (PLO) to verify whether EPA functions via GPR30 in ES2 cells. Next, to determine whether GPR30 is implicated in the anti-proliferative and pro-apoptotic action of EPA in ES2 cells, we attempted to reduce the expression level GPR30 in ES2 cells using RNA interference or G15, which is the inhibitor of GPR30. To determine the following pathways after GPR30 activation through western blot, cAMP detection and RT-PCR.Results In the absence of Ym25486, palmitic acid and EPA caused a rapid increase in [Ca2+]I by 4- and 1.5-fold, respectively. In the presence of Ym25486, the increase in [Ca2+]I by EPA did not change, while the increase in [Ca2+]I by palmitic acid was inhibited. Ym25486 did not affect EPA-induced anti-proliferation and pro-apoptosis. Quantitative PCR analysis showed that GPR30 expression was significantly higher that other FFARs. Additionally, the interaction between EPA and GPR30 existed and was dose-dependent. The decrease in cell number or pro-apoptotic effects following EPA treatment was significantly reversed in ES2 cells expressing the GPR30-specific siRNA. Similarly, the inhibitory effects of EPA in ES2 cells were also impaired when G15 was added. EPA blunts the activation of AKT and ERK1/2 through GPR30 in ES2 cells. GPR30-cAMP-PKA signaling also played an important role in anti-cancer effect of EPA in ES2 cells.Conclusion Classical G-protein coupled receptors for long-chain FFAs, GPR40 or GPR120, do not play essential role in mediating anti-cancer effect of EPA in ES2 cells. GPR30 functions as receptor of EPA in ES2 cells. GPR30 is involved in EPA-induced cell anti-proliferation and pro-apoptosis in ES2 cells.Part 3 EPA influences the growth of ES2 in vivo.Objective To evaluate the effect of EPA in ES2 in vivo.Methods To establish the subcutaneous tumor model of ES2 using BALC/C female nude mice. Inject 2*106/200ul ES2 cells on the right side of axillary subcutaneous of every mouse and observe the tumor formation everyday. Mice with tumor formation were randomly divided into four groups, that is NC group, NC+EPA group, shRNA group and shRNA+EPA group. Among them, the GPR30 expression in ES2 cells using for building tumors in shRNA group and shRNA+EPA group were firstly blocked by shRNA. Give peritumoral subcutaneous injection of 200ul 300uM EPA to every mouse every three days from 5th day on. Dissect the tumor on 14th day, measure the volume and weight of the tumors. And finally, make immunohistochemical detection.Results Tumors with diameter of 5mm were observed on 4th day. Tumors were significantly smaller after EPA treatment. Comparing the tumor in shRNA group and shRNA+EPA group, there was no significant change in tumor growth after EPA treatment. From the result of immunohistochemical detection, the expression of GPR30 could not be influenced by EPA. The expression of Ki67 significantly decreased after EPA treatment, but showed no change when GPR30 was blocked.Conclusion EPA could make effects on ES2 in vivo.