Yue Zhao, Meng-Fei Zhao, and Mei-Lin Yang contributed equally to this work.
While numerous epidemiological studies have indicated that omega-3 polyunsaturated fatty acids have anticancer properties in various cancers, the effects and mechanisms of eicosapentaenoic acid (EPA) in ovarian cancer cell growth are poorly understood.
ES2 ovarian clear cell carcinoma cells and SKOV3 adenocarcinoma cells were treated with palmitic acid or EPA, followed by flow cytometry and cell counting to measure apoptosis and proliferation, respectively. A modified protein lipid overlay assay was used to further verify whether EPA was a ligand of G protein–coupled receptor 30 (GPR30) in ES2 cells. The levels of apoptosis-related genes, phosphorylated AKT, and phosphorylated ERK1/2 were detected to explore the underlying mechanism. Finally, inhibitory effect of EPA on tumor growth via GPR30 was determined
EPA suppressed ES2 ovarian clear cell carcinoma cells growth via GPR30, a novel EPA receptor, by inducing apoptosis. As a ligand of GPR30, EPA activated the GPR30-cAMP–protein kinase A signaling pathway. When GPR30 was suppressed by siRNA or its inhibitor G15, the antiproliferative action of EPA was impaired. Furthermore, EPA inhibited tumor growth by blocking the activation of AKT and ERK. In the mouse xenograft model, EPA decreased tumor volume and weight through GPR30 by blocking tumor cell proliferation.
These results confirm that EPA is a tumor suppressor in human ovarian clear cell carcinoma cells and functions through a novel fatty acid receptor, GPR30, indicating a mechanistic linkage between omega-3 fatty acids and cancers.
The study of the association between diet and human cancer has drawn considerable attention in recent decades, especially which and how dietary factors might influence cancer risk. The new concept that changing diet can alter the epigenetic state of genes, thereby increasing or decreasing cancer risk, has been put forth in recent years [
Ovarian cancer is the most fatal gynecologic malignancy worldwide [
Compelling evidence shows that FAs could achieve their biological effect by governing the fluidity and configuration of membrane receptors, cell signaling pathways, transcription of genes, and inflammatory response [
In this study, we found that EPA suppressed ES2 ovarian clear cell carcinoma (OCCC) cells growth through GPR30 by activating caspase-3 and inducing apoptosis. Furthermore, GPR30 was expressed in patients with various ovarian cancers, and 300 μM EPA suppressed human ovarian cancer cell growth via GPR30 without influencing normal cells. All these findings open a new front in the battle against ovarian cancer.
FAs, FA-free bovine serum albumin (BSA), and forskolin were purchased from Sigma-Aldrich (St. Louis, MO). Fura2/AM was purchased from Dojindo (Kumamoto, Japan). G1 and G15 were purchased from TOCRIS Bioscience (Ellisville, MI) and Merck (San Diego, CA), respectively. YM254890 was from Yama-nouchi Pharmaceutical Co., Ltd. (Ibaraki, Japan).
ES2 and SKOV3 cell lines were obtained from the American Type Culture Collection (ATCC). ES2 and SKOV3 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum and antibiotics. Cells were cultured in Opti-MEM (Gibco, Grand Island, NY) without fetal bovine serum. FAs were first dissolved in pure ethanol and then freshly diluted to the culture medium containing 1% BSA. The ethanol and BSA were 0.05% (v/v) and 0.1% (v/v), respectively.
For the assay of ES2 and SKOV3 cell proliferation, 5×103 cells per well in Opti-MEM with different types and concentrations of FAs were seeded in triplicate, and cell numbers were monitored after 24, 48, and 72 hours using the Cell Counting Kit-8 (Dojindo). The cytotoxicity of various FAs at different concentrations on ES2 and SKOV3 cells was measured with the Cytotoxicity LDH Assay Kit-WST (Dojindo). All experiments were performed in triplicate wells for each condition and repeated three times independently.
A total of 0.5×106 cells were cultured into 6-well culture plates and cocultured with or without 300 μM EPA for 48 hours. For cell apoptosis analysis, cells were determined with the Annexin V/PI Apoptosis Detection Kit (Dojindo) and analyzed by a FACSCalibur (BD Bioscience, San Jose, CA).
Cells were trypsinized in 3.5-cm Petri dishes, centrifuged, and resuspended in phosphate buffered saline (PBS) with 3 mM Fura-2/AM. Cells were treated with the Ca2+ indicator Fura-2/AM (5 μM) to measure [Ca2+]i. After washed for three times to remove extracellular Fura-2/AM, the Fura-2 was detected using Felix fluorescence data acquisition as previously shown [
Cells were seeded and treated with G15 or GPR30 small interfering RNA (siRNA). The dose of G15 was based on Wang et al. [
The nitrocellulose membrane was spotted with EPA (1, 3, and 5 mM) and then blocked in buffer as previously reported [
Ovarian cancer xenograft models were generated in old female nude mice at 4 to 6 weeks of age by injecting 1×107 ES2 cells treated with or without adenoviral GPR30 shRNA into subcutaneous fat. Animals were divided into four groups with 10 per group. For the experimental group, 10 μL EPA was added to 90 μL aqueous vehicle. For the control group, 10 μL ethanol was added to 90 μL aqueous vehicle. Mice were given EPA (4 μg) once daily. For the treatment of G15, ovarian cancer xenograft models were generated in old female nude mice at 4 to 6 weeks of age by injecting 1×107 ES2 cells into subcutaneous fat. Animals were divided into four groups with 10 per group. Nude mice bearing ovarian tumors were received dimethyl sulfoxide (DMSO) in combination with MeOH as a control, EPA in combination with DMSO, MeOH in combination with G15 or EPA in combination with G15. Mice were given EPA (4 μg) once daily, while G15 (10 μg) once daily from day 5. Tumor volumes were calculated as 1/2×length×width2. Ki67 and GPR30 were stained to assay the inhibitory effects of EPA and GPR30 expression, respectively.
Ovarian cancer patient (n=173) samples were collected from the Department of Pathology of Nanjing Drum Tower Hospital. The tissue microarray (TMA) was made as previously reported [
Quantitative real-time PCR was performed using SYBR Select Master Mix (Vazyme, Nanjing, China) in an ABI 7300 sequence detector. The results were normalized to the 18S rRNA gene expression level in each sample. The primers were from PrimerBank (
The antibodies used were cleaved caspase-3, AKT, p-AKT, ERK, p-ERK, phosphorylated protein kinase A (p-PKA) substrate (Cell Signaling Technology, Danvers, MA), actin (Sigma-Aldrich), and GPR30 (Santa Cruz Biotechnology, Santa Cruz, CA).
The siRNA against GPR30 was GCACCUGUGGCUGACGAAUUU. ES2 cells were transfected with RNA interference (RNAi) oligonucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA).
The GPR30 shRNA sequence was GCACCTGTGGCTGACGAATTT. Adenovirus was amplified and purified using Sartorius Adenovirus Purification kits (Sartorius, Germany).
Data from at least three independent experiments were analyzed and are expressed as means±standard deviation. The nonpaired Student’s t test was used for these analyses. A difference was considered significant at p < 0.05, p < 0.01, and p < 0.001. R was used for the correlation analysis of GPR30 expression level with overall survival of ovarian cancers patients. Then packages “survival” and “survminer” were used for survival analysis using Kaplan-Meier method and generate the survival curve.
All animal procedures were carried out in accordance with the approval of the Animal Care (#CS20) and Use Committee at the Model Animal Research Center of Nanjing University in Nanjing.
FAs have an effect on cancer by altering cell membrane synthesis and saturation and via cholesterol lipid hormones that affect the function and distribution of signaling macromolecules [
As previously reported, n-3 PUFAs (docosahexaenoic acid or EPA) inhibit colorectal cancer cell proliferation by inducing cell apoptosis [
Previous findings suggest the receptors of long-chain FAs are GPR40 and GPR120 [
Similarly, we studied the effect of YM254890 on palmitic acid proliferation as well as the EPA antiproliferative effect in ES2 cells. As shown in
Based on the above results, we hypothesized that there exists some GPR whose Gαs subunit plays a dominant role in the EPA effect in ES2 cells. Thus, we detected various GPR proteins expression levels in ES2 cells. Quantitative PCR analysis showed that GPR30, coupled to Gαs protein [
To determine whether GPR30 mediates the effect of EPA in ES2 cells, we knocked down GPR30 using RNAi and found that GPR30 expression was reduced by 80% without changes in GPR40 or GPR120 (
Furthermore, we knocked down GPR30 to examine its effect on the EPA proapoptotic action in ES2 cells. The number of apoptotic cells dramatically declined when ES2 cells were treated with GPR30 siRNA compared with negative-control siRNA (
The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is the most common pathway related to cancer proliferation [
The results in
To confirm the above observations, we also conducted a mouse xenograft model to further verify the role of GPR30 in ES2 cells. In accordance with previous findings, EPA alone inhibited tumor growth significantly (
Extensive research implies that dysregulation of lipid metabolism is correlated with ovarian cancer progression [
Classical free fatty acid receptors, such as GPR40, and GPR120, might also mediate the function of EPA in ovarian cancer cells. Since Gαq is the α subunit of both GPR40 and GPR120, whose activation leads to a rapid increase in Ca2+, we detected the Ca2+ concentration after adding EPA, and an approximately 1.5-fold increase was observed. Importantly, YM254890, a specific inhibitor of the Gαq unit, did not inhibit the increase in Ca2+ caused by EPA, suggesting that neither GPR40 nor GPR120 is the specific receptor of EPA. We found a novel EPA receptor, GPR30, in ovarian cancer cells, confirmed by a modified protein lipid assay [
Oxidative stress has been reported to affect cancer cell development. For example, reactive oxygen species (ROS) participate in cancer cell progression and proliferation, cell apoptosis, and energy metabolism [
Our observations suggest that in ES2 OCCC cells, EPA functions through the GPR30-Gαs-cAMP-PKA signaling pathway. Originally, phosphorylation of ERK1/2 is increased by Gβγ, which is dissociated from Gα. Previous work also demonstrated that PKA stimulated by the α subunit of GPR30 could decrease the phosphorylation of ERK1/2 [
Overall, our study provides evidence that GPR30 is a novel receptor of EPA in ES2 cells that mediates its anticancer effects. EPA might inhibit proliferation by inducing apoptosis. Additionally, EPA might influence the GPR30-ERK or GPR30-AKT signaling pathway. In our future work, we will examine the mechanism of the effects that EPA has on other backgrounds. In addition, because other researchers have provided evidence that EPA can improve the sensitivity to chemotherapy [
Supplementary materials are available at Cancer Research and Treatment website (
Numbering order of ovarian cancer patients on tissue microarray
GPR30 expression grade with the classification of ovarian cancer
Clinical information of patients
Eicosapentaenoic acid (EPA) promotes cell growth in CAOV3 and A2780. Dose-response curves of EPA in CAOV3 cells (A) and A2780 cells (B). The cells were treated with the indicated concentrations of EPA for 24 hours, and cell growth was evaluated by CCK8. Values are normalized to the percentage of cell growth in control wells (n=3). *p < 0.05, **p < 0.01.
GPR119 does not mediate antitumor function of eicosapentaenoic acid (EPA) in ES2 cells. The expression of proapoptotic genes and antiapoptotic genes in ES2 cells treated with 300 μM EPA and control RNAi or 300 μM EPA and GPR119 RNAi for 48 hours was analyzed by quantitative reverse transcription polymerase chain reaction. Values are presented as mean±standard deviation from three independent experiments.
Conflict of interest relevant to this article was not reported.
We thank Prof. Zhongren Ding from Shanghai Medical College of Fudan University for kindly providing YM254890 from Yamanouchi Pharmaceutical Co., Ltd. (Ibaraki, Japan). This work was supported by the Chinese National Science Foundation (31601153 to Y.Z., 81770861 and 31571401 to X. Li), the Nature Science Foundation of Jiangsu Province (BK20160619 to Y.Z.), the Social Development Fund of Jiangsu Province (BE2017708 to C.L.), the Fundamental Research Funds for the Central Universities (14380269, 14380343, 14380469 to Y.Z.), and by China Postdoctoral Science Foundation (2016M601778 to Y.Z.). Chong Qing Science and Technology Foundation (cstc2018jcyjAX0232 to X. Li), Chong Qing Education Foundation (KJZD-K201800402 to X. Li).
Eicosapentaenoic acid (EPA) inhibits cell proliferation in ovarian cancer cells. (A) Dose-response curves of palmitic acid, palmitoleic acid, oleic acid, linoleic acid, arachidonic acid, linolenic acid, and EPA in ES2 cells and SKOV3 cells. (B) The toxicity of palmitic acid and EPA at different concentrations on ES2 and SKOV3 cells. (C) Cell proliferation of ES2 and SKOV3 cells in time course for various fatty acids in ES2 cells and SKOV3 cells were treated with 300 μM fatty acids. (D) Apoptosis by EPA in ES2 cells. The cells were treated with or without 300 μM EPA for 48 hours. Apoptosis was detected by cleaved caspase-3. PI, propidium iodide. (E) The expression of proapoptotic genes and antiapoptotic genes in ES2 cells treated with or without 300 μM EPA for 48 hours was analyzed by quantitative reverse transcription polymerase chain reaction. (F) The toxicity of EPA on normal ovarian epithelial cells (OEC), ES2, and SKOV3 cells. (G) The apoptosis rate of normal OEC, ES2, and SKOV3 cells treated with EPA. Values are presented as mean±standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
G protein–coupled receptor 30 (GPR30) acts as a novel eicosapentaenoic acid (EPA) receptor in ES2 cells. (A, B) ES2 cells were loaded with 3 μM of the fluorescent calcium probe fura-2A/M. (A) Cells were stimulated in phosphate buffered saline alone (control), 300 μM palmitic acid bound to bovine serum albumin (BSA) (0.1%), 300 μM EPA bound to BSA (0.1%), or BSA alone. (B) ES2 cells were incubated with YM254890 (Gq protein inhibitor) at 37°C for 5 minutes before the addition of fatty acids. The arrows indicate the onset of stimulation. (C) Cell proliferation of ES2 cells treated with 0.1% BSA, 300 μM palmitic acid (PAL), or 300 μM EPA in the absence or presence of YM254890 for 24 hours. (D) Apoptosis of ES2 cells treated with 300 μM EPA in the absence or presence of YM254890. Apoptosis was detected by cleaved caspase-3. PI, propidium iodide. (E) The expression of proapoptotic genes and antiapoptotic genes in ES2 cells treated with 300 μM EPA in the absence or presence of YM254890 for 48 hours was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR). (F) Intracellular cAMP by EPA in ES2 cells. Cells were treated with 0.1% BSA, 300 μM PAL, or 300 μM EPA for 24 hours for cAMP measurement. (G) The relative expression of various GPRs in ES2 cells by RT-PCR. Data were normalized to the expression level of free fatty acid receptor 3 (FFAR3). (H) Interaction of GPR30 and EPA was detected by a protein lipid overlay assay. Increasing amounts of EPA were incubated with increasing concentrations of purified GFP-GPR30 harvested from ES2 cell lysates. (I) Relative expression of GPR30 was measured by quantitative RT-PCR in different ovarian cancer cell lines. Data were normalized to the expression level in SKOV3. Values are presented as mean±standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.
G protein–coupled receptor 30 (GPR30) is expressed in patients with various ovarian cancers. The expression level of GPR30 in patients with various ovarian cancers according to a tissue microarray and immunohistochemistry. (A) Immunohistochemical staining of GPR30 in ovarian tissues with various ovarian cancers. (B) The expression grade of GPR30 in patients with various ovarian cancers (n=173). HGSC, high-grade serous carcinoma; LGSC, low-grade serous carcinoma; MC, mucinous carcinoma, CCC, clear cell carcinoma; EC, endometrioid adenocarcinoma. (C) The correlation analysis of GPR30 expression level with overall survival of patients (The grade score 0 to 3 represents the intensity of GPR30 from lowest to highest).
G protein–coupled receptor 30 (GPR30) is involved in eicosapentaenoic acid (EPA)–induced cell antiproliferation and proapoptosis in ES2 cells. (A) The efficacy of GPR30 small interfering RNA (siRNA). (B) Cell proliferation of ES2 cells treated with 0.1% bovine serum albumin (BSA), 300 μM palmitic acid (PAL), or 300 μM EPA with or without GPR30 siRNA treatment. (C) Cell proliferation of ES2 cells treated with 0.1% BSA, 300 μM PAL, or 300 μM EPA with or without G15. (D) Apoptosis of ES2 cells treated with 300 μM EPA and control RNA interference (RNAi) or 300 μM EPA and GPR30 RNAi for 48 hours. Apoptosis was detected by cleaved caspase-3. PI, propidium iodide. (E) The expression of proapoptotic genes and antiapoptotic genes in ES2 cells treated with 300 μM EPA or 300 μM EPA and GPR30 siRNA for 48 hours was analyzed by quantitative reverse transcription polymerase chain reaction. Values are presented as mean±standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Eicosapentaenoic acid (EPA) participates in the G protein–coupled receptor 30 (GPR30)–cAMP–protein kinase A signaling pathway in ES2 cells. (A) After 24 hours of serum starvation in Opti-MEM, cells were incubated for 24 hours in OptiMEM and stimulated with phosphate buffered saline alone (control), 0.1% bovine serum albumin (BSA), 300 μM palmitic acid (PAL) bound to BSA (0.1%) or 300 μM EPA bound to BSA (0.1%) with or without GPR30 siRNA treatment. (B) p-AKT/AKT and p-ERK/ERK were analyzed by Western blotting and quantified by ImageJ. (C, D) ES2 cells were treated with or without 300 μM EPA in the absence and presence of G15 (C) or GPR30 siRNA (D) for 24 hours for cAMP measurement. (E) Phospho-(Ser/Thr) substrate was detected by Western blotting in ES2 cells treated with EPA, EPA+GPR30 siRNA, EPA+G15, EPA+G1 (500 nM, the specific agonist of GPR30) or EPA+G1+GPR30 siRNA. (F) PKA activities were quantified by ImageJ, and the data are expressed as PKA activity (n=3). Values are presented as mean±standard deviation from three independent experiments. **p < 0.01, ***p < 0.001.
Eicosapentaenoic acid (EPA) blocks tumor growth via G protein–coupled receptor 30 (GPR30) in mouse xenografts. (A, B) Nude mice bearing ovarian tumors (ES2 cells) were received ethanol in combination with LacZ shRNA as a control, EPA in combination with LacZ shRNA, ethanol in combination with GPR30 shRNA or EPA in combination with GPR30 shRNA. (A) Xenograft tumors (scale bar=1 cm). (B) Ki67 and GPR30 expression (scale bar=50 μm). Tumor volume (C) and tumor weight (D) in (A). (E, F) Nude mice bearing ovarian tumors (ES2 cells) were received dimethyl sulfoxide (DMSO) in combination with MeOH as a control, EPA in combination with DMSO, MeOH in combination with G15 or EPA in combination with G15. (E) Xenograft tumors (scale bar=1 cm). (F) Ki67 and GPR30 expression (scale bar=50 μm). Tumor volume (G) and tumor weight (H) in (E). Values are presented as mean±standard deviation from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.