Proliferation and apoptosis regulation by G protein‑coupled estrogen receptor in glioblastoma C6 cells

Gutiérrez‑Almeida,Coral Estefania; Santerre,Anne; León‑Moreno,Lilia Carolina; Aguilar‑García,Irene Guadalupe; Castañeda‑Arellano,Rolando; Dueñas‑Jiménez,Sergio Horacio; Dueñas‑jiménez,Judith Marcela

doi:10.3892/ol.2022.13338

Proliferation and apoptosis regulation by G protein‑coupled estrogen receptor in glioblastoma C6 cells

Authors:
- Coral Estefania Gutiérrez‑Almeida
- Anne Santerre
- Lilia Carolina León‑Moreno
- Irene Guadalupe Aguilar‑García
- Rolando Castañeda‑Arellano
- Sergio Horacio Dueñas‑Jiménez
- Judith Marcela Dueñas‑jiménez
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Affiliations: Department of Physiology, University Center of Health Sciences, University of Guadalajara, Guadalajara, 44340 Jalisco, Mexico, Department of Cellular and Molecular Biology, University Center of Biological and Agricultural Sciences, University of Guadalajara, Zapopan, 45510 Jalisc, Mexico, Department of Neuroscience, University Center of Health Sciences, University of Guadalajara, Guadalajara, 44340 Jalisco, Mexico, Department of Biomedical Sciences, University Center of Tonala, University of Guadalajara, Tonala, 45425 Jalisco, Mexico
Published online on: May 18, 2022 https://doi.org/10.3892/ol.2022.13338
Article Number: 217
Copyright: © Gutiérrez‑Almeida et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Glioblastoma is the most frequent primary tumor in the human brain. Glioblastoma cells express aromatase and the classic estrogen receptors ERα and ERβ and can produce estrogens that promote tumor growth. The membrane G protein‑coupled estrogen receptor (GPER) also plays a significant role in numerous types of cancer; its participation in glioblastoma tumor development is not entirely known. The present study investigated the effect of the agonists [17β‑estradiol (E2) and G1] and antagonist (G15) of GPER on proliferation and apoptosis of C6 glioblastoma cells. GPER expression was evaluated by immunofluorescence, western blotting and reverse transcription‑quantitative PCR. Cell proliferation was determined using Ki67 immunopositivity. Cell viability was examined using the MTT assay and apoptosis using caspase‑3 immunostaining and ELISA. C6 cells express GPER, and the immunopositivity increased after exposure to E2, G1, or their combination. GPER protein expression increased after treatment with E2 combined with G1. However, GPER mRNA expression decreased in treated cells compared with control. The percentage of Ki67 immunopositive C6 cells increased under the effect of E2 in combination with G1 or G1 alone. G15 significantly reduced Ki67 immunopositivity. Pearson's correlation analysis revealed a positive relationship between GPER and Ki67 immunopositivity across the study conditions. Additionally, the MTT assay showed a significant reduction in C6 cell viability after G15 treatment, alone or in combination with G1. The exposure to G15 increased the percentage of caspase‑3 immunopositivity cells and caspase‑3 levels. Pearson's correlation analysis demonstrated a negative correlation between GPER and caspase‑3 immunopositivity across the study conditions. Glioblastoma C6 cells express GPER, and this receptor modulates cell proliferation and apoptosis. The GPER agonists E2 and G1 favored cell proliferation; meanwhile, the antagonist G15 reduced cell proliferation, viability and favored apoptosis. Therefore, GPER may be used as a biomarker of glioblastoma and as a target to develop new therapeutic strategies for glioblastoma treatment.

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1	Ostrom QT, Patil N, Cioffi G, Waite K, Kruchko C and Barnholtz-Sloan JS: CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2013-2017. Neuro Oncol. 22:iv1–iv96. 2020. View Article : Google Scholar
2	Stupp R, Taillibert S, Kanner A, Read W, Steinberg D, Lhermitte B, Toms S, Idbaih A, Ahluwalia MS, Fink K, et al: Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: A randomized clinical trial. JAMA. 318:2306–2316. 2017. View Article : Google Scholar : PubMed/NCBI
3	Ostrom QT, Rubin JB, Lathia JD, Berens ME and Barnholtz-Sloan JS: Females have the survival advantage in glioblastoma. Neuro Oncol. 20:576–577. 2018. View Article : Google Scholar
4	Yague JG, Lavaque E, Carretero J, Azcoitia I and Garcia-Segura LM: Aromatase, the enzyme responsible for estrogen biosynthesis, is expressed by human and rat glioblastomas. Neurosci Lett. 368:279–284. 2004. View Article : Google Scholar
5	Dueñas Jiménez JM, Candanedo Arellano A, Santerre A, Orozco Suárez S, Sandoval Sánchez H, Feria Romero I, López-Elizalde R, Alonso Venegas M, Netel B, de la Torre Valdovinos B and Dueñas Jiménez SH: Aromatase and estrogen receptor alpha mRNA expression as prognostic biomarkers in patients with astrocytomas. J Neurooncol. 119:275–284. 2014. View Article : Google Scholar
6	Prossnitz ER and Maggiolini M: Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol. 308:32–38. 2009. View Article : Google Scholar
7	Maggiolini M and Picard D: The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol. 204:105–114. 2010. View Article : Google Scholar
8	Hsu LH, Chu NM, Lin YF and Kao SH: G-protein coupled estrogen receptor in breast cancer. Int J Mol Sci. 20:3062019. View Article : Google Scholar
9	Filardo EJ: Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: A novel signaling pathway with potential significance for breast cancer. J Steroid Biochem Mol Biol. 80:231–238. 2002. View Article : Google Scholar
10	Vivacqua A, Bonofiglio D, Recchia AG, Musti AM, Picard D, Andò S and Maggiolini M: The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells. Mol Endocrinol. 20:631–646. 2006. View Article : Google Scholar
11	Smith HO, Arias-Pulido H, Kuo DY, Howard T, Qualls CR, Lee SJ, Verschraegen CF, Hathaway HJ, Joste NE and Prossnitz ER: GPR30 predicts poor survival for ovarian cancer. Gynecol Oncol. 114:465–471. 2009. View Article : Google Scholar
12	Tu G, Hu D, Yang G and Yu T: The correlation between GPR30 and clinicopathologic variables in breast carcinomas. Technol Cancer Res Treat. 8:231–234. 2009. View Article : Google Scholar
13	Bai LY, Weng JR, Hu JL, Wang D, Sargeant AM and Chiu CF: G15, a GPR30 antagonist, induces apoptosis and autophagy in human oral squamous carcinoma cells. Chem Biol Interact. 206:375–384. 2013. View Article : Google Scholar
14	Yang DL, Xu JW, Zhu JG, Zhang YL, Xu JB, Sun Q, Cao XN, Zuo WL, Xu RS, Huang JH, et al: Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia. Biochem Biophys Res Commun. 487:517–524. 2017. View Article : Google Scholar : PubMed/NCBI
15	Liu C, Liao Y, Fan S, Fu X, Xiong J, Zhou S, Zou M and Wang J: G-protein-coupled estrogen receptor antagonist G15 decreases estrogen-induced development of non-small cell lung cancer. Oncol Res. 27:283–292. 2019. View Article : Google Scholar : PubMed/NCBI
16	Giakoumettis D, Kritis A and Foroglou N: C6 cell line: The gold standard in glioma research. Hippokratia. 22:105–112. 2018.
17	Grobben B, De Deyn PP and Slegers H: Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion. Cell Tissue Res. 310:257–270. 2002. View Article : Google Scholar : PubMed/NCBI
18	Liu C, Liao Y, Fan S, Tang H, Jiang Z, Zhou B, Xiong J, Zhou S, Zou M and Wang J: G protein-coupled estrogen receptor (GPER) mediates NSCLC progression induced by 17β-estradiol (E2) and selective agonist G1. Med Oncol. 32:1042015. View Article : Google Scholar
19	Irecta-Nájera CA, del Rosario Huizar-López M, Casas-Solís J, Castro-Félix P and Santerre A: Protective effect of lactobacillus casei on DMH-induced colon carcinogenesis in mice. Probiotics Antimicrob Proteins. 9:163–171. 2017. View Article : Google Scholar
20	Trejter M, Jopek K, Celichowski P, Tyczewska M, Malendowicz LK and Rucinski M: Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats. Folia Histochem Cytobiol. 53:133–144. 2015. View Article : Google Scholar
21	Villalpando-Vargas F, Medina-Ceja L, Santerre A and Enciso-Madero EA: The anticonvulsant effect of sparteine on pentylenetetrazole-induced seizures in rats: A behavioral, electroencephalographic, morphological and molecular study. J Mol Histol. 51:503–518. 2020. View Article : Google Scholar
22	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
23	Üçeyler N, Riediger N, Kafke W and Sommer C: Differential gene expression of cytokines and neurotrophic factors in nerve and skin of patients with peripheral neuropathies. J Neurol. 262:203–212. 2015. View Article : Google Scholar
24	Castracani CC, Longhitano L, Distefano A, Anfuso D, Kalampoka S, La Spina E, Astuto M, Avola R, Caruso M, Nicolosi D, et al: Role of 17β-estradiol on cell proliferation and mitochondrial fitness in glioblastoma cells. J Oncol. 2020:23146932020. View Article : Google Scholar
25	Molina L, Bustamante F, Ortloff A, Ramos I, Ehrenfeld P and Figueroa CD: Continuous exposure of breast cancer cells to tamoxifen upregulates GPER-1 and increases cell proliferation. Front Endocrinol (Lausanne). 11:5631652020. View Article : Google Scholar : PubMed/NCBI
26	Chevalier N, Vega A, Bouskine A, Siddeek B, Michiels JF, Chevallier D and Fénichel P: GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation. PLoS One. 7:e346722012. View Article : Google Scholar : PubMed/NCBI
27	Hirtz A, Lebourdais N, Rech F, Bailly Y, Vaginay A, Smaïl-Tabbone M, Dubois-Pot-Schneider H and Dumond H: GPER agonist G-1 disrupts tubulin dynamics and potentiates temozolomide to impair glioblastoma cell proliferation. Cells. 10:34382021. View Article : Google Scholar : PubMed/NCBI
28	Bustos V, Nolan ÁM, Nijhuis A, Harvey H, Parker A, Poulsom R, McBryan J, Thomas W, Silver A and Harvey BJ: GPER mediates differential effects of estrogen on colon cancer cell proliferation and migration under normoxic and hypoxic conditions. Oncotarget. 8:84258–84275. 2017. View Article : Google Scholar
29	Ariazi EA, Brailoiu E, Yerrum S, Shupp HA, Slifker MJ, Cunliffe HE, Black MA, Donato AL, Arterburn JB, Oprea TI, et al: The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells. Cancer Res. 70:1184–1194. 2010. View Article : Google Scholar : PubMed/NCBI
30	Rajagopal S and Shenoy SK: GPCR desensitization: Acute and prolonged phases. Cell Signal. 41:9–16. 2018. View Article : Google Scholar
31	Cheng S Bin, Quinn JA, Graeber CT and Filardo EJ: Down-modulation of the G-protein-coupled estrogen receptor, GPER, from the cell surface occurs via a trans-Golgi-proteasome pathway. J Biol Chem. 286:22441–22455. 2011. View Article : Google Scholar : PubMed/NCBI
32	Jean-Alphonse F and Hanyaloglu AC: Regulation of GPCR signal networks via membrane trafficking. Mol Cell Endocrinol. 331:205–214. 2011. View Article : Google Scholar
33	Wang C, Lv X, Jiang C and Davis JS: The putative G-protein coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. Am J Transl Res. 4:390–402. 2012.PubMed/NCBI
34	Roque C, Mendes-Oliveira J and Baltazar G: G protein-coupled estrogen receptor activates cell type-specific signaling pathways in cortical cultures: Relevance to the selective loss of astrocytes. J Neurochem. 149:27–40. 2019. View Article : Google Scholar : PubMed/NCBI
35	Ulhaq ZS, Soraya GV, Milliana A and Tse WKF: Association between GPER gene polymorphisms and GPER expression levels with cancer predisposition and progression. Heliyon. 7:e064282021. View Article : Google Scholar : PubMed/NCBI
36	Sjöström M, Hartman L, Grabau D, Fornander T, Malmström P, Nordenskjöld B, Sgroi DC, Skoog L, Stål O, Leeb-Lundberg LM and Fernö M: Lack of G protein-coupled estrogen receptor (GPER) in the plasma membrane is associated with excellent long-term prognosis in breast cancer. Breast Cancer Res Treat. 145:61–71. 2014. View Article : Google Scholar
37	Ino Y, Akimoto T, Takasawa A, Takasawa K, Aoyama T, Ueda A, Ota M, Magara K, Tagami Y, Murata M, et al: Elevated expression of G protein-coupled receptor 30 (GPR30) is associated with poor prognosis in patients with uterine cervical adenocarcinoma. Histol Histopathol. 35:351–359. 2020.