Department of Oncology, The People's Hospital of Ma Anshan, Ma Anshan, Anhui, P.R. China.
Eur Rev Med Pharmacol Sci. 2017 Dec;21(24):5668-5676. doi: 10.26355/eurrev_201712_14011.
To determine whether the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR).
In this experiment, we selected epithelial cells from normal esophageal mucosa as the negative control group, and the ESCC EC109 and TE-1 cell strain as the observation group. Real-time PCR and Western-blotting were used to detect the expression of EP2, EGFR and phosphorylated EGFR (p-EGFR). The pre-treatment of ESCC cell strains was carried out using Butaprost (special agonist of PGE2 and EP2) and RNAi of EP2, and we observed the expression of EP2, EGFR, and p-EGFR. WST-8 (CCK-8) was applied for the detection of the cell proliferation rate. The transwell invasion experiment was conducted for the detection of the invasion capability of cells. The expression of MMP-9 (matrix metalloproteinase-9), VEGF (vascular endothelial growth factor), pro-inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected using ELISA.
The high mRNA and protein expression of EP2, EGFR, and p-EGFR were found in the EC109 and TE-1 cell strains in the observation group, which were higher than those in the control group (p < 0.05). After the intervention of PGE2, EP2 expression was decreased and the p-EGFR expression was increased (p < 0.05). There was no variation found in the expression of EGFR (p > 0.05). After cells were intervened using Butaprost, the expressions of EP2 and p-EGFR were increased (p < 0.05), and there were no changes identified in the expression of EGFR (p > 0.05). After the intervention of RNAi, the expression of EP2 and p-EGFR was decreased (p < .05), and no changes were identified in the expression of EGFR (p > 0.05). After the intervention of PGE2 and Butaprost, great increases were seen in the cell proliferation rate, invasion capability, and the expression of MMP-9, VEGF, IL-6, and TNF-α in EC109 and TE-1 cell strains (p < 0.05), however, the intervention of RNAi could reduce above indexes (p < 0.05).
Through cell experiments, we verified that the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR) to regulate the proliferation and invasion capability of esophageal squamous cell carcinoma (ESCC) cells, and secrete and express multiple cytokines, thus discovering the pathological mechanism of inflammation to carcinoma transition in the occurrence of ESCC, and providing the experimental evidence for the search of new target in the treatment of ESCC. ESCC cells can highly express the receptor subtype EP2 of PGE2 that can transactivate the EGFR, through which PGE2 is involved in the transition mechanism from inflammation to cancer.
确定前列腺素 E2 (PGE2) 与 PGE2 的亚型受体 EP2 的组合是否可以转激活表皮生长因子受体 (EGFR)。
本实验选择正常食管黏膜上皮细胞作为阴性对照组，选择 ESCC EC109 和 TE-1 细胞株作为观察组。采用实时 PCR 和 Western-blotting 检测 EP2、EGFR 和磷酸化 EGFR (p-EGFR) 的表达。用 Butaprost（PGE2 和 EP2 的特殊激动剂）和 EP2 的 RNAi 预处理 ESCC 细胞株，观察 EP2、EGFR 和 p-EGFR 的表达。用 WST-8（CCK-8）检测细胞增殖率。Transwell 侵袭实验检测细胞侵袭能力。用 ELISA 检测细胞上清液中 MMP-9（基质金属蛋白酶-9）、VEGF（血管内皮生长因子）和促炎因子（IL-6 和 TNF-α）的表达。
观察组 EC109 和 TE-1 细胞株中 EP2、EGFR 和 p-EGFR 的高 mRNA 和蛋白表达高于对照组（p < 0.05）。干预 PGE2 后，EP2 表达减少，p-EGFR 表达增加（p < 0.05）。EGFR 的表达无变化（p > 0.05）。用 Butaprost 干预细胞后，EP2 和 p-EGFR 的表达增加（p < 0.05），EGFR 的表达无变化（p > 0.05）。用 RNAi 干预后，EP2 和 p-EGFR 的表达减少（p < 0.05），EGFR 的表达无变化（p > 0.05）。用 PGE2 和 Butaprost 干预后，EC109 和 TE-1 细胞株的细胞增殖率、侵袭能力以及 MMP-9、VEGF、IL-6 和 TNF-α的表达均显著增加（p < 0.05），而 RNAi 的干预可降低上述指标（p < 0.05）。
通过细胞实验，我们验证了前列腺素 E2 (PGE2) 与 PGE2 的亚型受体 EP2 的组合可以转激活表皮生长因子受体 (EGFR)，调节食管鳞状细胞癌 (ESCC) 细胞的增殖和侵袭能力，并分泌和表达多种细胞因子，从而发现 ESCC 发生过程中炎症向癌转变的病理机制，为 ESCC 治疗中寻找新靶点提供了实验依据。ESCC 细胞可高度表达 PGE2 的受体亚型 EP2，可转激活 EGFR，通过 PGE2 参与从炎症到癌症的转化机制。
