Prostaglandin E2 accelerates invasion by upregulating Snail in hepatocellular carcinoma cells

• Published in: Tumor biology 2014-07, Vol.35 (7), p.7135-7145
• Main Authors: Zhang, Min, Zhang, Hai, Cheng, Shanyu, Zhang, Dengcai, Xu, Yan, Bai, Xiaoming, Xia, Shukai, Zhang, Li, Ma, Juan, Du, Mingzhan, Wang, Yipin, Wang, Jie, Chen, Meng, Leng, Jing
• Format: Article
• Language: English
• Subjects: Animals; Biomedical and Life Sciences; Biomedicine; Cancer Research; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - pathology; Cell Line, Tumor; Cell Movement - genetics; Dinoprostone - genetics; Dinoprostone - metabolism; Humans; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; Neoplasm Invasiveness - genetics; NF-kappa B - metabolism; Phosphorylation; Proto-Oncogene Proteins c-akt; Research Article; Signal Transduction; Snail Family Transcription Factors; Transcription Factors - biosynthesis; Transcription Factors - metabolism
• ISSN: 1010-4283; 1423-0380
• DOI: 10.1007/s13277-014-1963-4

Our previous studies showed that prostaglandin E 2 (PGE 2 ) promotes hepatoma cell growth and migration, as well as invasion; however, the precise mechanism remains elusive. Snail and p65 protein levels were detected in human samples with hepatocellular carcinoma (HCC) by immunohistochemistry (IHC) staining. HCC cell lines (Huh-7 and Hep3B) were used for in vitro experiments. PGE 2 /Akt/NF-κB pathway was investigated in Huh-7 and Hep3B cells after treatment with PGE 2 , EP4 receptor (EP4R) agonist, Akt inhibitor, and NF-κB inhibitor, respectively, by real-time reverse transcription (RT)-PCR, Western blotting, and immunofluorescence (IF) staining. In vitro cell invasion assay was performed to evaluate the effect of PGE 2 on tumor invasiveness. Knockdown of EP4R was carried out in Huh-7 cells through plasmid-based small interfering RNA (siRNA) approach to confirm the regulation of PGE 2 on Snail by EP4R. Dual luciferase reporter assay was performed to assess Snail promoter activity in Huh-7 cell after treatment with EP4R agonist. We found that the protein levels of Snail were higher in HCC tissues than those in control and that PGE 2 and EP4R agonist treatment significantly increased Snail expression in Huh-7 and Hep3B cells. EP4R agonist also profoundly promoted invasiveness of Huh-7 cells. Knockdown of the EP4R by siRNA completely blocked the PGE 2 -induced upregulation of Snail expression and reduced invasiveness of Huh-7 cells. We failed to find that EP4R-induced upregulation of Snail was reversed by inhibition of cAMP response element-binding protein (CREB), a canonical downstream target of EP4R. Alternatively, EP4R agonist treatment significantly increased the levels of phosphorylated EGFR and Akt both in Huh-7 and Hep3B cells. AG1478, an EGFR inhibitor, blocked the phosphorylation of Akt. The levels of phosphorylated IκB increased in Huh-7 cells after treatment with EP4R agonist for 30 min. The levels of phosphorylated p65 started to increase in Huh-7 cells treated with EP4R agonist for 4 h, and p65 translocated into the nucleus. In EP4R-agonist-treated Hep3B, the levels of phosphorylated p65 were also increased compared to the control group. The phosphorylation levels of p65 were significantly decreased in Huh-7 and Hep3B cells after treatment with the Akt signaling inhibitor LY294002 and EP4R agonist for 24 h. Treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) at 10 μM for 24 h blocked EP4R-agonist-induced Snail upregulation in Hu