Tumor Biology

, Volume 35, Issue 7, pp 7135–7145

Prostaglandin E2 accelerates invasion by upregulating Snail in hepatocellular carcinoma cells

  • Min Zhang
  • Hai Zhang
  • Shanyu Cheng
  • Dengcai Zhang
  • Yan Xu
  • Xiaoming Bai
  • Shukai Xia
  • Li Zhang
  • Juan Ma
  • Mingzhan Du
  • Yipin Wang
  • Jie Wang
  • Meng Chen
  • Jing Leng
Research Article

DOI: 10.1007/s13277-014-1963-4

Cite this article as:
Zhang, M., Zhang, H., Cheng, S. et al. Tumor Biol. (2014) 35: 7135. doi:10.1007/s13277-014-1963-4

Abstract

Our previous studies showed that prostaglandin E2 (PGE2) 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. PGE2/Akt/NF-κB pathway was investigated in Huh-7 and Hep3B cells after treatment with PGE2, 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 PGE2 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 PGE2 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 PGE2 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 PGE2-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 Huh-7 and Hep3B cells. Furthermore, we obtained human Snail promoter sequence from TRED-Promoter Database and identified a putative binding site of NF-κB in the sequence through TFSEARCH analysis. Subsequently, we treated Huh-7 cells with EP4R agonist or EP4R agonist and PDTC (NF-κB antagonist) and found significantly increased Snail promoter activity after EP4R agonist treatment for 12 h. The increased Snail promoter activity could be partially abolished by additional PDTC treatment. In addition, p65 protein levels were found increased together with Snail in HCC tissues compared to normal liver tissues. In conclusion, PGE2 activates Akt/NF-κB signaling and then upregulates Snail via the EP4R/EGFR to promote migration and invasion in hepatoma cells. These findings may help future evaluation of novel chemo-preventive strategies for HCC.

Keywords

PGE2 EP4 receptor Snail NF-κB Akt Hepatoma cell 

Supplementary material

13277_2014_1963_MOESM1_ESM.ppt (1.1 mb)
Suppl. Figure 1Detection of EP4R/EGFR/Akt/p65/Snail pathway in Hep3B cells. (A) The changes in Snail expression at the protein level in Hep3B cells treated with 0, 0.1, 1, 5, or 10 μM EP4R agonist (EP4Ra) for 24 hours. (B)Detection of Snail levels in Hep3B cells treated with PGE2 or EP4R inhibitor, respectively, through Western blotting.(C)Hep3B cells were treated with 10 μM EP4Ra for 0, 15, 30, or 60 minutes. As detected by western blotting, phospho-Akt levels started to increase at 15-minutes. (D)EP4Ra treatment significantly increased phosphorylation levels of EGFR at 45-minutes in Hep3B cells. (E) The level of phosphorylated p65 significantly increasedin Hep3B cells treated with EP4Ra. After treatment with the Akt signaling inhibitor LY294002, Hep3B cells were exposed to 10 μM EP4Ra for 24 hours. The increase in phosphorylated p65 levels by EP4Ra was blocked by LY294002.(F)Treatment with the NF-κB inhibitor PDTC at 10 μM blocked EP4Ra-induced Snail upregulation in Hep3B cells. Hep3B cells were exposed to 10 μM PDTC, followed by 10 μM EP4Ra for 24 hours. Levels of β-actin served as a loading control. (PPT 1124 kb)
13277_2014_1963_MOESM2_ESM.ppt (110 kb)
Suppl. Figure 2Prediction of binding site of NF-κB at human Snail promoter Promoter sequence of human Snail from -813 to 59 and putative binding site of NF-κB was marked in blue. (PPT 110 kb)
13277_2014_1963_MOESM3_ESM.ppt (122 kb)
Suppl. Figure 3Detection of p-GSK3βlevels in HUH-7 treated by EP4Ra HUH-7 cells were exposed to 10 μM EP4Ra for 0, 30, or 60 minutes. Phospho-GSK3β (Ser9) levels started to increase at 30-minutes. Total protein was isolated, and equal amounts of protein were separated by SDS-PAGE and visualized with the anti-phospho-GSK3β antibody. Levels of GAPDH served as a loading control. (PPT 122 kb)

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Min Zhang
    • 1
  • Hai Zhang
    • 1
  • Shanyu Cheng
    • 1
  • Dengcai Zhang
    • 2
  • Yan Xu
    • 3
  • Xiaoming Bai
    • 1
  • Shukai Xia
    • 1
  • Li Zhang
    • 1
  • Juan Ma
    • 1
  • Mingzhan Du
    • 1
  • Yipin Wang
    • 1
  • Jie Wang
    • 1
  • Meng Chen
    • 1
  • Jing Leng
    • 1
  1. 1.Cancer Center, Department of PathologyNanjing Medical UniversityNanjingPeople’s Republic of China
  2. 2.Pathology InstituteLanzhou UniversityLanzhouPeople’s Republic of China
  3. 3.Taizhou Vocation and Technical InstituteTaizhouPeople’s Republic of China

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