Aryl hydrocarbon receptor pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of matrix metalloproteinase-9

Abstract

Background: Abberant aryl hydrocarbon receptor (AhR) expression and AhR pathway activation are involved in gastric carcinogenesis. However, the relationship between AhR pathway activation and gastric cancer progression is still unclear. In present study, we used 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), a classic and most potent ligand of AhR, to activate AhR pathway and investigated the effect of AhR pathway activation on human gastric cancer AGS cell invasion and explored the corresponding mechanism.

Results: To determine whether AhR pathway can be activated in AGS cells, we examined the expression of CYP1A1, a classic target gene of AhR pathway, following TCDD exposure. RT-PCR and western blot analysis showed that both CYP1A1 mRNA and protein expression were increased in a dose-dependent manner following TCDD treatment and AhR antagonist resveratrol (RSV) could reverse this TCDD-induced CYP1A1 expression. To determine whether TCDD treatment of AGS cells results in an induction of MMP-9 expression, we detected MMP-9 mRNA using RT-PCR and detected MMP-9 enzymatic activity using gelatin zymography. The results showed that both MMP-9 mRNA expression and enzymatic activity were gradually increased with the concentration increase of TCDD in media and these changes could be reversed by RSV treatment in a dose-dependent manner. To examine whether AhR activation-induced MMP-9 expression and activity in AGS cells results in increased migration and invasion, we performed wound healing migration assay and transwell migration and invasion assay. After TCDD treatment, the migration distance and the migration and invasion abilities of AGS cells were increased with a dose-dependent manner. To demonstrate AhR activation-induced MMP-9 expression is mediated by c-Jun, siRNA transfection was performed to silence c-Jun mRNA in AGS cells. The results showed that MMP-9 mRNA expression and activity in untreated control AGS cells were very weak; After TCDD (10 nmol/L) treatment, MMP-9 mRNA expression and activity were significant increased; This TCDD-induced MMP-9 expression and activity increase could be abolished by c-Jun siRNA transfection.

Conclusion: AhR pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of MMP-9. Our results provide insight into the mechanism and function of the AhR pathway and its impact on gastric cancer progression.

Figure 1

AhR agonist TCDD and AhR antagonist RSV regulated CYP1A11 expression in AGS cells. After different concentrations (as shown above) of TCDD treatment for 24 hours, (A) RT-PCR analysis of CYP1A1 mRNA expression in a concentration-response. (B) Western blot analysis of CYP1A1 protein expression in a concentration-response. After co-treatment with TCDD (1 nM) and RSV (at different concentrations as shown above) for 24 hours, (C) mRNA expression of CYP1A1 was detected by RT-PCR. (D) Protein expression of CYP1A1 was detected by Western blot.

Figure 2

AhR agonist TCDD and AhR antagonist RSV regulated MMP-9 expression and activity in AGS cells. After different concentrations (as shown above) of TCDD treatment for 24 hours, (A) RT-PCR analysis of MMP-9 mRNA expression in a concentration-response. (B) Gelatin zymography analysis of MMP-9 protein activity in a concentration-response. After co-treatment with TCDD (1 nM) and RSV (at different concentrations as shown above) for 24 hours, (C) mRNA expression of MMP-9 was detected by RT-PCR. (D) Protein activity of MMP-9 was detected by gelatin zymography.

Figure 3

The effect of AhR agonist TCDD on AGS cells migration and invasion. (A) Wound healing migration assay. (B) Transwell migration assay. (C) Transwell invasion assay.

Figure 4

c-Jun mediates TCDD-induced MMP-9 expression and activity. (A) TCDD induces c-Jun mRNA expression in a dose-dependent manner. (B) TCDD induces c-Jun protein expression in a dose-dependent manner. (C) TCDD induces c-Jun mRNA expression in a time-dependent manner. (D) TCDD induces c-Jun protein expression in a time-dependent manner. (E) RSV reverses TCDD-induced c-Jun mRNA expression. (F) RSV reverses TCDD-induced c-Jun protein expression. (G) c-Jun siRNA silences c-Jun mRNA expression. (H) c-Jun siRNA silences c-Jun protein expression. (I) c-Jun siRNA inhibits TCDD-induced MMP-9 mRNA expression. (J) c-Jun siRNA inhibits TCDD-induced MMP-9 activity increase.