Canolol inhibits gastric tumors initiation and progression through COX-2/PGE2 pathway in K19-C2mE transgenic mice

Abstract

4-Vinyl-2, 6-dimethoxyphenol (canolol) is an antioxidant phenolic compound extracted from crude canola oil. In current research, K19-C2mE transgenic mice, developing hyperplastic tumors spontaneously in the glandular stomach, were used to study the mechanisms involved in the anti-inflammation and anti-tumor effects of canolol. Tg mice receiving canolol diet had a reduced tumor incidence, to 41.2%, compared with Non-treatment Tg mice, 77.8% of which had gastric tumor (P=0.002). Besides that, the mean tumor diameter was decreased from 6.5 mm to 4.5 mm (P<0.001) after canolol administration. COX-2/PGE2 pathway is known to play pivotal role in inflammation-induced gastric tumorigenesis. The neutrophils and lymphocytes infiltration was suppressed significantly, and the mRNA levels of the proinflammatory cytokines COX-2, IL-1β and IL-12b were also downregulated in gastric mucosa. Additionally, immunohistochemical analysis showed that COX-2, EP2, Gαs and β-catenin, key factors involving in PGE2 signal transduction, were positive staining with higher H scores in Non-treatment Tg mice, while the expressions were suppressed significantly by 0.1% canolol (P<0.001). In addition, tumor-suppressor miR-7 was reactivated after canolol administration, and COX-2 was showed to be a functional target of miR-7 to suppress the tumor progression. In conclusion, canolol could inhibit the gastritis-related tumor initiation and progression, and the suppression effect was correlated with the blocking up of canonical COX-2/PGE2 signaling pathway and might be regulated by miR-7.

Fig 1. Experiment design in this study.

4 weeks old mice were subjected to genotyping PCR, after identified, 6 weeks old Tg mice were randomly divided into Non-treatment group and Canolol-treated group. All the mice were fed with modified AIN93G diet with(out) 0.1% canolol till 52 weeks.

Fig 2. The macroscopic photograph in wild mice (A), Non-treatment Tg mice (B), Canolol-treated Tg mice (C), and the scatter plot of the tumor size (D).

The arrows indicate gastric tumors occurred in Tg mice. Bold horizontal bar indicates the median with interquartile range. **, P<0.01.

Fig 3. Histological examination showed the grades of gastritis.

The normal gastric epithelial in wild type mice (grade 0, A), moderate (grade 2, C) and marked (grade 3, D) gastritis in Non-treatment Tg mice, and the alleviation of inflammatory responses in Canolol-treated Tg mice (grade 1, B). The arrow indicates mononuclear cell infiltration into the gastric glands or lymphoid follicle formed in submucosa. HE×20

Fig 4. Immunohistochemical staining of COX-2 (A, B), EP2 (C, D), Gαs (E, F) and β-catenin (G, H) in Non-treatment mice (left panel) and the Canolol-treated mice (right panel).

Note that the intensity of COX-2, EP2, Gαs and β-catenin immunoreactivity in the Canolol-treated Tg gastric mucosa is weaker than that in Non-treatment Tg group. IHC×40.

Fig 5. The β-catenin expression in cytoplasm and nuclear in Non-treatment (1&3) and Canolol-treated (2&4) gastric tumor tissue.

Note the β-catenin nuclear accumulation was decreased in Canolol-treated groups (Lane 4) compare to Non-treatment groups (Lane 3).

Fig 6. The effects of canolol on the COX-2/PGE2 pathway genes (A) and miR-7 expression (B).

The studied genes involved proinflammatory cytokines (IL-12b, IL-1β, IL-6), oxidative responding gene HO-1, PGE2 synthases genes (COX-2, mPGES-1) and Gαs that relays PGE2 signal into cytoplasm. The horizontal bar in B indicates the 1.0 *, P<0.05; * *, P<0.01.

Fig 7. COX-2 is the direct target of miR-7.

The relative luciferase activity was reduced significantly in 3’-UTR-1 but not in 3’-UTR-2 (A). miR-7 and its putative two binding sequence in the 3’-UTR of COX-2, 86–92 sites (B) and 1111–1118 sites (C). The mutants of 3’-UTR were indicated by the arrows. *, P<0.05