Inhibitory effect of linoleic acid on transformation of IEC6 intestinal cells by in vitro azoxymethane treatment

Abstract

The effect of linoleic acid (LA) on growth and transformation of IEC6 intestinal cells was examined. IEC6 cells expressed mRNAs of 15-lipooxygenase (LOX15) and peroxisome proliferator-activated receptor (PPAR)c but not COX-2. Cell growth was suppressed by LA in a dose-dependent manner in IEC6 cells. Threeweek treatment with LA provided IEC6 cells a quiescent state. LA-induced growth inhibition was abrogated by exposure to antisense S-oligodeoxynucleotides (S-ODNs) for LOX15 and/or PPARc. In an in vitro carcinogenesis model, IEC6 cells, which had confirmed CYP2E1 expression and activity, were continuously treated with AOM and/or LA for 40 weeks. DNA injury in AOMtreated cells was suppressed to the control level by concurrent LA treatment. Colony formation of AOM-treated cells in soft agar was suppressed by treatment with LA, which was reversed by exposure to antisense S-ODNs for LOX15 and/or PPARc. AOMtreated IEC6 cells formed s.c. tumors in 9 of 12 mice, whereas AOM1LA-treated cells formed no tumor. IEC6 cells showed no remarkable alteration of protein production by AOM treatment, whereas cells treated with AOM1LA showed decreased epidermal growth factor receptor (EGFR) and phospho-EGFR and increased BAX. These findings suggest that LA inhibited AOMinduced transformation of COX-2-negative IEC6 cells, which was possibly mediated with PPARc ligands generated by LOX15 from LA. ' 2005 Wiley-Liss, Inc.Key words: linoleic acid; azoxymethane; colon cancer; transformation; peroxisome proliferator-activated receptor Colorectal cancer is the third most common malignant neoplasm worldwide and the third leading cause of cancer deaths in Japan. 1 The frequency of colon cancer in Japan is continuously increased along with the alteration of lifestyle from traditional Japanese to Western. In particular, the increase of fat intake and decrease of fiber intake have been regarded as the most important nutritional influence on colorectal cancer development. 2,3 Prostaglandins are bioactive lipids derived from the metabolites of membrane PUFAs and play important roles in a number of biologic processes. 4 COX-2-dependent overproduction of PGE 2 is hypothesized to be an important part of sustained proliferative and chronic inflammatory conditions. 5,6 Several in vivo studies hypothesize that a high amount of x-6 PUFA, such as LA, might enhance colorectal carcinogenesis via stimulation of colonic epithelial cell proliferation. 7-10 Indeed, rats treated with AOM and fed a diet supplemented with LA developed more tumors than animals treated with AOM alone. 11 AOM is a potent carcinogen, which induces colorectal cancers at high incidence in rats and mice. Adduct formation (mainly O 6 -methylguanine) by AOM induces point mutations in multiple genes such as K-ras and b-catenin. 12 In the present study, we used AOM as a carcinogen in the in vitro carcinogenesis model.The oxidative metabolites of LA, particularly 9-HODE, 13-HODE and 13-OXO, have biologic effects as PPARg ligands. 13 CLA, a strong ligand for PPARg, has a substantial anticarcin...

Peroxisome Proliferator-Activated Receptor γ Pathway Targeting in Carcinogenesis: Implications for Chemoprevention

“…Others have shown PPARg activation with 9,11-conjugated linoleic acid (45,46), and because PPARg is known to induce itself, this is a simple explanation for why elevated PPARg levels were observed. In another study that supports an anticarcinogenic role for PPARg in colon carcinogenesis, immortalized rat intestinal IEC6 cells experience reduced proliferation with linoleic acid (47). These changes are reversible when PPARg expression is inhibited by RNA knockdown techniques, thus suggesting a tumor-suppressive role for PPARg constitutive expression.…”

Linoleic-Acid-Induced Growth Suppression Induces Quiescent Cancer Cell Nests in Nude Mice

“…Exposure to PPAR-␥ antisense S-oligodeoxynucleotide reversed the LA-induced quiescence in both MKN28 and Colo320 cells. PPAR-␥ antisense treatment reversed LA antitumor effects in azoxymethane-induced carcinogenesis and peritoneal metastasis [15,29] . In the present study, LA-treated cells showed the same alteration of protein levels to those in cells treated with PPAR-␥ ligands, TGZ and CLA.…”

“…Activation of PPAR-␥ affects expression of EGFR, cyclin D 1 [34] and Bax [35,36] . We previously reported that LA and CLA decrease the expression of EGFR and transforming growth factor ␣ , and increase Bax expression [15,29,37] . These alterations might be associated with cell growth inhibition and induction of apoptosis.…”

“…Levels of Bak, EGFR and VEGF were partially recovered by LA withdrawal. Our previous studies show that the antitumoral effect of LA depends on PPAR-␥ activation by LA metabolites, which have PPAR-␥ ligand activity [15] . In the final set of experiments, we treated 5-week LA-treated MKN28 and Colo320 cells with PPAR-␥ ligands, CLA and TGZ in an LA-free condition ( fig.…”

15-Lipoxygenase-1 in Colorectal Cancer: A Review

“…Interestingly, studies show that PPAR-␥ activation by troglitazone and rosiglitazone (synthetic PPAR-␥ ligands) synergises with simultaneous inhibition of X-linked inhibitor of apoptosis protein (endogenous apoptotic inhibitor, overexpressed in colon cancer) in treatments of colon cancer in mice xenograft models [101,102] . Growth of IEC6 colon cancer cells (expressing 15-LOX-1 and PPAR-␥ but not COX-2), induced by a potent carcinogen, azoxymethane, was successfully controlled by linoleic acid in a dose-dependent manner in in vitro studies [103] . Taken together, these results strongly support the notion that both linoleic acid and synthetic PPAR-␥ ligands may be beneficial in colon cancer, but remains to be confirmed in man.…”