Conjugated linoleic acid stimulates an anti-tumorigenic protein NAG-1 in an isomer specific manner

Conjugatedlinoleicacids(CLAs),naturallyoccurringfattyacids in ruminant food products, have anti-tumorigenicand pro-apoptotic properties in animal as well as in vitromodels of cancer. However, the cellular mechanism hasnot been fully understood. NAG-1 (non-steroidal anti-inflammatory drug-activated gene-1) is induced by severaldietary compounds and belongs to a TGF-b superfamilygene associated with pro-apoptotic and anti-tumorigenicactivities. The present study was performed to elucidatethe molecular mechanism by which CLA stimulates anti-tumorigenic activity in human colorectal cancer (CRC)cells. The trans-10, cis-12-CLA (t10,c12-CLA) repressedcell proliferation and induced apoptosis, whereas linoleicacid or c9,t11-CLA showed no effect on cell proliferationandapoptosis.Wealsofoundthatt10,c12-CLAinducedtheexpression of a pro-apoptotic gene, NAG-1, in human CRCcells. Inhibition of NAG-1 expression by small interferenceRNA(siRNA)resultsinrepressionoft10,c12-CLA-inducedapoptosis. Microarray analysis using t10,c12-CLA-treated HCT-116 cells revealed that activating transcrip-tion factor 3 (ATF3) was induced and its expression wasconfirmedbywesternanalysis.Thet10,c12-CLAtreatmentfollowed by the overexpression of ATF3 increased NAG-1promoter activity in HCT-116 cells. We further providethe evidence that t10,c12-CLA inhibited the phosphoryla-tion of AKT and the blockage of GSK-3 by siRNA abol-ished t10,c12-CLA-induced ATF3 and NAG-1 expression.The current study demonstrates that t10,c12-CLA stimu-lates ATF3/NAG-1 expression and subsequently inducesapoptosis in an isomer specific manner. These effects maybe through inhibition of AKT/GSK-3b pathway in humanCRC cells.IntroductionCancer is second only to heart disease as the leading causeof death in the USA and colorectal cancer (CRC) is one ofthe most prevalent causes of cancer-related mortality in theWestern world (1). Among the strategies to reduce coloncancer risk, chemoprevention by dietary factors has receivedconsiderable attention as an effective approach for cancerprevention. A possible correlation between dietary fat intakeand cancer incidence has been hypothesized because the largeinternational variation in rates of cancers of the breast, colonandprostatearestronglycorrelatedwithapparentpercapitafatconsumption.Conjugated linoleic acid (CLA) is composed of isomers ofoctadecadienoic acid with conjugated double bonds and isfound naturally in dairy products and ruminant meats (2).CLA has been known as a novel dietary component for theprevention of increased risk of cancer due to its chemo-protectiveproperties.DietaryCLAinhibitschemicallyinduceddevelopment of colon cancer in animal models (3) and inhibitsgrowth rates of human CRC cells in vitro (4). CLA inhibitsPI3K/AKT and ERK-1/2 signaling pathways (5) and compro-mises the expression of cyclins required for G1/S progressionand the phosphorylation status of Rb in vitro (6). In contrast,CLA was not effective when given only during the promotionphase of colon carcinogenesis (7), or in the Min mouse model(8).Itisverylikelythatthisconflictpersistsduetothefactthatthe chemopreventive mechanism of CLAs is isomer specificand the efficacy of the CLA is dependent on cell or tissuecontext.Using PCR-based subtractive hybridization, our groupidentified a non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) as a pro-apoptotic and anti-tumorigenic protein induced by NSAIDs (9). The humanNAG-1 cDNA encodes a secreted protein with homology tomembers of the TGF-b superfamily and has been previouslyidentified as macrophage inhibitory cytokine-1 (MIC-1), pla-cental transformation growth factor-b (PTGFB), prostatederived factor (PDF), growth differentiation factor-15 (GDF-15) and placental bone morphogenetic protein (PLAB) (9).NAG-1 is highly expressed in mature intestinal epithelialcells, but is significantly reduced in human CRC samples andneoplastic intestinal polyps of Min mice (10). In addition, exp-ression of the full-length NAG-1 protein resulted in increasedapoptosis in HCT-116 cells and NAG-1 expression results in adecrease of tumor sizein the xenograft mouse model and leadsto an inhibition of growth on soft agar (9). In prostate cancercells, DU-145, exposure to NAG-1 resulted in an increasedlevel of caspase-dependent apoptosis (11). We have recently