The Mechanism Of Axin On ATRA-induced Growth Inhibition And Apoptosis In Glioma Cell Lines

Retinoids, including retinoic acid, retinamide and retimid ester, arederivants of vitamin A (retinol). It is essential for life and has important roles inembryonic development, spermatogenesis, vision and cellular differentiation.Early researches with vitamin A-deficient animals revealed a connectionbetween this vitamin and susceptibility to carcinogenesis. In the diet,pharmacological retinol can reduce the incidence of carcinogen-inducedcarcinoma. Retinoids play an important role in prevention and therapy of sometumors such as leukecythemia, breast cancer, skin cancer, cervix cancer andCNS tumors. Retinoids reduces the growth of certain tumor cells and inducestheir apoptosis and differentiation. All-trans retinoic acid is the third generationof retinoids. There are two isomerides classified as cis-retinoic acid and all-transretinoic acid according to the lateral chain of polyalkene peptide and the locationof carboxy. All-trans isoform is frequent and stable constitution. Retinoic acidand its derivations induce cells apoptosis and differentioation and reduceproliferation by nuclear receptor (NR) signaling pathway (RARs and RXRs).Many studies demonstrated it has a crosstalk between Wnt/beta-canetinsignaling and NR signaling pathway. They synergistically regulate development, proliferation and apoptosis.Axin (Axis inhibition), the product of the mouse Fused (Fu) gene found in1997, is an important scaffold protein of the Wnt signaling pathway. It transmitscellular signaling to downstream effective molecule to regulate the signalingtransduction between different signaling pathways. To date, Axin has beenimplicated in at least four different signaling pathways: the Wnt, JNK, p53, andTGF-βsignaling pathways and take part in embryonic development, neurondifferentiation, carcinogenesis, cell apoptosis and glycometabolism. EspeciallyAxin as a tumor suppor has arose more peopleís interest to study the relationshipbetween Axin and human tumor. Over-expression of Axin in transgenic miceleads to massive cell death in different organs. Other studies demonstrated thatectopic expression of Axin induced apoptosis in some tumor cells. Our groupfound that over-expression of Axin by stable transfection induced apoptosis ofglioma C6 cell lines. Recent study reveals that Axin take part in ATRA-induceddifferentiation of embryonic carcinoma cells, suggesting Axin may play animportant role in ATRA-mediated cell fate. The introduction of wild-type Axininto heptocellular and colorectal cancer cells induces apoptosis.Gliomas are the most common tumors in central nervous system (CNS)(account for 42% of primary CNS tumors and 3/4 of them are malignant). Theclinical and experimental researches have shown that retinoids cut down therecrudescent rate of glioma and improved its survival rate. It is not clear whetherAxin as a regulator of ATRA-mediated cell growth.Objectives:To investigate in glioma cells whether Axin takes part inATRA-induced cell proliferative inhibition and apoptotic cell death.Methods: (1) C6 and U251 cell lines were treated with ATRA. The effect ofATRA on the proliferation of cells was determined by 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay. Todetermine the effect of ATRA on clonal proliferation, plate colony formationassays were performed. The ability of ATRA to inhibit DNA synthesis wasdetermined by estimating the amount of BrdUrd incorporation into DNA byimmunocytochemistry staining. The change of cell cycle was analyzed by flowcytometry. (2) The subcellular localization ofβ-catenin was detected byimmunofluorescent staining. The mRNA and protein of Axin, p53,β-cateninwere examined by RT-PCR and Western blot respectively. (3) To investigatewether over-expression of Axin can mimice the role of ATRA, glioma C6 cellline was stably or transiently transfected with rAxin. Cell cycle were examinedby FCM. The subcellular localization ofβ-catenin was detected byimmunofluorescent staining. The mRNA and protein of Axin, p53,β-cateninwere examined by RT-PCR and Western blot respectively. (4) To investigatewhether Axin RNAi attenuate ATRA-induced cell cycle arrest and apoptosis,siRNA oligos of Axin were transfected into C6 cells. FCM, RT-PCR andWestern blot was use to examine the cell cycle and the expression of p53,respectively.Results: (1) ATRA inhibited cell proliferation and induced cell apoptosis. ATRAactivated the expression of Axin and p53. ATRA accumulated cytoplasmicbeta-catenin without changing the level of total protein. (2) Over-expression ofAxin could mimice the role of ATRA: (i) inhibited cell proliferation and inducedapoptosis; (ii) accumulated cytoplasmic beta-catenin without altering the levelof protein; (iii) activated the expression of Axin and p53. (3) Axin RNAiattenuated ATRA-induced G1/S arrest and apoptosis and down-regulated theexpression of p53.Conclusions: ATRA-activated the expression of Axin result in the accumulation of cytoplasmic beta-catenin and the activation of p53. This activationcontributed to ATRA-induced cell proliferative inhibition and apoptosis.Intereatingly, Axin-RNAi caused only moderate decrease of p53 with the ATRAtreatment, implying that there are other mechanisms to regulate ATRA-activatedp53.