Mechanisms Between Axin Regulating β-Catenin And Lung Cancer

Objectiveβ-Catenin is a key molecule of both Wnt signaling pathway and E-cadher-in/catenin complex. When accumulating in nucleus, p-catenin can activate Wnt pathway and the transcription of target genes, then result in the proliferation and invasion of tumor cells. Axin ( Axis inhibition protein) is an important negative regulator of Wnt signaling pathway which induce the phosphorylation and degradation of p-catenin.The regulations of these molecules are still unclear in human lung cancer. To analysis their expressions and explore their regulation mechanisms is very important for preventin human lung cancer. In the present study, we detected the expressions of Axin, p-catenin and the mutation of p-catenin gene;analyzed the relationships among Axin, p-catenin and clinicopathological factors, and then analyzed the mechanisms of Axin regulating p-catenin and other down-stream molecules and inducing apoptosis.Methods(1) The expressions of Aixn protein and mRNA were detected with western blot and reverse-transcription polymerase chain reaction ( RT-PCR) in 44 lung cancer and corresponding normal lung tissue samples.(2) The expressions of Axin and p-catenin were detected with immunohis-tochemistry using anti-Axin antibody (1:50) and anti-β-catenin antibody (1: 600) in 100 lung cancer samples. The mutation of p-catenin gene exon 3 was detected with polymerase chain reaction (PCR) and direct sequencing.(3) The Axin gene was transfected into lung cancer PG cell line which hasvery low Axin expression. The expressions and locations of Axin, p-catenin and TCF-4 before and after transfection were detected with immunofluorescence. The mRNA expressions of Axin, p-catenin and TCF-4 before and after transfection were examined with RT-PCR. The cell cycle and apoptosis of lung cancer cells before and after transfection was examined with flow cytometry.(4) The SPSS 13.0 software was employed to ayalyze the data. P <0.05 was considered as statistical significance.Results(1) The expression quantities of Axin protein and mRNA were both significantly lower in 44 lung cancers than in corresponding normal lung tissues ( t = -2. 819, P =0. 007 for Axin protein and t = -2. 513, P =0. 016 for Axin mRNA ). The expressions of Axin protein and mRNA were significantly correlated with each other ( P =0.047, Correlation coefficient 0.301).(2) The reduced membranous expression of p-catenin was 80. 0% while aberrant nuclear expression was 26. 0%. Well and moderately differentiated lung cancers showed significantly higher membranous expression than poorly differentiated ones ( P = 0. 012). Lymph node metastasis was also significantly associated with such reduced p-catenin expression ( P =0.044). Positive Axin expression, normally seen in cytoplasma, in well and moderately differentiated cancers was significantly higher than poorly differentiated cases. Lower levels of positive Axin expression significantly related to higher nuclear p-catenin expression ( P <0.001). However, this study failed to detect any exon 3 mutation of P-catenin gene in 100 lung squamous carcinomas and adenocarcinomas.(3) After transfected Axin gene into PG cell line, the expressions of Axin mRNA and protein were significantly higher than that before transfection. The expression of p-catenin protein was reduced significantly after Axin transfection than before, but the expression of p-catenin mRNA had not obviously change. Meanwhile, the expressions of TCF-4 mRNA and protein were decreased. The flow cytometry revealed that the apoptosis rate was much higher after Axin transfection than before, but there was not any obvious change in cell cycle.Conclusion(1) The expression of Axin was much lower in lung cancer tissues than in normal lung tissues. The expression of Axin was negatively correlated with high TNM stage and poor differentiation of lung squamous cell carcinomas and adeno-carcinomas, and negatively correlated with p-catenin nuclear expression. Hie up-regulation of Axin can promote the degradation of ($-catenin, inhibit the expression of TCF-4 and induce the apoptosis of lung cancer cells. The defective of Axin expression would be one of the important reasons responsible for proliferation of lung cacer cells.(2) Abnormal expression of p-catenin was correlated with poor differentiation and lymph node metastasis. The mutation of (3-catenin gene exon 3 may not be an important reason for its abnormal expression in lung cancers.