The Role And Possible Mechanism Of Axin In The Progression Of Astrocytoma

Astrocytoma is one of the most common malignant brain tumors, and is also the most common glioma type of pathology. Modern molecular genetic analyses have revealed that the progression of astrocytic tumors results from accumulating inactivation of different tumors suppressor genes and/or amplification of certain oncogene, while the molecular mechanisms of the carcinogenesis and progression of astrocytoma have not been clarified.Axin was found in 1997, initially be considered as an inhibitor of the Wnt signaling pathway, and later studies have showed that Axin be able to negatively control axis development. Further research showed that Axin is a very important tumor suppressor, with a wide variety of tumor is closely related to the occurrence and development. Until now, the role of Axin in astrocytoma and its underlying mechanism are still unknown. In this study, we detected the expression of Axin in astrocytoma and explored the relationship between Axin expression and the clinicopathologic factors of astrocytoma. Furthermore, the role and possible mechanism of Axin in astrocytoma were investigated by Axin overexpression and Axin silencing.【Objectives】1. To investigate the relationship between Axin expression and progression of astrocytoam by RT-PCR and immunohistochemistry staining; 2. To study the effects of Axin on astrocytoma cell growth and apoptosis; 3. To explore the possible mechanism of Axin on cell growth and apoptosis in astrocytoma; 4. To study the localization ofβ-catenin in astrocytoma,and evaluate the patient survival with the level ofβ-catenin expression.【Methods】1. The expression of Axin in human astrocytoma tissues was detected by RT-PCR and immunohistochemical staining; 2. The eukaryotic expression vector with Axin was constructed and the C6 cell line stably expressing Axin gene was established; 3. The Axin gene was silenced in C6 cells by treating with Axin siRNA; 4. The effects of Axin on growth and apoptosis of astrocytoma C6 cells were respectively investigated by MTT assay, flow cytometry, colony formation assay, TUNEL staining and Hoechst staining; 5. The mRNA expression was determined by RT-PCR or Real-time PCR; 6. The protein expression level was determined by Western blot; 7. The expression and localization ofβ-catenin in astrocytoma was detected by immunohistochemistry staining.【Results】1. Axin expression correlated with the grades of astrocytoma To investigate the relationship between the expression of Axin and progression of astrocytoma, the RT-PCR and immunohistochemistry were employed to investigate the expression of Axin in astrocytoma. The Axin mRNA was detected in 2 of 2 samples of grade II astrocytoma. However, only 1 of 2 samples of grade III and 0 of 2 samples of grade IV astrocytoma showed Axin mRNA expression. The immunohistochemistry results showed that the localization of Axin was mainly preserved in cytoplasma. Thirty-three cases in the 96 samples of astrocytoma showed Axin positive expression (34.4%). Twenty-three cases of 49 grade II samples (46.9%), 8 of 31 grade III samples (25.8%), 2 of 16 grade IV samples (12.5%) showed Axin positive staining. The spearman's correlation test revealed that Axin positive expression was inversely correlated with the grades of astrocytoma significantly. The expression level of Axin didn't differ significantly by age, sex, and location.2. Axin could induce apoptosis, and inhibite proliferative activity in astrocytoma cellsThe flow cytometry results indicated that overexpression of Axin could reduce the distribution of G2+ S-phase cells and led to G1-phase cell cycle arrested. Furthermore, silencing of Axin in C6 cells could increase the distribution of G2+ S-phase cells. MTT assay showed that the level of proliferation in C6 cells with overexpressed Axin was significantly lower than that of C6 cells or C6 cells transfected with pIRES2-EGFP. Similarly, the colony-forming ability markedly reduced in the cells overexpressed with Axin compared with the C6 cell transfected with EGFP. TUNEL staining showed that apoptotic index of cells with overexpressed Axin significantly increased compared to the pCMV5 group and the blank control.3. Axin induce C6 cell apoptosis and reduce cell proliferation mainly by activating p53 pathwayThe preliminary experiment showed that overexpression of Axin could increase p53 mRNA level significantly, and the total level mRNA level ofβ-catenin was not changed. And then Real-time PCR and Western blot were used to detect p53 and its downstream genes mRNA and protein expression. The results showed that the mRNA level of p53 in C6 cells overexpressed with Axin markedly increased compared with the C6 cells transfected with pIRES2-EGFP. Meanwhile, the mRNA level of p21 significantly increased, and the Cyclin D1 mRNA decreased compared with the control groups. Similarly, the immunoblotting analysis revealed that Axin could induce the expression of p53 and p21 in C6 cells, and reduce the expression of Cyclin D1. In addition, the apoptosis relative molecule Bax was increased after transfected with Axin gene.After treated the cells with pifithrin-α, the results showed that the C6 cells with endogenous or overexpressed Axin treated with p53 inhibitor resulted in significantly reduction of p53, p21 and Bax, and induction of Cyclin D1 compared with C6 cells overexpressed with Axin, suggesting the p53 inhibitor could counteract the effect of Axin.Furthermore, the apoptotic index of C6-Axin treated with p53 inhibitor was 19.5%. Howere, There are not obviously changes between the groups which treated with and non-treated with pifithrin-αin C6-EGFP cells.The distribution of G2+ S-phase cells treated with pifithrin-αincreased, and G1-phase cells reduced compared to untreated cells.All results suggested the p53 inhibitor could antagonize the cell growth and death-inducing effect of Axin. Collectively, these results demonstrate that p53 pathway plays an importrant role in reduced cell ploliferation and induced apoptosis by Axin in astrocytoma. 14 Real-time PCR was used to identify theβ-catenin mRNA level in astrocytoma, and showed that the total mRNA was not changed. And then, we detected theβ-catenin levels in cytoplasma and nucleus respectively by Western blot. This result indicates thatβ-catenin level in C6 cell cytoplasma higher than that in nuclear, but lower than that in nuclear after transfected with Axin gene. The immunocytochemical staining result showed that theβ-catenin nucleus positive cells in C6-Axin were higher than that in C6-Vector cells.4. Increased expression ofβ-catenin with poor prognosisAs one of the most important molecule in Wnt signal pathway,β-catenin might play role on the progression of astrocytoma. 65 astrocytoma samples were detected by immunohistochemistry staining. The result showed that the expression ofβ-catenin was preserved in cytoplasma, membrane and nucleus. Spearman analysis showed that the distribution ofβ-catenin (cytoplasma, membrane and nucleus) was not correlated with the grades of astrocytoma, indicated thatβ-catenin translocation was not influence the malignance of astrocytoma. We also found that there were significant correlation betweenβ-catenin expression and 2-year survival. The expression ofβ-catenin was not correlated with the grades, tumor size, age, sex, and tumor location. Moreover, the survival curves for patients with astrocytoma showing low expression ofβ-catenin tend to be associated with good prognosis, but high expression ofβ-catenin tend to be associated with poor prognosis.【Conclusion】We provide evidence that Axin is associated with the progression of astrocytoma, with the increase of astrocytoma grades the expression of Axin decreased. Meanwhile, we found that, in astrocytoma, Axin induces cell death and reduces cell proliferation by activiting p53 pathway. As a downstream gene of Axin,β-catenin expression in cytoplasma, membrane and nucleus was not correlated with the grades of astrocytoma, but the expression ofβ-catenin was correlated with the prognosis of patients.