Role Of STAT3 Signal Pathway In Glioma Cell Lines Of Human Beings

The critical reason for glioma recurrence following the resection was the invasiveness of progression. It has been discovered that there was accumulation of genetic lesions in astrocytoma. Without regulation in proliferation, differentiation and apoptosis, cells of tissue tended to proliferate without limitation and progress invasively. Thus, many researches focused on the molecular mechanism of glioma for the final destination of control the disease effectively. STAT3 (Signal Transducer and Activator of Transcription 3) regulated gene expression in response to the signaling from cytokine and growth factor receptors, which correlated with the biological process of cell proliferation, differentiation and immunoloregulation. Constitutive activation of STAT3 protein has been detected in a variety of malignant tumors of human beings. It has also been reported that constitutive activation of STAT3 contributed to oncogenesis or tumor promotion through its intimate connection to the signaling relating to growth, apoptosis or angiogenesis. Thus, STAT3 as a novel target for therapeutic intervention of human cancers has been shown to be valuable.Though the constitutive activation of STAT3 has been found in astrocytoma, study on mechanisms of STAT3 signaling around the development of glioma did not actually expend. And the reports on STAT3 as a new therapeutic target for tumor of central nervous system were quite few. Therefore, we studied the function of STAT3 signaling pathway in astrocytoma in genesis and progress. We observed the expression of STAT3 in astrocytoma resection specimens. By inhibiting the STAT3 signaling pathway in U251 and U87 cell lines, we manage to reveal the effects of STAT3 on cell proliferation, cell cycle, apoptosis and invasiveness of astrocytoma in vitro. Our study tried to provid a basis for further evaluation of STAT3 as a novel target of glioma therapy and lead to a new path to understand the mechanism of astrocytoma invasiveness and progress as well as other biological features.1, The expression of STAT3 in astrocytoma specimen30 cases of astrocytoma resection specimens were selected and sliced. Immunohistochemistry staining was applied to observe the STAT3 expression in astrocytoma in tissue section. We found that every astrocytoma case we checked expressed STAT3. In most cases, STAT3 stain located in the plasma of the tumor cells. The level of STAT3 expression was significantly higher in the tumor area than in the peritumoral regions (P < 0.01). However, there was no significant difference of STAT3 expression in groups of histolpathalogical grade, sex and age (P > 0.05). The STAT3 positive cells seemed more even in group of low pathological grade than that of high grade, not only in distribution but also stain intensity. More, we found that the cytoplasm of the mitotic cells as well as multinucleated giant cells in astrocytoma appeared to be STAT3 positive. Moreover, the endothelial cells of neoplastic capillaries commonly expressed STAT3 protein in evidence.2, The expression of STAT3 in glioma cell lines U251 and U87We checked STAT3 and p-STAT3 expression in glioma cell lines U251 and U87 by way of immunocytochemistry staining and found that both U251 and U87 intensely expressed STAT3 as well p-STAT3 protein. The STAT3 protein was located in cytoplasm of tumor cells while p-STAT3 in cell nucleus. There seemed to be more STAT3 positive cells than p-STAT3 positive ones.3, The STAT3 signaling pathway being blocked by AG490 in glioma cells in vitroBoth U251 and U87 were treated with AG490 at concentrations of 12.5uM, 25uM and 50uM. The groups of no AG490 treating were set as control. At the time point of 72 hours, cells of all groups were harvested and both cytoplasmic and nuclear protein was obtained by way of whole cell extract. Western Blot was applied to detect and evaluate the expression of STAT3 and p-STAT3 in the cell extract, by which we found that both U251 and U87 expressed STAT3 as well as p-STAT3. The level of the two antigens was a little bit higher in U251 than in U87. Also in both of the glioma cell lines, the expression of STAT3 appeared be not influenced by AG490 but the level of p-STAT3 declined progressively till none in accompany with elevating of AG490 concentration. Thus, we proved that AG490 prevented the STAT3 protein from activating so that the STAT3 signaling pathway in glioma cells was blocked.4, The proliferation and survival rate of U251 and U87 cells were depressed after STAT3 signaling pathway inhibitionU251 and U87 were treated with AG490 respectively at the concentrations of 12.5uM, 25uM and 50uM in 96-well plates and groups of no AG490 treating were set as control. At the time point of 24 hours, 48 hours and 72 hours, cells were fixed and stained by method of SRB (Sulforhodamine B Protein Stain). The proliferation and survival rate of the two glioma cell lines were evaluated according to the OD values checked thereafter. The data were analyzed in SPSS 11.0 by way of Univariate Analysis of Variance. The result showed that both the proliferation and survival rate of U251 and U87 cells were significantly depressed by AG490 in a time- and concentration- dependent way (P < 0.01). The tests of between-subjects effects further revealed that both facts, time and concentration were responsible for the drop of cell proliferation and survival rate (P < 0.01) and the effect enhanced when both of them functioned simultaneously (P < 0.01).5, That AG490 inhibiting STAT3 induced apoptosis in glioma cell lines U251 and U87U251 and U87 cells were pretreated with AG490 for 48 and 72 hours respectively. All groups of tumor cells including control were harvested and stained according to the protocol of Annexin V-FITC apoptosis detection kit. The stained cells were then measured by flow cytometry. We found that the apoptosis rates of tumor cells were markedly higher in groups of AG490 treatment than those of control. More, the higher AG490 concentration applied, the more apoptosis tumor cells detected in the group.6, Cell cycle of U251 and U87 were arrested after AG490 applicationU251 and U87 cells were pretreated with AG490 of 50uM for 48 hours and harvested with groups of control. Then we stained the cells by method of PI staining and measured them with flow cytometry to see the change of DNA cell cycle of the glioma cell lines. It was observed that the cell cycle of both U251 and U87 showed arrested in G1-S phase after AG490 treatment.7, Inhibition of invasiveness of glioma Cell lines by blocking the STAT3 signaling pathway in vitroCell invasion was assayed in a 24-well Transwell cell culture chambers. Tumor cells were suspended into the upper chamber with AG490, while the controls without. After 10 hours of incubation, the invading tumor cells on the lower surface of the chamber were stained with Giemsa. The number of invading cells were counted and analyzed by student's t test. Furthermore, we analyzed the secretion of MMP-2 and MMP-9 via a zymography assay. The result showed that AG490, which blocked the STAT3 signaling pathwa, significantly decreased the number of invading tumor cells (P < 0.01) and down regulated the secretion of both MMP-2 and MMP-9. Therefore, AG490 inhibited the invasiveness of glioma cell lines cells in vitro.8, Change of mRNA expression of some related genes in U251 cell in case of STAT3 pathway being blockedU251 cells were pretreated with AG490 for 72 hours. The whole cell RNA of GBM cell was extracted. mRNA expression of several related genes was initially assessed by RT-PCR using corresponding specific primers. The PCR result indicated that mRNA expression of apoptosis inhibitors genes Mcl-1, Bcl-XL and Survivin were down-regulated in respond to AG490 treatment. Similarly, angiogenesis gene VEGF decreased too. While cell cycle relevant gene Cyclin D1 also came down to some extent with p21 kept unchanged. The STAT3 mRNA expression did not affected by AG490.Conclusion:The findings that STAT3 widely expressed in astrocytoma and involved in the cell proliferation, survival, cell cycle, apoptosis, invasiveness and angiogenesis of glioma cell lines contributed to the conception that STAT3 signaling pathway is important in the pathogenesis of glioma and, therefore, possible to be a promising target in glioma therapy.