Janus Kinase Inhibitor AG490 Blocks Stat3 Signal Transduction And The Antitumor Effects In C6 Cells

PrefaceGlioma is the most common primary central nervous system cancer, its extensive infiltration, surgery is extremely difficult to cut the whole, supplemented by radiotherapy and chemotherapy effects are poor, so finding a new target for glioma therapy to become the current study hot spots, these studies focused on tumor-related gene cloning and functional analysis, cell signal transduction and cell cycle regulation and other fields. Signal transducers and activators of transcription factors (signal transducers and activators of transcription, STAT3) is the study of a hot spot, its excessive activation will result in abnormal cell proliferation and apoptosis of obstacles, directly involved in the malignant transformation of cells. STAT3 mediated by various cytokines and growth factors caused by a wide range of cellular responses, roles related to cell proliferation, differentiation and immune regulation in many areas, research in a variety of human malignancies found in abnormal activation of STAT3 signaling pathway phenomenon, abnormal STAT3 activation through growth factor signaling system and apoptosis-related factors, promoting angiogenesis factors such as a result of interaction between tumor generate and promote tumor development.Materials and methodsIn this study, using cultured rat C6 glioma cell line, and build different concentrations (25μM,50μM, 100μM) tyrosine kinase inhibitor AG490 blocked STAT3 signaling pathway in C6 cell line at different times (24 hours,48 hours and 72 hours) model. Immunocytochemistry were used to approach and observe the difference between the fluorescence microscope STAT3 and p-STAT3 in glioma cell line C6 in the expression; extraction of total cellular protein, each group, through the quantitative detection of protein, protein concentration, Western Blot semi-quantitative method of Detection of STAT3 and p-STAT3 expression; by MTT method STAT3 signaling pathway in various cell model of blocking cell proliferation test, and to Excel and SPSS 17.0 software, computing and statistical analysis; Transwell cell invasion by blocking STAT3 trials examine signaling pathway glioma cell line impact of invasive characteristics; in each group AG490 blocked STAT3 signaling pathway in cell model, and with the cellular DNA content of KGI Detection Kit (cell cycle), KGI Annexin V-FITC cells wither death detection kit and flow cytometry cell cycle DNA of tumor cells and apoptosis detection.Results1. The STAT3 protein was located in cytoplasm of tumor cells while p-STAT3 in cell nucleus. There seemed to be STAT3 positive cells than p-STAT3 positive ones.2. The STAT3 signaling pathway being blocked by AG490 in C6 cells in vitro C6 cells were treated with AG490 at concentrations of 25μM,50μM and 100μM. 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 C6 expressed STAT3 as well as p-STAT3. 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.3. The result of method of MTT showed that both the proliferation and survival rate of c6 cells were significantly depressed by AG490 in concentration-dependent way (P<0.05). Concentration were responsible for the drop of cell proliferation and survival. 4. Inhibition of invasiveness of glioma Cell lines by blocking the STAT3 signaling pathway in vitro Cell 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 24 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 test. STAT3 signaling pathway significantly decreased the number of invading tumor cells (P<0.01). Therefore AG490 inhibited the invasiveness of glioma cell lines cells in vitro.5. C6 cells were pretreated with AG490 100μM 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 showed arrested in G1-S phase after AG490 treatment.ConclusionSTAT3 in human brain glioma in a wide range of expression, STAT3 signaling pathway and glioma cell line proliferation, apoptosis, cell cycle, tumor invasion and angiogenesis are closely linked, and many other areas. Thus, STAT3 signaling pathway may be inhibiting the growth of a new target for glioma invasion.