Effect Of TRKs Inhibitors On Biological Behaviors Of Human Glioblastma Cells

Backgrounds: Glioma is a major disease that threat human health and life, due to its high fatality rate. Development of anti-glioma drugs, especially the new antitumor drugs with high specificity and low side effects, is a hotspot for anticancer research. Molecular targeted therapy by targeting specific signaling pathways of carcinogenesis and cancer development, is an efficient way to prevent the growth of tumor cells with low cytotoxicity. Tropomyosin receptor kinase family(TRKs) is a member of the tyrosine kinase families, its inhibitors play an anti-neoplastic effect in a wide variety of tumors. They can bind with nerve growth factors and then activate a series of signaling transduction cascades, including PI3K/AKT/m TOR, MAPK/ERK and so on, causing changes of cell phenotypes. It is reported that abnormal expression of TRKs family in glioma cells is a potential target for treatment of glioma.Objectives: To screen the synthetic inhibitors of tropomyosin receptor kinase with preliminary anti-tumor activity, and to further explore the impact of its biological behaviors on glioma cells.Methods: U251 and U87 glioma cells as well as HUVEC cells were treated with different compounds, and proliferation potential of treated cells was detected by MTT assay so as to screen active tropomyosin receptor kinase(TRKs) inhibitors with high anti-tumor activity and specificity. The inhibition activity of the compounds was evaluated by ADP-Glo Kinase Assay. After the candidate compound was selected,Hoechst33258/PI double staining and TUNEL methods were used to analyze the changes of cell morphology, cell apoptosis and DNA fragmentations of candidate inhibitor treated cells. The effect of TRKs inhibitors on tumor proliferation was observed by clonal formation experiment. Inhibitors induced changes in apoptosis and cell cycle distribution of glioma cells was determined by FACS; Western Blot was employed to detect the level of anti-apoptotic proteins(pro-Caspase3 、 Bcl-2),apoptotic protein(BAD) and phosphorylated AKT( key molecule of PI3K/AKT/m TOR pathway), so as to explore the anti-tumor mechanisms of candidate compound.Results: The candidate TRKs inhibitor(No.6 compound) with high anti-tumoractivity and specificity was selected, and its inhibitory activity on TRKA kinase was evaluated. The results showed that the candidate compound had high inhibiting activity on TRKA kinase. Cell proliferation and clonal formation experiments showed that the NO.6 compound could significantly inhibit the proliferation of glioma cells.When the concentration of NO.6 compound is about 9μM, the inhibition rate was50%, and the cytotoxicity was much lower than the positive control. Hoechst33258/PI double staining and TUNEL staining analysis showed that the No.6 compound treated glioma cells exhibited obvious characteristics of apoptosis: cytoplasmic shrinkage,nuclear membrane cracking, marginalization, and DNA fragmentations etc. Apoptosis detected by flow cytometry showed that the inhibitor could block the cell cycle of tumor cells and promote apoptosis. Subsequent Western Blot analysis confirmed a significant increase in the expression of pro-apoptosis protein BAD, and decrease in the expression of anti-apoptotic proteins(Bcl-2, pro-caspase3)as well as phosphorylated AKT levels in NO.6 compound treated U251 cells, suggesting that inhibitor may exert its anti-tumor effect by modulation of AKT signaling pathways.Conclusions: NO.6 compound is a candidate TRKs inhibitor with high antitumor activity and specificity. It may exert its antitumor activity by inhibiting TRKA kinase activity, modulation of PI3K-AKT signaling pathway and the phosphorylated AKT levels in treated cells, which may increase in the expression of pro-apoptosis proteins BAD and decrease in the expression of anti-apoptotic proteins(pro-Caspase3、Bcl-2), leading to cell cycle blocking and apoptosis. We hope our study can provide new ideas for anti-glioma drug development and the candidate TRK inhibitor will play a role in clinical glioma therapy.