Background:Despite newly,progresses have been made in multimodal therapy of glioma,the overall treatment effect is poor,and the long-term survival rate is very low,especially for the most invasive glioblastoma multiforme.The recurrence rate following surgical treatment is still high.Drug resistance and metastasis are easy to occur after radiotherapy and chemotherapy,and the prognosis is poor.Therefore,in order to find more effective therapeutic drugs,new research is urgently needed.As a member of the chalcone family,Butein plays a role in many chronic diseases.Recent studies have shown that Butein has antitumor activity in a variety of tumors.However,there is no report on the effect of Butein on glioma cells at present.Objective:In this study,we explored the direct-action targets of Butein on glioma cells and the effects of this target on glioma cells in vitro and in vivo models,thus providing new ideas and theoretical basis for the treatment of clinical glioma patients.Methods:1.The effect of Butein on the activity of human glioma cell lines U87 MG,LN229,U251,A172 in vitro was detected by CCK-8 method.2.The effect of Butein on the proliferation of U87 MG and LN229 cells was observed by Colony formation assay and Ed U staining test.3.The effects of Butein on the cell cycle of U87 MG and LN229 were detected by flow cytometry,and the effects of Butein on the cell cycle related proteins were detected by Western blotting.4.The effects of Butein on the migration and invasion of U87 MG and LN229 cells were observed by Wound healing assay and Transwell invasion assay.Western blotting was used to detect the effect of Butein treatment on migration and invasion related proteins.5.The death mode of U87 MG and LN229 cells induced by Butein was detected by Flow cytometry.Western blotting was used to detect the effect of Butein treatment on apoptosis related proteins of glioma cells.6.The possible mechanism of Butein inhibiting glioma was analyzed through network pharmacology-based approach.7.After the U87 MG and LN229 cells were treated with Butein and PI3 K activator 740 YP alone or jointly,the expression of the pathway and downstream related proteins was detected by Western blotting.8.Molecular docking method was used to analyze the potential targets of Butein in inhibiting glioma,and drug affinity response target stability(DARTS)was used to verify the results;RT-q PCR combined with Western blotting were used to detect the mechanism of action of Butein on the target.9.The selective inhibitor of AKR1B1,Epalresta,was used to observe its inhibitory effect on U87 MG cells and the expression of related proteins.10.A cell line stably overexpressing AKR1B1 was established in A172 cells by lentivirus transfection.We detected the effect of Butein on its activity by CCK-8,and the effect of Butein on AKR1B1 and its downstream protein was detected by Western blotting.11.The therapeutic effect of Butein on glioma in vivo was tested by establishing U87 MG nude mice subcutaneous xenograft tumor model.Results:1.CCK-8 experiment results showed that Butein inhibited the activity of U87 MG,LN229,U251,A172 cells.2.Ed U staining showed that Butein inhibited the proliferation of U87 MG and LN229 cells.The Colony formation assay showed that Butein inhibited the formation of U87 MG and LN229 cell clones.3.Flow cytometry(PI staining)showed that Butein induced U87 MG and LN229 cell cycle arrest at G2/M phase;Western blotting showed that Butein induced down-regulation of cyclin B1 and CDK1 and up-regulation of p21 in glioma cell cycle related proteins.4.The results of Wound healing assay showed that Butein inhibited the migration of U87 MG and LN229 cells.The results of Transwell invasion assay showed that Butein inhibited the invasion ability of U87 MG and LN229 cells,and Western blotting results showed that Butein reduced the levels of migration and invasion related proteins Slug and MMP-2 of glioma cells.5.The results of flow cytometry showed that Butein induced apoptosis in U87 MG and LN229 cells.Western blotting showed that Butein significantly upregulated the levels of Cleared Caspase-3 and Cleared PARP1 proteins in glioma cells,and decreased the level of MCL-1 protein.6.Analysis methods based on network pharmacology show that Butein can inhibit glioma through multiple targets and multiple signal pathways,including PI3K/AKT signal pathways.7.Western blotting results showed that 740Y-P reversed the effect of Butein on the PI3K/AKT signaling pathway and downstream proteins.8.Molecular docking analysis showed that Butein may inhibit glioma by directly acting on EGFR,AKR1B1,TERT,PTGS2,MCL-1,ALOX5,and XDH.DARTS showed that Butein directly binds to AKR1B1.RT-q PCR and Western blotting results showed that Butein had no effect on the m RNA and protein expression levels of AKR1B1 in glioma cells.9.CCK-8 and Western blotting results showed that Epalrestat inhibited the activity of U87 MG cells and inhibited the activation of PI3K/AKT signal pathway within a certain concentration and time range.10.Overexpression of AKR1B1 in glioma cell line A172 can enhance the sensitivity of cells to Butein inhibition and affect PI3K/AKT signal pathway.11.U87 MG nude mice subcutaneous xenograft tumor model showed that Butein played a safe and effective role in anti-glioma in vivo.Conclusions:1.Butein can inhibit the proliferation,migration,and invasion of glioma cells and induce cell cycle arrest and apoptosis of glioma cells both in vivo and in vitro experiments.2.Butein inhibits the development of glioma by inhibiting the activation of PI3K/AKT signaling pathways.3.Butein inhibits the activation of PI3K/AKT signaling pathways in glioma cells by directly binding to AKR1B1.4.Glioma cells with high expression of AKR1B1 are more sensitive to Butein therapy. |