Biochanin A Inhibiting Autophagy Enhances Tmz Chemosensitivity In Glioblastoma

Background:Glioma is the most common malignancy in the central nervous system and the most malignant glioma in adults.Due to its rapid infiltration growth and high recurrence rate,the surgery can not be completely removed.The average median survival of GBM is only 12-15 months.Temozolomide(TMZ)is the first-line drug for glioma chemotherapy.Because the decrease of chemosensitivity and drug resistance,TMZ is less than 45% effective for malignant glioma.Biochanin A(BCA)is a naturally occurring isoflavone compound that has an estrogen-like structure similar to estrogen and has a broad antitumor effect.Objective:To study the effects of BCA on proliferation,apoptosis,invasion and migration and autophagy of GBM cells,and explore the molecular mechanism of BCA inhibiting GBM cell proliferation and increasing TMZ sensitivity.Methods:1.U251 cells were used as research objects,and interventions were performed with different concentrations of BCA.The activity of U251 cells was detected by CCK-8.Invasion and migration were detected by scratch and Transwell chamber assay.Apoptosis was detected by flow cytometry.Autophagy was detected by pyridine orange staining,and MDC staining,and transmission electron microscopy,and the expression of apoptosis and autophagy related proteins were detected by western blotting.2.Observe the effects of BCA and TMZ single and combination drugs on U251 cells.Cell viability was detected by CCK-8,colony formation was detected by colony formation assay,apoptosis was detected by flow cytometry,and expression of apoptosis and autophagy-related proteins were detected by western blotting.3.Construct a subcutaneous xenograft model of nude mice and an orthotopic xenograft model of rat glioma.In vivo intervention by single and combined use of BCA and TMZ,observation of tumor size by head perfusion CT scan,HE staining pathological changes.Immunohistochemistry was used to observe the expression of proliferation,apoptosis and autophagy-related proteins.Results:1.BCA can inhibit the proliferation of U251 cells in a time-and dose-dependent manner,and decrease cell invasion and migration,and increase the content of p62,and reduce the expression of Beclin-1 and the conversion of LC3 BII to LC3 BI.The results of transmission electron microscopy showed that the autophagosomes in the BCA intervention group were significantly reduced.At the same time,it promotes the expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic protein.2.The results of CCK-8 and colony formation experiments showed that the combined drug group inhibited the proliferation of U251 cells more obviously,compared with the TMZ single-drug group.the combination drug group had a higher apoptosis rate than the single drug group.TMZ can increase the ratio of LC3BII/LC3 BI,and the combination group can significantly reduce the ratio of LC3BII/LC3 BI.3.BCA and TMZ can inhibit the growth of xenografted tumor and rat glioma in vivo,and the inhibitory effect of TMZ on tumors is more obvious than that of BCA.BCA can enhance the chemosensitivity of TMZ.Immunohistochemistry results showed that TMZ could enhance the expression of LC3 B protein,and the combination group could significantly reduce the expression of LC3 B and Ki-67.TUNEL assay showed that the apoptosis of the drug intervention group was significantly increased,and the combination group was the most obvious.Conclusion : BCA can inhibit the proliferation,invasion and migration,autophagy and induce apoptosis of glioblastoma U251 cells.In vivo and in vitro BCA may enhance the chemosensitivity of TMZ to glioblastoma by inhibiting autophagy.