Effects Of ZYH005 On Proliferation And Apoptosis Of Malignant Meningioma And Its Mechanism

Object:Meningioma is the most common intracranial tumor in neurosurgery.However,the prognosis of malignant meningioma is worse.The five-year survival rate is only between 12%and 57%.At present,there is no effective drug available for the treatment of malignant meningioma.ZYH005 is a newly synthesized phenanthridinone derivative from The College of Pharmacy,Tongji Medical College.Preclinical studies have shown that it has favorable anti-tumor effects.The purpose of this study was to clarify the biological effects of phenanthridinone derivative ZYH005 on malignant meningioma and to explore its mechanism.Methods:CCK-8 assay was used to detect the effects of ZYH005 on the proliferation of human malignant meningioma cells and normal cells.DAPI staining and flow cytometry were used to detect the apoptosis level of malignant meningioma cells treated with ZYH005.Western blot was used to detect the changes in apoptosis-related protein expression levels after treatment with ZYH005.The subcutaneous xenograft tumor model of nude mice was established to explore the effect of ZYH005 on human malignant meningioma cell IOMM-Lee in vivo.The tumor volume and weight were measured,and the proliferation and apoptosis levels of tumor tissues were detected by Ki-67immunofluorescence staining and TUNEL staining.Single-cell gel electrophoresis experiments showed that the malignant meningioma cells treated with ZYH005 appeared comet-like tailing,and the tail DNA content and tail length of the drug-treated group were significantly greater than those of the control group.Western blot analysis showed that ZYH005 could up-regulate the expressions of P-ATM andγ-H2AX and down-regulate the expression of BRCA1,but had no effect on the expressions of P-ATR and XRCC4.The effects of ZYH005 on migration and invasion of human malignant meningioma cells and human umbilical vein endothelial cells（HUVEC）were detected by scratch test and transwell migration assay.The effect of ZYH005 on tubule formation of HUVEC was studied in vitro.The changes in phosphorylation levels of receptor tyrosine kinase in IOMM-Lee cells after ZYH005 treatment were analyzed by using a human receptor tyrosine kinase phosphorylation antibody chip.Results:After 48 h treatment,the IC₅₀ of ZYH005 to CH157-MN and IOMM-Lee cells were 0.071μM and 0.050μM,respectively.While SVG P12,NCM460,and MEFs were0.428μM,0.291μM and 0.227μM,respectively.After treatment with ZYH005,DAPI staining was performed,and pyknotic nuclei were observed.The results of flow cytometry showed that the total apoptotic rates of CH157-MN and IOMM-Lee cells were as high as54.6%±3.0%and 79.8%±3.2%,respectively,after ZYH005 treatment for 72 h.Western blot showed that Cleaved PARP and Cleaved caspase-3 were up-regulated after ZYH005treatment,but the expressions of anti-apoptotic protein BCL-XL and pro-apoptotic protein BAX were not affected.The results of animal experiments showed that the tumor volume and weight of the ZYH005 group were significantly lower than those of the control group on the 28th day.At the same time,the proportion of Ki-67 positive cells in the ZYH005treatment group was lower than that in the control group,while the proportion of TUNEL positive cells was higher than that in the control group.In the single cell gel electrophoresis assay,a large number of comet-like trailing phenomena were observed in malignant meningioma cells treated with 0.1μM ZYH005 for 12 h.The tail DNA content and tail length of the drug treatment group were significantly higher than that of the blank control group.Western blot analysis showed that ZYH005 up-regulated the expression of P-ATM andγ-H2AX,down-regulated the expression of BRCA1,but had no effect on the expression of P-ATR and XRCC4.Flow cytometry showed that ZYH005 treated with 0.1μM ZYH005 for 12 h significantly increased the proportion of cells in G₂/M phase.Western blot analysis showed that ZYH005 up-regulated the expression of p21 protein,and down-regulated the expression of CDK1 and P-CDK1,but had no effect on the expression of Cyclin A2 and Cyclin B1.ZYH005 treatment of CH157-MN,Iomm-Lee and HUVEC cells can significantly delay the healing of scratch and reduce the number of invasive cells.The tubule formation assay showed that ZYH005 significantly reduced the node number and the total length of tubules.Using human receptor tyrosine kinase phosphorylation antibody chip analysis,it was found that the EGFR phosphorylation level of malignant meningioma cells was significantly down-regulated after ZYH005 treatment.Conclusions:ZYH005 can inhibit the proliferation of malignant meningioma in vitro and in vivo,and the inhibitory effect is related to the induction of apoptosis,which may be transmitted by non-endogenous pathways.ZYH005 can cause DNA double-strand break damage in malignant meningioma cells and inhibit the repair of DNA damage by down-regulating BRCA1 expression level.In addition,ZYH005 induces G₂/M arrest of malignant meningioma cells by up-regulating p21 expression and inhibiting downstream CDK1 activity.In vitro experiments showed that ZYH005 may inhibit malignant biological behaviors such as migration,invasion,and angiogenesis of malignant meningioma cells by down-regulating EGFR phosphorylation.