The Inhibitory Effect And Mechanism Of Glaucocalyxin A On Glioma Cells

Objective:To observe the effects and explore the mechanism of glaucocalyxin A (GLA) on glioma cell apoptosis and microenvironment.Methods:Cell proliferation and cell migration were analyzed by cell real-time analyzer and MTT assay. Changes in cell morphology were observed by microscope. The ability of colony formation was detected by plate colony assay. Cell apoptosis, cell cycle and mitochondrial membrane potential were detected by FCM. SILAC quantitative proteomic was used to identify potential targets. Using SiRNA, PCR and western blot technique to detect and verify the compound induces apoptosis molecular signaling mechanisms. Glioma culture supernatant or interleukin-10(IL-10) treated BV-2microglia (simulate glioma-associated macrophages/microglia) and detect the compound effects and molecular mechanisms of GLA on M2transformation in tumor-related microglia.Results:1. GLA can significantly inhibit the C6, GL261, U251, U87MG, T98G five glioma cells growth in a dose-dependent manner. The IC50of GLA were15.03±1.44μM (C6cells),14.14±1.35μM (GL261cells),12.42±0.68μM (U251cells),18.49±1.36μM (U87MG cells),44.32±1.92μM (T98G cells). Lower concentration of GLA prominently inhibited the colony formation of C6cells. C6cells became round and fall off from dish, chromatin condensation and nuclear fragmentation treated with GLA. GLA induced cell apoptosis of C6glioma cells in a does-dependent manner and blocked its cell cycle in G2/M phase. GLA obviously reduced mitochondrial membrane potential, increased the release of Cytochrome c from mitochondria to cytoplasm, decreased the expression of Bcl-2, activated Caspase3and cleaved PARP to execute cell apoptosis in C6cells. GLA lead to the upregulation of262proteins and the downregulation of581proteins in C6cells. There are16proteins down-regulation, and12proteins occur up-regulated associated with cell cycle. Apoptosis-related proteins occur down-regulation has13proteins, up-regulated has7proteins, especially the significant upregulation of GEF-H1. After the silence of GEF-H1could alleviate GLA-induced apoptosis. GLA can cause MAPK family protein phosphorylation in a dose-dependent manner, and ERK inhibitor U0126pretreatment can partly block GLA-induced Caspase3activation and PARP cleaved.2. GLA can significantly inhibited mRNA of Argl, CD163, IL-10, TGF-p, TNF-a induced by tumor conditioned medium or IL-10on BV2microglial by inhibiting the expression of NF-κB (p65) activation.Conclusion:1. GLA and its derivatives inhibited cell proliferation and induced glioma cells apoptosis in vitro. The basic activity structure is α,β-unsaturated ketone diterpenes.2. GLA-induced glioma cells apoptosis through activation of GEF-H1/RhoA/ERK pathway.3. GLA inhibited glioma cell supernatants or IL-10-induced M2transformation of microglial cells by inhibiting NF-κB (p65) activation.