Inhibitory Effect Of Fatostatin On Glioblastoma And Its Molecular Mechanism

Background:Glioblastoma is the most common high-grade glioma of the nervous system and has a poor prognosis.After comprehensive treatment including surgery,radiotherapy and chemotherapy,the average survival time of glioblastoma is only 18 months.Recurrence and drug resistance of the tumor are the main reasons for poor prognosis.A management approach combining multi-agent,multi-treatment regimens may become an effective way to prolong the survival of glioblastoma patients.Fatostatin was first studied in metabolic syndrome as a specific inhibitor of SREBPs,and was found to suppress a variety of tumors via SREBPs dependent or-independent pathways.SREBPs independent pathways include cell cycle arrest,unfolded protein response（UPR）induced apoptosis,and others.Another study found that fatostatin could induce autophagy in breast cancer cells.Currently,there is no related research of fatostatin in glioblastoma,and the mechanism of fatostatin in glioblastoma remains unclear.Objective:To verify the inhibitory effect of fatostatin in glioblastoma and the related specific molecular mechanism.Methods:The CCK-8 assay was used to detect cell viability.The cloning assay was used to detect cell proliferation ability.The scratch healing assay was used to detect cell migration ability.Flow cytometry was paired with various fluorescent staining kits for the detection of cell cycle,apoptosis,mitochondrial membrane potential and intracellular neutral fat content.Transfection of small interfering RNAs was used to silent gene at key nodes of relevant pathways.Western blotting was used to detect the levels of related proteins including cell cycle,apoptosis,UPR and autophagy.Results:1.Fatostatin suppresses cell survival in multiple glioblastoma cell lines in a time or concentration gradient,and inhibits cell proliferation and migration abilities of glioblastoma cells in a concentration gradient.2.Fatostatin could arrest the cell cycle of glioblastoma cells at G2/M phase.The expression of cell cycle related proteins cyclin B1 and CDK1 increased.3.Treated with fatostatin,apoptosis rate of glioblastoma cells increased and the mitochondrial membrane potential decreased;as for apoptosis-related proteins,the cleavage of PARP and Caspase 3 increased and the ratio of BAX/Bcl-2 increased.4.Fatostatin did not result in statistically significant differences of glioblastoma cells in SREBP-1 activation levels,fatty acid synthase（FASN）expression levels and fluorescence intensity of intracellular neutral fat.5.Fatostatin activated UPR in glioblastoma cells,in which the expression of activating transcription factor 4（ATF4）and its downstream C/EBP homologous protein（CHOP）were increased.Silencing ATF4 decreased the expression of CHOP,decreased apoptosis rate,and decreased cleavage of apoptosis related protein Caspase 3 in fatostatin treated cells.6.Fatostatin induced autophagy in glioblastoma cells,with corresponding alterations in autophagy related proteins.Silencing autophagy related gene 7（Atg7）to reduce autophagy increased apoptosis rate and increased cleavage of apoptosis related protein Caspase 3 in fatostatin treated cells.After silencing ATF4,autophagy induced by fatostatin were observed to be reduced.Conclusion:1.Fatostatin can inhibit glioblastoma cell proliferation,migration,arrest the glioblastoma cell cycle in the G2/M phase,and induce mitochondrial mediated apoptosis.2.Fatostatin activates the unfolded protein response,upregulates ATF4/CHOP expression and thereby leads to glioblastoma cell apoptosis.3.ATF4/CHOP upregulated by fatostatin can activate protective autophagy in glioblastoma cells.Fatostatin-induced apoptosis will increase if autophagy is genetically inhibited.