Study On The Mechanisms Of An Autophagy Inhibitor,Spautin-A41 In The Treatment Of Renal Carcinoma Cells

Objective:The study focuses on the mechanisms of an autophagy inhibitor,spautin-A41,in the treatment of renal carcinoma cells,which provides a new idea of the target of renal cell carcinoma.Methods:Part I:In order to evaluate the levels of autophagy in single cell,stable H4 human neuroglioma cells expressing the autophagy marker LC3-GFP was used.Spautin-1 was reported as a potent autophagy inhibitor in previous study.By imaging-based highthrough screening,the current study identifies a novel spautin-1 derivative spautin-A41.Phosphatidylethanolamine-conjugated microtubule-associated protein light chain 3(LC3 II)was analyzed using western blot.The stabilities of spautin-A41 and spautin-1 were measured by metabolic assessment.Under different conditions,CCK-8 was used to detect the effect of spautin-1 or spautin-A41 on the proliferation of 786-O cells.Part II:Renal carcinoma cell line 786-O was used as cellular model and western blot was used to detect the effect of spautin-A41 on sunitinib-induced autophagy;CCK-8 and colony formation assay were used to detect the effect of sunitinib on the proliferation of 786-O cells in the presence or absence of spautin-A41.Wound-healing and transwell assays were used to determine the effects of sunitinib on the migration of 786-O cells in the presence or absence of spautin-A41.Flow cytometry was used to detect the effect of spautin-A41 on sunitinib-induced apoptosis.Western Blot was used to detect the effects of spautin-A41 on the PI3K/AKT/GSK3β signaling pathway and the expression of the anti-apoptotic proteins Bcl-2 and Mcl-1.Results:Part I:Spautin-A41 significantly decreased LC3-GFP puncta and the protein levels of LC3 II.Spautin-A41 also showed improved stabilities and effectiveness in inhibiting autophagy when compared with spautin-1.Under serum or glucose starvation conditions,spautin-A41 resulted in more effective inhibition of tumor growth of renal carcinoma cells.Part II:Spautin-A41 markedly inhibited sunitinib-induced autophagy by downregulating the autophagy protein beclin-1.Co-treatment with spautin-A41 enhanced sunitinibinduced inhibition of cell proliferation and colony formation.Co-treatment with spautin-A41 enhanced sunitinib-induced inhibition of cell migration.Spautin-A41 enhanced sunitinib-induced apoptosis.Spautin-A41 can decrease the phosphorylation levels of AKT and GSK3β in the PI3K/AKT/GSK3β signaling pathway,co-treatment of spautin-A41 and sunitinib downregulated the anti-apoptotic proteins including Bcl-2 and Mcl-1.Conclusions:Part I:Spautin-A41 was more effective and stable than spautin-1;Under serum or glucose starvation conditions,proliferation of renal cancer cells was more effectively inhibited by spautin-A41.Part II:Co-treatment of spautin-A41 and sunitinib enhanced cell apoptosis by inhibiting sunitinib-induced autophagy as well as inactivating PI3K/AKT and activating downstream GSK3β,which downregulated the expression of anti-apoptotic proteins Bcl-2 and Mcl-1.