Aloe-emodin Targets Multiple Signaling Pathways By Blocking Ubiquitin-mediated Degradation Of DUSP1 In Nasopharyngeal Carcinoma Cells

Objective: The aim of this study was to evaluate the inhibitory effect of Aloe-emodin(AE)on NPC cells and investigate the molecular mechanism of this phenomenon.Methods: 1.CCK-8 assay was used to detect the effect of AE on the viability of NPC cells 5-8F and CNE1,and appropriate concentrations were selected for subsequent verification experiments on function and mechanism.2.Colony experiment and EdU incorporation experiment was used to detect the effect of AE on the proliferation of NPC cells.3.Flow cytometry was used to detect the effect of AE on cell cycle and apoptosis of NPC cells.4.The effect of AE on the migration of NPC cells was detected by scratch test.5.The expression changes of DUSP1 and its downstream signaling pathway related proteins ERK1/2,AKT and p38-MAPK after the intervention of AE were detected by Western Blot.6.BCI hydrochloride,DUSP1 selective inhibitor,was used to investigate its effects on the expression of ERK1/2,AKT,p38-MAPK pathway related proteins in NPC cells.7.The effect of BCI intervention on the inhibitory effect of AE on NPC was detected by CCK-8,EdU incorporation experiment,scratch and flow cytometry.8.Molecular docking simulated the spatial binding of AE and DUSP1 protein;The binding affinity of AE to DUSP1 was detected by MST assay.The DUSP1 ubiquitination site was predicted to explore whether the binding of AE with DUSP1 has potential steric effect on its ubiquitination.9.Ubiquitin antibody was used for immunoco-precipitation assay to detect the effect of AE on DUSP1 ubiquitination modification.Results: 1.CCK-8 results showed that AE could significantly reduce the viability of NPC cells in a time-and dose-dependent manner.Select the corresponding concentration(10 μM)of cell vitality around 70% for the follow-up function and mechanism verification.2.AE inhibits the proliferation and migration,blocking cycle and inducing apoptosis of NPC cells.3.AE up-regulates DUSP1 expression and inhibits the activation of ERK1/2,AKT and p38-MAPK pathways.4.BCI partially reversed ERK1/2,AKT,p38-MAPK pathway block induced by AE.5.BCI partially reversed the proliferation inhibition,migration inhibition,cycle arrest and apoptosis induction induced by AE.6.Molecular docking simulated the binding of AE with DUSP1,and MST experiment was used to confirm this result.The binding amino acid residues were spatially close to the ubiquitination site of DUSP1 predicted by GPS-Uber.IP experiments showed that the binding of AE with DUSP1 inhibited ubiquitin-proteasome mediated degradation of DUSP1.Conclusions: AE can inhibit the malignant biological behavior of NPC cells by inhibiting DUSP1 ubiquitination and up-regulating its intracellular protein level,resulting in the inactivation of ERK1/2,AKT and p38-MAPK signaling pathways associated with NPC.