Antitumor Effects And Mechanism Of Naphthalimide-polyamine Conjugate On ESCC Via Targeting Lysosomes

Objective:Esophageal squamous cell carcinoma is a common malignant tumor in the digestive tract,which is with a high morbidity and mortality worldwide.Patients are usually diagnosed at the middle and late stages with poor prognosis and low five-year survival rate.Patieents often suffer from side effects and toxic effects caused by those chemotherapy drugs.The naphthalimide-polyamine conjugates TZY2 and TZY with good antitumor activity were synthesized by our research group in recent years.In this study,human esophageal squamous cell carcinoma cells KYSE-30,KYSE-150,and EC-9706 were taken as the main research objects to investigate the role and mechanism of naphthalimide-polyamine conjugates TZY2 and TZY in inhibiting esophageal squamous cell carcinoma.Methods:The effects of compounds TZY2 and TZY on the viability of ESCC were detected by the MTT assay and the CCK-8 assay.The effects of compounds on the cell clonies were detected by clony formation experiments.The fluorescence and intensity of TZY2 and TZY were detected by the inverted fluorescence microscope,laser confocal microscope and HCS.Cells stained with lyso-Tracker Red,Golgi-Tracker Red,and ER-Tracker Red were used to detect the localization of the compounds in the cells and their effects on lysosomes.Lyso Sensor ? Green DND-189 staining was used to detect the fluorescence intensity of lysosomes in cells.Apoptosis of ESCC was detected by Trypan blue and Caspase-3 Assay Kit staining.Apoptosis of ESCC stained by Annexin ? and PI was detected by flow cytometry.Rh123 staining was used to detect the mitochondrial membrane potential in cells.Cells treated by TZY2 were then stained by MDC to detect the autophagy in cells.The proliferation of cells pretreated with Baf-A1 was detected by MTT assays and Trypan blue.The effect of TZY2 on the migration of ESCC at different times was detected using scratch assays.The expression levels of proteins which are related to apoptosis,autophagy and lysosome in ESCC treated by TZY2 were detected by western blot.Results:1.The results of the MTT assay and the CCK-8 assay showed that TZY2 and TZY could inhibitthe proliferation of ESCC depending on the concentration and time.At the same time,compared to TZY,TZY2 had a stronger effect.Colony formation experiments showed that TZY2 inhibited colony formation.2.Both TZY2 and TZY showed blue and green fluorescence in ESCC.Compared to TZY,TZY2 showed stronger fluorescences intensity.3.Lyso-Tracker Red,Golgi-Tracker Red,and ER-Tracker Red staining results showed that TZY2 and TZY localized in the lysosomes.Lysosomes changed from diffuse distribution to a dot-like aggregation,and its volume increased;the effects of TZY2 were greater than that of TZY.Lyso Sensor ?Green DND-189 staining results showed that the fluorescence of ESCC increased and the p H decreased after the addition of TZY2.4.Trypan blue staining showed that TZY2 induced cell death.Annexin ?/PI and Caspase-3Assay Kit staining results showed that TZY2 induced apoptosis in ESCC.5.Rh123 staining showed that TZY2 could cause the decrease the mitochondrial membrane potential in cells.6.TZY2 could induce autophagy in esophageal squamous cell carcinoma.The survival rate of esophageal squamous cell carcinoma was increased after cells were pretreated with Baf-A1.7.The scratch test results showed that TZY2 inhibited the migration of ESCC.8.Western blot results showed that the apoptosis-associated protein PARP was cleaved,the expression of cleaved-PARP was up-regulated,and p21 was highly expressed in esophageal squamous carcinoma cells which were treated by TZY2 for 24 h,.Autophagy-related protein Beclin1 was significantly up-regulated,p62 protein expression was down-regulated,and the ratio of LC3-II / LC3-I was increased significantly.The expression of lysosomal related proteins LAMP1 and LMAP2 were up-regulated,pro-Cathepsins D expression was up-regulated and mature-Cathepsins D expression was down-regulated,pro-Cathepsins L expression was up-regulated and mature-Cathepsins L expression was down-regulated.Conclusions:Both TZY2 and TZY showed blue and green fluorescence in ESCC,and the fluorescences of TZY2 were much stronger than TZY.Compound TZY2 could target lysosomes and affect lysosomal function.Naphthalimide-polyamine conjugates TZY2 and TZY could inhibit the proliferation of ESCC,and the effects of TZY2 were better than the compound TZY.TZY2 could induce apoptosis and autophagyof ESCC,and could inhibit the migration of ESCC.Compound TZY2 could inhibit ESCC by regulating proteins related to apoptosis,autophagy and lysosome.