Molecular Mechanism Of TZY7,a Novel Polyamine Conjugate,mediated ROS-related Autophagy On Liver Cancer Cells

Objective:The clinical application of naphthalimides was limited for severe adverse reaction,such as myelotoxicity,despite of their potent antitumor effects.Polyamines had favorable tumorous target,so naphthalimide-polyamine conjugate may be exert potent antitumor effects.In this study,we evaluated the antitumor effect and molecular mechanism of TZY7,a novel naphthalimide-polyamine conjugate,in vitro and in vivo.Methods:MTT assay was used to explore the antiproliferative effects of TZY7 on liver cancer Hep G2,SMMC-7721 cells.The morphological changes of cells were observed under microscope after treatment with TZY7 at different time intervals.The autophagy of Hep G2 and SMMC-7721 cells induced by TZY7was detected by flow cytometry after MDC staining.Western blotting was used to further verify the expression of autophagy-related proteins.DCFH-DA fluorescent probe was used to detect the changes of reactive oxygen species of Hep G2 and SMMC-7721 cells treated by TZY7 for 24 h and 48 h.The migration ability of Hep G2 and SMMC-7721 cells after treatment with TZY7 was detected by wound healing test.Furthermore,the inhibitive effects of tumor growth and lung metastasis of TZY7 were evaluated in vivo.Results:TZY7 had potent antiproliferative effects on Hep G2,SMMC-7721 cells in a dose-and time-dependent manner,the IC₅₀ values were?6.34±1.41??M,?5.13±1.15??M after treated with 48 h,respectively.TZY7 induced Hep G2 and SMMC-7721 cells autophagy but not apoptosis in a dose-dependent manner after treatment with 48 h.Western blotting results demonstrated that TZY7 up-regulated the proteins expression of LC3,ATG5,ATG7,BECN1,but down-regulated the p62 expression.The ROS was significantly increased in a dose-dependent manner on Hep G2 and SMMC-7721 cells after treated with TZY7.However,NAC,an active oxygenscavenger,not only reversed ROS produce but also attenuated the antiproliferative effects and autophagy after treatment with TZY7.Furthermore,TZY7 can inhibit the migration of Hep G2 and SMMC-7721 cells in a dose-dependent manner and down-regulate the protein expression of MMP-9 and?-catenin.TZY7 inhibited the solid tumor growth?inhibition rate 57.3%?and lung metastasis?inhibition rate53.49%?in Kunming mice H22 models and BALB/c mice CT-26 lung metastases model?inhibition rate53.22%?in vivo.Conclusions:TZY7 exerted potent antitproliferative and antimetastatic effects in vitro and in vivo.The molecular mechanism was related with TZY7-mediated ROS-related autophagy and down-regulation the protein expression of MMP-9 and ?-catenin.