Study On Antitumor Activities And Mechanisms Of Thiosemicarbazone

Objectives:Thiosemicarbazones are a kind of important imine derivatives that have kinds of pharmacological activities,such as antitumor,antibacterial,antiviral and antimalarial.It has found that compounds with thiosemicarbazone as the parent nucleus usually show excellent antitumor activity,which have attracted wide attention.In our previous study,we found that thiosemicarbazone is used as the parent nucleus and the introduction of halogen atom such as fluorine and bromine has positive effects on several properties of molecules,including drug-target interactions and specificity,metabolic stability,membrane permeability and toxicity.Based on this foundation,2-?3,4-difluorobenzylidene?hydrazinecarbothioamide,a new fluorinated derivative of thiosemicarbazone was synthesized.Its antitumor activities were tested and the underlying molecular mechanisms were investigated.It provides a theoretical basis for the development of fluorinated thiosemicarbazone derivatives as antitumor drugs.Methods:MTT method was used to detect the anti-proliferative effects of2-?3,4-difluorobenzylidene?hydrazinecarbothioamide on various tumor cell lines and normal cell lines,including human lung cancer cell A549,human colorectal adenocarcinoma cell Caco-2,human glioma cell U87,human hepatoma cell HepG2 and human umbilical vein endothelial cell HUVEC.The effects of 2-?3,4-diflorobenzylidene?hydrazinecarbothioamide on the proliferation and migration of A549 cells were studied by clone formation and cell invasion experiment.Flow cytometry and PI staining were used to explore the effect of 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide on the A549 cell cycle and Western Blot was used to detect its effect on the expression of related proteins.The effects of 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide on the apoptosis morphology of A549 cells were detected by Hoechst33258 staining.Annexin V/PI staining was used to analyze the effects on the apoptosis of A549 cells and Western Blot was used to detect the expression levles of apoptosis proteins.Then we focus on the effect of 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide on A549 cell mitochondrial apoptosis pathway.Intracellular ROS level in A549 cells was assessed by DCFH-DA dye and the mitochondrial membrane potential of A549 cells was detected by JC-1 kit.Results:MTT results showed that 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide could inhibit the growth of A549 cells.The IC₅₀0 of A549,Caco-2,U87 and HepG2 was28.52?g/m L,30.83?g/mL,86.37?g/mL and 134.15?g/mL respectively.A549 cells were much more sensitive than the other cell lines.Moreover,it showed better anti-proliferative activity on A549(IC₅₀=28.52?g/mL)than HUVEC(IC₅₀=86.37?g/mL).The clone formation experiment revealed that the colony-forming ability of A549cellswassignificantlysuppressedafter2-?3,4-difluorobenzylidene?hydrazinecarbothioamide treatment.It also significantly inhibited the invasion of A549 cells.The results of cell cycle study found that2-?3,4-difluorobenzylidene?hydrazinecarbothioamide could arrest A549 cells in G0/G1phase,the percentage of G0/G1 phase cells was increased from 32.93%to 50.97%.At the same time,it caused a significant decrease in the protein levels of Cyclin D1 and CDK4.Results showed that 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide could significantly induce A549 cell apoptosis.When treated with 2-?3,4-difluorobenzylidene?hydrazinecarbothioamide at the different concentrations,the percentage of apoptotic cells was increased from 5.5%to 33.7%.2-?3,4-difluorobenzylidene?hydrazinecarbothioamide changed the expression level of apoptotic-related proteins.The Bcl-2/Bax ratio was reduced while the contents of Cleaved-PARP,Cleaved-Caspase 3 and p53 were increased.Inthestudyofmitochondrialapoptoticpathway,wefoundthat2-?3,4-difluorobenzylidene?hydrazinecarbothioamide could induce A549 cells to produce reactive oxygen species and destroy the mitochondrial membrane potential of A549,resulting in the decrease of mitochondrial membrane potential.These results suggest that2-?3,4-difluorobenzylidene?hydrazinecarbothioamide could induce apoptosis and G0/G1arrest of A549 cells via reactive oxygen species mediated mitochondrial-dependent pathway.Conclusion:2-?3,4-difluorobenzylidene?hydrazinecarbothioamide showed excellent antitumor activity and induced apoptosis of A549 cells via reactive oxygen species mediated mitochondrial-dependent pathway.The results of this study can provide theoretical and experimental bases for the antitumor research of fluorinated thiosemicarbazone derivatives.