Study On The Cellular Effects And Mechanism Of The Schiff Base Copper Complexes Of Symmetrical Thiosemicarbazide

Copper,as one of the essential trace elements in human body,is the active center of many proteins and enzymes,and plays an important role in the maintenance of human physiological function.As a transition metal,copper can be combined with ligands of different structures to form stable complexes.Many studies have shown that copper complexes have biological activities such as anti-tumor,antibacterial and antioxidant.Therefore,the biological effects of copper complexes have been widely concerned.The preliminary studies showed that the thiosymmetric diaminourea Schiff base copper complexes 1,2 and 3 could effectively inhibit the activity of PTP1B and TCPTP.Based on this,the cellular effects and mechanism of copper complexes 1,2 and 3 are discussed.The main work and research results of this paper are as follows:(1)The effects of copper complexes 1,2 and 3 on the proliferation of three kinds of tumor cells,including SW-480,MCF-7 and Hep G2,were studied by MTT method.The results showed that copper complex 1-3 could inhibit the proliferation of three tumor cells,and the inhibition of proliferation was positively correlated with time and concentration.Copper complex 2 strongest inhibited the three cells especially SW-480 cells.The IC₅₀ values of complex 2 on the cells were lower than 5?M,and the lowest IC₅₀for SW-480 cells was 2.4?M.(2)The effect of copper complex 1-3 on the apoptosis of SW-480 cells was analyzed by flow cytometry,and their intake in SW-480 cells was measured.The results of apoptosis experiment showed that all the three copper complexes could induce apoptosis of SW-480 cells at early stage,and the order of inducing apoptosis was 2>3>1.The ability of complex 2inducing SW-480 cells was the strongest with the apoptosis rate of 96%when dealing with 5?M.The results indicated that the inhibition of copper complexes on cell proliferation mainly stems from apoptosis.ICP-MS analysis showed that the intake of three copper complexes was higher than that of Cu Cl₂·2H₂O.The amount of copper complex 1 entering the cell was less while copper complexes 2 and 3 were more,possibly due to 2 and 3 had better lipid solubility.The amount of copper complex 2 entering cells was the largest.Therefore,the effect of copper complex 2 on cell proliferation and apoptosis is closely related to cell intake.(3)Western blotting studies showed that high concentration copper complex 2 reduced almost all protein contents detected,including protein tyrosine phosphatase substrates p-src(Y529),p-src(Y418)and p-IRS-1;PTP1B,TCPTP;EGFR and internal reference proteins GAPDH and?-Actin;and apoptosis factors Bax,Bcl-2 and Caspase 3;only p-EGFR increased significantly.The results indicated the inhibition on cell proliferation and induction of apoptosis by copper complex 2 do not resulted from the improved phosphorylation level by inhibiting PTPs.The decrease of a large amount of proteins suggested that copper complex 2 might promote apoptosis by activating some factors in the cells to improve the degradation of proteins.(4)Because proteasome is closely related to the degradation of proteins in cells,we studied the effect of copper complex 2 on proteasome activity in SW-480 cells.The results showed that copper complex 2 significantly increased the proteasome chymotrypsin-like activity and reduced the amount of ubiquitin-protein in SW-480 cells.Because the proteasome chymotrypsin-like activity of SW-480 cell extracts cultured with high concentration copper complex 2 was much higher than that of cell extracts directly dealing with the high concentration of copper complex 2,we speculated that copper complex 2 might interact with the upstream targets of proteasome,and induced the expression of proteasome and improved its activity.In conclusion,our research shows that copper complex 2 may affect the proliferation and apoptosis of SW-480 cells by enhancing proteasome activity,which provides a new way to study the biological effects and mechanism of copper complexes.