Synthesis And Characterization Of Novel Amino Acid Contained Ternary Copper Drugs And Its Activity In Human Lung Cancer

Part I The preparation,characterization,interaction with DNA and anti-lung cancer A549 cell line activity of novel amino acid contained ternary copper drugsObjective: Three novel amino acid contained ternary copper drugs(Hereinafter referred to as ternary copper drugs 1,2,3 or TCD-1,2,3)have been synthesized using amino acid with reduced Schiff base ligand,N,N?-chelating ligands and copper(II)salts and characterized through the necessary spectrum methods.The ability of the interactions to CT-DNA and inhibiting the proliferation and invasion of lung cancer A549 cells were also investigated.Methods: 1)The ligand HL was synthesized through a classical chemical reaction,the formation of Schiff Base ligand,which coupled the amino group of amino acids and the aldehyde group of the another raw material.The as-synthesized Schiff based ligand was then reduced by Na BH4,which formed reduced Schiff base ligand HL.The ternary copper drugs 1-3 were synthesized by mixing the difference liands and the cooper salts in room temperature for some time and collected as the final products.2)The structures of the ternary copper drugs 1-3 have been characterized by elemental analysis,IR and Gaussian calculations.3)We investigated the interactions between the complexes and DNA through various spectrophotometric methods and viscosity measurement.4)The proliferation and invasion of the three drugs to lung cancer A549 cells have been tested by MTT assay and Transwell method.Results: 1)The yield of the three ternary copper complexes 1-3 was 86.5%,88.2% and 89.8% respectively.2)The structure and molecular formula of the Schiff base ligand HL and three ternary copper complexes 1-3 were in agreement with the theoretical values.3)The red shift and the hypochromism on the ultraviolet spectrum,the quenching of the EB emission intensity on the fluorescence spectrum appeared after the addition of the three copper drugs.Further more,the viscosity of the CT-DNA system increased a lot with increase of CT-DNA concentration.The binding constants of the three ternary copper drugs 1-3 and DNA were1.37?105 M-1,1.81?105 M-1 and 3.21?105 M-1,respectively,resulting in a fluorescence quenching constant Kq of 1.67?104 M-1,2.95?104 M-1 and 5.04? 104 M-1.4)The IC50 values of the three drugs to A549 cell line were(7.48±0.86)mmol/L,(3.93±0.28)mmol/L and(1.47±0.14)mmol/L,which were on an order of magnitude of the IC50 value of cisplatin(2.49±0.28)mmol/L.Compared with the control group,the TCD 1-3 had the ability to anti lung cancer invasion(p<0.05).Among them,the biological activities of the TCD-3 were the strongest(p<0.05),while it was not statistical significance compared with cisplatin(p>0.05).Conclusion: The yield of the three ternary copper drugs 1-3 is reasonable,and the results are in accordance with the original design.The TCD 1-3 can bind to CT-DNA through the insertion mode,and the order of the DNA binding efficiency of the three drugs was 3> 2> 1.All of the three ternary copper drugs can inhibit the cell growth(A549 cell),of which the drug 3 is the best one.Part II Study on the mechanism of novel amino acid contained ternary copper drugs against lung cancer A549 cellsObjective: To investigate the mechanism of TCD 1-3 against lung cancer A549 cells.Methods: 1)After the A549 cells were treated with 1?mol/L TCD 1-3 and PBS for 12 hours,the DNA damage was observed by comet electrophoresis.2)After treated with TCD 1-3(1?mol/L)and PBS for 12 hours,the effect of the drug on cell cycle was investigated by flow cytometry.3)After treated with 1?mol/L TCD 1-3 and PBS for 24 hours,cell apoptosis was studied by Annexin V/PI and Tunel assay.4)After treated with1?mol/L TCD 1-3 and PBS for 24 hours,the effect of the three drugs on the expression of p53,Bax and Bcl-2 protein was observed by western-blotting.Results: 1)Compared with the control group,all of the TCD 1-3 had DNA damage to A549 cells(p<0.05),and the order of the damage ability was 3>2>1(p<0.05).2)Compared with the control group,the proportion of G0/G1 phase in the TCD 1-3 was significantly increased(p<0.05)while the proportion of S phase decreased significantly(p<0.05).The order of the cell block effect was 3>2>1(p<0.05).3)Compared with the control group,Annexin V/PI assay and Tunel assay indicated that TCD 1-3 could induce the apoptosis of lung cancer cells(p<0.05),and the ability of TCD-3 was more effective than that of TCD-1(p<0.05).There were no significant difference between TCD-3 and TCD-2(p>0.05).4)Compared with the control group,TCD 1-3 could up-regulate the level of p53,Bax protein,and down--regulate the level Bcl-2 protein in A549 cells.Conclusion: TCD 1-3 could interfere with the DNA replication by DNA damage,block cells in G0 / G1 phase and promote the apoptosis to inhibit A549 cells proliferation,in which TCD-3 is the strongest ability to use.They also could up-regulate the expression of Bax,p53 protein and down-regulate the expression of Bcl-2 protein to induce apoptosis.Part III Study on the anti-lung cancer activity and side effects of novel amino acid contained ternary copper drugs in vivoObjective: To investigate the anti-lung cancer activity and the side effects of TCD 1-3 in vivo.Methods: 1)Nude mice models carring A549 cells were created and divided randomly into five groups(TCD-1,TCD-2,TCD-3,DDP and PBS control groups,6 nude mice in each group).The nude mice in each group received corresponding 4mg/kg drug by caudal vein injection every week and lasted for 3 weeks.The volume of the tumor was measured every week after the first injection and the mice were killed at last.The growth curves of the tumor were drawn and the tumor growth inhibition rates were calculated.2)After the experiment,the general conditions,bone marrow function(RBC,WBC,PLT),renal function(CREA,BUN)and liver function(ALT,?-GT)of the nude mice were observed.Results: 1)The TCD 1-3 significantly inhibited the growth of human lung cancer A549 cells in vivo(p<0.05).The anticancer ability of TCD-2 and 3 was stronger than that of TCD-1(p<0.05),and there were no significant difference with cisplatin(p>0.05).The inhibitory rates of TCD 1-3 and cisplatin were 26.64%,50.03%,54.89% and 50.70% respectively.2)The mice carring A549 cells were well tolerant to TCD1-3,and there were no significant effect on body weight(p>0.05).TCD 1-3 had a certain inhibitory effect on bone marrow function,renal function and liver function(p<0.05),but their overall side effects were lower than that of cisplatin(p<0.05).Conclusion: The TCD1-3 have good anti-lung cancer activity in vivo,among which the antitumor activity of TCD-2 and 3 are equivalent to that of cisplatin.The side effects of TCD 1-3 on bone marrow,kidney function,liver function are less than cisplatin.