Study And Development Of Several Schiff-based Copper Complexes For Cisplatin-resistant NSCLC

Objective:A549/DDP cells were treated with the copper-based complexes to elucidate the mechanism of pro-apoptosis and anti-tumor angiogenesisin vitro and vivo,for providing theoretical basis in cisplatin-resistant non-small cell lung cancer treatment.Methods:Chapter 1:The cytotoxicity of three copper-based complexes containing Schiff base ligand and 1.10’-phenanthroline against A549/DDP cells were determined by a MTT method.Flow cytometry was used to detect the apoptosis induced by these complexes and the mechanism by investigating the changes of reactive oxygen species and mitochondrial membrane potential in vitro.The proteins VEGFR2,Fak and Akt and their phosphorylated proteins p-VEGFR2,p-Fak and p-Akt are important in VEGF/VEGFR2 signal pathway.The expressions of these proteins were investigated by WB assay to expound the molecular mechanism of which copper-based complexes inhibited the development of A549/DDP cells in vitro.The tumor model was established by subcutaneous injection of A549/DDP cells,and A549/DDP tumors were treated with the copper-based complexes by intraperitoneal injection.The inhibitory effect on tumor growth was evaluated.The morphological changes of tumor tissues were observed after HE staining.The inhibitory effect on angiogenesis was analyzed by immunohistochemical staining with CD31.The expressions of apoptosis-related factors Bcl-2,Bax and Caspase-9 were also investigated to reveal the mechanism of inhibition of A549/DDP tumor growth.Chapter 2:The cytotoxicity of the other three copper-based complexes containing Schiff base ligands were determined by a MTT method.Flow cytometry was used to detect the apoptosis in A549/DDP cells,and the mechanism of apoptosis was investigated.The expressions of VEGFR2,Fak,Akt and Erk and their phosphorylated proteins in the downstream of VEGF/VEGFR2 signal pathway were investigated by WB assay to verify the mechanism of anti-A549/DDP cells in vitro.Chapter 3:A new copper-based complex Cu-3 was synthesized with5-methyl-2-hydroxy-1,3-benzenedialdehyde,L-amphetamine,sodium hydroxide and Cu（NO₃）₂·3H₂O in ethyl alcohol.Its structure was characterized by infrared spectroscopy,X-ray crystal diffraction and X-ray powder diffraction.The apoptosis and mechanism were detected by flow cytometry,and the inhibition of tumor angiogenesis-related pathway factor expression was confirmed by WB assay.Result:Chapter 1:In vitro the results showed that all three copper-based complexes inhibited A549/DDP cell growth,decreased mitochondrial membrane potential,induced apoptosis,regulated intracellular reactive oxygen species,and down-regulated important factors in the downstream of VEGF/VEGFR2signaling pathway.In vivo experiments showed that these copper-based complexes significantly inhibited the growth of A549/DDP tumor,up-regulated the expression of Bax and Caspase-9 and down-regulated the expression of Bcl-2 to play a pro-apoptotic effect,and reduced the number of vascular endothelial cells in tumor to inhibit angiogenesis.Chapter 2:All the copper-based complexes inhibited the growth of A549/DDP and C33A cells,induced the apoptosis by decreasing mitochondrial membrane potential in A549/DDP cells,and regulated the expressions of important proteins VEGFR2,Fak,Akt and Erk and their phosphorylated proteins in VEGF/VEGFR2signaling pathway.Chapter 3:A novel Schiff base copper-based complex was synthesized and characterized.It had toxic effects on various tumor cells,and ability to decreases the mitochondrial membrane potential of A549/DDP cells to induce apoptosis.It down-regulated the expression of important proteins in the VEGF/VEGFR2 signaling pathway.Conclusions:Several of copper-based complexes had inhibitory effect on A549/DDP cells in vitro,which induced apoptosis by activating mitochondrial membrane potential and VEGF/VEGFR2signaling pathway.These inhibitory effects were related to the concentration and treatment time of copper-based complexes,and even were due to the difference in structure.In vivo experiments showed that some copper-based complexes caused tumor cell apoptosis by interfering with the Bax/Bcl-2/Caspase-9 signal axis,and reduced the number of vascular endothelial cells to inhibit tumor angiogenesis.These copper-based complexes had potential therapeutic effects on cisplatin-resistant non-small cell lung cancer,and were the promising candidates for the treatment of cisplatin-resistant non-small cell lung cancer.