Study And Application Of Four New Copper-Based Complexes Against Human Triple-Negative Breast Cancer In Vitro

Objective: By detecting the inhibitory effect of four copper-based complexes on MDA-MB-231 cells in vitro,the mechanism of inducing apoptosis and anti-tumor angiogenesis was preliminarily investigated,providing theoretical reference for clinical drug candidates for the treatment of triple negative breast cancer.Methods: Chapter 1: 1.Preparation nanoparticles of tetranuclear copper-based complexes with L-methionol-derived Schiff base as ligand.ICP-MS was used to determine that the compounds could be better absorbed by cells.2.The inhibitory effects of two copper-based complexes on MDA-MB-231 cells and HUVECs were verified by MTT assay.3.Flow cytometry,tube formation experiment,and western blotting were used to study the effects of two tetranucleate complexes on inducing apoptosis of MDA-MB-231 cells in vitro and regulating the expression of tumor angiogenesis related pathway factors.Chapter 2: 1.The nanoparticles of the other two copper-based complexes were prepared 2.MTT method was used to verify the inhibitory effect of these two copper-based complexes on the viability of MDA-MB-231 cells and HUVEC cells.3.The apoptosis of MDA-MB-231 cells and HUVECs induced by these two copper-based complexes in vitro was analyzed by flow cytometry.Result: Chapter 1: 1.Transmission electron microscopy showed that the nanoparticles of the two compounds were spherical with good dispersion.The diameter of N(TNCu-1)was(88.33±16.07)nm and the diameter of N(TNCu-2)was(308.00±14.43)nm.ICP-MS results showed that both compounds could be absorbed by MDA-MB-231 cells and HUVECs.2.MTT method results showed that in MDA-MB-231 cells,N(TNCu-2)showed comparable toxicity to cisplatin group,while N(TNCu-1)toxicity was slightly weaker than N(TNCu-2).In HUVECs,the cytotoxicity of N(TNCu-1)and N(TNCu-2)was less than cisplatin.3.Apoptosis experiments and MMP experiments confirmed that these compounds could reduce MMP and induce apoptosis in MDA-MB-231 cells and HUVECs.In MDA-MB-231 cells,N(TNCu-1)and N(TNCu-2)could down-regulate ROS,while in HUVECs N(TNCu-1)and N(TNCu-2)could increase ROS.N(TNCu-2)is stronger than N(TNCu-1)in terms of its ability to induce apoptosis.However,both of them could make the cells show concentration dependent apoptosis,and had stronger effect on MDA-MB-231 cells than on HUVECs.In the tube formation experiment of HUVECs,both copper-based complexes showed strong inhibitory effects compared with blank control group and the positive control group.Erk1/2,AKT and FAK proteins and their phosphorylated proteins p-Erk1/2,p-AKT and p-FAK are key signal molecules located downstream of the VEGFR2 pathway.Western blot showed that both copper-based complexes had regulatory effects on the VEGF/VEGFR2 signaling pathway.Chapter 2: 1.Transmission electron microscopy showed that nanoparticles of the two copper-based compounds had good dispersion,and the N(Cu-3)form was more spherical.The diameter of N(Cu-3)was(56.14±6.28)nm and the diameter of N(Cu-4)was(74.98±12.50)nm.2.MTT test results showed that N(Cu-3)was more toxic than cisplatin and N(Cu-4)in MDA-MB-231 cells and HUVECs,while N(Cu-4)toxicity was weaker than cisplatin.3.In the apoptotic experiment,N(Cu-3)had certain effect on both MDA-MB-231 cells and HUVECs.Under the same conditions,N(Cu-3)had a stronger effect on MDA-MB-231 cells and showed a significant dose-dependent effect on HUVECs.The effect of N(Cu-4)on MDA-MB-231 cells was obviously dose dependent,but had little effect on HUVECs.Conclusions: Chapter 1: Both N(TNCu-1)and N(TNCu-2)nanoparticles had a certain inhibitory effect on MDA-MB-231 cells.They significantly induced MDA-MB-231 cell apoptosis and inhibited angiogenesis by destroying intracellular reactive oxygen levels,reducing mitochondrial membrane potential levels,activating signaling molecules in the VEGF/VEGFR2 signaling pathway.These inhibitory effects were related to the concentration and time of N(TNCu-1)and N(TNCu-2).However,due to the difference of its structure,the effect was different.Chapter 2: N(Cu-3)and N(Cu-4)nanoparticles had different inhibitory effects on MDA-MB-231 cells.Meanwhile,apoptosis experiments showed that both of them could induce cell apoptosis and had higher selectivity to MDA-MB-231 cells.