Study Of The Mechanism Of Apoptosis Induced By Novel Naphthalimide Derivatives In Human Breast Cancer MCF-7Cells

The naphthalimide derivative has become a hot research topic due to its significant effect in treating tumor. Thereinto, Elinafide and Bisnafide have entered into the clinical research stage of phase Ⅱ, but its toxic-side effect limite its further clinical research of phase III. Therefore, developing a novel naphthalimide derivative with less toxic-side effect and high anti-tumor activity and confirming its molecular mechanisms and target for inducing apoptosis are becoming an important research topic.The inhibition of five novel bis-naphthalimide derivatives DN2-DN6with alkyl isothiorea cation of different lengths as the linker on the growth of human’s breast cancer MCF-7cells and its target were investigated in this study. A novel compound with high anti-tumor activity was separated and its effects of apoptosis for MCF-7cells as well as molecular mechanism were also further investigated.The inhibition of DN2-DN6for the growth of MCF-7cells was determined via MTT test. The results showed that DN2-DN6could significantly inhibit the growth of MCF-7cells, decreasing the survival rate of cells. The ascending order of IC50value of DN2-DN6was DN6<DN5<DN4<DN3<DN2, indicating that the inhibition of DN2-DN6for the growth of MCF-7cells will be enhanced with the increasing length of DN2-DN6alkyl isothiourea linker i.e. the length of linker has positive correlation to cell toxic. Thereinto, the inhibition of DN6on the growth of cell was the strongest. After acting on MCF-7cells in12,24and36h, IC50value of DN6were11.70±1.84,9.42±2.07and8.04±0.23μmol/L, respectively. Therefore, DN6was selected with the aim to investigate its effect for the apoptosis of MCF-7cells and molecular mechanisms.Through the observation of cellular morphology via DAPI staining, the determination of cell cycle distribution by PI single straining and rate of apoptosis by Annexin V-FITC/PI double staining. The result indicated that DN6could induce the apoptosis of MCF-7cells in the concentration-dependent way and disturb the synthesis of DNA, which further made the cycle of MCF-7cells stagnate at S phase.Through the co-localization between DN2-DN6and neutral red (NR) dye using fluorescence confocal microscopy, the targets of these five compounds in the MCF-7cells were observed.The determination of lysosomal membrane stability by acridine orange (AO) staining, the measurement of Cathepsin B and cytochrome C releasing using ELISA. the changes of Bcl-2and Bax protein expression levels and mitochondrial membrane potential by flow cytometric analysis and Caspase-9and Caspase-3expression by spectrophotometry. The results showed that DN2-DN6was located in the lysosome. DN6could cause the increase of lysosomal membrane permeability with concentration and time-dependent way and release of Cathepsin B which acted on Bcl-2protein and decreased the ratio of Bcl-2to Bax. And then it caused the decline of mitochondrial membrane potential and the release of cytochrome C. which activated Caspase-9and Caspase-3and finally led to the apoptosis of MCF-7cells via lysosomal-mitochondrial pathway.