Effects Of Isomycin Derivant On Inducing Apoptosis Of Human Breast Carcinoma MCF-7 Cell Line In Vitro

Objective:To investigate the effect of isomycin derivant on inhibiting proliferation and inducing apoptosis of human breast carcinoma MCF-7 cell line in vitro.Methods:MTT assay was used to measure the growth inhibition rate of human breast carcinoma MCF-7 cells which were treated with isomycin derivant; Morphological changes of the apoptosis were observed by inverted microscope, HE staining, AO/EB double staining and Scanning electron microscope; Flow cytometry was used to investigate the apoptosis and the changes of cell cycle; TUNEL assay was used to detect the rate of the apoptosis of the MCF-7 cells; The expression of nuclear proliferating antigen Ki-67, apoptosis related genge such as Bcl-2,Bax,Fas, apoptosis protien kinase such as Caspase-3,-8,-9, IAP family members such as Livi,Survivin and nuclear factor kappa B (NF-?B) were detected by immunohistochemitry.Results:(1) MTT assay showed that isomycin derivant (Br-, I-, OAC-) all could significantly inhibit the growth of MCF-7 cells and the effect was in a dose-dependent and time-dependent manner.The effect of the derivant which retouched by Br was the strongest; (2) Under inverted microscope MCF-7 cells showed the classical apoptosis changes like apoptotic bodies, the cells showed necrosis when treated with isomycin derivant after 72h; HE staining showed that the apoptotic bodies were stained by the classical navy blue and pompadour color,the changes could be observed under the optical microscope; The results detected by AO/EB demonstrated that the nuclear chromatins of living cells were green but dead ones were orange; Scanning electron microscopy identified that there were rich microvilli on the surface of normal cells. the volume of the cells which were treated with 5μg/ml isomycin derivant decreased apparently, and the microvilli on the surface ruptured and decreased. Finally, the microvilli on the surface of the cells were disappeared completely and the cell membrane surface became smooth; (3)Flow cytometry showed isomycin derivant could induce the apoptosis of MCF-7 cells, some typical sub-dipioid peaks were observed, the percentage of cells in S phase increased and the percentage of cells in Go/Gi phase decreased after treated with isomycin derivant, isomycin derivant could arrest MCF-7 cells in S phase; (4) TUNEL assay showed that isomycin could induce the apoptosis of MCF-7 cells, after 72h we could see the necrosis of cells, AI of experiment group was significantly different from control group(P<0.05); (5)Isomycin derivant dowm-regulated the expression of Bc1-2,Ki-67,Livin and Survivin, it also up-regulated the expression of Caspase-9,Caspase-8,Caspase-3,Fas,Bax, and they were significantly different from control groups(P<0.05).Conclusions:1. Isomycin derivant (Br-,I-,OAC-) all could significantly inhibit the growth of MCF-7 cells and the effect was in a dose-dependent and time-dependent manner.2. Isomycin derivant could arrest the MCF-7 cells in the S phase.3. Isomycin derivant induced apopsis via two main pathways:the mitochondrial pathway and the death receptor-mediated pathway.4. Inhibition of the signal transduction pathway of NF-?B may be the mechanism of inducing apoptosis.5. Isomycin derivant had a significant effect of anti-tumor, we hoped it would be exploded as a new anti-tumor medicine.