| Breast cancer remains a major public health challenge .More than one million women worldwide are diagnosed with breast cancer annually . Epidemiology data showed the breast cancer risk is attributed to dietary factors.Equol, is gut bacterial metabolic product of the isoflavones,that is more biologically active thantheir precursor daidzein. The effects of treatment and prevention ontumor are probably stronger than isoflavones. However , the mechanism is unclear.Autophagy is a cell death mechanism defined as typeâ…¡programmed cell death (PCDâ…¡). It is a kind of cellular catabolic degradation response to nutrient starvation or metabolic stress. Now , more and more attentionsare paid on the relationship between the autophagy and tumor , but the exact mechanism is still unclear. Obejective:To observe the effects of equol on the estrogen receptor-independent human breast cancer MDA-MB-231 cells and observe the effects of autophagy which is induced by equol to the tumor cells . Further investigatedits possible mechanism.Method:1. MDA-MB-231 cells are treated with equol for 48h.Cell viability , morphological characters, apoptosis and the damage of DNA were measured by MTT, microscope, flow cytometry, and Comet assay. Concentration of LDH in the medium was determined by colorimetric method;2. MDA-MB-231 cells were treated by equol for 48h . Autophagosome was observed via transmission electron microscopy and the expressionof the significantly protein Beclin-1was detected byimmunocytochemistryand Western-Blot;3. MDA-MB-231 cells were treated by equol for 48h . The expression of MAPKs and phosphorylation were measured via Western-Blot.4. The MDA-MB-231 cells were treated with equol and inhibitors of ERK1/2 and c-JNK. To detect the expression of beclin-1 by Western-Blot;5. The MDA-MB-231 cells were treated with equol and inhibitor of autophagy .The apoptosis was measured via flow cytometry and TUNEL kit.The expression of Caspase family and p53 ,which are the significantly protein to apoptosis, are detected by Western-Blot.Effects of equol on mitochondrial activity were measured using the mitochondrial membrane potential sensitive fluorescence probe JC-1;6. The MDA-MB-231 cells were treated with equol and inhibitor of Caspase family to detect the level of Beclin-1via Western-Blot.Result :1. Equol had significant anti-proliferation effects on MDA-MB-231cells.As compared with control group, the concentration of LDH in medium increased significantly(p<0.05),apoptosis rate significant enhanced (p<0.05),and damage of DNA increased siganificantly(p<0.05);2. The formation of autophagosome and autophagolysome can be observed in equol-treated cells via the transmission electron microscopy, and the number of autophagosome increased in response to the concentration of equol. With immunocytochemistry, more beclin-1 stained cells were observed after equol treatment, and expression of beclin-1 increased significantly (p<0.05) as the increasing of equol concentration.;3. Treatment with equol resulted in the significant(p<0.05) increase of ERK1/2 phosphorylation and JNK phosphorylation in MDA-MB-231cells. In contrast. p38 phosphorylation was significantly (p<0.05) downregulated .Meanwhile, the MDA-MB-231 cells were treated with equol and inhibitors of ERK1/2 and c-JNK, the expression of Beclin-1 decreased significantly(p<0.05);4. Autophagy induced apoptosis: treatment with inhibitor of autophagy and equol, apoptosis rate decreased and mitochondrial membrane potential significant(p<0.05) increased. The expression of Caspase 3,9,Bax and p53 significant (p<0.05)increased .In contrast Bcl-2 significant(p<0.05) decreased.; 5. Different effects of Caspase lead to different results: the expression of autophagy marker protein Bcelin-1 was abolished (p <0.05) when the activity of Caspase family was blocked by the inhibitor. ?Conclusions :1. Equol injure MDA-MB-231 cells via the autophagy ;2. Equol-induced autophagy in MDA-MB-231 cells through the MAPK pathway;... |
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