Effects And Mechanism Of Autophagy Induced By Curcumin In Human Breast Cancer MCF-7 Cells

According to the report of International Agency for Research on Cancer,breast cancer accounted for about 23% of all cancers in womenall over the world,and it became one of the most important reasons for leading to death of women.With the constant development of modern medicine,the treatment of breast cancer has been made great achievements.However,western medicine for the treatment of breast cancer,such as radiotherapy,chemotherapy,surgery,etc.also gave rise to drug resistance and toxicity of chemotherapeutic drugs.With the deepening of the research of traditional chinese medicine,the role of traditional chinese medicine in the treatment of breast cancer will be increasingly important.Curcumin is a yellow phenolic pigment extracted from Curcuma longa L.of Zingiberaceae.Curcuma longa L.distributes widely in the world.Previous researches demonstrated that curcumin contains phenolichydroxyl and methoxy groups,and curcumin could inhibit the growth of tumor cells,it also has the role of anti inflammation,anti-oxidation,anti bleeding,anti virus,reducing blood fat and so on.The previous study also showed that curcumin could inhibit the proliferation of breast cancer cells by the way of inducing apoptosis and inhibiting the formation of tumor vessel.There are few studies on the autophagy induced by curcumin on breast cancer cells,for example,Chen Jian found that curcumin could induce autophagic death in breast cancer cells,but its mechanism of autophagy was not fully elucidated.In this study,transmission electron microscopea and MDC staining were used to observe the morphology of autophagy in human breast cancer cell line MCF-7,the determination of autophagy relatedproteins and signal pathways were finished by western blot method.In short,the possible mechanism of autophagy was clarified,and the theoreticalbasis for developing new drugs against breast cancer was provided.There were three groups in this experiment: normal control group,solvent(DMSO)group and drug group(10,20,40,80 and 160 ?mol·L-1),time was fixed at 24,48 and 72 h.The inhibitory effect of curcumin on cell proliferation was detected by MTT assay.TEM demonstrated autophagy phenomenon directly from cell ultrastructure.The changes of autophagic vacuoles in cells were observed by MDC staining.Western blot method could measure: the expression of autophagy related proteins Beclin1 and LC3?,pathway related proteins Akt and p-Akt;the expression of Beclin1,LC3?,Akt and p-Akt when joined in LY294002(inhibitor of PI3K/Akt pathway);the expression of Beclin1 and LC3? when joined in 3-methyladenine(3-MA,inhibitor of autophagy)and rapamycin(RAP,inducer of autophagy).The experimental results are as follows:1 The inhibition effect of curcumin on breast cancer MCF-7 cellsCurcumin had inhibition effect on breast cancer MCF-7 cells at different concentrations.At the same time,the inhibitionratio increased as curcumin concentration increased,and there were significant differences between drug group and normal group(P<0.05);there were significantdifferences when different drug concentrations compared with each others(P<0.05).The inhibition ratios of curcumin groups withinthe same concentrations increased as time goes on(P<0.05).In solvent group,the maximum inhibition ratio of DMSO was below 1% compared with normal control group.DMSO had no effect on the proliferation of breast cancer MCF-7 cells.So in the following experiments,there was no solvent group.cells 2 The curcumininducedmorphologicalphenomenon of autophagy in MCF-72.1 TEM was used to observe ultrastructure of autophagic vacuole TEM results showed,MCF-7 cells in normal group were intact.But autophagic vacuoles appeared in the cells after treatment with curcumin.2.2 The result of MDC staining Green fluorescence of MDC enhanced after treatment with curcumin,and the fluorescence intensity increased with the increasing of curcumin concentration.These results suggested that curcumin can promote the formation of autophagic vacuoles in MCF-7 cells.3 The function mechanism of MCF-7 cell autophagy induced by curcuminThe results of western blot indicated that,compared with the normal group,curcumin can reduce the level of p-Akt protein in MCF-7 cells,and up-regulatte the expression of Beclin1 and LC3 IIwith the increasing of drug concentration.Compared with LY294002(LY)group,the group of LY plus curcumin could down-regulate the expression of p-Akt(P<0.01).The group of 3-MA plus curcumin could down-regulate the expression of Beclin1 and LC3 II(P<0.05),on the contrary,the group of RAP plus curcumin could up-regulate the expression of Beclin1 and LC3 II(P<0.01).From the results we can conclude that:1 Curcumin could induce the autophagicphenomenon in MCF-7cells and inhibit the proliferation of MCF-7cells.2 Curcumin may up-regulate the expression of Beclin1 and LC3 ?to induce the autophagy of cells by inhibiting the p-Akt protein.