The Mechanisms Of ApoG2 Induced Apoptosis And Autophagy On Breast Cancer Cell Line MCF-7

1. Background:Breast cancer is the most common cancer to life and health of women recently. China is one of the fastest growing incidence of breast cancer,Chinese Anti-Cancer Association released statistics showed that:in recent years,the incidence of breast cancer increased at an annual rate of 3%,and became the fastest growing cancer in cities.In addition, the age of patient is more and more younger.Despite the treatments were great improved in recent years, there were still many patients emerged metastasis or recurrence during or post-treaments. Therefore,we need to continue studing the pathogenesis of breast cancer,and looking for more and brtter method of treatment for breast cancer.Bcl-2 is one of the important anti-apoptotic protein in Bcl-2 family, and overexpressed in many tumor cells. In recent years,the gene of Bcl-2 was considered to be an oncogene, can inhibited the apoptosis or autophagy, induce the tumor. Most studies show that Bcl-2 is overexpressed in breast cancer cells. Therefore, we can make bcl-2 protein as a target spot for therapeutic approaches. Inhibited the activity of bcl-2 or lower the expression of bcl-2 may be a new treatment for breast cancer.About 65% of breast cancer expressed ER, and half of the tumor growth dependent estrogen (estradiol,17β-estradiol, E2).E2 could partially inhibit tumor necrosis factor (TNF)-induced ER (+) MCF-7 cell apoptosis,but there was no such effect on MDA-MB-231 cells. The expression of Bcl-2 and ER in the tumor tissue containing a significant correlation:85% of the Bcl-2 positive tumor were ER-positive.28% of the Bcl-2 negative tumors were ER negative. The relationship between Bcl-2 with ER instructed that Bcl-2 protein was regulated through ER by estrogen. ER may be the induction agent of Bcl-2. The patients with ER-positive tumors receiving anti-estrogen drugs such as tamoxifen can affect the expression of Bcl-2. This suggest that the use of anti-estrogen drug therapy decreased Bcl-2 protein, result in cellular automata to apoptosis.Therefore, bcl-2 protein as a target for therapeutic approach, targeted inhibition of bcl-2 activity or reduced expression of bcl-2 may be an effective way to treat breast cancer.Bcl-2 (B-Cell lymphoma/leukemia 2) was first found in a high incidence of t (14.18) chromosomal translocation of B-cell lymphoma by Tsujimoto in 1984. Bcl-2 was an important anti-apoptotic protein in apoptotic Bcl-2 related protein Bcl-2 family, overexpressed in a variety of tumor cells. In recent years, it has been considered to be a cancer gene in the field of anti-tumor research,it can inhibit the tumor cell apoptosis or autophagy process, and thus lead to tumor development, drug resistance and tumor recurrence. Most studies show that breast cancer cells overexpressing the Bcl-2, and more vulnerable to recurrence, metastasis or drug resistance.With the development of relationship between Bcl-2 family and tumor, the anticancer drugs have been developed depended on the mechanism of action. The biological treatment and advantages of peptides, small organic molecules and other drugs have been used for some appropriate anti-apoptotic protein based on Bcl-2. The small organic molecules derivatives of Bcl-2 inhibitor are the important direction of development.Gossypol is a yellow polyphenolic compound high contented in the Malvaceae Gossypium cotton plant seeds, leaves, stems and roots and other organs. It is a small molecule inhibitor of Bcl-2. Studies sowed that L-gossypol is the active ingredient of gossypol, and play a major role in the Bcl-2/Bcl-XL gene, and prevent its pro-apoptotic genes such as Bak, Bax, Bad, etc. to form heterodimers,induce apoptosis of tumor cells. The vivo and vitro experiments have demonstrated that gossypol has the effective anti-tumor activity. However, it was considered that the aldehyde in the ends of the molecular structure of L-gossypol are related with toxicity, although the effect is weak, but limits the body's maximum tolerance dose. In addition, this mixture into the body is easily combined with other proteins, which weaked the effectiveness of drug treatment, increased the toxicity. Apogossyplone (ApoG2), removed the two aldehyde groups, is a new derivative of gossypol. Its cytotoxicity tests showed that the inhibition of tumor cell growth is more than the L-gossypol, and showing the better prospects. In our study, we choose the human breast cancer cell line MCF-7 as the research object, discuss its possible mechanism for treatment of ApoG2 in MCF-7 cells, provide some new research directions of breast cancer.Objeetive:This experiment to make use of high expression of Bcl-2 protein in breast cancer cell line MCF-7 as the research object.Research growth inhibition in vitro of Apogossypolone (ApoG2) on human breast cancer cell line MCF-7, and to explore its possible mechanism. Provide experimental evidence and theoretical basis for further research of ApoG2 in clinical application.Methods:1. Using MTT proliferation studies research the growth inhibition of ApoG2 act on the breast cancer cell line MCF-7 in vitro;2. Using colony forming assay research the inhibition of colony formation of ApoG2 on MCF-7 breast cancer cell line in vitro;3. Using Hoechst 33258 fluorescent staining research the nucleus morphological changes of breast cancer cell line MCF-7, which was induced by ApoG2;4. Flow cytometry(FCM) was used to measure the apoptosis and cell cycle changes in breast cancer MCF-7 cells induced by ApoG2;5. Using the TEM detected the morphological changes of breast cancer cell line;6. Using Western blot analysis detected the expression of apoptosis related proteins Bcl-2 and Bax.Results1. Apoossypol act on the growth of MCF-7 cells growth inhibition, and this effect was time and dose—dependent effect. 10μmol/L of Apogossypol act over the role of 24h can significantly inhibit the growth of MCF-7 cells(P <0.05).2. Cloning formation experiments show that with different concentrations (5,10,20μmol/L) ApoG2 cultured with breast cancer cells MCF-7 cells for 2 weeks, the clone of MCF-7 cells were significantly inhibited, compared with the control group (P<0.05).3. Transmission electron microscopy detection can see that in the control group MCF-7 cell morphology were well, but the group of 20μmol/LApoG2 treated MCF-7 cells appeared The phenomenon of autophagy:a large number of intracellular vacuoles, cell volume increases, but the cell membrane, nuclear membrane were complete, chromatin condensation is not obvious.4. Hoechst 33258 fluorescence staining shows that MCF-7 cells with 20umol/L of ApoG2 after 72h co-culture showing a clear nuclear condensation and fragmentation, chromatin condensation, apoptotic body formation of apoptosis; and control cells are presented uniform dispersion, the normal light blue nucleus.5. Flow cytometry shows that the apoptosis rate of MCF-7 cells with 20μmol/L of ApoG2 after 72h co-culture increased and the cell cycle S phase and G2/M phase was higher than the control group(P<0.05).6. Western blot test results suggest that MCF-7 cell lines co-culture with ApoG2 (20μmol/L) of 72h, the intracellular expression of bcl-2 less than the control group (P<0.05); while the expression of Bax protein increased over the previous(P<0.05).Conclusion:ApoG2 can inhibit the proliferation and colony formation of breast cancer cell line MCF-7, blocked the cell cycle arrest, and induced the programmed cell death. The mechanism may be that ApoG2 can inhibit the expression of anti-apoptotic protein Bcl-2, increase the expression of pro-apoptotic protein Bax protein, thereby inducing the Bcl-2-related apoptosis pathway, causing the death of tumor cells. Meanwhile, when the expression of Bcl-2 decreased, the binding of Bcl-2 and Beclin-1 protein reduced too, indirectly increase Beclin-1 protein expression, promote autophagy mechanisms occur, and inhibited the tumor occurrence and development.