Inhibitory Effect Of RApoptin On MCF-7 Breast Cancer Cells And Their Stem Cells

Breast cancer is the most common malignant tumor in women. Its morbidity and mortality rank No. 1 among the female malignant tumors around the world, and the corresponding statistical figures are still continually increasing. Compared with the rapidly growing morbidity, lack of safe and effective treatment for breast cancer is still a worldwide problem. Therefore, the researches and development on more effective anti-tumor reagents not only have great social benefits, but also bring huge economic profits.Apoptin is the only functional protein of chicken anemia virus(CAV). Compared with the existing antitumor reagents, Apoptin has the following characteristics: Firstly, Apoptin induces tumor cells to apoptosis independent of P53 pathway, and has theraputic effects on P53 mutation tumor cells. Secondly, the over-expression of Bcl-2 cannot inhibit the apoptosis effects of Apoptin on tumor cells. On the contrary, it can be used to enhance its apoptosis function. Thirdly, Apoptin has tumor-selective apoptosis function to multiple cancers.At present, there are still two important technical bottlenecks hindering large-scale production and wide clinical application of Apoptin. Firstly, there is no related protein receptor on human cell membrane, so it cannot integrate with human cells to exert its killing effect. Secondly, it is difficult to obtain large quantities of high purity products of Apoptin, because its prokaryotic expression protein in vitro is difficult to be dissolved and stably preserved. In order to investigate and verify better technology methods on Apoptin application, our research group uses recombinant tumor specific apoptosis factor(r Apoptin) to study the killing effect and related mechanism of r Apoptin on MCF-7 human breast cancer cells and its cancer stem cells. r Apoptin is a recombinant protein of GST-Apoptin which is chemically modified by folic acid. A recent study finds out that the number of folate receptors on tumor cells is dozens of normal cells, which indicates that r Apoptin protein modified by folic acid can obviously enrich in tumor cells and plays killing effect. At present, there is no research about whether r Apoptin can effectively inhibit breast cancer, breast cancer stem cells and its related mechanisms. Therefore, studying on the killing effect and related mechanism of r Apoptin is likely to enhance the clinical therapeutic effects of breast cancer and brings new hope for prognosis.Phosphorylation of related factors in Apoptin pathway is an important regulatory mechanism during the process of apoptosis. PI3K/Akt is the core factor in the upstream of Apoptin-apoptosis signaling pathway. After entering into cytoplasm, Apoptin can cause Akt phosphorylation and combine with it to "hijack" Akt into nucleus through which executes its inducing apoptosis function. The phosphorylation of Nur77 is the core event of the downstream signaling pathways compared with Akt. After phosphorylated by Apoptin, Nur77 exports from nucleus to cytoplasm, triggers mitochondrial apoptosis pathway and accomplishes apoptosis function. Caspase-3 is the final executor in Apoptin signal pathway, which ultimately exerts the apoptosis effect.In this study, we use the MCF-7 human breast cancer tumor cells as the experimental objects at the very beginning. Firstly, we use CCK-8 method to detect the r Apoptin inhibition rate curve of MCF-7 cell and confirm the subsequent drug treatment period and concentration gradient. Then, colony formation assay is used to detect the anti-proliferation effects of r Apoptin. Annexin V / PI double staining method is used to detect the efficiency of r Apoptin inducing-apoptosis. Western Blot technique is used to detect the level changes of phosphorylated Akt(p-Akt), phosphorylated Nur77(p-Nur77) and Caspase-3 p17 activation fragment in Apoptin signaling pathway after treatment. On this basis, we establish nude mice transplanted tumor model of MCF-7 cells, and use it as the second stage experimental object. After the tumor-bearing mice are treated by r Apoptin, we observe the morphological changes of transplantation tumor by measuring tumors weight and HE staining. We observe the activated levels of the Apoptin signaling pathway in transplanted tumor tissues by immunohistochemical staining and Western Blot technique. Finally, we use the breast cancer stem cells in MCF-7 cells as the experimental objects. Flow cytometric analysis is used to test the level changes of CD44+/CD24- breast cancer stem cells. Serum-free culture mammosphere formation assay is used to test self-renewal ability of breast cancer stem cells. Western Blot and RT-PCR analysis on ALDH1 is used to identify whether r Apoptin can effectively inhibit breast cancer stem cells of MCF-7 cells.,Part ⅠThe study of growth inhibition effects of r Apoptin on MCF-7 human breast cancer cellObjective: To investigate whether r Apoptin can effectively inhibit the growth of MCF-7 cells, induce apoptosis and related mechanisms in order to lay a foundation for further study of r Apoptin in vivo.Methods: CCK-8 method is used to detect the MCF-7 growth inhibition rate curve by r Apoptin. Colony formation assay is used to test r Apoptin anti-proliferation effect. Annexin V / PI double staining method is used to detect the efficiency of r Apoptin inducing-apoptosis. Western Blot is used to detect activation levels of p-Akt(p-Akt), p-Nur77(p-Nur77) and Caspase-3 p17 after dosing gradient treatment.Results: r Apoptin can inhibit the growth of MCF-7 cell and induce it to apoptosis. r Apoptin mechanism is executed by Akt and Nur77 phosphorylation and Caspase-3 p17 activation. CCK-8 experiment result shows that after MCF-7 cell is treated by r Apoptin for 24 h, its IC50 is 3.2μg/ml. When the drug concentration is 1.0μ g/ml, 2.0μ g/ml, 3.0μ g/ml, 3.5μ g/ml, 4.0μ g/ml, 5.0μ g/ml, 6.0μ g/ml and 7.0μ g/ml, MCF-7 cell growth inhibition rates gradually increase, followed by 6.67±1.10%, 38.13±2.48%, 45.09±3.60%, 56.30±4.59%, 68.79±6.08%, 85.59±4.36%, 92.34±1.00% and 94.56±0.99%. Except for 6.0μ g/ml group and 7.0μ g/ml group, the growth inhibition rate of each group has statistical difference(P<0.05)among others. Colony formation assay result shows that after 6 day drug treatment, cloning efficiency of groups gradually decreases, followed by 93.67±1.86%, 85.56±1.90%, 42.56±6.08%, 27.22±4.68%, 14.56±3.86% and 8.67±1.33%, when the drug concentration is 0μ g/ml, 0.6μ g/ml, 1.2μ g/ml, 1.8μ g/ml, 2.4μ g/ml and 3.0μ g/ml. Cloning efficiency of all the experimental groups has obvious differences(P<0.01)compared with control group. Except for 2.4μ g/ml group and 3.0μ g/ml group, there are statistical differences(P<0.05)among the other experimental groups. Annexin V / PI double staining method shows that after treatment for 24 hours, early apoptosis rate of 0μ g/ml group, 1.6μ g/ml group, 3.2μ g/ml group and 4.8μ g/ml group increases gradually, followed by 1.36±0.14%, 4.24±0.74%, 4.69±0.60% and 12.84±1.24%. There is no obvious difference(P>0.05)between 1.6μ g/ml group and 3.2μ g/ml group. There are obvious statistical differences(P<0.05)among the other groups. Meanwhile, the later period apoptosis and necrosis rate is 0.78±0.42%, 2.00±0.52%, 1.93±0.76%, 2.30±0.79%, and there are no statistical differences(P<0.05)among groups. Western Blot result shows that after 24 hours treatment, the activated levels of p-Akt, p-Nur77 and Caspase-3 p17 in MCF-7 cell rise gradually with the increase of drug concentration, there are statistical differences(P < 0.05) among groups.Conclusion: Apoptin can inhibit the growth of MCF-7 cell and induce it apoptosis. There is a positive dose effect relation between drug concentration and apoptosis effect. r Apoptin can cause Akt and Nur77 phosphorylation and Caspase-3 activation, and the mechanism of apoptosis may be achieved through the inducing-apoptosis signal pathway of Apoptin. In addition to the function of inducing apoptosis, r Apoptin may have the ability to inhibit the cell cycle of MCF-7 cell. Part II Inhibition effects of r Apoptin on MCF-7 human breast cancercell transplanted to nude miceObjective: To investigate whether r Apoptin can effectively inhibit the transplanted tumor growth of MCF-7 cells in nude mice and induce its apoptosis, then discuss the related mechanism of this process.Methods: Transplanted tumor is built by injection of MCF-7 cells in nude mice breast pad. After the treatment with r Apoptin, we observe the morphological changes of transplanted tumor by measuring weight and HE staining. Immunohistochemical staining and Western Blot techniques are used to detect the level changes of p-Akt, p-Nur77 and Caspase-3 p17 activation fragment in transplanted tumor tissues.Results: r Apoptin can effectively inhibit the growth of transplanted tumor and induce transplanted tumor to apoptosis. The apoptosis mechanism of r Apoptin is realized by Akt and Nur77 phosphorylation and Caspase-3 p17 activation. Transplanted tumor measuring result shows that after r Apoptin injection, the tumor weight of 0mg/kg group, 0.8mg/kg group and 1.6 mg/kg group decrease gradually, followed by 2.50±0.27 g, 1.92±0.60 g and 1.18±0.29 g, and there are statistical differences(P<0.05)among the groups. HE staining result shows that some focal tissue structures are frequently missing, most cell volumes decrease and nucleus are pyknosis and darkened in treatment groups, no obvious inflammation reaction is observed at the same time. Immunohistochemical staining result shows that p-Akt, p-Nur77 and caspase-3 p17 in all experimental groups are all stained brown and located in the cytoplasm. The grade of positive staining increases gradually with the drug concentration. Western Blot result shows that after 24 hours treatment, the levels of p-Akt, p-Nur77 and Caspase-3 p17 in transplanted tumor tissues rise gradually with drug concentration, there are statistical differences(P < 0.05) among groups. The results of Western Blot are almost in accordance with the results of immunohistochemical staining.Conclusion: r Apoptin can inhibit transplanted tumor of MCF-7 human breast cancer cells in nude mice and induce it to apoptosis. There is a positive dose effect relation between drug concentration and apoptosis effect. The mechanism of r Apoptin inducing-apoptosis is achieved through the signal pathway of Apoptin via Akt, Nur77 phosphorylation and Caspase-3 activation. r Apoptin can effectively pass through MCF-7 cell membrane and nuclear membrane. Folic acid modification does not influence Apoptin inducing-apoptosis. r Apoptin has no obvious toxic and side effects on bearing-tumor nude mice.Part III The study on inhibition ability of r Apoptin to MCF-7 breastcancer stem cellsObjective: To investigate whether r Apoptin can effectively inhibit MCF-7 breast cancer stem cells.Methods: Flow cytometric analysis on CD44+/CD24- cell proportion, serum-free culture mammosphere formation assay, Western Blot and q RT-PCR on ALDH1 are used to identify whether r Apoptin can effectively inhibit breast cancer stem cells in MCF-7 cells.Results: r Apoptin has no significant inhibition effects on MCF-7 breast cancer stem cells. Flow cytometry results shows that after 24 hours treatment, the proportion of CD44+/CD24- in 0μ g/ml group, 1.6μ g/ml group, 3.2μ g/ml group and 4.8μ g/ml group rise gradually, followed by 2.10±0.16%, 7.61±1.11%, 10.38±0.97% and 13.21±1.90%. There are statistical differences( P<0.05) among the groups. Mammosphere formation assay result shows that after 14 days treatment, there are no obvious mammosphere volume differences among 0μ g/ml group, 1.2μ g/ml group and 2.4μ g/ml group, and the cells in mammosphere in all groups connect tightly. The numbers of mammosphere are 116.33±5.03, 122.33±4.93 and 124.67±6.03 successively, no statistical differences(P>0.05)among the groups. The results of Western Blot and q RT-PCR show that the expression level of ALDH1 protein and m RNA rise gradually along with the increasing drug concentration, and there are statistical differences(P<0.05)among the groups.Conclusion: r Apoptin cannot effectively inhibit breast cancer stem cells in MCF-7 cells, which might be resisted by the mechanism of multidrug resistance in breast cancer stem cells.