ApoG2 Inhibits Proliferation And Induces Aptosis In Human Breast Cancer Cell In Vitro

BackgroundBreast Cancer, at the moment, is one of the most common malignant tumors occurred among women worldwide, which accounts for 29% of the cancer incidents among American women in 2014, also comes out No.1 of female tumor sufferers in the States. The death rate of breast cancer takes up to 15% of all the female malignant tumors, second only to lung cancer in US.The statistics from the Chinese Anti-cancer Association shows that the (morbidity) of breast cancer in China have been gaining a great expansion year by year, which takes the list top of the female cancer rate, also the No.4 of the tumor mortality. At the same time, the patient age becomes younger and younger. Though, research approaches and solutions towards breast cancer have been pouring out and therapies being updated, yet, drug resistance, recurrence and metastasis are the major difficulty and hotspot during and after regular treatment, especially for the patients of advanced and terminal breast cancer stage. Therefore it is very pressing to find a way to negative control occurrence of the recurrence and metastasis in the process of the treatment.At present, breast cancer treatments can be classified to surgical therapy, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy, etc. Comprehensive application of the above therapies, based on the biological behavior of the patients’ tumor, can significantly improve the living quality of the patients, and prolong their lives, whether the therapies are carried out either regionally or systemically. Medical treatment is the key step for patients in the advanced stage, whose purpose is to keep the tumor from recurrence and metastasis. However, the by-effects of chemotherapy can not be neglected during the treatment. The standard chemotherapy of CAF, TAC, CMF often produce the following side effects:nausea and vomitting, marrow suppression, nerve damage, immune dysfunction, and injury of mucocutaneous membranes, which make the patients unable to endure the pains, and sometimes fail the treatment purpose. So the patients and medical staff urgently demand a suitable drug or remedy to be found to alleviate the side effects above while killing the breast tumor cells.Gossypol, a yellow compound of double naphthalene hydroxyl polyphenol aldehyde mainly exits in the roots, stems, leaves and seeds of cotton plants, a mallow plant. Researches have proved, ever since 1960s, that gossypol can block the growth of tumor cells and has some anti-cancer function. Gossypol, composed of two isomers, R-gossypol and L-gossypol, L-gossypol being the effective part, can stop Bcl-2 family anti-apototic protein like Bcl-2, Bcl-xL and anti-apototic protein like Bax and Bad from compoundingdimers, which can induce cell death. But the aldehydes ofL-gossypol, exited at both ends of the molecules, with some toxicity, will prevent the L-gossypol from playing a full role in anti-tumor cells. Under this circumstance we restructure L-gossypol by removing the aldehydes at both ends of the molecules, and compound the Apogossypolone, ApoG2 in short. Compared with aldehydes, the new element offers a better cure for the tumor cells with less side effect. This research paper targets on overexpression of Bcl-2 genetic MCF-7、 MDA-MB-231 cells and MCF-7/ADR cells, mainly focuses on how ApoG2 works on the proliferation inhibition and Apoptosis induction of the three cells of the breast cancer. An initial explore is also presented on reversal of drug resistance of MCF-7/ADR.ObjectiveThe experiment chose overexpressive Bcl-2 protein celllines MCF-7-. MDA-MB-231 and MCF-7/ADR as the object in the purpose to see how ApoG2 works on the proliferation inhibition and Apoptosis induction of the above the 3 cell lines, so as to probe into the mechanism of action, and at the same time initial explore has been made on reversal of drug resistance of MCF-7/ADR.Methods1. With in vitro proliferation method of CCK8 to see how ApoG2 has the growth inhibition effect on MCF-7、MDA-MB-231 and MCF-7/AD if applied with the concentration of 0 μ mol/L、10 μmol/L、20 μmol/L、40 μmol/L和80 μmol/L of ApoG2 after 24 h,48 h, and 72 h.2. Observation is made to see how the apoptotic morphological changes is produced with the help of Hoechst 33342 and PI double fluorescent staining method on MCF-7、MDA-MB-231 and MCF-7/ADR if applied with the concentration of 20 μmol/L和40 μmol/L of ApoG2 after 48 hours.3. Using tablet cloning test to observe the cell proliferation ability with the application of the ApoG2’s concentration of 5 μmol/and 10 μmol/L on MCF-7、 MDA-MB-231 and MCF-7/ADR after 14 days.4. Using Double staining kit of Annexin V/FITC, with the help of Flow cytometry, to observe the apoptotic rate of changes of MCF-7、MDA-MB-231 and MCF-7/ADR, affected by the ApoG2 under the concentration of 20 μmol/L 和 40 μmol/L after 48 hours.5. Using JC-1 Mitochondrial membrane potential detection kit, with the help of flow cytometry, to test and observe the changes of activity of mitochondria of MCF-7, MDA-MB-231 and MCF-7/ADR, affected by ApoG2 under the concentration of 20 μmol/L 和 40 μmol/L after 48 hours.6. Using PI staining, with the help of flow cytometry, to observe cell cycle changes of MCF-7、MDA-MB-231 and MCF-7/ADR, affected by ApoG2 under the concentration of 20 μmol/L和40 μmol/L after 24 hours.7. Using scratch wound healing assay to observe the cell migration ability with the effect of ApoG2 upon MCF-7/ADR if used with the concentration of 5 μmol/or 10 μmol/L, after 12 hours and 24 hours.8. Using Western Blotting to observe the protein expression changes of Bcl-2, Bax and Caspase 3 protein from MCF-7、MDA-MB-231 and MCF-7/ADR, if applied with 20 μmol/L or 40 μmol/L ApoG2 after the application of 24 hours and 48 hours. Using Western Blotting to observe the protein expression changes of MDR1 protein from MCF-7/ADR, if applied with 20 μmol/L or 40 μmol/L ApoG2 after 24 hours and 48 hours.Results1. The in vitro-proliferation test of CCK8 suggests that different results between groups come out with statistical significance if different concentrations of ApoG2 are used upon MCF-7. MDA-MB-231 and MCF-7/ADR (P<0.05). The same concentration of ApoG2 can produce different degrees of proliferation inhibition when used upon MCF、MDA-MB-231 and MCF-7/ADR after 24,48 and 72 hours (P<0.05). The thicker the concentration of the ApoG2 is, or the longer the application of the dose used, the better effect the proliferation inhibition can achieve.2. Using Hoechst 33342 and PI double staining fluorescent method on MCF-7、 MDA-MB-231 and MCF-7/ADR if applied with the concentration of 20 umol/L and 40 μmol/L of ApoG2 after 48 hours, we can observe the light blue of the visible negative cells of the compared group under the fluorescence microscope, the bright blue of the dead cells and the dark red and light purple of the overlapped red. The dying phenomena of cell’s nuclear condensation and fragmentation, chromatin condensation, apoptotic body formation of apoptosis can be easily seen under the fluorescent microscope. The more the dose applied, the more apoptotic cells would be found.3. Using tablet cloning test with the application of the ApoG2’s concentration of 5 μmol/L and 10 μmol/L on MCF-7、MDA-MB-231 and MCF-7/ADR after 14 days, we can observe that the cell’s cloning ability drop in accordance with the dosage decrease of ApoG2, the results have a significant statistic value (P<0.05), which implies that low concentration of ApoG2 can lower the formation of cell’s cloning ability.4. Using Double staining kit of Annexin V/FITC, with the help of flow cytometry, we can observe the apoptosis rate of changes of MCF-7、MDA-MB-231 and MCF-7/ADR, affected by the ApoG2 under the concentration of 20 μmol/L和40 μmol/L after 48 hours. The results between groups come out with statistical significance(P<0.05). With dose increased, the rising cell apoptotic rate suggests the application of ApoG2 can cause the cells’dying.5. Using JC-1 Mitochondrial membrane potential detection kit, with the help of flow cytometry, we can observe mitochondrial membrane potential drop of each treatment groups, when the ApoG2 used upon MCF-7、MDA-MB-231 and MCF-7/ADR, under the concentration of 20 μmol/L and 40 μmol/L after 48 hours. The mitochondrial membrane potential drop or rise has a close relation with the drop or rise of ApoG2’s concentration used.6. Using PI staining, with the help of flow cytometry, we can observe the cell cycle blockings of MCF-7, MDA-MB-231 and MCF-7/ADR, affected by ApoG2 under the concentration of 20 μmol/L和 40 μmol/L after 48 hours. Compared with negative control group, the application of ApoG2 can cause the cycle blockings for the cancer cells, which has a significant statistic value (P<0.05)7. Using scratch wound healing assay, with the concentration of 5μmol/L or 10 μmol/L of ApoG2’s application on MCF-7/ADR after 12 hours and 24 hours, we can observe that the ApoG groups wounds healed slower, compared with negative control group, suggests that low concentration of ApoG2 applied can restrain the cell moving ability of the MCF-7/ADR.8. Using Western Blotting to test the protein expression changes of Bcl-2 protein and Caspase 3 protein, coated with 20 μmol/L or 40 μmol/L ApoG2 after the application of 24 hours and 48 hours, we found that Bcl-2 protein expression drop (P<0.05), compared with the negative control group, while Bax protein arise (P<0.05). So the conclusion is that after the treatment of ApoG2 on the two cell lines, Bcl-2 protein expression drops and Bax expression arise. Compared with control group, Caspase-3 protein expression arise in the 3 cell lines after treated with ApoG2, suggesting that apoptosis occur. Protein expression changes of MDR1 from MCF-7/ADR, coated with 20 μmol/L or 40 μmol/L ApoG2 after the application of 24 hours and 48 hours respectively, we found that ApoG2’s application could cause the changes of drug resistance of MCF-7/ADR while the protein expression of MDR1 drops (P<0.05) compared with negative control group.ConclusionApoG2 can remarkably inhibit the proliferation of MCF-7 cells and MDA-MB-231 cells, reduce the moving ability of MCF-7/ADR cells, and induce Apoptosis to the three kinds of cells above, whose organism has been related with expression changes of BCL-2 family proteins and the changes of mitochondrial membrane potential.