Effect Of Ad-p53 Combined With Fulvestrant On Induction Of Apoptosis In MCF-7 Breast Cancer Cells

ObjectiveSince 1979, p53 gene was found, and its structure and function has been widely recognized.p53 protein coded by p53gene has become an important participant in the development process of tumor cells.In more than 100 kinds of human tumors,More than 60% of tumors were happened with p53 gene mutation,Now known,20% of human breast cancer p53 gene were mutations, while the remaining 80 percent of breast cancer contain wild-type p53,And Breast cancer is different from gastric cancer, colorectal cancer, lung cancer and other tumor types,On the one hand, the development of breast cancer is due to genetic mutation of the tumor suppressor gene p53,The other is due to the nucleotide sequence of the normal p53 gene does not play inhibition of tumor cell proliferation and repair of DNA damage.it has become the new direction the new era of cancer treatment that The exogenous tumor suppressor gene (p53 gene) is introduced into the recombinant tumor after processing, handling.and Inducing apoptosis of tumor cells, and then play the role of inhibition of tumor tissue growth.Fulvestrant (fulvestrant) is a new endocrine therapy for breast cancer,known as selective estrogen receptor down transfers.It can be high affinity binding to the estrogen receptor, reducing the activity of receptors, while reducing the number of receptors,resulting in losing their activity, and it can not play its role in promoting proliferation.We combined p53 and fulvestrant,and then observed the changes in Morphological of breast cancer cell line MCF-7 and the expression levels of p53 protein and ER proteins, apoptosis of MCF-7 cells and the proliferation rate.We try to investigate the inhibition of cell proliferation both in terms of whether it has a synergistic effect,providing a new method for the clinical treatment of breast cancer, a new direction.Methods1. Observing the growth and morphological changes of control group and the experimental groupcells:control group (viral load group), fulvestrant (1 μmol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI)+fulvestrant (1 μmol/l) group acting on MCF-7 cells after 72h, using an inverted microscope control group, the experimental group of cell growth conditions and changes in morphology.2. Calculate the rate of cell proliferation inhibition of control group and the experimental group:control group (no load virus group), fulvestrant (1 μmol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI)+fulvestrant (1 μmol/l) group acting on MCF-7 cells after 72h, then calculating the control group, the experimental group cell proliferation rate.3. Comparing the apoptosis rate of control group and the experimental group:MCF-7 cells were dealed with control group (no load virus group), fulvestrant (1 μmol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI)+fulvestrant (1μmol/l) group after 72h, Detect apoptosis with AnnexinV-FITC/PI double staining successful double standard method using flow cytometry in the control group, the experimental group MCF-7 cells.4. Detect the expression levels of ER and p53 protein of control group and the experimental group:MCF-7 cells were dealed with the control group (viral load group), fulvestrant (1 μmol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI)+ fulvestrant (1 μmol/l) group after 72h, then detected the expression levels of p53 protein and ER protein.Results1.Inverted microscope observe the growth state of the cells:MCF-7 cells in the control group of adherent normally, it has no clear turbid medium, and cell growth state is good.MCF-7 cells dealed with Fulvestrant group may adherent, the medium is slightly turbid,Appearing a certain suspension cells.MCF-7 cells dealed with Ad-p53 were observed in poor state of adherent cell growth, low cell density, cell irregular, appear cellular debris, cell loss and other changes in apoptosis;.MCF-7 cells dealed with Ad-p53 and fulvestrant were low-density, the number of adherenting cells significantly reduced, a number of suspended cells appears.2. Effect of the combine of Ad-p53 and Fulvestrant on apoptosis of MCF-7 cell:Using flow cytometry analysis, the apoptosis rate of control, Fulvestrant (1μM) alone, Ad-p53 (50MOI) alone, Ad-p53 (50MOI)plus Fulvestrant (1μM) were 6.3±1.5%, 13.3±1.2%、14.5±2.9% and 38.1±5.9%, respectively.3. After being treated for 24h-96h by Ad-p53 alone or fulvestrant alone, the inhibition of cell proliferation rate respectively was(8.21±0.54)%, (28.5±1.42)%, (50.14±0.78)% and (58.25±2.92)% or(9.73±1.68)%, (25.26±0.82)%, (35.25±3.94)% and (46.37± 2.56)%.Simultaneously,the inhibition of cell proliferation rate of combined group was (12.42±1.76)%, (35.20±0.58)%, (62.08±2.56)% and (75.43%±3.56)%.4. Expressions of p53 protein were significantly upregulated in the cells treated with Ad-p53 plus Fulvestrant or Ad-p53 alone, but not Fulvestrant alone, Compared with the control group, ER protein levels were significantly upregulated in cells treated with Ad-p53 (50MOI) alone and downregulated in the cells treated with Ad-p53 plus Fulvestrant or Fulvestrant alone.Conclusions1. Obvious morphological change in MCF-7 cells were discovered by an inverted microscope when infected by Ad-p53 gene. An extremely low proliferation and early appearance of morphological change occurred when using fulvestrant with Ad-p53.2. MTS assay showed that while the proliferation inhibition rate of MCF-7 cells was increased with a time-dependent manner under all treatments, the simultaneous use of p53 with Fulvestrant showed the greatest result to inhibit the cell proliferation compared with p53 or Fulvestrant alone3-Ad-p53 plus Fulvestrant significantly increased the number of apoptotic cells compared with Ad-p53 alone (P< 0.05), suggesting a possible synergic effect of Ad-p53 and Fulvestrant on induction of apoptosis of MCF-7 cell.4.These results indicate that in MCF-7 cells, infection of Ad-p53 up-regulates ER expression and Fulvestrant blocks this upregulation, demonstrating that there may be a possible synergic effect between Ad-p53 and Fulvestrant.