Study On ADAMTS8 Inhibits MDA-MB-453 Cell Proliferation And Invasion,and Induces Apoptosis In Breast Cancer And Its Mechanism

Part One Effect of ADAMTS8 overexpression on proliferation and apo-ptosis of MDA-MB-453 cell and its mechanismObjective: To investigate the effect of ADAMTS8 on proliferation and apoptosis in breast cancer and its mechanism.Methods:1.Western blot assays were used to test the expression of ADAMTS8 in tumor tissues and adjacent non-tumor tissues of patients with breast cancer.2.Western blot assays were used to examine the expression of ADAMTS8 in the breast cancer cells lines(MDA-MB-231,MDA-MB-468,MDA-MB-453,MCF-7,T47D).3.Designed and transcriptive synthesized a overexpression plasmid targeting the ADAMTS8,which were transfected into MDA-MB-453 cells.Western blot assays were used to examine the expression of ADAMTS8 in MDA-MB-453 cells.4.CCK-8 assays were performed to examine the effect of ADAMTS8 overexpression on proliferation of the MDA-MB-453 cells.5.CCK-8 assays were performed to examine the effect of ADAMTS8 overexpression on chemosensitivity of the MDA-MB-453 cells.6.Flow cytometry were performed to detect the effect of ADAMTS8 overexpression on apoptosis of the MDA-MB-453 cells.7.Western blot assays were used to detect the effect of ADAMTS8 overexpression on the expression of P53 and C-Myc proteins in MDAMB-453 cells.Results:1.The ADAMTS8 protein level was evaluated in 20 tumor tissues and adjacent non-tumor tissues of patients with breast cancer by Western blotting.The result showed that the expression level of ADAMTS8 protein in patients with breast cancer was significantly lower than that in the adjacent normal tissues tested(P<0.05).2.The ADAMTS8 protein level was evaluated in five breast cancer cell lines by Western blot.MDA-MB-453 cells exhibited lower ADAMTS8 protein levels compared with other BC cell lines.Therefore,these MDA-MB-453 cells were used for further experiments.3.The MDA-MB-453 cells stably transfected with ADAMTS8 overexpression were successfully constructed.4.The ADAMTS8 protein level was elevated dramatically after ADAMTS8 overexpression plasmid transfection compared with that of the control vector and blank control group.5.Cell proliferation assays showed that overexpression of ADAMTS8 could significantly inhibit the proliferative ability,compared with the control vector and blank control groups(P<0.05).6.Chemosensitivity assays showed that overexpression of ADAMTS8 could significantly enhanced the chemosensitivity of MDA-MB-453 cells to paclitaxel(P<0.05).7.Flow cytometry showed that the percentage of apoptotic cells in the ADAMTS8 overexpression group was significantly higher than that of the control vector and blank control groups(P<0.05).8.Compared with the control vector and blank control groups,P53 and c-Myc proteins levels signifcantly decreased upon ADAMTS8 overexpression(P<0.05).Conclusions:1.Stably transfected with ADAMTS8 overexpression plasmid could effectively increased the expression of ADAMTS8 in breast cancer MDAMB-453 cell line.2.ADAMTS8 overexpression significantly inhibited the ability of proliferation,enhanced the chemosensitivity of MDA-MB-453 cells to cisplatin,and promoted apoptosis of the MDA-MB-453 cells.3.Decreased the expression of P53 and c-Myc might be the key mechanism for regulating apoptosis of the MDA-MB-453 cells.Part Two Effect of ADAMTS8 overexpression on migration and invasion of MDA-MB-453 cell and its mechanismObjective: To investigate the effect of ADAMTS8 on migration and invasion in breast cancer and its mechanism.Methods:1.A ADAMTS8 overexpression or control vector plasmid was transfected into MDA-MB-453 cells,and stably transfected MDA-MB-453 monoclonal cells was selected by puromycin.2.The effects of ADAMTS8 on the migration ability of MDA-MB-453 cells were investigated by wound-healing assays.3.The effects of ADAMTS8 on the migration ability of MDA-MB-453 cells were further investigated by Transwell migration assays.4.The effects of ADAMTS8 on the invasion ability of MDA-MB-453 cells were further investigated by Transwell invasion assays.5.Western blot assays were used to detect the effect of ADAMTS8 overexpression on the expression of MMP-7 proteins in MDA-MB-453 cells.Results:1.Wound-healing assays showed that the migration ability was markedly decreased in the MDA-MB-453 cells that overexpressed ADAMTS8 when compared with that of the control cells(P<0.05).2.Transwell migration assays showed that overexpressing ADAMTS8 inhibited the migration of MDA-MB-453 cells(P<0.05).3.Transwell invasion assays showed that overexpressing ADAMTS8 attenuated the invasiveness of MDA-MB-453 cells(P<0.05).4.MMP-7 proteins levels signifcantly decreased upon ADAMTS8 overexpression,compared with the control vector and blank control groups.Conclusions:1.ADAMTS8 overexpression significantly inhibited the ability of migration and invasion of MDA-MB-453 cells.2.Decreased the expression of MMP-7 might be the key mechanism for inhibiting the migration and invasion of the MDA-MB-453 cells.Part Three Effect of ADAMTS8 on the growth of MDA-MB-453 in vivoObjective: To investigate the effect of ADAMTS8 on the growth of breast cancer,a xenograft tumor assay was performed in nude mice.Methods:1.For the xenograft tumor growth assay,200μl MDA-MB-453 cells(2×106 cells per mouse)were stably transfected with a negative control,or an ADAMTS8 overexpressing plasmid was injected subcutaneously into the subcutaneous of each mouse.2.The size and growth rate of tumors was oberserved every week,and the volume of tumors were calculate.After 4 weeks,the mice were euthanized,and the tumors were removed,then the tumor growth curve was plotted.Results: Tumors formed from MDA-MB-453 cells stably transfected with ADAMTS8 overexpression were much smaller than those from the control vector and blank control groups.The volume of the tumors that stemmed from MDA-MB-453 cells stably transfected with p EZ-M90-ADAMTS8 were markedly reduced compared to that of the control vector and blank control groups 28 days after implantation.Conclusions: The present results showed that ADAMTS8 can inhibit the MDA-MB-453 cells xenograft tumor growth of the MDA-MB-453 cells.It is suggested that ADAMTS8 has potential as a new target for the treatment of breast cancer.