BMP-2-reduced Expression Of VEGF Via P38MAPK Pathway

IntroductionBreast cancer is one of female universal malignant tumor.In developed country,it is the second of all cause of death.The similar with carcinoma of prostate,breast cancer has prone to osseous metastasis.Tumor vessel certainly is a important factor of tumor genesis,many vascular growth factors are expressed in tumor.VEGF is one of most effective factor,it may directed or undirected participate in every process of neo-blood vessel.There is research indicated that BMP has important effect in onset and transfer of bone tumor and BMP-2 especially outstanding.The mitogen-activated protein kineses(MAPKs)are serine/threonine kinases,which generally exist in various cells.p38MAPK is one important member of mitogen-activated protein kinase(MAPK) family.Recently P38MAPK pathway.Therefore we research BMP-2,VEGF and p-p38 expression in breast cancer tissues,and in vitro research the correlation between p38MAPK pathway and BMP-2 proteinum in order to offer corresponding theory of treatment of breast cancer.Material and Methods1.SamplesA total of 68 paraffin-embedded breast cancer tissues obtained from the surgical resection at the pathological department of the First Affiliated Hospital of China Medical University between the years 2003 to 2006.According to the World Health Organization breast carcinoma histological classification criteria(2003),all specimens were infiltrating ducal carcinoma.Human breast cancer cells MDA-MB-435s and MCF-7 and human breast cells 10A were obtained from Chinese Peking Union University.2.ReagentsThe first antibodies were murine monoclonal antibody to p-p38 and rabbit polyclonal antibody to integrin BMP-2 and VEGF was purchased from Sigma and Cell signaling.DMEM and RPMI 1640 were purchased from Gibco.3.Methods(1)Immunohistochemistry(S-P):IHC was performed according to the indirect streptavidin-biotin-hyperoxidase method,as manufacture protocol.For the negative controls, the primary antibody was omitted by PBS,but all incubation steps were identical.Previously identified strongly staining tumor tissue sections were used as positive controls.We used an intensity-adjusted scoring system to evaluate immunostaining indices.The sections were scanned by light microscope.Five fields were randomly selected and 100 tumor cells in each field were counted.The staining correspond to negative and positive,respectively.VEGF and p-p38:With respect to the invasive cell count,＜50%positive cells or no color were negetive,＞50%were positive,no color or less yellow were negative,yellow or strong yellow were positive.(2)Western blot:Protein concentration was measured by Bradford method.Tissue lysates were electrophoresed in polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes,Protein bands were blocked and incubated with the first and second antibody and visualized with DAB kit.Protein contents were calculated by densitometry.(3)Transwell:in vitro Cell Migration and Invasion Assays48 h after transfectd,cells from culture flasks was resuspend in serum-free media at a density of 5×105 cells/mL, then 100 ul of the cell suspension was pipetted onto the matrigel(1:3 dilution,BD Biosciences)prepared 2h before according the manufacturer's protocol.Lower chamber of the 24-transwell is filled with 600 ul of culture media containing 10%FBS as a chemoattractant.The chambers were incubated for 24h followed by washed with PBS,fixed in 100%methanol,stained with Hematoxylin,photographed,and counted. 4.Statistical analysisThe results were compared using chi-squared test and T-test.p＜0.05 was considered statistically significant.Results1.BMP-2,VEGF and p-p38 expression in breast cancer tissues and correlation with lymph node statusImmunohistochemical results showed that p-p38 protein was observed in nucleus of breast cancer cells and stained darkly and its positive rate is 57.50%(46/80).BMP-2 was observed in cytoplasm of breast cancer cells and stained darkly and its positive rate is 56.25%(45/80).VEGF was in breast cancer cells and stained darkly and its positive rate is 86.25%(69/80).Statistical analysis suggested a positive relationship between expression of p-p38,BMP-2,VEGF(p＜0.05).2.Expression of BMP-2,VEGF and p-p38 in three differently metastatic breast cancer cellsWestern blot analysis indicated that the expression of BMP-2,VEGF andp-p38 in the highly metastatic MDA-MB-435s cells was higher than that in lowly metastatic MCF-7 cells and higher than that in nomal metastatic 10A cells.3.Change of VEGF and p-p38 protein expression after blocked by Nogin(specific inhibitor of BMP-2)The expression level of VEGF and p-p38 protein in breast cancer MDA-MB-435Ss cells decreased after Nogin was added into the culture fluid of MDA-MB-435s cells.4.Change of VEGF protein expression after blocked by SB203580 (specific inhibitor of p-p38)The expression level of VEGF protein in breast cancer MDA-MB-435Ss cells decreased after SB203580 was added into the culture fluid of MDA-MB-435s cells.5.Cell invasion ability changed by Nogin and SB203580Transwell results showed that MDA-MB-435Ss cells decreased 48%and 57% after nogin and SB203580 was added into the culture fluid of MDA-MB-435s cells.DiscussionAt first,we researched the expression of BMP-2,p-p38,VEGF in breast cancer. We discovered that BMP-2,VEGF and p-p38 expression in breast cancer tissues and correlation with lymph node status.This prompted that the expression of BMP-2,VEGF and p-p38 expression in breast cancer tissues may correlated with the malignant advancement and invasion of breast cancer.Then we supposed that BMP-2 may reduce the expression of VEGF via p38MAPK pathway.We detected expression of three proteinum in three different metastasis level cell lines.Western blot analysis indicated that the expression of BMP-2,VEGF and p-p38 in the highly met static MDA-MB-435s cells was higher than that in lowly met static MCF-7 cells and higher than that in normal met static 10A cells.At the same time we discovered that the expression level of VEGF and p-p38 protein in breast cancer cells decreased by blocked BMP-2;the expression level of VEGF protein in breast cancer cells decreased by blocked p38MAPK pathway.All of this explained that BMP-2 may reduce the expression of VEGF via p38MAPK Pathway in breast cancer.All of our experiment demonstrated that BMP-2 may reduce the expression of VEGF via p38MAPK Pathway in breast cancer.It may in offer corresponding theory for p38MAPK pathway acted breast cancer treatment target.Conclusion1.BMP-2,VEGF and p-p38 expression in breast cancer tissues and correlation with lymph node status. 2.BMP-2-reduced Expression of VEGF via p3 8MAPK Pathway.