Lps Induces Angiogenesis And Increases Lung Metastasis In Breast Cancer Via Pge2-ep2Pathway

BACKGROUNDAccording to the American cancer statistics, the United States will have1665,540new cancer cases in2014, among which585,720patients will die of malignant tumor. In American women, the incidence of breast cancer accounted for the first in malignant tumor, and metastasis is a major cause of death in patients with breast cancer. Cancer metastasis is a complicated process affected by factors including the tumor cell and host environment, In1889, Paget first proposed the theory of "seed and soil", namely the cancer cells (seed) need proper microenvironment (soil) for growth, infiltration and metastasis. It was believed that the tumor microenvironment plays an important role in the process of tumor progression. Inflammation is proved to be an important environment factor which promotes tumor metastasis by regulating tumor cell activity via cytokines, chemokines, activated oxygen and so on. The relationship between inflammation and tumor metastasis become one of research focus. The aim of this study is to explore the role of inflammation in the process of pulmonary metastasis of breast cancer and a possible mechanism.METHODS50female BALB/c mice were randomly divided into5groups:(1) PBS group: intraperitoneal injection of phosphate buffer solution (PBS);(2) LPS groups: intraperitoneal injection of lipopolysaccharide (LPS) for3days (5mg/Kg/d);(3) PT group:intraperitoneal injection of PBS+tail vein injection of4T1cells(1×105) on the4th day;(4) LT group:intraperitoneal injection of LPS+tail vein injection of4T1 cells;(5) LTC group:intraperitoneal injection of LPS+tail vein injection of4T1cells+intraperitoneal injection of celecoxib (Cele,5mg/kg/d). Among them, the PBS group and LPS group were sacrificed on the4th day; PT group, LT group and LTC group were sarcrificed after4weeks. Vascular endothelial growth factor (VEGF) and Prostaglandin-E2(PGE2) levels in serum of all mice were assayed by enzyme-linked immunoabsorbent assay (ELISA). Lung tissue were collected for pathological examination to observe pulmonary metastases and blood vessel density in lungs were analyzed with immunofluorescence CD31staining. Cyclooxygenase (COX-2) expression of lung tissue proteins were determined by Western blotting. In vitro, proliferation of4T1cells treated with LPS was determined by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Mice pulmonary vascular endothelial cells (MPVEC) were stimulated with PGE2, PGE2receptor agonist, PGE2receptor antagonist, and culture supernatant were collected to assay VEGF expression. We also examined VEGF proteins produced by PGE2-treated cells by Western blotting. MPVECs were stimulated with VEGF, PGE2, Celecoxib and in-vitro angiogenic of endothelial cells was observed with tube forming test.RESULTS1.LPS induces systemic inflammatory in mouse modelLungs of LPS mice were edema, congestion and the volume of lungs was larger than that of PBS group. Hematoxylin-eosin (HE) staining showed that alveolar walls were interrupted in lungs of LPS group. There were visible capillary hyperemia and inflammatory exudation in alveolar space in lungs of LPS mice.2.LPS increases angiogenesis in lungsPulmonary angiogenesis of mice was assayed by confocal laser scanning microscope. It showed that pulmonary vessel density in LPS-treated mice was remarkably increased than PBS-treated mice. After treatment with celecoxib (5mg/kg/d), CD31positive cells in lung tissue of LTC group were obviously reduced compared to LT group.3.LPS increases pulmonary metastasis of breast cancer Pulmonary metastasis was observed with HE staining. The number and size of metastatic lesions in lungs of LPS-treated mice(LT group) were significantly greater than lungs of PBS-treated mice(PT group). Treatment with celecoxib significantly decreased pulmonary metastasis in LTC group compared to LT group.4.LPS has no obvious effect on4T1cell proliferationThe proliferation of4T1cells was assayed by MTT experiments. It turned out that treatment with different concentrations of LPS (0,10ug/ml,100ug/ml) for24hours had no significant difference on the proliferation of4T1cells (P<0.05).5.LPS increases VEGF productionIn vitro, MPVECs were treated with various concentrations of PGE2(10nM,100nM,200nM) for24hours, the release of VEGF by MPVECs into monolayer culture medium and VEGF expression in endothelial cell were significantly increased in a concentration dependent manner. In vivo, VEGF and PGE2levels in the serum of LPS-treated mice were higher than PBS-treated mice. Treatment with celecoxib could counteract the effects of LPS.6.Celecoxib inhibits the angiogenesis on MPVECsEffects of celecoxib, VEGF and PGE2on angiogenesis was observed with tube forming test on MPVECs. The result showed that treatment with celecoxib alone could significantly reduce MPVECs to form tube-like structure. PGE2or VEGF alone promoted formation of tube-like structure, and such effect was counteracted by celecoxib.7.LPS increases VEGF production by PGE2-EP2pathwayVEGF production was increased by ONO-AE1-259-01, a specific EP2receptor agonist. However, such increases could be counteracted by AH6809, a specific EP2receptor antagonist. Other EP receptor agonists had no significant effects on VEGF production.CONCLUSION1. LPS promotes angiogenesis and pulmonary metastasis of breast cancer by increasing the production of VEGF. 2. PGE2can regulate the release of VEGF by MPVECs, and it is mainly mediated by PGE2-EP2pathway.