The Expression Of UPA,uPAR Of Breast Cancer Cells In Hypoxia

OBJECTIVE: To study the effect ofhypoxia on the breast cancer cellsexpressing uPA, uPAR and HIF-la and to investigate its potentialmechanism in vitro. To study the contribution of celecoxib to the hypoxicbreast cancer tissues expressing uPA, uPAR and HIF-1a and to studyinfluence of apoptosis and angiogenesis of celecoxib to the hypoxic breastcancer in vivo,and further to explore feasibility that celecoxib was used totreat hypoxic tumors.METHODS:The hypoxic model was simulated by COCl₂ in vitro.Thehuman breast cancer MDA-MB-231 cells were exposed to the differentconcentration COCl₂(0, 50, 100, 150, 200μmol/L)for 16h respectively,to investigate the dose-efficiency relation.Meanwhile MB-231 cells wasexposed to CoCl₂(100μmol/L)for different time(0, 8, 16, 24,32h)respectively to investigate the time-effcicency relation.The uPA mRNA and uPAR mRNA levels were analyzed using RT-PCR.The uPA, uPAR and HIF-1αprotein were deteced through the immuno-cytochemical technique. MB-231 human breast cancer xenografts in nudemice were constructed successfully. These bearing tumor mice wereadministered with celecoxib(10mg/kg/d) which was a selective COX-2 inhibitor for 30 days. The expression ofuPA, uPAR and HIF-1αproteinwas detected by the immunohistochemical technique in the hypoxictissues of the xenografts and MVD was also assessed by theimmunohistochemical technique. Apoptosis index(AI)was detected by theTUNEL technique in the hypoxic tissues of the xenografts.RESULTS: In the dose-efficiency relation group, the expression ofuPA mRNA and uPAR mRNA in MB-231 cells treated with COC12(50, 100,150, 200μmol/L for 16h)was higher than that of control group (0μmol/LCOCl₂,16h) (P＜0.05). In the time-efficiency relation group, the expressionof uPA mRNA and uPAR mRNA in MB-231 cells treated with COCl₂(100μmol/L for 8, 16, 24, 32h) was higher than that of controlgroup(100μmol/L CoCl₂, Oh) (P＜0.05). The data indicated that expressionof uPA mRNA and uPAR mRNA up-regulated with increasing concentra-tions of CoCl₂ or the prolongation of treated time (P＜0.05).And the highexpression of uPA mRNA and uPAR mRNA was observed with 100μmol/Lin dose-dependent manner and for 16h in time-dependent manner, thendown-regulated gradually in the both dose-dependent and time-dependentmanners (P＜0.05).The similar effects of uPA, uPAR and HIF-1αwerealso observed in protein level. The expression of uPA, uPAR and HIF-1αof celecoxib treatment group in protein level was 0.207±0.049,0.203±0.048 and 0.181±0.058 respectively in vivo, the expression of uPA,uPAR and HIF-1αof control group in protein level was 0.298±0.071, 0.289±0.068 and 0.281±0.075 respectively in vivo. The expression ofuPA,uPAR and HIF-1αin protein level was significantly decreased in thehypoxic tissues of celecoxiib treatment group(P＜0.05).The MVD and AIof the celecoxiib treatment group was 19.80±2.59 and 5.616±0.892respectively and the MVD and AI of control was 26.80±3.42 and3.089±0.609 respectively.Celecoxib induced apoptosis of MB-231 cells andsuppressed angiogenesis of MB-231 cells in the hypoxic tissues of thexenografts (P＜0.01).CONCLUSION:â‘ The up-regulation of uPA,uPAR and HIF-1a is induced by hypoxiain the breast cancer MB-231 cells simulated by COCl₂ and the optimalcondiction is required for high expression of uPA,uPAR and HIF-1a in vitro.HIF-1a is presumed to be involved in the regulation of the uPA and uPARof MB-231 cells.â‘¡The celecoxib not only could induce apoptosis of breast cancerMB-231 cells but also suppress angiogenesis in the hypoxic tissues ofxenografts, which is probably correlated with the down-regulation ofuPA,uPAR and HIF-1a treated with celecoxib.â‘¢Celecoxib is hopefully a new choice in treatment hypoxic tumors.