中性粒细胞分泌的组织蛋白酶C对乳腺癌侵袭转移的影响

Effect of cathepsin C secreted by neutrophils on invasion and metastasis of breast cancer cells

目的探讨中性粒细胞分泌的组织蛋白酶C对乳腺癌侵袭、转移的影响。方法将MDA-MB-231乳腺癌细胞与中性粒细胞上清液共培养, 以组织蛋白酶C抑制剂(AZD7986)处理共培养细胞24 h, Transwell法检测3组细胞(MDA-MB-231细胞组、共培养组、共培养+AZD7986组)侵袭、迁移能力的变化。蛋白质印迹法(Western blot)检测3组细胞中CTSC、上皮-间充质转化(EMT)相关蛋白表达变化。将MDA-MB-231乳腺癌细胞经肠系膜上静脉注射入BALB/c雌性小鼠体内, 随机分为两组, 分别给予口服磷酸盐缓冲液(PBS, 100 μl)(对照组)或AZD7986(5 mg/kg)(实验组), 4周后观察小鼠肝脏成瘤情况。3组间比较采用单因素方差分析, 两组间比较采用独立样本t检验。结果 MDA-MB-231细胞组、共培养组、共培养+AZD7986组侵袭细胞数[(67.67±2.52)、(79.67±4.04)、(56.33±3.21)个, F(2, 6)=37.131, P<0.01]差异有统计学意义, MDA-MB-231细胞组、共培养组、共培养+AZD7986组迁移细胞数[(98.33±3.79)、(107.00±4.58)、(82.33±4.04)个, F(2, 6)=27.277, P<0.01]差异有统计学意义。共培养+AZD7986组、共培养组、MDA-MB-231细胞组CTSC蛋白水平(0.53±0.03、1.05±0.08、0.75±0.06, F(2, 6)=49.600, P<0.01)差异有统计学意义, 共培养+AZD7986组E-钙黏蛋白(E-cadherin)蛋白水平高于共培养组(1.05±0.05比0.58±0.06, t=16.060, P<0.01), 高于MDA-MB-231细胞组(1.05±0.05比0.94±0.08, t=4.882, P<0.05), N-钙黏蛋白(N-cadherin)蛋白水平低于共培养组(0.58±0.07比0.83±0.03, t=8.503, P<0.01), 低于MDA-MB-231细胞组(0.58±0.07比0.77±0.05, t=6.391, P<0.01), 波形蛋白(Vimentin)蛋白水平低于共培养组(0.52±0.08比0.75±0.05, t=7.866, P<0.01), 低于MDA-MB-231细胞组(0.52±0.08比0.67±0.03, t=5.173, P<0.01), Snail蛋白水平低于共培养组(0.66±0.05比0.83±0.05, t=5.997, P<0.01), 低于MDA-MB-231细胞组(0.66±0.05比0.77±0.02, t=4.711, P<0.05), ZEB蛋白水平低于共培养组(0.30±0.02比0.81±0.02, t=17.390, P<0.01), 低于MDA-MB-231细胞组(0.30±0.02比0.82±0.02, t=17.692, P<0.01)。在体内实验中, AZD7986组小鼠肝脏肿瘤体积低于对照组[(1.98×106±1.90×105)比(6.49×106±3.41×105), t=37.080, P<0.01]。结论通过抑制组织蛋白酶C的表达, MDA-MB-231乳腺癌细胞的侵袭迁移能力明显下降, 小鼠肝脏肿瘤体积明显减小。

Objective To investigate the effect of cathepsin C(CTSC)secreted by neutrophils on invasion and metastasis of breast cancer cells.Methods MDA-MB-231 cells were co-cultured with neutrophil supernatant for 24 h before being treated with the cathepsin C inhibitor(AZD7986).The Transwell method was utilized to assess the invasion and migration abilities of three groups(the MDA-MB-231 group,the co-culture group,and the co-culture+AZD7986 group).The Western blotting was utilized to detect changes in CTSC and epithelial-mesenchymal transition(EMT)protein expression.MDA-MB-231 cells were injected into BALB/c female mice via the superior mesenteric vein and randomly divided into two groups that were given either oral phosphate buffer saline(PBS,100μL)(the control group)or AZD7986(5 mg/kg)(the experimental group),and tumor formation in the liver of mice was observed after 4 weeks.The one-way analysis of variance(ANOVA)was used to compare the three groups,and the independent sample t-test was used to compare the two groups.Results The number of invasive cells varied significantly among the MDA-MB-231 group,the co-culture group,and the co-culture+AZD7986 group[(67.67±2.52),(79.67±4.04),(56.33±3.21),F(2,6)=37.131,P<0.01],and the number of migrating cells varied significantly among the MDA-MB-231 group,the co-culture group,and the co-culture+AZD7986 group[(98.33±3.79),(107.00±4.58),(82.33±4.04),F(2,6)=27.277,P<0.01].There were significant differences in CTSC protein levels among the co-culture+AZD7986 group,the co-culture group,and the MDA-MB-231 group(0.53±0.03,1.05±0.08,0.75±0.06,F(2,6)=49.600,P<0.01).In the co-culture+AZD7986 group,E-cadherin protein levels were higher than in the co-culture group(1.05±0.05 vs.0.58±0.06,t=16.060,P<0.01)and in the MDA-MB-231 group(1.05±0.05 vs.0.94±0.08,t=4.882,P<0.05).N-cadherin protein levels were lower in the co-culture+AZD7986 group than in the co-culture group(0.58±0.07 vs.0.83±0.03,t=8.503,P<0.01)and the MDA-MB-231 group(0.58±0.07 vs.0.77±0.05,t=6.391,P<0.01).Vimentin protein levels were lower in the co-culture+AZD7986 group than in the co-culture group(0.52±0.08 vs.0.75±0.05,t=7.866,P<0.01)and the MDA-MB-231 group(0.52±0.08 vs.0.67±0.03,t=5.173,P<0.01).Snail protein levels in the co-culture+AZD7986 group were lower than in the co-culture group(0.66±0.05 vs.0.83±0.05,t=5.997,P<0.01)and the MDA-MB-231 group(0.66±0.05 vs.0.77±0.02,t=4.711,P<0.05).ZEB protein levels in the co-culture+AZD7986 group were lower than in the co-culture group(0.30±0.02 vs.0.81±0.02,t=17.390,P<0.01)and the MDA-MB-231 group(0.30±0.02 vs.0.82±0.02,t=17.692,P<0.01).In the in vivo experiment,the liver tumor volume of mice in the AZD7986 group was smaller than that in the control group[(1.98×106±1.90×105)vs.(6.49×106±3.41×105),t=37.080,P<0.01].Conclusion The invasion and migration capacity of MDA-MB-231 cells was significantly reduced by inhibiting cathepsin C expression,as was the volume of liver tumors in mice.