血管紧张素Ⅱ受体拮抗剂调节乳腺癌脑转移肿瘤微环境抑制肿瘤增殖的机制研究

Mechanism of angiotensinⅡreceptor antagonist modulating the tumor microenvironment of breast cancer brain metastases and inhibiting tumor proliferation

目的探讨血管紧张素Ⅱ受体拮抗剂(氯沙坦)对乳腺癌脑转移肿瘤微环境的调节作用及其抑制肿瘤增殖的潜在机制。方法取鼠源性4T1乳腺癌细胞株通过蛋白质免疫印迹(WB)法检测其血管紧张素Ⅱ(AngⅡ)、AngⅡ受体1型(ATIR)的表达水平;通过CCK-8实验检测对照组、氯沙坦组(氯沙坦)、紫杉醇组(化疗药,紫杉醇)、联合用药组(氯沙坦联合紫杉醇)4组中4T1乳腺癌细胞的增殖情况。建立4T1乳腺癌脑转移荷瘤小鼠模型,同样随机分为4组(每组n=4),通过小鼠头颅MRI检查观察颅内肿瘤最大径及药物调节肿瘤的增殖情况;采用免疫组织化学及免疫荧光染色方法检测肿瘤微环境中胶原变化相关的α-1I型胶原蛋白(COL1A1)、血管分布相关的血小板-内皮细胞黏附分子(CD31)及缺氧程度相关的缺氧诱导因子1α(HIF-1α)的表达情况,评估氯沙坦对乳腺癌脑转移肿瘤微环境的作用。结果WB结果显示,与健康小鼠的脑组织相比,4T1乳腺癌细胞中AngⅡ的表达量显著增高(分别为0.29±0.07和0.50±0.11,t=3.28,P=0.017);AT1R基因表达为阳性。CCK-8实验结果显示,4组相对吸光度值的差异具有统计学意义(F=556.40,P<0.001)而对照组与氯沙坦组、紫杉醇组与联合用药组的差异均无统计学意义(均P>0.05)。小鼠头颅MRI结果显示,4组颅内肿瘤最大直径均呈线性增长趋势,差异具有统计学意义(F=3.27,P=0.049);在药物治疗28d,与对照组相比,氯沙坦组的肿瘤最大直径较小,与紫杉醇组相比,联合用药组的肿瘤最大直径较小,差异均具有统计学意义(均P<0.05)。免疫组织化学染色结果显示,4组COL1A1阳性率的差异具有统计学意义(F=64.47,P<0.001);免疫组织荧光染色结果显示,4组CD31、HIF-1α阳性率的差异均具有统计学意义(均P<0.05)。结论AngII受体抗剂可能是通过调节乳腺癌脑转移肿瘤的微环境,降低缺氧程度,增强瘤内血管分布,提高化疗药物的治疗效果进而抑制肿瘤细胞的增殖,而非直接作用于肿瘤细胞。

ObjectiveeTo investigate the regulatory role of Angiotensin Ⅱ(Ang Ⅱ)receptor antagonist(Losartan)in the tumor microenvironment of breast cancer brain metastases and the potential mechanism by which it inhibits tumor proliferation.Methods Western bltting(WB)was used to detect the expression levels of Ang Ⅱ and Ang Ⅱ receptor(ATIR)in the 4TI breast cancer cell line.The CCK-8 assay was used to evaluate the effects of Control group,Losartan group(Losartan)Paclitaxel group(chemotherapy drug,Paclitaxel),and Combination drug group(Losartan combined with Paclitaxel)on the proliferation of 4T1 breast cancer cells.A 4T1 animal model of breast cancer cerebral metastasis was developed.Randomly,the animals were separated into four groups:Control,Losartan,Paclitaxel,and Combination drug(each n=4).Based on the head MRI results,tumor diameters and the drug effects on tumor growth were monitored.Immunohistochemistry and immunofluorescence staining were used to assess the gene expression of collagen type I alpha 1 chain(COLIA1),platelet endothelial cell adhesion molecule(CD31)and hypoxia-inducible factor 1-alpha subunit(HIF-1α)in the tumor microenvironment.This study evaluated the function of Losartan in the tumor microenvironment of brain metastases from breast cancerR.esults The relative expression of Ang I in the 4T1 breast cancer cell line was significantly higher than in the brain tissue of normal rodents(respectively:0.29±0.07,0.50±0.11,t=3.28,P=0.017);the 4T1 breast cancer cell line expressed AT1R.The CCK-8 assay revealed a difference in the relative absorbance of the four groups(F=556.40,P<0.001).There was no significant difference between Control group and Losartan group,Paclitaxel group and Combination group(all P>0.05).The MRI results revealed that the tumor size in all four groups grew linearly relative to the size at the time of entry,with a difference between the four groups(F=3.27,P=0.049).After 28 days of medication,compared with the Control group,the maximum tumor diameter of Losartan group was smaller,and that of Combination group was smaller than that of Paclitaxel group,and the differences were statistically significant(both P<0.05).The immunohistochemical staining results showed the positive rates of COLIA1 was significantly different among the four groups(F=64.47,P<0.001).The immunofluorescence staining results showed the positive rates of CD31 and HIF-1αamong four groups were statistically significant(all P<0.05).Conclusion Ang I receptor antagonist enhances the efficacy of chemotherapeutic agents and inhibits tumor cell proliferation by modulating the tumor microenvironment of breast cancer brain metastases,reducing hypoxia,and enhancing intra-tumoral vascular distribution,instead of directly acting on tumor cells.