5-对氟苄氧基杨梅醇对人肺腺癌A549-Homo BIRC5体内增殖的抑制作用及机制研究

Study on Inhibitory Effect and Mechanism of Myricanol 5-Fluorobenzyloxy Ether on Proliferation of Human Lung Adenocarcinoma Cells A549-Homo BIRC5 in Vivo

关键词:  5-对氟苄氧基杨梅醇  A549-Homo BIRC5  移植瘤  凋亡

Key words:  myricanol 5-fluorobenzyloxy ether  A549-Homo BIRC5  xenograft tumor  apoptosis

摘要:

目的 研究5-对氟苄氧基杨梅醇(myricanol 5-fluorobenzyloxy ether，5FEM)对人肺腺癌A549-Homo BIRC5体内增殖的抑制作用及机制。方法 构建A549-Homo BIRC5裸鼠移植瘤模型，随机分为模型组、溶剂(聚乙二醇400，2.5 mL·kg^-1)组和5FEM低、中、高剂量(20，40，80 mg·kg^-1)组，每组8只。各组每日腹腔注射给药，连续5周，每周测量移植瘤体积用于绘制肿瘤生长曲线，实验结束后处死裸鼠，剥离瘤体并称重。TUNEL法检测移植瘤组织细胞凋亡情况；RT-qPCR测定移植瘤组织中Caspase-9、PARP、Bax、Bcl-2和Survivin的mRNA表达水平；ELISA法检测瘤组织中Caspase-9、Survivin和PARP蛋白含量；免疫组化法检测瘤组织中Survivin、Caspase-9、Cleaved caspase-9、PARP和Cleaved PARP蛋白水平。结果 与模型组相比，5FEM(20，40，80 mg·kg^-1)能够显著减小移植瘤体积和减轻瘤重，呈一定剂量依赖性。TUNEL染色结果显示5FEM干预后肿瘤细胞凋亡较模型组明显增多。RT-qPCR结果显示，5FEM(20，40，80 mg·kg^-1)可以诱导瘤组织中Caspase-9和Bax的mRNA表达显著升高，并诱导Bcl-2、PARP和Survivin的mRNA表达降低。ELISA结果显示，5FEM(40，80 mg·kg^-1)可以显著增加瘤组织中Caspase-9蛋白表达量，并显著降低瘤组织中PARP和Survivin蛋白的表达量。免疫组化结果显示，5FEM(80 mg·kg^-1)处理后瘤组织中Caspase-9、Cleaved caspase-9和Cleaved PARP蛋白表达均显著升高，而PARP和Survivin蛋白表达被不同程度抑制。结论 5FEM可下调Survivin表达，促进Caspase-9和PARP活化，诱导肿瘤细胞凋亡，抑制肺腺癌A549-Homo BIRC5裸鼠移植瘤生长。

Abstract:

OBJECTIVE To investigate the inhibitory effect and mechanism of myricanol 5-fluorobenzyloxy ether(5FEM) on the proliferation of human lung adenocarcinoma A549-Homo BIRC5 in vivo.METHODS The model of A549-Homo BIRC5 xenograft tumors in nude mice was constructed and randomly divided into model group, solvent(polyethylene glycol 400, 2.5 mL·kg^-1) group and 5FEM low, medium, and high dose(20, 40, 80 mg·kg^-1, respectively) groups, with 8 mice in each group. Each group received daily intraperitoneal injection for 5 weeks, and the tumor volume was measured weekly to draw the tumor growth curve, and the nude mice were sacrificed after the experiment ended, solid tumors were harvested and weighed. The apoptosis of tumor tissue was analysed by TUNEL-staining. The mRNA expression level of Caspase-9, PARP, Bax, Bcl-2 and Survivin in tumor tissues were measured by RT-qPCR; ELISA assay was used to detect the protein levels of Caspase-9, Survivin and PARP in tumor tissues. Immunohistochemistry was adopted to detect the levels of Survivin, Caspase-9, Cleaved caspase-9, PARP and Cleaved PARP proteins in tumor tissues. RESULTS Compared with the model group, 5FEM(20, 40, 80 mg·kg^-1) could significantly reduce the volume and weight of the xenograft tumors in a dose-dependent manner. TUNEL staining showed that the apoptosis of tumor cells after 5FEM intervention was significantly higher than that in model group. RT-qPCR showed that 5FEM(20, 40, 80 mg·kg^-1) could significantly increase the mRNA expression of Caspase-9 and Bax, and decrease the mRNA expression of Bcl-2, PARP and Survivin in tumor tissues. ELISA results showed that 5FEM(40, 80 mg·kg^-1) could significantly increase the expression of Caspase-9 protein in tumor tissues, and significantly decrease the expression of PARP and Survivin protein in tumor tissues. Immunohistochemical results showed that the expressions of Caspase-9, Cleaved caspase-9 and Cleaved PARP protein in tumor tissues were significantly increased after 5FEM(80 mg·kg^-1) treatment, while the expression of PARP and Survivin protein were inhibited in different degrees. CONCLUSION 5FEM can down-regulate the expression of Survivin, promote the activation of Caspase-9 and PARP, induce the apoptosis of tumor cells, and inhibit the growth of lung adenocarcinoma A549-Homo BIRC5 xenograft tumors in nude mice.