Jia Yongsen, Yan Xin, Cao Ying, Song Wei, Zhang Guangji, Hu Xueqin
Chinese Medicine College, North China University of Science and Technology, Tangshan 063210, China.
Postgraduate School, North China University of Science and Technology, Tangshan 063210, China.
Evid Based Complement Alternat Med. 2020 Sep 29;2020:3053506. doi: 10.1155/2020/3053506. eCollection 2020.
To explore the activity of (MTD) against Eca109 esophageal cancer (EC) cell invasion and metastasis and to ascertain the mechanism of its anticancer activity during the epithelial-mesenchymal transition (EMT) as mediated by the HIF-1-Snail axis.
Herbal compounds were prepared by ethanol extraction, and 6 herbs composing into MTD were dipped in water-free ethanol and filtered. The filtrate was collected and centrifuged. The remains were concentrated into a paste which was adjusted to 5000mg/mL concentration with DMSO. PBS was used to dilute the herbal solution to the half maximal inhibitory concentration. A hypoxic microenvironment was induced with CoCl in RPMI 1640 medium, in which Eca109 cells were cultured. The cytotoxicity of MTD was determined with CCK-8 assay. The activity of MTD against cell invasion and metastasis was explored with scratch assay and transwell assay. Western blot analysis was conducted to analyze the anticancer effects of MTD on the expression of HIF-1-Snail axis- and EMT-related proteins. Quantitative RT-PCR was used to assess the mRNA expression of Snail. Immunofluorescence labeling was performed to examine how MTD affected the coexpression of Snail and HIF-1.
The fifty percent inhibitory dose of MTD was 1410 g/mL in the normoxic environment and 1823 g/mL in the hypoxic environment based on the CCK-8 assay. The scratch assay showed that MTD significantly inhibited cell migration in both the normoxic and hypoxic microenvironments compared with the control groups ( < 0.05). The transwell assay showed that MTD significantly inhibited cell invasion in both the normoxic and hypoxic environments compared with the control groups ( < 0.05). Western blot showed that MTD significantly inhibited the expression of the HIF-1, Snail, Vimentin, MMP-2, MMP-9, and VE-cadherin proteins and significantly induced the expression of E-cadherin in both the normoxic and hypoxic microenvironments compared with the control groups ( < 0.05). qRT-PCR indicated that MTD significantly inhibited Snail mRNA expression compared with that in the control groups ( < 0.05). Immunofluorescence assay showed that MTD significantly inhibited the coexpression of HIF-1 and Snail in both the normoxic and hypoxic microenvironments compared with the control groups ( < 0.05).
MTD downregulated HIF-1-Snail axis- and EMT-related proteins to inhibit EC cell invasion and metastasis in both the normoxic and hypoxic environments.
探讨（MTD）对Eca109食管癌细胞侵袭和转移的作用，并确定其在由HIF-1-Snail轴介导的上皮-间质转化（EMT）过程中抗癌活性的机制。
通过乙醇提取制备草药化合物，将组成MTD的6种草药浸入无水乙醇中并过滤。收集滤液并离心。将残留物浓缩成糊状物，用二甲基亚砜将其浓度调整为5000mg/mL。用PBS将草药溶液稀释至半数最大抑制浓度。在含有Eca109细胞的RPMI 1640培养基中用氯化钴诱导低氧微环境。用CCK-8法测定MTD的细胞毒性。用划痕试验和Transwell试验探讨MTD对细胞侵袭和转移的作用。进行蛋白质免疫印迹分析以分析MTD对HIF-1-Snail轴和EMT相关蛋白表达的抗癌作用。用定量逆转录聚合酶链反应评估Snail的mRNA表达。进行免疫荧光标记以检查MTD如何影响Snail和HIF-1的共表达。
基于CCK-8试验，MTD在常氧环境中的半数抑制浓度为1410μg/mL，在低氧环境中为1823μg/mL。划痕试验表明，与对照组相比，MTD在常氧和低氧微环境中均显著抑制细胞迁移（P<0.05）。Transwell试验表明，与对照组相比，MTD在常氧和低氧环境中均显著抑制细胞侵袭（P<0.05）。蛋白质免疫印迹显示，与对照组相比，MTD在常氧和低氧微环境中均显著抑制HIF-1、Snail、波形蛋白、基质金属蛋白酶-2、基质金属蛋白酶-9和血管内皮钙黏蛋白的表达，并显著诱导E-钙黏蛋白的表达（P<0.05）。定量逆转录聚合酶链反应表明，与对照组相比，MTD显著抑制Snail mRNA表达（P<0.05）。免疫荧光试验表明，与对照组相比，MTD在常氧和低氧微环境中均显著抑制HIF-1和Snail的共表达（P<0.05）。
MTD下调HIF-1-Snail轴和EMT相关蛋白，从而在常氧和低氧环境中抑制食管癌细胞的侵袭和转移。
