Liu Qian, Zhao Shanshan, Meng Fanguang, Wang Hankang, Sun Liwei, Li Guijie, Gao Feng, Chen Feng
Department of Radiology, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan, China.
Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China.
Front Oncol. 2021 Jun 9;11:661157. doi: 10.3389/fonc.2021.661157. eCollection 2021.
Higher oxidant stress capacity could promote invasion and metastasis. A previous study showed hepatocellular carcinoma (HCC) expressed more Nrf2 than para-carcinoma tissue. The chemotherapeutics such as epirubicin (EPI) could increase Nrf2 expression, while Camptothecin (CPT) could inhibit tumor growth by down-regulating the key molecule of antioxidant stress signal-Nrf2. The role of Nrf2 in invasion and metastasis was still unclear. In this study, we use EPI and CPT to determine the invasion and metastasis in Huh7 cells, H22 and Huh7 mouse models. In Huh7 cells, Nrf2 expression and ROS level were found increased after incubation with EPI by western blot and flow cytometry assay. But with the combination of EPI and CPT, inhibition of Nrf2 could decrease proliferation, invasion, and metastasis, which were investigated by CCK8 assay, wound healing, and Transwell assays. In Huh7 and H22 mouse models, EPI promoted Nrf2 up-regulation and nucleus translocation. Tumor growth was obviously inhibited with a single application of EPI or CPT. The combination of EPI and CPT could inhibit Nrf2 expression but demonstrated more suppressing effect of tumor growth than EPI. Western blot and immunohistochemical staining study revealed that Nrf2 inhibition was beneficial in decreasing the expression of N-cadherin, MMP9, Snail as well as Twist, and increasing E-cadherin, which were associated with epithelial-mesenchymal transition (EMT). Nrf2 down-regulation promoted lung metastasis of H22 cells . In addition, H&E staining and immunofluorescence staining of VEGFR suggested angiogenesis of Huh7 and H22 tumors was reduced. In conclusion, down-regulation of Nrf2 demonstrated inhibition of invasion, metastasis, and angiogenesis of hepatoma, which may provide a potential therapy in HCC.
更高的氧化应激能力可促进侵袭和转移。先前的一项研究表明，肝细胞癌（HCC）中Nrf2的表达高于癌旁组织。表柔比星（EPI）等化疗药物可增加Nrf2的表达，而喜树碱（CPT）可通过下调抗氧化应激信号的关键分子Nrf2来抑制肿瘤生长。Nrf2在侵袭和转移中的作用仍不清楚。在本研究中，我们使用EPI和CPT来确定Huh7细胞、H22和Huh7小鼠模型中的侵袭和转移情况。在Huh7细胞中，通过蛋白质免疫印迹法和流式细胞术检测发现，与EPI孵育后Nrf2表达和ROS水平升高。但联合使用EPI和CPT时，抑制Nrf2可降低增殖、侵袭和转移，这通过CCK8检测、伤口愈合实验和Transwell实验进行了研究。在Huh7和H22小鼠模型中，EPI促进Nrf2上调和核转位。单独使用EPI或CPT均可明显抑制肿瘤生长。EPI和CPT联合使用可抑制Nrf2表达，但对肿瘤生长的抑制作用比EPI更强。蛋白质免疫印迹法和免疫组织化学染色研究表明，抑制Nrf2有利于降低N-钙黏蛋白、基质金属蛋白酶9、Snail以及Twist的表达，并增加E-钙黏蛋白的表达，这些均与上皮-间质转化（EMT）相关。Nrf2下调促进了H22细胞的肺转移。此外，VEGFR的苏木精-伊红染色和免疫荧光染色表明，Huh7和H22肿瘤的血管生成减少。总之，Nrf2的下调表明对肝癌的侵袭、转移和血管生成具有抑制作用，这可能为HCC提供一种潜在的治疗方法。
