异牡荆黄素通过调节 MnSOD 和 FoxM1 降低肝癌干细胞的致癌性和干性。

Isovitexin reduces carcinogenicity and stemness in hepatic carcinoma stem-like cells by modulating MnSOD and FoxM1.

机构信息

Department of Pharmaceutical Science, Medical College, Hunan Normal University, Changsha, 410013, Hunan, China.

Key Laboratory of Study and Discover of Small Targeted Molecules of Hunan Province, Changsha, 410013, Hunan, China.

出版信息

J Exp Clin Cancer Res. 2019 Jun 17;38(1):264. doi: 10.1186/s13046-019-1244-6.

DOI:10.1186/s13046-019-1244-6

PMID:31208440

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6580799/

Abstract

BACKGROUND

Manganese superoxide dismutase (MnSOD) upregulating FoxM1 have previously been demonstrated promoting lung cancer stemness. Isovitexin exhibits antitumor activities in various cancers. This study aimed to assess whether isovitexin inhibits hepatic carcinoma stem-like cells (HCSLCs) features via regulating MnSOD and FoxM1 expression.

METHODS

Second-generation spheres from the hepatic carcinoma cell lines, respectively, were used as HCSLCs. Protein amounts of MnSOD, FoxM1 and stemness-associated markers (CD133, CD44, ALDH1, Bmi1, Nanog and Oct4) were determined by immunoblotting. In vitro carcinogenicity was evaluated by sphere- and colony-formation assays. The effects of isovitexin on HCSLC carcinogenicity and stemness were examined in vitro and in xenograft models. An adenoviral delivery system was employed to manipulate MnSOD and/or FoxM1. Luciferase reporter assay was performed to verify isovitexin downregulated FoxM1 by inhibiting MnSOD-mediated effects of E2F1 and/or Sp1 on activation of FoxM1 promoter.

RESULTS

FoxM1 upregulation by MnSOD contributed to carcinogenicity and stemness, with increased sphere- and colony-formation capabilities, upregulated stemness-associated markers and CD133 subpopulation as well as elevated oncogenicity in vivo in HCSLCs compared with hepatic carcinoma cells. Isovitexin substantially decreased sphere and colony formation rates, and stemness-associated markers in cultured HCSLCs by suppressing MnSOD and FoxM1 expression. Importantly, isovitexin significantly inhibited tumor growth of in nude mice bearing HCSLCs and reduced CD133 protein expression of xenograft in nude mice. MnSOD or FoxM1 knockdown enhanced the effects of isovitexin suppression on carcinogenicity and stemness in HCSLC. MnSOD or FoxM1 overexpression attenuated the effects of isovitexin. Additionally, isovitexin and MnSOD knockdown could inhibit FoxM1 reporter activity via a decreased binding of E2F1 and/or Sp1 onto FoxM1 promoter. FoxM1 overexpression reversed the effects of isovitexin combined with MnSOD knockdown, without affecting MnSOD expression. Moreover, MnSOD knockdown plus thiostrepton, a FoxM1 specific inhibitor, cooperated with isovitexin to repress xenograft tumor growth and downregulate MnSOD and FoxM1 in nude mice bearing HCSLCs from MHCC97H cells.

CONCLUSIONS

Isovitexin inhibits carcinogenicity and stemness in HCSLCs by downregulating FoxM1via inhibition of MnSOD.

摘要

背景

锰超氧化物歧化酶（MnSOD）上调 FoxM1 先前已被证明可促进肺癌干细胞特性。异荭草苷在各种癌症中具有抗肿瘤活性。本研究旨在评估异荭草苷是否通过调节 MnSOD 和 FoxM1 表达来抑制肝癌干细胞样细胞（HCSLCs）的特征。

方法

分别使用肝癌细胞系的第二代球体作为 HCSLCs。通过免疫印迹法测定 MnSOD、FoxM1 和干细胞相关标记物（CD133、CD44、ALDH1、Bmi1、Nanog 和 Oct4）的蛋白含量。通过球体和集落形成测定评估体外致癌性。在体外和异种移植模型中研究异荭草苷对 HCSLC 致癌性和干性的影响。采用腺病毒传递系统来操纵 MnSOD 和/或 FoxM1。进行荧光素酶报告基因测定以验证异荭草苷通过抑制 MnSOD 介导的 E2F1 和/或 Sp1 对 FoxM1 启动子激活的影响来下调 FoxM1。

结果

MnSOD 上调 FoxM1 促进了致癌性和干性，与肝癌细胞相比，HCSLCs 的球体和集落形成能力增强，干细胞相关标记物和 CD133 亚群上调，体内致瘤性增强。异荭草苷通过抑制 MnSOD 和 FoxM1 表达，显著降低培养的 HCSLC 中的球体和集落形成率以及干细胞相关标记物。重要的是，异荭草苷显著抑制裸鼠携带 HCSLC 的肿瘤生长，并降低裸鼠异种移植中的 CD133 蛋白表达。MnSOD 或 FoxM1 敲低增强了异荭草苷对 HCSLC 致癌性和干性的抑制作用。MnSOD 或 FoxM1 过表达减弱了异荭草苷的作用。此外，异荭草苷和 MnSOD 敲低可通过减少 E2F1 和/或 Sp1 与 FoxM1 启动子结合来抑制 FoxM1 报告基因活性。FoxM1 过表达逆转了异荭草苷与 MnSOD 敲低联合的作用，而不影响 MnSOD 表达。此外，MnSOD 敲低加硫代丝菌素，一种 FoxM1 特异性抑制剂，与异荭草苷合作抑制裸鼠携带 MHCC97H 细胞的 HCSLC 异种移植肿瘤生长，并下调裸鼠中的 MnSOD 和 FoxM1。