中国癌症杂志 ›› 2020, Vol. 30 ›› Issue (2): 113-121.doi: 10.19401/j.cnki.1007-3639.2020.02.005

• 论著 • 上一篇    下一篇

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

温培楠 1 ,林明恕 1 ,金 冕 1 ,徐贤绸 1 ,陈龙云 2   

  1. 1. 浙江省平阳人民医院普外科,浙江 温州 325400 ;
    2. 湖北中医药大学基础医学院,湖北 武汉 430065
  • 出版日期:2020-02-29 发布日期:2020-03-06
  • 通信作者: 徐贤绸 E-mail: songtaoqi2015@126.com
  • 基金资助:
    湖北中医药大学青苗计划(2016ZZX023,XJ2014KJ014)。

Dihydromyricetin inhibits gastric cancer angiogenesis in vitro and in vivo through ERK/VEGFA/VEGFR2 signaling pathway

WEN Peinan 1 , LIN Mingshu 1 , JIN Mian 1 , XU Xianchou 1 , CHEN Longyun 2   

  1. 1. Department of General Surgery, the People’s Hospital of Pingyang, Wenzhou 325400, Zhejiang Province, China; 2. Basic Medical College School of Laboratory Medicine, Hubei University of Chinese Medicine, Wuhan 430065, Hubei Province, China
  • Published:2020-02-29 Online:2020-03-06
  • Contact: XU Xianchou E-mail: songtaoqi2015@126.com

摘要: 背景与目的:二氢杨梅素(dihydromyricetin,DHM)通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用,并研究其可能作用机制。方法:不同剂量DHM作用体外人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)和胃癌细胞系MKN28后,采用细胞计数试剂盒(cell counting kit-8,CCK-8)法筛选无毒剂量的DHM,Transwell小室实验测定细胞侵袭;小管形成实验测定HUVEC体外血管生成能力;采用蛋白质印迹法(Western blot)检测血管内皮生长因子A(vascular endothelial growth factor A, VEGFA)、phospho-血管内皮生长因子受体(vascular endothelial growth factor receptor, VEGFR)2、细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、phospho-c-Jun氨基端激酶(c-Jun NH2-terminal kinase,JNK)和phospho-p38表达水平;基质胶塞实验测定DHM对体内血管形成的影响;通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型,观察DHM给药对体内胃癌生长的影响,Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果:(0.5~2.5 μmol/L)的DHM对HUVEC和MKN28细胞生长无明显抑制作用,该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成,VEGFA(20 μg/L)可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果;DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降,而对p-p38和p-JNK的表达无明显影响;DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降;低剂量(30 mg/kg)DHM对裸鼠无明显的不良反应,但可抑制体内血管形成,同时有效延缓体内胃癌生长,体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用,但低剂量DHM对Ki-67无明显作用。论:低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长,该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。

关键词: 二氢杨梅素, 胃癌, 细胞外信号调节激酶, 血管生成

Abstract: Background and purpose: Dihydromyricetin (DHM) exerts anti-cancer effects by inhibiting cell cycle, promoting apoptosis and decreasing angiogenesis. The aim of this study was to investigate the anti-angiogenic effect of DHM on the gastric cancer in vitro and in vivo and its mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) and human gastric cancer MKN28 cells were exposed to increasing doses of DHM. Nontoxic doses of DHM were screened by cell counting kit-8 (CCK-8) assay. The migratory capacity of HUVECs was analyzed using transwell migration chambers. The capillary tube formation assay was used to evaluate the angiogenic activity of HUVEC. Western blot was employed to analyze the levels of vascular endothelial growth factor A (VEGFA), phospho-vascular endothelial growth factor receptor (VEGFR) 2, phospho-extracellular signal-regulated kinase (ERK), phospho-c-Jun NH2-terminal kinase (JNK) and phospho-p38 in the HUVEC and MKN28 cells. Matrigel plug assay was employed to analyze the anti-angiogenic potential of DHM in vivo. MKN28 cells were implanted subcutaneously into right flank of nude mice to observe the anti-cancer effect of DHM in vivo. Immunohistochemistry was employed to analyze the levels of Ki-67, CD34 and VEGFA in the xenografts after DHM treatment. Results: DHM exerted no cytotoxic effects on the growth of both HUVECs and MKN28 cells from the concentration of 0.5 μmol/L to 2.5 μmol/L. However, nontoxic doses of DHM treatment significantly inhibited HUVEC migration and tube formation, and the suppressive effects of DHM on HUVEC migration and tube formation were partly reversed by the addition of VEGFA. In MKN28 cells, the levels of VEGFA and phospho-ERK declined in a dosage-dependent manner after DHM treatment, whereas the addition of DHM did not change the expression levels of phospho-JNK and phospho-p38. DHM treatment also triggered a dosage-dependent phospho-VEGFR2 down-regulation in HUVEC. In vivo experiment showed the angiogenesis and the growth of subcutaneously implanted tumors were notably inhibited by DHM at low dosage with almost no chemotoxic side effects. The protein level of CD34 and VEGFA in tumor tissues were obviously decreased by the treatment of DHM. Conclusion: A low dosage of DHM inhibits the angiogenesis of gastric cancer both in vitro and in vivo. And the anti-angiogenic function of DHM, at least partly, is realized by inactivating ERK/VEGFA/VEGFR2 signaling pathway.

Key words: Dihydromyricetin, Gastric cancer, Extracellular signal-regulated kinase, Angiogenesis