乙氧基二氢血根碱靶向EGFR抑制结直肠癌细胞恶性生物学行为并诱导凋亡的机制研究
The Mechanism of Ethoxydihydrosanguinarine Targeting EGFR to Inhibit Malignant Progression and Induce Apoptosis of Colorectal Cancer
谭苗 1钱琛 1向可 2段钟琪 3万芳 3刘莹1
作者信息
- 1. 湖北医药学院 基础医学院,十堰 442000;湖北医药学院 武当特色中药研究湖北省重点实验室,十堰 442000
- 2. 湖北文理学院附属谷城医院,襄阳 441700
- 3. 湖北医药学院 基础医学院,十堰 442000
- 折叠
摘要
目的 探讨乙氧基二氢血根碱(ethoxydihydrosanguinarine,ESG)对结直肠癌细胞的抑制及具体分子机制.方法 选用结直肠癌细胞系RKO和HRT-18.实验分为空白对照组和加药组(1.0、1.5、2.0 μmol/L ESG).通过CCK-8法、实时无标记细胞分析技术(real time cellular analysis,RTCA)及克隆形成实验检测ESG对细胞增殖能力的影响;以流式细胞术、DAPI染色、JC-1线粒体膜电位和活性氧检测试剂盒检测ESG对细胞凋亡的影响;通过高内涵成像分析技术及Transwell侵袭实验检测ESG对细胞侵袭、迁移的影响;Western blot检测细胞凋亡及侵袭、迁移相关蛋白的表达,表皮生长因子受体(epidermal growth factor receptor,EGFR)以及下游Akt/ERK信号通路蛋白的表达及磷酸化水平;通过过表达EGFR和敲低EGFR探讨EGFR在ESG诱导结直肠癌细胞凋亡、抑制细胞增殖及侵袭迁移过程中的作用;通过分子对接、靶点稳定性药物亲和反应实验以及微量热泳动实验检测ESG与EGFR的直接结合作用.结果 ESG显著抑制结直肠癌细胞的增殖及克隆形成能力;ESG诱导结直肠癌细胞发生线粒体途径凋亡并抑制其侵袭、迁移能力;ESG直接结合EGFR的激酶活性区而抑制其活性,进而下调其下游与肿瘤恶性进展相关的信号分子Akt、ERK的磷酸化水平.结论 ESG靶向结合并抑制EGFR,进而通过其下游Akt/ERK信号通路抑制结直肠癌细胞侵袭、迁移并诱导线粒体途径的细胞凋亡,有望成为靶向EGFR治疗结直肠癌的新型靶向药物.
Abstract
Objective To investigate the antitumor effects of ethoxydihydrosanguinarine(ESG)on colorectal cancer cells and its possible mechanisms.Methods Colorectal cancer cell lines RKO and HRT-18 were used.The cells were divided into blank control group and drug treatment groups(1.0,1.5,2.0 μmol/L ESG).CCK-8 assay,real time cellular analysis(RTCA)and colo-ny formation assay were used to detect the effect of ESG on cell proliferation and survival.Flow cytometry,DAPI staining,JC-1 mitochondrial membrane potential assay,and reactive oxygen species detection were performed to detect the effect of ESG on cell apoptosis.High-content analysis and Transwell invasion assay were used to detect the effect of ESG on cell invasion and mi-gration.Western blot was used to detect the expression of apoptosis,invasion and migration-related proteins,and the expression and phosphorylation of epidermal growth factor receptor(EGFR)and Akt/ERK signaling pathway proteins.The roles of EGFR in ESG-induced apoptosis,proliferation,invasion,and migration inhibition in colorectal cancer cells were detect through overex-pression and knockdown of EGFR.The direct binding between ESG and EGFR was detected by molecular docking and drug af-finity responsive target stability assay.Results The result showed that the proliferation and colony formation ability of colorec-tal cancer cells were significantly inhibited by ESG.Apoptosis through mitochondrial pathway was induced by ESG.The inva-sion and migration ability of colorectal cancer cells were inhibited by ESG.Moreover,ESG directly bound to EGFR and down-regulated the phosphorylation levels of Akt and ERK.Conclusion ESG targets and inhibits EGFR,thereby inhibiting the malig-nant progression of colorectal cancer cells and inducing mitochondrial apoptosis through its downstream Akt/ERK signaling pathway.This study provides new targeted drugs for targeting EGFR in the treatment of colorectal cancer.
关键词
结直肠癌/乙氧基二氢血根碱/表皮生长因子受体/侵袭/迁移/凋亡Key words
colorectal cancer/ethoxydihydrosanguinarine/epidermal growth factor receptor/invasion/migration/apoptosis引用本文复制引用
基金项目
国家自然科学基金资助项目(82072928)
湖北省卫生健康委指导性项目(WJ2023F079)
湖北医药学院研究生科技创新项目(YC2022032)
2022年大学生创新创业训练计划项目(202210929007)
2023年大学生创新创业训练计划项目(S202310929001)
出版年
2024
NETL
NSTL
万方数据