新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制*

doi:10.12047/j.cjap.5968.2020.071

中国应用生理学杂志 ›› 2020, Vol. 36 ›› Issue (4): 330-335.doi: 10.12047/j.cjap.5968.2020.071

新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制^*

黄娇娇, 王艳林, 孙丽丹, 曹春雨, 秦烨, 杨建林^△

三峡大学医学院肿瘤微环境与免疫治疗湖北省重点实验室, 湖北宜昌 443002

收稿日期:2019-11-05 修回日期:2020-05-12 发布日期:2020-11-09
通讯作者: ^△Tel: 0717-6397179; E-mail: 435203858@qq.com
基金资助:
^*国家自然科学基金资助项目(81372265,30772590);湖北省卫生健康科研基金(WJ2019H532)肿瘤微环境与免疫治疗湖北省重点实验室开放基金项目(2019KZL01,2016KZL04)

Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism

HUANG Jiao-jiao, WANG Yan-lin, SUN Li-dan, CAO Chun-yu, QIN Ye, YANG Jian-lin^△

Medical College of China Three Gorges University, Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Yichang 443002, China

Received:2019-11-05 Revised:2020-05-12 Published:2020-11-09

摘要/Abstract

摘要： 目的: 探讨本实验室新发现的新型多胺代谢酶小分子抑制剂SI-4650对结肠癌CT-26细胞增殖、自噬和凋亡的影响。方法: 体外培养CT-26细胞,以0 μmol·L^-1SI-4650处理48 h细胞为正常对照组,单独2.5 mmol·L^-13-MA处理细胞为自噬抑制对照组,40、80 μmol·L^-1SI-4650处理48 h细胞以及3-MA联合40、80 μmol·L^-1SI-4650处理48 h细胞为4个实验组,化学发光法检测CT-26细胞中多胺代谢酶SMO和APAO酶活性的变化,HPLC法检测细胞中多胺含量的变化,CCK8法检测CT-26细胞增殖能力变化;PI单染结合流式细胞术分析细胞周期;Western blot法分析细胞自噬;PI/FITC-Annexin V双染、JC-1荧光探针和Fluo-3 AM钙离子荧光探针分别结合流式细胞术以及Western blot法分析细胞凋亡。结果: 与正常对照组比较,40、80 μmol·L^-1SI-4650实验组细胞生长抑制率分别为36.98%、46.91%,有效抑制肿瘤细胞增殖(P＜0.01);同时细胞中SMO和APAO酶活性下降(P＜ 0.01);细胞中多胺总含量减少(P＜0.01);CT-26细胞被阻滞在G0/G1期(P＜0.01);凋亡细胞数分别为7.69%和 16.87%,细胞中钙浓度增加(P＜0.01)、线粒体膜电位下降(P＜0.01),c-PARP、Bax表达增加(P＜0.01)、Bcl-2含量减少(P＜0.01);以及CT-26细胞中自噬相关蛋离子白Beclin-1、LC3-Ⅱ以及P62含量显著上升(P＜0.01)。与单独40、80 μmol·L^-1SI-4650处理组相比,2.5 mmol·L^-13-MA联合40、80 μmol·L^-1SI-4650实验组细胞自噬水平下降(P＜0.01),凋亡相关蛋白、线粒体膜电位和钙离子浓度变化均减弱(P＜0.01),凋亡细胞数减少(P＜0.01)。结论: SI-4650有效抑制结肠癌CT-26细胞增殖,机制可能与抑制多胺代谢酶活性,干扰多胺代谢,减少多胺总含量以及诱导细胞周期阻滞、细胞自噬和凋亡相关。

关键词: 多胺代谢酶抑制剂SI-4650, 结肠癌, CT-26细胞, 凋亡, 自噬

Abstract: Objective: To investigate the effects of a novel polyamine metabolism enzyme inhibitor SI-4650 on autophagy and apoptosis of colon cancer CT-26 cells as well as their correlation. Methods: CT-26 cells treated with 40, 80 μmol·L^-1 SI-4650 alone or in combination with 3-MA were used as experimental group. CT-26 cells treated with 0 μmol·L^-1 SI-4650 alone or in combination with 3-MA were used as control group. Chemiluminescence was used to analyze the effect of SI-4650 on spermine oxidase (SMO) and acetylpolyamine oxidase(APAO) activity. High performance liquid chromatography (HPLC) was performed to detect cellular polyamine content.The CCK8 method was used to detect the inhibitory effect of SI-4650 on proliferation of CT-26 cells. PI single-staining/flow cytometry (FCM) were used to analyze cell cycle. Western blot were used to analyze autophagy. Apoptosis was analyzed by PI/FITC-Annexin V double staining, JC-1 fluorescent probe and Fluo-3 AM calcium ion fluorescent probe combined with flow cytometry and Western blot. Results: CCK8 assay showed that 24-,48-,72-hours treated with SI-4650 all could inhibit the proliferative activity of CT-26 cells in a dose- and time-dependent manner (P＜0.01) . The inhibition rate was 36.98% and 46.91% in 40 μmol·L^-1 SI-4650 group and 80 μmol·L^-1 SI-4650 group respectively. SI-4650 could significantly inhibit the activities of SMO and APAO interfere with polyamine metabolism and reduce the content of total polyamine in CT-26 cells (P＜0.01). SI-4650 could block CT-26 cells in G0/G1 phase, significantly reduce the number of cells in S phase(P＜0.01), and lead to a significant increase in the contents of autophagy-related Beclin-1, LC3-II in CT-26 cells(P＜0.01); At the same time, the concentration of calcium in CT-26 cells was increased, the mitochondrial membrane potential was decreased, the expressions of c-PARP and Bax were increased, the content of Bcl-2 was decreased, and the number of apoptotic cells was increased. After SI-4650 combined with autophagy inhibitor 3-MA treatment of CT-26 cells, the level of autophagy, the apoptosis-related protein, mitochondrial membrane potential and calcium ion concentration were decreased, and the number of apoptotic cells was decreased. Conclusion: SI-4650 has the pharmacological activity of killing colon cancer CT-26 cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy. In this process, autophagy is inhibited to block apoptosis, autophagy and apoptosis combined to kill tumor cells.

Key words: polyamine metabolic enzyme inhibitor, CT26, apoptosis, autophagy

中图分类号:

R966

黄娇娇, 王艳林, 孙丽丹, 曹春雨, 秦烨, 杨建林. 新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制^*[J]. 中国应用生理学杂志, 2020, 36(4): 330-335.

HUANG Jiao-jiao, WANG Yan-lin, SUN Li-dan, CAO Chun-yu, QIN Ye, YANG Jian-lin. Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism[J]. CJAP, 2020, 36(4): 330-335.

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新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制^*

Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism

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