葫芦素B联合奥沙利铂对人结肠癌SW480细胞增殖和凋亡的抑制作用及其机制

doi:10.13481/j.1671-587x.20200114

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (01): 78-83.doi: 10.13481/j.1671-587x.20200114

葫芦素B联合奥沙利铂对人结肠癌SW480细胞增殖和凋亡的抑制作用及其机制

冯杰^1,2, 任立群³, 陈素贤², 万义增², 徐培斌⁴

1. 锦州医科大学基础医学院病理学与病理生理学教研室, 辽宁锦州 121000;
2. 锦州医科大学附属第三医院病理科, 辽宁锦州 121000;
3. 吉林大学药学院实验药理与毒理教研室, 吉林长春 130021;
4. 长春中医药大学中医学教研室, 吉林长春 130117

收稿日期:2019-02-27 出版日期:2020-01-28 发布日期:2020-02-03
通讯作者: 万义增,教授,硕士研究生导师(Tel:0416-3999089,E-mail:wanyz@hmu.cn) E-mail:wanyz@hmu.cn
作者简介:冯杰(1992-),女,山东省济宁市人,医学硕士,主要从事胃肠道肿瘤方面的研究。
基金资助:
辽宁省科技厅自然科学基金资助课题（2013022007）；吉林省科技厅重点科技攻关项目资助课题（20150204015YY）；国家中医药管理局第四批全国中医（临床，基础）优秀人才研修项目资助课题（国中医药人教法（2017）24号）

Inhibitory effects of cucurbitacin B combined with oxaliplatin on proliferation and apoptosis of human colon cancer SW480 cells and their mechanisms

FENG Jie^1,2, REN Liqun³, CHEN Suxian², WAN Yizeng², XU Peibin⁴

1. Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Jinzhou Medical Universsity, Jinzhou 121000, China;
2. Department of Pathology, Third Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, China;
3. Department of Experimental Pharmacology and Toxicology, School of Pharmacy, Jilin University, Changchun 130021, China;
4. Department of Traditional Chinese Medicine, Changchun Universsity of Chinese Medicine, Changchun 130117, China

Received:2019-02-27 Online:2020-01-28 Published:2020-02-03

摘要/Abstract

摘要： 目的：探讨葫芦素B（CUB）联合奥沙利铂（OXA）对人结肠癌SW480细胞增殖及凋亡的影响，并阐明其作用机制。方法：将SW480细胞分为对照组，10、20和40μmol·L^-1CUB组，OXA组（100μmol·L^-1）及联合组（40μmol·L^-1 CUB+100 μmol·L^-1OXA）。采用MTT法检测SW480细胞增殖率，Hoechst33258染色法观察SW480细胞形态表现，流式细胞术检测SW480细胞的细胞周期及细胞凋亡率，Western blotting法检测SW480细胞中半胱氨酸天冬氨酸特异性蛋白酶3（caspase-3）、活化后的caspase-3（cleavedcaspase-3）、B淋巴细胞瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的蛋白表达水平。结果：MTT实验，与对照组比较，CUB组和OXA组细胞增殖率明显降低（P<0.05）；与不同剂量CUB组和OXA组比较，联合组细胞增殖率明显降低（P<0.05）。Hoechst33258染色，与对照组比较，不同剂量CUB组和OXA组细胞蓝色荧光更亮，细胞核可见颗粒块状荧光；与不同剂量CUB组和OXA组比较，联合组细胞蓝色荧光明显变亮，颗粒状荧光明显增加。流式细胞术检测细胞周期，与对照组比较，不同剂量CUB组和联合组G₂/M期细胞百分率明显升高（P<0.05），40 μmol·L^-1 CUB组、OXA组和联合组S期细胞百分率明显升高（P<0.05）。流式细胞术检测细胞凋亡率，与对照组比较，不同剂量CUB组和OXA组细胞凋亡率明显升高（P<0.05）；与不同剂量CUB组和OXA组比较，联合组细胞凋亡率明显升高（P<0.05）。Western blotting法检测，与对照组比较，不同剂量CUB组、OXA组和联合组细胞中caspase-3和Bcl-2蛋白表达水平降低（P<0.05），cleavedcaspase-3和Bax蛋白表达水平升高（P<0.05），cleavedcaspase-3/caspase-3比值升高（P<0.05），Bcl-2/Bax比值降低（P<0.05）；与不同剂量CUB组和OXA组比较，联合组细胞上述蛋白表达水平及cleavedcaspase-3/caspase-3和Bcl-2/Bax比值变化更明显（P<0.05）。结论：CUB联合OXA可有效抑制结肠癌SW480细胞增殖，其机制可能与细胞阻滞在S期、G₂/M期及上调cleavedcaspase-3/caspase-3比值、下调Bcl-2/Bax比值有关。

关键词: 葫芦素B, 奥沙利铂, 结肠肿瘤, 细胞增殖, 细胞凋亡

Abstract: Objective: To expore the effects of cucurbitacin B(CUB) combined with oxaliplatin(OXA)on the proliferation and apoptosis of human colon cancer SW480 cells, and to clarify their mechanisms. Methods: The SW480 cells were divided into control group, 10,20, and 40 μmol·L^-1 CUB groups, OXA group(100 μmol·L^-1) and combination group (40 μmol·L^-1 CUB +100 μmol·L^-1 OXA). The proliferation rate of the SW480 was determined by MTT assay. Hoechst33258 staining was used to observe the morphology of SW480 cells. The cell cycle and apoptotic rates of SW480 cells were detected by flow cytometry. The expressions levels of caspase-3,cleaved caspase-3,Bax and Bcl-2 proteins were measured by Western blotting method. Results: The MTT results showed that compared with control group, the proliferation rates of the SW480 cells in different doses of CUB groups and OXA group were significantly decreased(P<0.05); compared with different doses of CUB groups and OXA group, the proliferation rate of the SW480 cells in combination group was significantly decreased(P<0.05). The results of Hoechst 33258 staining showed that the blue fluorescence in the SW480 cells in different doses of CUB groups and OXA group were brighter than that in control group,which showed granular blue fluorescence in the cell nucles. Compared with different doses of CUB groups and OXA group, the blue fluorescence in the SW480 cells in combination group was more significantly bright, and granular blue fluorescence was significantly increased. The cell cycle detection results of flow cytometry showed that compared with control group, the percentages of the SW480 cells in G₂/M phase in different doses of CUB groups and combination group were incresaed (P<0.05); the percentages of SW480 cells in S phase in 40 μmol·L^-1 CUB group, OXA group and combination group were incresaed (P<0.05). The apoptosis detection results of flow cytometry showed that compared with control group, the apoptotic rates of the SW480 cells in different doses of CUB groups and OXA group were increased (P<0.05); compared with different doses of CUB groups and OXA group, the apoptotic rate of the SW480 cells in combination group was significantly increased(P<0.05).The Western blotting results showed that compared with control group, the expressions levels of caspase-3 and Bcl-2 in the SW480 cells in different doses of CUB groups and OXA group were decreased (P<0.05), the expression levels of cleaved caspase-3 and Bax were increased (P<0.05),and the Bcl-2/Bax ratios were decreased(P<0.05); compared with different doses of CUB groups and OXA group, the changes of the above protein expression levels,the cleaved caspase-3/caspase-3 ratio and the Bcl-2/Bax ratio in combination group were more obvious (P<0.05). Conclusion: CUB combined with OXA can effectively inhinbit the proliferation of colon cancer SW480 cells, and its mechanism may be related to the S and G₂/M phase arrest,up regulation of the ratio of cleaved-caspase-3/caspase-3 and down-regulation of the ratio of Bcl-2/Bax.

Key words: cucurbitacin B, oxaliplatin, colon neoplasms, cell proliferation, apoptosis

中图分类号:

R735.35

冯杰, 任立群, 陈素贤, 万义增, 徐培斌. 葫芦素B联合奥沙利铂对人结肠癌SW480细胞增殖和凋亡的抑制作用及其机制[J]. 吉林大学学报(医学版), 2020, 46(01): 78-83.

FENG Jie, REN Liqun, CHEN Suxian, WAN Yizeng, XU Peibin. Inhibitory effects of cucurbitacin B combined with oxaliplatin on proliferation and apoptosis of human colon cancer SW480 cells and their mechanisms[J]. Journal of Jilin University(Medicine Edition), 2020, 46(01): 78-83.

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葫芦素B联合奥沙利铂对人结肠癌SW480细胞增殖和凋亡的抑制作用及其机制

Inhibitory effects of cucurbitacin B combined with oxaliplatin on proliferation and apoptosis of human colon cancer SW480 cells and their mechanisms

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