摘要
本研究利用alamarBlue~?测定细胞活力法、流式细胞术(annxin V-FITC)/PI双染色测定细胞凋亡法和高效液相色谱(high performance liquid chromatography,HPLC)测定5α-还原酶活性法评价灵芝酸A对前列腺癌的体外抗肿瘤活性,并进一步利用流式细胞术2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorofluorescindiacetate,H2DCFDA)染色测定ROS释放量法、实时荧光定量PCR(quantitative real-time PCR,qPCR)和蛋白质免疫印迹(western blot,WB)检测雄激素受体(androgen receptor,AR)基因和凋亡相关基因表达的方法,探讨其作用机理。研究结果表明灵芝酸A可通过抑制5α-还原酶活性,抑制睾酮诱导的前列腺癌LNCaP细胞增殖,诱发细胞早期凋亡来发挥抗肿瘤活性;进一步研究表明,灵芝酸A降低前列腺癌细胞中AR的表达,并通过引发细胞线粒体功能障碍释放过量活性氧(reactive oxygen species,ROS)诱发细胞凋亡,而qPCR和WB的数据进一步表明细胞线粒体功能障碍与抑癌基因caspase-3、bad和aifm1的高表达密切相关。
AlamarBlue■ method, flow cytometry(Annxin V-FITC)/PI double staining method and HPLC method were used to evaluate anti-tumor activity of ganoderic acid A(GA-A) against prostate cancer in vitro. Flow cytometry 2’,7’-dichlorofluorescindiacetate(H2 DCFDA) staining, quantitative real-time PCR(qPCR) and western blot(WB) were used to explore the mechanism of anti-tumor action. The results indicated that ganoderic acid A(GA-A) could exert its anti-tumor effect by reducing 5-reductase activity, inhibiting the proliferation and inducing early apoptosis of prostate cancer LNCaP cells. The results further showed that GA-A could reduce the expression of androgen receptor and induce apoptosis by triggering mitochondrial dysfunction to release excessive reactive oxygen species(ROS). Realtime PCR and WB data indicated that cell mitochondrial dysfunction was closely related to the high expression of tumor suppressor genes caspase-3,bad and aifm1.
作者
徐宾
贾薇
王忠
张赫男
吴迪
唐传红
杨焱
刘振东
张劲松
汪雯翰
XU Bin;JIA Wei;WANG Zhong;ZHANG He-Nan;WU Di;TANG Chuan-Hong;YANG Yan;LIU Zhen-Dong;ZHANG Jing-Song;WANG Wen-Han(Key Laboratory of Agricultural Gen etics and Breeding of Shanghai, Shanghai 201403, China;Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences,Key Laboratory of Edible Fun gal Resources and Utilization (South), Ministry of Agriculture, China, National Engineering Research Center of Edible Fungi,National R & D Center for Edible Fun gal Processing, Key Laboratory of Agricultural Gen etics and Breeding of Shanghai, Shanghai 201403, China;Department of Urology, Sixth People's Hospital South Campus Affiliated with Shanghai Jiaotong University, Shanghai 201400, China;Tibet Agriculture and Animal Husbandry College, Linzhi, Tibet 860000, China)
出处
《菌物学报》
CAS
CSCD
北大核心
2019年第5期717-727,共11页
Mycosystema
基金
上海市科技兴农重点攻关项目[沪农科攻字(2016)第5-4号]
上海市农业科学院卓越团队[农科创2017(A-06)]~~
关键词
灵芝酸A
LNCAP细胞
细胞凋亡
活性氧
雄激素受体
作用机理
ganoderic acid A
LNCaP cells
cell apoptosis
reactive oxygen species (ROS)
androgen receptor
mechanism of action
