扶正抗癌方联合吉非替尼对A549细胞的协同抗癌作用及机制  

Synergistic Anti-cancer Effect and Mechanism of FZKA Decoction Combined with Gefitinib on A549 Cells

作　　者：方芳 石玮[1] 金晓炜 吴万垠[2] FANG Fang;SHI Wei;JIN Xiao-wei;WU Wan-yin(First Clinical College,Guangxi University of Chinese Medicine,Nanjing 530000,China;Fangcun Branch of Guangdong Province Hospital of Traditional Chinese Medicine,Guangzhou 510370,China;Yunnan Cancer Hospital,Kunming 650018,China)

机构地区：[1]广西中医药大学第一附属医院,南宁530000 [2]广东省中医院芳村分院,广州510370 [3]云南省肿瘤医院,昆明650018

出　　处：《中国实验方剂学杂志》2020年第4期95-101,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81803919);广西壮族自治区卫生厅中医药科技专项(GZPT13-49);广西中医药大学自然科学研究课题项目(2017QN030)。

摘　　要：目的:通过体外体内实验观察扶正抗癌(FZKA)方联合吉非替尼(Gefitinib)对人肺腺癌A549细胞增殖,凋亡及侵袭转移能力的影响,并探讨研究其相关作用机制。方法:采用噻唑蓝(MTT)比色法检测空白组,FZKA方组(0.2,0.4,0.8,1.6,3.2 g·L^-1),Gefitinib组(10,20,40,60,80,100μmol·L^-1)的干预A549细胞24,48,72 h及FZKA方(2 g·L^-1)联合Gefitinib(40μmol·L^-1)组干预A549细胞24 h的增殖能力;流式细胞术分析检测联合用药组细胞凋亡及周期的变化;划痕实验和transwell小室实验检测联合用药组的侵袭转移能力变化;蛋白免疫印迹法检测细胞联合用药组活化的半胱氨酸蛋白酶-3(cleaved-Caspase-3),B淋巴细胞瘤-2(Bcl-2),B淋巴细胞瘤-2相关X蛋白(Bax),F-盒和含蛋白7的WD重复结构域(FBW7)及髓样细胞白血病-1(MCL-1)蛋白表达的变化情况。结果:与空白组比较,FZKA方,Gefitinib呈剂量依赖性、时间依赖性显著抑制A549细胞的增殖(P<0.05);与空白组比较,FZKA方组,Gefitinib组均能减弱细胞划痕愈合能力和侵袭能力,促进细胞凋亡(P<0.05),均能上调Bax,Caspase-3,FBW7蛋白表达,下调Bcl-2,MCL-1蛋白表达(P<0.05);与Gefitinib单独给药比较,FZKA方联合Gefitinib抑制A549细胞增殖能力和诱导细胞凋亡更明显(P<0.05);细胞划痕愈合能力与侵袭能力明显减弱(P<0.05);上调Bax,Caspase-3,FBW7蛋白表达,下调Bcl-2,MCL-1蛋白表达效果明显(P<0.05)。结论:FZKA方与Gefitinib合用后比单用Gefitinib更能诱导A549细胞凋亡,抑制其增殖及侵袭转移能力更强,两者具有协同抗癌作用,其机制可能与调控FBW7/MCL-1通路相关。Objective:To observe the effect of Fuzheng Kangai(FZKA)decoction combined with gefitinib on the cells proliferation,apoptosis,invasion and metastasis of human lung adenocarcinoma A549 cells in vitro and in vivo,and relevant mechanisms.Method:The A549 cell proliferation of the control group,FZKA decoction groups(0.2,0.4,0.8,1.6,3.2 g·L^-1),Gefitinib groups(10,20,40,60,80,100μmol·L^-1)for 24,48,72 hours,and FZKA decoction(2 g·L^-1)combined with Gefitinib(10μmol·L^-1)groups for24 hours was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay.The changes of cell apoptosis,invasion and metastasis abilities of A549 cells were analyzed by flow cytometry,Wound Healing,transwell invasion assay.Western blot assay was used to examine the protein expressions of cleaved Caspase-3,B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2 associated X(Bax),F-box and WD repeat domain-containing(FBW7)and myeloid cell leukemia-1(MCL-1)in vitro.Result:Compared with control group,FZKA decoction group and Gefitinib group inhibited the cell proliferation,cell apoptosis,cell invasion and metastasis abilities in a dose-dependent and time-dependent manner,and improve the protein expressions of Bax,Caspase-3,FBW7,but decreased the protein expressions of Bcl-2,MCL-1(P<0.05).Compared with treatment with Gefitinib alone,FZKA combined with Gefitinib inhibited the proliferation of A549 cells,and induced apoptosis more significantly(P<0.05).Compared with treatment with Gefitinib alone,the cell scratch healing and invasion abilities were significantly reduced after combined treatment(P<0.05).FZKA decoction combined with Gefitinib up-regulated Bax,Caspase-3 and FBW7 protein expressions,and down-regulated Bcl-2 and MCL-1 protein expressions compared with treatment with Gifitinib alone(P<0.05).Conclusion:FZKA decoction combined with Gefitinib can inhibit the proliferation,invasion and metastasis,and induce apoptosis on A549 cells.The mechanism may be associated with the FBW7/MCL-1 pathway.

关 键 词：扶正抗癌方 吉非替尼 协同作用 F-盒和含蛋白7的WD重复结构域(FBW7)/髓样细胞白血病-1(MCL-1) 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]