南葶苈子乙酸乙酯浸膏对人非小细胞肺癌H1975细胞增殖、凋亡的影响
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摘要
目的探讨南葶苈子的乙酸乙酯浸膏对人非小细胞肺癌H1975细胞增殖、凋亡的影响。方法采用系统溶剂萃取法对南葶苈子甲醇浸膏进行初步分离。MTT法检测各萃取浸膏(5、10、20、40、80μg/mL)的细胞毒性。集落克隆法检测不同浓度的南葶苈子乙酸乙酯浸膏(0.625、1.25、2.5μg/mL)对人非小细胞肺癌H1975细胞增殖的影响。Annexin V-FITC/PI双染及流式细胞术检测不同浓度的乙酸乙酯浸膏(0、10、20、40μg/mL)对人非小细胞肺癌H1975细胞凋亡的影响。免疫印迹法检测不同浓度乙酸乙酯浸膏(0、10、20、40μg/mL)对凋亡相关蛋白Akt、Bax及Bcl-2蛋白表达的影响。建立人非小细胞肺癌H1975细胞裸鼠移植瘤模型,设置空白组,给药组(100 mg/kg),阳性对照组,观察乙酸乙酯浸膏的体内抗肿瘤活性。结果 MTT和集落克隆结果显示,乙酸乙酯浸膏呈剂量依赖、时间依赖方式抑制人非小细胞肺癌H1975细胞增殖。与对照组相比,差异均具有统计学意义(P<0.05)。流式细胞术结果表明,乙酸乙酯浸膏能显著诱导人非小细胞肺癌H1975细胞发生凋亡(P<0.05),并且呈现浓度依赖性。Western blot结果显示,乙酸乙酯浸膏可降低蛋白Bcl-2、Akt的表达,增加蛋白Bax的表达(P<0.05)。另外,南葶苈子乙酸乙酯浸膏表现出较好的体内抗肿瘤活性,对裸鼠体质量和器官的毒性较小。结论南葶苈子的乙酸乙酯浸膏具有较强的体内、体外抗肿瘤作用,具有成为抗肺癌药物的潜力。
Objective To investigate the effects of methanol-ethyl acetate partitioned fractions from Descurainia sophia(MEDS) on the proliferation and apoptosis of human non-small cell lung cancer H1975 cells.Methods The systemic solvent extraction method was used to preliminary separation of the effective fractions in the methanol extract of Descurainia sophia.The cytotoxicity of each extract(5,10,20,40,and 80 μg/mL) was tested using MTT assay.Colony cloning method was used to assess the effect of different concentrations of methanol-ethyl acetate partitioned fractions from MEDS(5,10,20,40,and 80 μg/mL) on the proliferation of H1975 cells.Flow cytometric analysis with Annexin V-FITC/PI staining was performed to detect the apoptosis of the cells after treatment with different concentrations of MEDS fractions(10,20,and 40 μg/mL).Western blotting was used to evaluate the effects of MEDS fractions on the expressions of apoptosis-related proteins Akt,Bax,and Bcl-2.The anti-tumor activity of 100 mg/kg MEDS fractions was tested in a nude mouse model bearing H1975 cell xenografts.Results MTT assay and colony forming experiment showed that MEDS fractions significantly inhibited the proliferation of H1975 cells in a dose-and time-dependent manner(P<0.05).The results of flow cytometry showed that MEDS fractions induced obvious apoptosis of H1975 cells in a concentration-dependent manner(P<0.05).MEDS fractions also significantly decreased the expressions of Bcl-2 and Akt protein and increased the protein expression of Bax(P<0.05).In the tumor-bearing nude mouse model,MEDS fractions showed potent anti-tumor effects with a low toxicity to affect the body weight and organs of the mice.Conclusion The methanol-ethyl acetate partitioned fractions from MEDS show potent anti-tumor activity both in vivo and in vitro,suggesting their value as promising therapeutic agents against lung cancer.
Objective To investigate the effects of methanol-ethyl acetate partitioned fractions from Descurainia sophia(MEDS) on the proliferation and apoptosis of human non-small cell lung cancer H1975 cells.Methods The systemic solvent extraction method was used to preliminary separation of the effective fractions in the methanol extract of Descurainia sophia.The cytotoxicity of each extract(5,10,20,40,and 80 μg/mL) was tested using MTT assay.Colony cloning method was used to assess the effect of different concentrations of methanol-ethyl acetate partitioned fractions from MEDS(5,10,20,40,and 80 μg/mL) on the proliferation of H1975 cells.Flow cytometric analysis with Annexin V-FITC/PI staining was performed to detect the apoptosis of the cells after treatment with different concentrations of MEDS fractions(10,20,and 40 μg/mL).Western blotting was used to evaluate the effects of MEDS fractions on the expressions of apoptosis-related proteins Akt,Bax,and Bcl-2.The anti-tumor activity of 100 mg/kg MEDS fractions was tested in a nude mouse model bearing H1975 cell xenografts.Results MTT assay and colony forming experiment showed that MEDS fractions significantly inhibited the proliferation of H1975 cells in a dose-and time-dependent manner(P<0.05).The results of flow cytometry showed that MEDS fractions induced obvious apoptosis of H1975 cells in a concentration-dependent manner(P<0.05).MEDS fractions also significantly decreased the expressions of Bcl-2 and Akt protein and increased the protein expression of Bax(P<0.05).In the tumor-bearing nude mouse model,MEDS fractions showed potent anti-tumor effects with a low toxicity to affect the body weight and organs of the mice.Conclusion The methanol-ethyl acetate partitioned fractions from MEDS show potent anti-tumor activity both in vivo and in vitro,suggesting their value as promising therapeutic agents against lung cancer.
引文
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[2]Molina JR,Yang P,Cassivi SD,et al.Non-small cell lung cancer:epidemiology,risk factors,treatment,and survivorship[J].Mayo Clin Proc,2008,83(5):584-94.
[3]Wang LJ,Yu CH,Liu Y,et al.Lung cancer mortality trends in China from 1988 to 2013:new challenges and opportunities for the government[J].Int J Environ Res Public Health,2016,13(11):1052.
[4]Reck M,Heigener DF,Mok T,et al.Management of non-small-cell lung cancer:recent developments[J].Lancet,2013,382(9893):709-19.
[5]Tan DS,Camilleri-Bro雝S,Tan EH,et al.Intertumor heterogeneity of non-small-cell lung carcinomas revealed by multiplexed mutation profiling and integrative genomics[J].Int J Cancer,2014,135(5):1092-100.
[6]D'incecco A.Cappuzzo F.Gefitinib for non-small-cell lung cancer treatment[J].Expert Opin Drug Saf,2011,10(6):987-96.
[7]王晋,尹军强,沈靖南,等.32种中草药提取物体外抗骨肉瘤作用的筛选[J].南方医科大学学报,2006,26(9):1293-6.
[8]Stone R.Biochemistry:lifting the veilon traditional Chinese medicine[J].Science,2008,319(5864):709-10.
[9]Newman DJ,Cragg GM,Snader KM.The influence of natural products upon drug discovery[J].Nat Prod Rep,2000,17(3):215-34.
[10]Kundranda MN,Niu J.Albumin-bound paclitaxel in solid tumors:clinical development and future directions[J].Drug Des Devel Ther,2015,9:3767-77.
[11]宋海星,胡洪华.羟基喜树碱抑制肺癌A549细胞的体外增殖并下调Bcl-2基因的表达[J].南方医科大学学报,2012,32(9):1341-5.
[12]王爱芹,王秀坤,赵海誉,等.南葶苈子化学成分与质量研究[J].中国药物与临床,2005,5(1):5-6.
[13]国家药典委员会.中华人民共和国药典》2015版(一部)[M].北京:中国医药科技出版社,2015:333-4.
[14]谢强,王萧.强心方对充血性心力衰竭的临床疗效观察[J].临床军医杂志,2001,29(3):34-7.
[15]周喜丹,唐力英,周国洪,等.南北葶苈子的最新研究进展[J].中国中药杂志,2014,39(24):4699-708.
[16]Nimrouzi M,Zarshenas MM.Phytochemical and pharmacological aspects of Descurainia sophia Webb ex Prantl:modern and traditional applications[J].Avicenna J Phytomed,2016,6(3):266-72.
[17]Lee YJ,Kim NS,Kim H,et al.Cytotoxic and anti-inflammatory constituents from the seeds of Descurainia sophia[J].Arch Pharm Res,2013,36(5):536-41.
[18]Zhou N,Sun YP,Zheng XK,et al.A Metabolomics-Based strategy for the mechanism exploration of traditional Chinese medicine:descurainia sophia seeds extract and fractions as a case study[J].Evid-Based Complement Alternat Med,2017:2845173
[19]陈建发,陈引香,李萍,等.半边旗提取物5F对人胃癌细胞凋亡的影响及其机制[J].南方医科大学学报,2011,31(8):1345-8.
[20]Han J,Goldstein LA,Hou W,et al.HSP90 inhibition targets autophagy and induces a CASP9-dependent resistance mechanism in NSCLC[J].Autophagy,2018,14(6):958-71.
[21]Mellatyar H,Talaei S,Pilehvar-Soltanahmadi YA,et al.Targeted cancer therapy through 17-DMAG as an Hsp90 inhibitor:Overview and current state of the art[J].Biomed Pharmacother,2018,102:608-17.
[22]周芳竹,王欣,代安亚,等.藏药桃儿七可促进慢性粒细胞白血病K562细胞的凋亡[J].南方医科大学学报,2017,37(2):226-31.
[23]Sun Y,Peng ZL.Programmed cell death and cancer[J].Postgrad Med J,2009,85(1001):134-40.
[24]Hassan M,Watari H,Abualmaaty A,et al.Apoptosis and molecular targeting therapy in cancer[J].Biomed Res Int,2014:150845.
[25]Broek RV,Mohan S,Eytan DF,et al.The PI3K/Akt/mTOR axis in head and neck cancer:functions,aberrations,cross-talk,and therapies[J].Oral Dis,2015,21(7):815-25.
[26]Sithanandam G,Fornwald LW,Fields J,et al.Inactivation of ErbB3by siRNA promotes apoptosis and attenuates growth and invasiveness of human lung adenocarcinoma cell line A549[J].Oncogene,2005,24(11):1847-59.
[27]Yamaguchi H,Wang HG.The protein kinase PKB/Akt regulates cell surival and apoptosis by inhibiting Bax conformational change[J].Oncogene,2001,20(53):7778-86.
[28]Carrara L,Lavezzi SM,Borella E,et al.Current mathematical models for cancer drug discovery[J].Expert Opin Drug Discov,2017,12(8):785-99.