细梗香草皂苷联合吉西他滨对胰腺癌细胞BxPC-3抑制作用的研究
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摘要
目的观察细梗香草皂苷(LC)和吉西他滨(GEM)单药及联合用药对人胰腺癌Bx PC-3细胞增殖和凋亡的影响,探究LC抗胰腺癌的作用机制。方法培养人胰腺癌Bx PC-3细胞时加入不同浓度梯度的LC与GEM,采用MTS法检测细胞增殖抑制率,计算两种药物的半抑制浓度(IC_(50))和联合作用指数(CI)。将细胞分为4组,A组(对照空白)、B组(GEM 1μmol/L)、C组(LC15μg/ml)和D组(GEM 1μmol/L+LC 15μg/ml联合用药),培养后采用AnnexinⅤ-FITC/PI法观察LC与GEM对Bx PC-3细胞的诱导凋亡作用;Western blot法检测抗多聚(ADP-核糖)聚合酶(PARP)、抗半胱氨酸蛋白酶-3(Caspase-3)的表达。结果8~64μg/ml的LC及1.25~20μmol/L的GEM均对Bx PC-3细胞的增殖有抑制作用,且呈浓度依赖性,其IC_(50)分别为16.55μg/ml和1.27μmol/L。15μg/ml的LC与各个浓度的的GEM协同作用最强,CI值为0.53~0.65。B、C、D组的早、晚期凋亡率与A组比较差异均有统计学意义(均P<0.01),且D组与B、C组的早期凋亡率比较差异均有统计学意义(均P<0.01)。C、D组与A组比较,PARP蛋白表达水平明显降低(均P<0.05);B、C、D组与A组比较,Caspase-3蛋白表达水平增加(均P<0.05)。结论 LC有抑制胰腺癌细胞生长的作用,与GEM联合作用效果更好;其作用机制可能是通过激活Caspase-3表达,分解PARP,从而诱导细胞凋亡。
Objective To investigate the effect of lysimachia capillipes(LC) and gemcitabine(GEM) on proliferation and apoptosis of human pancreatic cancer cell line BxPC-3 in vitro. Methods The cultured BxPC-3 cells were treated with GEM(1μmol/L, group B), LC(15μg/ml, group C) and GEM with LC(15μg/ml and 1μmol/L, group D) for 48 h, respectively. The untreated BxPC-3 cells served as controls(group A). The proliferation of BxPC-3 cells was measured by MTS method, half maximal inhibitory concentration(IC_(50)) and combined index(CI) of the two drugs were analyzed with Calcusyn software. Cell apoptosis was examined by using flow cytometry(FCM) with AnnexinⅤ/PI staining solution. The expression of proteins PARP and Capase-3 was detected by Western blot. Results The proliferation of BxPC-3 cells was inhibited by LC and GEM. The IC_(50) value of LC and GEM were16.55μg/ml and 1.27μmol/L, the CI value was 0.53-0.65, when BxPC-3 cells treated with 15-20μg/ml LC and 1-30μmol/L GEM. The apoptosis rate of BxPC-3 cells was increased in group B, C and D compared with group A(all P<0.01), and the effect of group D is stronger than that of group B and C(both P <0.01). The expression of Capase-3 protein was up-regulated in group B, C and D.Compared with A(all P<0.05), and the expression of PARP protein was down-regulated in group C and D Cormpared with A(both P<0.05). Conclusion LC and GEM can inhibit the proliferation of BxPC-3 cells, and the effect of combination of the two drugs is more marked. LC can induce apoptosis of BxPC-3 cells by activating the Caspase-3 protein expression and decomposing PARP protein.
Objective To investigate the effect of lysimachia capillipes(LC) and gemcitabine(GEM) on proliferation and apoptosis of human pancreatic cancer cell line BxPC-3 in vitro. Methods The cultured BxPC-3 cells were treated with GEM(1μmol/L, group B), LC(15μg/ml, group C) and GEM with LC(15μg/ml and 1μmol/L, group D) for 48 h, respectively. The untreated BxPC-3 cells served as controls(group A). The proliferation of BxPC-3 cells was measured by MTS method, half maximal inhibitory concentration(IC_(50)) and combined index(CI) of the two drugs were analyzed with Calcusyn software. Cell apoptosis was examined by using flow cytometry(FCM) with AnnexinⅤ/PI staining solution. The expression of proteins PARP and Capase-3 was detected by Western blot. Results The proliferation of BxPC-3 cells was inhibited by LC and GEM. The IC_(50) value of LC and GEM were16.55μg/ml and 1.27μmol/L, the CI value was 0.53-0.65, when BxPC-3 cells treated with 15-20μg/ml LC and 1-30μmol/L GEM. The apoptosis rate of BxPC-3 cells was increased in group B, C and D compared with group A(all P<0.01), and the effect of group D is stronger than that of group B and C(both P <0.01). The expression of Capase-3 protein was up-regulated in group B, C and D.Compared with A(all P<0.05), and the expression of PARP protein was down-regulated in group C and D Cormpared with A(both P<0.05). Conclusion LC and GEM can inhibit the proliferation of BxPC-3 cells, and the effect of combination of the two drugs is more marked. LC can induce apoptosis of BxPC-3 cells by activating the Caspase-3 protein expression and decomposing PARP protein.
引文
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[21]葛建彬,顾锦华,李梅,等.银杏内酯A对小鼠脑缺血/再灌注损伤的保护作用及其抑制NF-κB信号通路下调p53、Caspase-3表达的机制[J].中国药理学通报,2012,28(8):1105-1110.
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[2]Ko AH.Progress in the treatment of metastatic pancreatic cancer and the search for next opportunities[J].J Clin Oncol,2015,33(16):1779-1786.doi:10.1200/JCO.2014.59.7625.
[3]Stathis A,Moore MJ.Advanced pancreatic carcinoma:current treatment and future challenges[J].Nat Rev Clin Oncol,2010,7(3):163-172.doi:10.1038/nrclinonc.2009.236.
[4]Oettle H,Post S,Neuhaus P,et al.Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer:A randomized controlled trial[J].JAMA,2007,297(3):267-277.doi:10.1001/jama.297.3.267.
[5]Neuhaus P,Riess H,Schramm H,et al.CONKO-001:Final results of therandomized,prospective,multicenter phase III trial of adjuvant chemotherapy with gemcitabine versus observation in patients with resected pancreatic cancer(PC)[J].J Clin Oncol,2008,26(Suppl):Abstr LBA4504.doi:10.1200/jco.2005.23.16_suppl.lba4013.
[6]田景奎,邹忠梅,徐丽珍,等.细梗香草化学成分的研究[J].中国药学杂志,2006,41(3):171-173.doi:10.3321/j.issn:1001-2494.2006.03.004.
[7]费正华,陈云亮,马胜林.细梗香草总皂苷诱导肺癌细胞H460凋亡及其机制的研究[J].浙江医学,2014,36(9):742-746.
[8]徐燕,荣语媚,刘小保,等.细梗香草总皂苷的抗肿瘤活性研究[J].中国药理学通,2012,28(4):545-549.doi:10.3969/j.issn.1001-1978.2012.04.024.
[9]Wang D,Wang Z,Tian Y,et al.Two hour exposure to sodium butyrate sensitizes bladder cancer to anticancer drugs[J].Int J Urol,2008,15(5):435-441.doi:10.1111/j.1442-2042.2008.02025.x.
[10]Zhang Q,Zeng L,Huang K,et al.Pancreatic cancer epidemiology,detection,and management[J].Gastroenterol Res Pract,2016,2016:1-10.doi:10.1155/2016/8962321.
[11]Von Hoff DD,Ervin T,Arena PF,et al.Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine[J].N Engl J Med,2013,369(18):1691-1703.doi:10.1056/NEJMoa-1304369.
[12]Ueno H,Ioka T,Ikeda M,et al.Randomized phaseⅢstudy of gemcitabine plus S-1,S-1 alone,or gemcitabine alone in patients with locally advanced and metastatic pancreatic cancer in Japan and taiwai:GEST study[J].J Clin Oncol,2013,31(13):1640-1648.doi:10.1200/JCO.2012.43.3680.
[13]Altwegg R,Ychou M,Guillaumon V,et al.Second-line therapy for gemcitabine-pretreated advanced or metastatic pancreatic cancer[J].World J Gastroenterol,2012,18(12):1357-1364.doi:10.3748/wjg.v18.i12.1357.
[14]Reni M,Cereda S,Milella M,et al.Maintenance sunitinib or observation in metastatic pancreatic adenocarcinoma:a phase II randomised trial[J].Eur J Cancer,2013,49(17):3609-3615.doi:10.1016/j.ejca.2013.06.041.
[15]Lee HS,Park SW.Systemic chemotherapy in advanced pancreatic cancer[J].Gut Iver,2016,10(3):340-347.doi:10.5009/gnl15465.
[16]Vincent A,Herman J,Goggins AM,et al.Pancreatic cancer[J].Lancet,2011,378(9791):607-620.doi:10.1016/S0140-6736(10)62307-0.
[17]李峨,陈信义,王玉芝.九种中药活性成分抗耐药肿瘤细胞体外研究[J].北京中医药大学学报,2004,27(2):24-26.doi:10.3321/j.issn:1006-2157.2004.02.008.
[18]Srivastava SK,Bhardwaj A,Singh AP,et al.Micro RNA-345induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and-independent pathways[J].Br J Cancer,2015,113(4):660-668.doi:10.1038/bjc.2015.252.
[19]Geisen U,Zenthoefer M,Kalthoff H,et al.Molecular mechanisms by which a fucus vesiculosus extract mediates cell cycle inhibition and cell death in pancreatic cancer cells[J].Mar rugs,2015,13(7):4470-4491.doi:10.3390/md13074470.
[20]Jakubowska K,Guzińska-Ustymowicz K,Pryczynicz A,et al.Reduced expression of caspase-8 and cleaved caspase-3 in pancreatic ductal adenocarcinoma cells[J].Oncol Lett,2016,11(3):1879-1884.doi:10.3892/ol.2016.4125.
[21]葛建彬,顾锦华,李梅,等.银杏内酯A对小鼠脑缺血/再灌注损伤的保护作用及其抑制NF-κB信号通路下调p53、Caspase-3表达的机制[J].中国药理学通报,2012,28(8):1105-1110.
[22]Germain M,Affar EB,D'Amours D,et al.Cleavage of automodified poly(ADP-ribose)polymerase during apoptosis.Evidence for involvement of caspase-7[J].J Biol Chem,1999,274(40):28379-28384.doi:10.1074/jbc.274.40.28379.