益气解毒方通过MAPK/ERK信号通路抑制鼻咽癌细胞增殖
|
摘要
该文研究益气解毒方水提物对人鼻咽癌CNE2细胞增殖的抑制作用,并初步探讨其作用机制,为益气解毒方的临床应用提供新的理论依据。用不同质量浓度(0.125,0.25,0.5,1.0 g·L~(-1))的益气解毒方水提物、阳性对照药(顺铂,4.0 mg·L~(-1))、抑制剂PD98059(50μmol·L~(-1))、激活剂isoproterenol hydrochloride(ISO,20μmol·L~(-1))、激活剂ISO+益气解毒方水提物0.5 g·L~(-1)处理CNE2细胞,使用实时无标记细胞功能分析仪(RTCA)和CCK-8法检测细胞增殖活性,并计算出半数抑制浓度(IC50),用荧光双染流式细胞仪PI染色检测药物作用后细胞周期分布情况;Western blot法检测周期相关蛋白和MAPK/ERK信号通路相关蛋白的表达水平。RTCA和CCK-8法检测结果显示,与正常培养组比较,益气解毒方水提物能够有效抑制CNE2细胞增殖(P<0.01),呈剂量和时间依赖性,48 h IC50为0.5 g·L~(-1);细胞周期结果显示,水提物处理48 h后,G0/G1期比例降低,G2/M期比例增加,阻滞细胞周期于G2/M期(P<0.01);Western blot实验结果显示,经过不同浓度水提物处理48 h后,细胞周期相关蛋白cyclin D1,cyclin D3,CDK2表达下调,MAPK/ERK信号通路相关蛋白p-c-Raf,p-MEK,p-ERK1/2表达明显下调,与对照组相比差异具有统计学意义(P<0.05);在此基础上,加入MAPK/ERK信号通路的激活剂和抑制剂,结果显示,加入激活剂ISO,细胞增殖明显高于正常培养组,周期相关蛋白cyclin D1,cyclin D3,CDK2表达量均提高,同时,该信号通路关键蛋白p-c-Raf,p-MEK,p-ERK1/2的表达量也相对上升;而加入抑制剂PD98059后,细胞增殖明显低于正常培养组,相应蛋白的表达量亦降低,与对照组相比差异明显(P<0.05)。益气解毒方水提物主要通过下调MAPK/ERK信号通路关键蛋白p-c-Raf,p-MEK,p-ERK1/2的表达,阻滞细胞周期,抑制CNE2细胞增殖。
To study the effect of aqueous extracts of Yiqi Jiedu formula( YQ) on the proliferation of CNE2 cells in human nasopharyngeal carcinoma,and investigate its mechanism to provide a new theoretical basis for the clinical application of YQ. CNE2 cells were treated with different concentrations( 0. 125,0. 25,0. 5,0. 25 g·L~(-1)) of YQ,positive control medicine( cisplatin 4. 0 mg·L~(-1)),inhibitor PD98059( 50 μmol·L~(-1)),activator isoproterenol hydrochloride( 20 μmol·L~(-1)),activator isoproterenol hydrochloride( ISO) + YQ 0. 5 g·L~(-1). Then cell labeling by using real-time analyzer( RTCA) and CCK 8 method were used to detect cell proliferation activity,and the half inhibitory concentration( IC_(50)) was calculated. The cell cycle distribution was detected by fluorescence double dye flow cytometry PI staining,and Western blot method was used to detect the expression levels of related protein and MAPK/ERK signaling pathway. The results of RTCA and CCK-8 test showed that as compared with the control group,YQ group could effectively inhibit the proliferation of CNE2 cells( P < 0. 01),with a dose and time dependence,and 48 h IC_(50) value was 0. 5 g·L~(-1). The results of cell cycle showed that after 48 h of water extract treatment,the cell cycle was significantly changed,the proportion of G_0/G_1 was reduced,the ratio of G_2/M increased,and the cell cycle was in G_2/M period( P < 0. 01). Western blot results showed that after48 h treatment with different concentrations of aqueous extract,cell cycle-related proteins cyclin D1,cyclin D3 and CDK2 expression levels were down-regulated; MAPK/ERK signaling pathway related protein p-c-Raf,p-MEK,p-ERK1/2 expression level significantly lower as compared with the control group( P < 0. 05). After adding activator and inhibitor in MAPK/ERK signaling pathway on this basis,the results showed that after adding activator ISO,cell proliferation was significantly higher than that in the Control group; the cycle related proteins cyclin D1,cyclin D3,and CDK2 expression levels were increased; at the same time,key protein p-c-Raf,p-MEK,p-ERK1/2 expression levels in the signal pathways were relatively increased. While after the addition of inhibitor PD98059,the cell proliferation was significantly lower than that in the Control group,and the expression level of corresponding protein was decreased,which was significantly different from the Control group( P < 0. 05). So YQ could block cell cycle and inhibit the proliferation of CNE2 cells mainly by reducing the expression of MAPK/ERK signaling pathway key protein p-c-Raf,p-MEK and p-ERK1/2.
To study the effect of aqueous extracts of Yiqi Jiedu formula( YQ) on the proliferation of CNE2 cells in human nasopharyngeal carcinoma,and investigate its mechanism to provide a new theoretical basis for the clinical application of YQ. CNE2 cells were treated with different concentrations( 0. 125,0. 25,0. 5,0. 25 g·L~(-1)) of YQ,positive control medicine( cisplatin 4. 0 mg·L~(-1)),inhibitor PD98059( 50 μmol·L~(-1)),activator isoproterenol hydrochloride( 20 μmol·L~(-1)),activator isoproterenol hydrochloride( ISO) + YQ 0. 5 g·L~(-1). Then cell labeling by using real-time analyzer( RTCA) and CCK 8 method were used to detect cell proliferation activity,and the half inhibitory concentration( IC_(50)) was calculated. The cell cycle distribution was detected by fluorescence double dye flow cytometry PI staining,and Western blot method was used to detect the expression levels of related protein and MAPK/ERK signaling pathway. The results of RTCA and CCK-8 test showed that as compared with the control group,YQ group could effectively inhibit the proliferation of CNE2 cells( P < 0. 01),with a dose and time dependence,and 48 h IC_(50) value was 0. 5 g·L~(-1). The results of cell cycle showed that after 48 h of water extract treatment,the cell cycle was significantly changed,the proportion of G_0/G_1 was reduced,the ratio of G_2/M increased,and the cell cycle was in G_2/M period( P < 0. 01). Western blot results showed that after48 h treatment with different concentrations of aqueous extract,cell cycle-related proteins cyclin D1,cyclin D3 and CDK2 expression levels were down-regulated; MAPK/ERK signaling pathway related protein p-c-Raf,p-MEK,p-ERK1/2 expression level significantly lower as compared with the control group( P < 0. 05). After adding activator and inhibitor in MAPK/ERK signaling pathway on this basis,the results showed that after adding activator ISO,cell proliferation was significantly higher than that in the Control group; the cycle related proteins cyclin D1,cyclin D3,and CDK2 expression levels were increased; at the same time,key protein p-c-Raf,p-MEK,p-ERK1/2 expression levels in the signal pathways were relatively increased. While after the addition of inhibitor PD98059,the cell proliferation was significantly lower than that in the Control group,and the expression level of corresponding protein was decreased,which was significantly different from the Control group( P < 0. 05). So YQ could block cell cycle and inhibit the proliferation of CNE2 cells mainly by reducing the expression of MAPK/ERK signaling pathway key protein p-c-Raf,p-MEK and p-ERK1/2.
引文
[1]Kong F,Cai B,Lin S,et al.Assessment of radiotherapy combined with adjuvant chemotherapy in the treatment of patients with advanced nasopharyngeal carcinoma:a prospective study[J].J BUON,2015,20(1):206.
[2]尚云峰,田道法,YANG P C.益气解毒法不同组方对鼻咽癌CNE2Z细胞迁徙运动潜能及相关基因表达活性的影响[J].中国中西医结合耳鼻咽喉科杂志,2013(3):165.
[3]廖雪,蔺婷,罗晶婧,等.益气解毒方水提物对RAW264.7细胞免疫功能的影响[J].肿瘤基础与临床,2015(6):473.
[4]陈惠,渠景连,龚婕宁.现代医学对恶性肿瘤转移相关机制的研究进展[J].中国中药杂志,2014,39(15):2823.
[5]陈向军,刘国磊,郜元坤,等.顺铂诱导鼻咽癌CNE-2细胞自噬和凋亡的体外研究[J].现代肿瘤医学,2015,23(2):180.
[6]Sun X J,Zhang P,Li H H,et al.Cisplatin combined with metformin inhibits migration and invasion of human nasopharyngeal carcinoma cells by regulating E-cadherin and MMP-9[J].Asian Pac J Cancer P,2014,15(9):4019.
[7]郭瑞霞,张瑞芳,王新燕,等.信号传导通路抑制剂PD98059和LY294002对子宫内膜癌细胞和裸鼠移植瘤的抑制作用[J].中华妇产科杂志,2011,46(6):446.
[8]Ah L J,Yong-Sung J.Isoproterenol increases histone deacetylase6 expression and cell migration by inhibiting ERK signaling via PKA and Epac pathways in human lung cancer cells[J].Exp Mol Med,2016,48(1):e204.
[9]Kaschula C H,Hunter R,Stellenboom N,et al.Structure-activity studies on the anti-proliferation activity of ajoene analogues in WHCO1 oesophageal cancer cells[J].Eur J Med Chem,2012,43(33):236.
[10]王兵,侯炜.中医辨治鼻咽癌的几点认识[J].世界中西医结合杂志,2013,8(1):89.
[11]王磊,梁杰,阿提坎·卡吾力,等.复方苦参注射液治疗鼻咽癌放射性口腔黏膜损伤疗效观察[J].中草药,2015,46(6):875.
[12]胡梅,田道法,刘群良,等.益气解毒方提取物对CNE2细胞的体内外抑瘤作用[J].中国中医药信息杂志,2012(11):41.
[13]江洁琼,贺安意,田道法,等.益气解毒方对Tg N(p53mtLMP1)/HT转基因小鼠鼻腔和鼻咽黏膜上皮细胞增殖及凋亡相关基因表达的影响[J].中国组织工程研究,2008,12(42):8201.
[14]Wu A,Luo W,Zhang Q,et al.Aldehyde dehydrogenase 1,a functional marker for identifying cancer stem cells in human nasopharyngeal carcinoma[J].Cancer Lett,2013,330(2):181.
[15]Heilmann A M,Dyson N J.Phosphorylation puts the pRb tumor suppressor into shape[J].Gene Dev,2012,26(11):1128.
[16]Qiu C,Xie Q,Zhang D,et al.GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK Signaling[J].Cell Physiol Biochem,2014,33(3):784.
[17]Chen J C,Chen Y J,Lin C Y,et al.Amphiregulin enhances alpha6beta1 integrin expression and cell motility in human chondrosarcoma cells through Ras/Raf/MEK/ERK/AP-1 pathway[J].Oncotarget,2015,6(13):11434.
[18]Gutin J,Sadeh A,Rahat A,et al.Condition-specific genetic interaction maps reveal crosstalk between the c AMP/PKA and the HOG MAPK pathways in the activation of the general stress response[J].Mol Syst Biol,2015,11(10):829.
[19]Chen X H,Ling X M,Shi S.microRNA-106a induces the proliferation and apoptosis of glioma cells through regulating JNK/MAPK pathway[J].Eur Rev Med Pharmaco,2016,20(7):1221.
[2]尚云峰,田道法,YANG P C.益气解毒法不同组方对鼻咽癌CNE2Z细胞迁徙运动潜能及相关基因表达活性的影响[J].中国中西医结合耳鼻咽喉科杂志,2013(3):165.
[3]廖雪,蔺婷,罗晶婧,等.益气解毒方水提物对RAW264.7细胞免疫功能的影响[J].肿瘤基础与临床,2015(6):473.
[4]陈惠,渠景连,龚婕宁.现代医学对恶性肿瘤转移相关机制的研究进展[J].中国中药杂志,2014,39(15):2823.
[5]陈向军,刘国磊,郜元坤,等.顺铂诱导鼻咽癌CNE-2细胞自噬和凋亡的体外研究[J].现代肿瘤医学,2015,23(2):180.
[6]Sun X J,Zhang P,Li H H,et al.Cisplatin combined with metformin inhibits migration and invasion of human nasopharyngeal carcinoma cells by regulating E-cadherin and MMP-9[J].Asian Pac J Cancer P,2014,15(9):4019.
[7]郭瑞霞,张瑞芳,王新燕,等.信号传导通路抑制剂PD98059和LY294002对子宫内膜癌细胞和裸鼠移植瘤的抑制作用[J].中华妇产科杂志,2011,46(6):446.
[8]Ah L J,Yong-Sung J.Isoproterenol increases histone deacetylase6 expression and cell migration by inhibiting ERK signaling via PKA and Epac pathways in human lung cancer cells[J].Exp Mol Med,2016,48(1):e204.
[9]Kaschula C H,Hunter R,Stellenboom N,et al.Structure-activity studies on the anti-proliferation activity of ajoene analogues in WHCO1 oesophageal cancer cells[J].Eur J Med Chem,2012,43(33):236.
[10]王兵,侯炜.中医辨治鼻咽癌的几点认识[J].世界中西医结合杂志,2013,8(1):89.
[11]王磊,梁杰,阿提坎·卡吾力,等.复方苦参注射液治疗鼻咽癌放射性口腔黏膜损伤疗效观察[J].中草药,2015,46(6):875.
[12]胡梅,田道法,刘群良,等.益气解毒方提取物对CNE2细胞的体内外抑瘤作用[J].中国中医药信息杂志,2012(11):41.
[13]江洁琼,贺安意,田道法,等.益气解毒方对Tg N(p53mtLMP1)/HT转基因小鼠鼻腔和鼻咽黏膜上皮细胞增殖及凋亡相关基因表达的影响[J].中国组织工程研究,2008,12(42):8201.
[14]Wu A,Luo W,Zhang Q,et al.Aldehyde dehydrogenase 1,a functional marker for identifying cancer stem cells in human nasopharyngeal carcinoma[J].Cancer Lett,2013,330(2):181.
[15]Heilmann A M,Dyson N J.Phosphorylation puts the pRb tumor suppressor into shape[J].Gene Dev,2012,26(11):1128.
[16]Qiu C,Xie Q,Zhang D,et al.GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK Signaling[J].Cell Physiol Biochem,2014,33(3):784.
[17]Chen J C,Chen Y J,Lin C Y,et al.Amphiregulin enhances alpha6beta1 integrin expression and cell motility in human chondrosarcoma cells through Ras/Raf/MEK/ERK/AP-1 pathway[J].Oncotarget,2015,6(13):11434.
[18]Gutin J,Sadeh A,Rahat A,et al.Condition-specific genetic interaction maps reveal crosstalk between the c AMP/PKA and the HOG MAPK pathways in the activation of the general stress response[J].Mol Syst Biol,2015,11(10):829.
[19]Chen X H,Ling X M,Shi S.microRNA-106a induces the proliferation and apoptosis of glioma cells through regulating JNK/MAPK pathway[J].Eur Rev Med Pharmaco,2016,20(7):1221.