Bufalin在食管癌细胞中对MMP-2、MMP-9及MEK信号通路的作用机制研究
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摘要
目的：我国是食管癌的高发国家，根据调查研究显示,食管癌死亡率仅次于肺癌、胃癌、肝癌，居全国第4位。肿瘤的发生、发展是物理、化学、生物等环境因素与遗传因素共同作用的结果，是一个多因素、多阶段、累积渐进的过程。而在肿瘤细胞局部浸润、无限增殖及远处转移的背后，细胞信号转导异常与其表现出了一定的关系。因此进一步对与其相关的信号转导通路及临床抗肿瘤药物作用机制的研究，仍为食管癌治疗研究的目标。
细胞外信号调节激酶(ERK1/2)是丝裂原活化蛋白激酶((mitogen-activated protein kinase,MAPK)家族中的重要成员之一，其中，Ras/Raf/MEK/ERK是ERK信号转导通路的主要途径。而其中，MEK是ERK信号通路的关键蛋白，分为MEK1和MEK2两种亚型,其作用是磷酸化并激活下游底物ERK。MEK1/2广泛存在于各种生物体中，在细胞癌变过程中发挥重要作用，其可被多种丝裂原信号及癌基因产物所激活从而成为P-MEK1/2，进而转导胞外信号至细胞核内，进一步参与调节细胞的增长、分化、凋亡等多种生理活动，并在细胞的恶性转化过程中发挥重要作用。
国内外学者研究发现，基质金属蛋白酶(MMPS)是降解细胞外基质蛋白的最主要的蛋白水解酶，它在肿瘤的发生、发展过程中具有核心作用。大量研究表明，MMPS具有广泛的底物特异性，通过降解细胞外基质成分，促进肿瘤侵袭及转移，并且还可以通过新生血管生成等促进肿瘤的生长和扩散。其中，MMP-2与MMP-9是基质金属蛋白酶家族中分布最广、研究较清楚的主要成员。研究发现，MMP-2、MMP-9在多种实体瘤中（包括肺癌、乳腺癌、宫颈癌、结肠癌、骨肉瘤、乳头状甲状腺癌）均呈现高表达。另有学者研究认为，在纤维肉瘤细胞HT1080中，抑制ERK磷酸化，MMP-2、MMP-9的表达进而受到抑制，表明MMP-2和MMP-9的表达依赖ERK信号通路中ERK的磷酸化。而本实验中MEK又为ERK信号通路的重要成员。
本实验通过研究不同药物浓度的Bufalin(0、10、25、50、100nmol/L)作用于体外培养的食管癌细胞株(TE-13)后，并观察对MMP-2、MMP-9以及MEK1/2及其活化形式(P-MEK1/2)表达的影响，进一步探讨MEK信号通路与基质金属蛋白酶(MMPS)在食管癌发生中的相互作用关系，为MEK信号通路成为食管癌临床治疗的新靶点提供必要的实验依据。
方法：常规培养的人食管癌细胞TE-13经不同浓度的Bufalin（蟾蜍灵）进行处理后，通过MTT方法检测细胞药物毒性，采用逆转录聚合酶链反应RT-PCR方法对细胞中MMP-2、MMP-9mRNA的表达进行分析，采用蛋白印记Western-blot及免疫细胞化学方法对食管癌细胞进行MEK1/2、P-MEK1/2蛋白的相对定量检测，分析药物作用后其表达水平的变化。统计分析采用SPSS13.0软件，数据采用单因素方差分析。
结果：
1MTT检测结果
不同浓度的蟾蜍素（对照组，10nM/L，25nM/L，50nM/L，100nM/L，200nM/L，400nM/L，800nM/L）干预24h后平均OD值（0.991、0.869、0.735、0.561、0.499、0.371、0.265、0.258），蟾蜍素在100nM/L及其以下的药物浓度作用下，细胞存活率大于50%，即TD0为100nM/L。
2RT-PCR结果显示：
MMP-2mRNA的表达量在食管癌细胞TE-13中随着Bufalin药物浓度的升高而逐渐降低，100nmol/L时表达量最低。相对表达量对照组为（0.772±0.010），10、25、50、100nmol/L分别为（0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019），各浓度与对照组相比，差异具有显著的统计学意义（P<0.05）。
MMP-9mRNA的表达量随着Bufalin药物浓度的升高而呈现逐渐降低的趋势，并且100nmol/L时表达量最低。相对表达量对照组为（0.783±0.013），10、25、50、100nmol/L分别为（0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017），与对照组相比，差异具有显著的统计学意义（P<0.05），结果与MMP-2mRNA相一致。
3Western-blot结果显示：
MEK1/2的表达量随着Bufalin浓度的升高没有显著变化，相对表达量对照组、10、25、50、100nmol/L分别为（0.724±0.005、0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006），各组间差异没有统计学意义（P＞0.05）。
P-MEK1/2表达量随着Bufalin浓度的升高而呈降低趋势，100nmol/L时表达量最低。相对表达量对照组为（0.710±0.006），10、25、50、100nmol/L分别为（0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020），各组与对照组相比，差异具有显著的统计学意义（P<0.05）。说明Bufalin可以抑制MEK1/2的活化形式(P-MEK1/2)，而对MEK1/2没有显著影响。
4免疫细胞化学检测结果显示：
MEK1/2的阳性表达率随着Bufalin浓度的升高没有显著变化，对照组、10、25、50、100nmol/L分别76.58、76.47、76.04、75.40、74.98，差异没有统计学意义（P＞0.05）。
P-MEK1/2阳性细胞随着Bufalin浓度的升高而减少，棕黄色颗粒密度减低，阳性率分别为80.33、68.41、67.30、52.62、27.00，与对照组相比，差异具有统计学意义（P<0.05）。
结论：
1在食管癌细胞TE-13中，随着Bufalin药物浓度的升高，MEK1/2蛋白的表达水平没有显著变化，表明不能被Bufalin有效的抑制。
2MMP-2、MMP-9mRNA及P-MEK1/2蛋白在食管癌细胞TE-13中的表达能够被Bufalin有效的抑制，且随着药物浓度的升高，表达逐渐减弱，呈现药物剂量依赖性。表明Bufalin治疗食管癌的机制可能与下调MMP-2、MMP-9mRNA及P-MEK1/2蛋白的表达有关。
3在食管癌中，MMP-2、MMP-9与P-MEK1/2的表达呈现正相关，二者联合有可能成为食管癌治疗的新靶点。
Objective: China is a country of high incidence of esophageal cancer, thesurvey data show that the mortality rate of esophageal cancer ranked fouth inChina, after only to lung cancer, stomath cancer and liver cancer. Theoccurrence and developpment of tumer is a result of the role of genetic andenvironmental factors，and is a multi-factor, multi-stage, accumulated gradualprocess. Behind the local infiltration, unlimited proliferaton, distant metastasisin the tumor cells, the abnormal of cell sigal transduction show a certainrelationship. Therefore, the further study of the mechanism of action ofclinical anticancer drugs and associated with signal transduction pathways, isstill the goal in the treatment of esophageal cancer.
Extracellular signal-regulated kinase (ERK) is one of the importantmembers of mitogen activated protein kinase (MAPK) family. In the ERKsignal transduction pathway, the Ras/Raf/MEK/ERK is the main way. Amongthem, the MEK is the key protein of the ERK signal transduction pathway andit is divided into two subtypes, MEK1and MEK2. The role of MEK is tophosphorylate and activate ERK, which is the downstream substrates of MEK.MEK1/2widely exists in a variety of organisms, and plays an important role inthe process of carcinogenesis in the cell. MEK1/2can also be activated intothe state of phosphorylation (P-MEK1/2) by variety of mitogen-activatedsignaling and oncogene products. P-MEK1/2transducts signal to the nucleus,involves in regulating cell proliferation, differentiation, apoptosis and otherphysiological processes, and plays an important role in malignanttransformation of cell.
Domestic and foreign scholars have found that matrix metalloproteinase（MMPS）is the main protelytic enzymes to degradate the extracellular matrixproteins, and it has a central role in the process of the occurrence and developpment of tumer. Numerous studies show that MMPS has a broadsubstrate specificity, it can promote the invasion and metastasis of tumor bydegradating the extracellular matrix components. Also, it can promote thegrowth and spread of tumor through angiogenesis. Among them, MMP-2andMMP-9are the main members of most widely distributed and clearer researthin MMPS family. Studies have found that MMP-2and MMP-9showed a highexpression in a variety of solid tumors, including lung cancer, breast cancer,cervical cancer, colon cancer, osreosarcoma and papillary thyroid carcinoma.Some scholars think that the expression of MMP-2and MMP-9dependents onthe phosphorylation of ERK in the ERK signaling pathway in fibrosarcomacells HT1080. We can inhibit the expression of MMP-2and MMP-9byinhibiting the phosphorylation of ERK. In this experiment, MEK is animportant member in ERK signaling pathway.
In this experiment, by investigating Bufalin with different concentrationroles in esophageal carcinoma cell line in vitro (TE-13), we observe the effectof Bufalin on the expression of MMP-2, MMP-9, MEK1/2and its activatedform (P-MEK1/2). And to further explore the interaction between MEKsignaling pathway and matrix metalloproteinases (MMPS) in esophagealcancer. It may provide experimental evidence for whether MEK signalingpathway is a new target for treating esophageal carcinoma.
Methods: After dealing the esophageal cancer cell lines TE-13withBufalin of different concentration, we detected the expression of MMP-2andMMP-9by transcriptase-polymerase chain reaction RT-PCR and theexpression of MEK1/2and P-MEK1/2using Western-blot andImmunocytochemistry methods. And further more to detect the expression ofdrug effects. Statistical analysis used SPSS13.0software, date usesingle-factor analysis of variance.
Results：
1RT-PCR results showed that in esophageal cancer cells TE-13, theexpression level of MMP-2mRNA gradually decreased with the Bufalinconcentration increasing and100nmol/L was the lowest. The relative expression level of MMP-2mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019, compared with0.772±0.010of control group. Differences werestatistically significant (P＜0.05).
The expression level of MMP-9mRNA also gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of MMP-9mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017, compared with0.783±0.013of control group. Differences werestatistically significant (P＜0.05). The results was consistent withMMP-2mRNA.
2Western-blot results showed that the expression level of MEK1/2proteinhad no significant change with the Bufalin concentration increasing. Therelative expression level of MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006, compared with0.724±0.005of control group. The differencebetween the groups was not statistically significant (P＞0.05).
The expression level of P-MEK1/2protein gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of P-MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020, compared with0.710±0.006of control group. Differences werestatistically significant (P＜0.05). It explain Bufalin can inhibit the activationof MEK1/2(P-MEK1/2), and the inhibition was dose-dependent.
3The Immunocytochemistry method showed that the positive expressionrate of MEK1/2had no significant change with the Bufalin concentrationincreasing. The difference between the groups was not statistically significant(P＞0.05).
With the concentration of Bufalin increased, the number of positive cellsgradually reduced, the density of brown-yellow particles also graduallyreduced. Differences were statistically significant (P＜0.05).
Conclusion:
1In esophageal cancer cells TE-13, the expression of MEK1/2protein hadno siginificant change with the Bufalin concentration increasing, showingthat it can not be inhibited by Bufalin.
2MMP-2、MMP-9mRNA and P-MEK1/2protein expression in esophagealcancer cells TE-13can be effectively inhibited by Bufalin. And theexpression level gradually decreased with the Bufalin concentrationincreasing, showing dose-dependent. This study indicates the treatingmechanism of Bufalin may be related with down-regulation ofMMP-2,MMP-9mRNA and P-MEK1/2protein.
3In esophageal cancer, the expression between MMP-2、MMP-9and P-MEK1/2have a positive correlation. Thus, their combination may becomea new target for esophageal cancer treatment.
细胞外信号调节激酶(ERK1/2)是丝裂原活化蛋白激酶((mitogen-activated protein kinase,MAPK)家族中的重要成员之一，其中，Ras/Raf/MEK/ERK是ERK信号转导通路的主要途径。而其中，MEK是ERK信号通路的关键蛋白，分为MEK1和MEK2两种亚型,其作用是磷酸化并激活下游底物ERK。MEK1/2广泛存在于各种生物体中，在细胞癌变过程中发挥重要作用，其可被多种丝裂原信号及癌基因产物所激活从而成为P-MEK1/2，进而转导胞外信号至细胞核内，进一步参与调节细胞的增长、分化、凋亡等多种生理活动，并在细胞的恶性转化过程中发挥重要作用。
国内外学者研究发现，基质金属蛋白酶(MMPS)是降解细胞外基质蛋白的最主要的蛋白水解酶，它在肿瘤的发生、发展过程中具有核心作用。大量研究表明，MMPS具有广泛的底物特异性，通过降解细胞外基质成分，促进肿瘤侵袭及转移，并且还可以通过新生血管生成等促进肿瘤的生长和扩散。其中，MMP-2与MMP-9是基质金属蛋白酶家族中分布最广、研究较清楚的主要成员。研究发现，MMP-2、MMP-9在多种实体瘤中（包括肺癌、乳腺癌、宫颈癌、结肠癌、骨肉瘤、乳头状甲状腺癌）均呈现高表达。另有学者研究认为，在纤维肉瘤细胞HT1080中，抑制ERK磷酸化，MMP-2、MMP-9的表达进而受到抑制，表明MMP-2和MMP-9的表达依赖ERK信号通路中ERK的磷酸化。而本实验中MEK又为ERK信号通路的重要成员。
本实验通过研究不同药物浓度的Bufalin(0、10、25、50、100nmol/L)作用于体外培养的食管癌细胞株(TE-13)后，并观察对MMP-2、MMP-9以及MEK1/2及其活化形式(P-MEK1/2)表达的影响，进一步探讨MEK信号通路与基质金属蛋白酶(MMPS)在食管癌发生中的相互作用关系，为MEK信号通路成为食管癌临床治疗的新靶点提供必要的实验依据。
方法：常规培养的人食管癌细胞TE-13经不同浓度的Bufalin（蟾蜍灵）进行处理后，通过MTT方法检测细胞药物毒性，采用逆转录聚合酶链反应RT-PCR方法对细胞中MMP-2、MMP-9mRNA的表达进行分析，采用蛋白印记Western-blot及免疫细胞化学方法对食管癌细胞进行MEK1/2、P-MEK1/2蛋白的相对定量检测，分析药物作用后其表达水平的变化。统计分析采用SPSS13.0软件，数据采用单因素方差分析。
结果：
1MTT检测结果
不同浓度的蟾蜍素（对照组，10nM/L，25nM/L，50nM/L，100nM/L，200nM/L，400nM/L，800nM/L）干预24h后平均OD值（0.991、0.869、0.735、0.561、0.499、0.371、0.265、0.258），蟾蜍素在100nM/L及其以下的药物浓度作用下，细胞存活率大于50%，即TD0为100nM/L。
2RT-PCR结果显示：
MMP-2mRNA的表达量在食管癌细胞TE-13中随着Bufalin药物浓度的升高而逐渐降低，100nmol/L时表达量最低。相对表达量对照组为（0.772±0.010），10、25、50、100nmol/L分别为（0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019），各浓度与对照组相比，差异具有显著的统计学意义（P<0.05）。
MMP-9mRNA的表达量随着Bufalin药物浓度的升高而呈现逐渐降低的趋势，并且100nmol/L时表达量最低。相对表达量对照组为（0.783±0.013），10、25、50、100nmol/L分别为（0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017），与对照组相比，差异具有显著的统计学意义（P<0.05），结果与MMP-2mRNA相一致。
3Western-blot结果显示：
MEK1/2的表达量随着Bufalin浓度的升高没有显著变化，相对表达量对照组、10、25、50、100nmol/L分别为（0.724±0.005、0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006），各组间差异没有统计学意义（P＞0.05）。
P-MEK1/2表达量随着Bufalin浓度的升高而呈降低趋势，100nmol/L时表达量最低。相对表达量对照组为（0.710±0.006），10、25、50、100nmol/L分别为（0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020），各组与对照组相比，差异具有显著的统计学意义（P<0.05）。说明Bufalin可以抑制MEK1/2的活化形式(P-MEK1/2)，而对MEK1/2没有显著影响。
4免疫细胞化学检测结果显示：
MEK1/2的阳性表达率随着Bufalin浓度的升高没有显著变化，对照组、10、25、50、100nmol/L分别76.58、76.47、76.04、75.40、74.98，差异没有统计学意义（P＞0.05）。
P-MEK1/2阳性细胞随着Bufalin浓度的升高而减少，棕黄色颗粒密度减低，阳性率分别为80.33、68.41、67.30、52.62、27.00，与对照组相比，差异具有统计学意义（P<0.05）。
结论：
1在食管癌细胞TE-13中，随着Bufalin药物浓度的升高，MEK1/2蛋白的表达水平没有显著变化，表明不能被Bufalin有效的抑制。
2MMP-2、MMP-9mRNA及P-MEK1/2蛋白在食管癌细胞TE-13中的表达能够被Bufalin有效的抑制，且随着药物浓度的升高，表达逐渐减弱，呈现药物剂量依赖性。表明Bufalin治疗食管癌的机制可能与下调MMP-2、MMP-9mRNA及P-MEK1/2蛋白的表达有关。
3在食管癌中，MMP-2、MMP-9与P-MEK1/2的表达呈现正相关，二者联合有可能成为食管癌治疗的新靶点。
Objective: China is a country of high incidence of esophageal cancer, thesurvey data show that the mortality rate of esophageal cancer ranked fouth inChina, after only to lung cancer, stomath cancer and liver cancer. Theoccurrence and developpment of tumer is a result of the role of genetic andenvironmental factors，and is a multi-factor, multi-stage, accumulated gradualprocess. Behind the local infiltration, unlimited proliferaton, distant metastasisin the tumor cells, the abnormal of cell sigal transduction show a certainrelationship. Therefore, the further study of the mechanism of action ofclinical anticancer drugs and associated with signal transduction pathways, isstill the goal in the treatment of esophageal cancer.
Extracellular signal-regulated kinase (ERK) is one of the importantmembers of mitogen activated protein kinase (MAPK) family. In the ERKsignal transduction pathway, the Ras/Raf/MEK/ERK is the main way. Amongthem, the MEK is the key protein of the ERK signal transduction pathway andit is divided into two subtypes, MEK1and MEK2. The role of MEK is tophosphorylate and activate ERK, which is the downstream substrates of MEK.MEK1/2widely exists in a variety of organisms, and plays an important role inthe process of carcinogenesis in the cell. MEK1/2can also be activated intothe state of phosphorylation (P-MEK1/2) by variety of mitogen-activatedsignaling and oncogene products. P-MEK1/2transducts signal to the nucleus,involves in regulating cell proliferation, differentiation, apoptosis and otherphysiological processes, and plays an important role in malignanttransformation of cell.
Domestic and foreign scholars have found that matrix metalloproteinase（MMPS）is the main protelytic enzymes to degradate the extracellular matrixproteins, and it has a central role in the process of the occurrence and developpment of tumer. Numerous studies show that MMPS has a broadsubstrate specificity, it can promote the invasion and metastasis of tumor bydegradating the extracellular matrix components. Also, it can promote thegrowth and spread of tumor through angiogenesis. Among them, MMP-2andMMP-9are the main members of most widely distributed and clearer researthin MMPS family. Studies have found that MMP-2and MMP-9showed a highexpression in a variety of solid tumors, including lung cancer, breast cancer,cervical cancer, colon cancer, osreosarcoma and papillary thyroid carcinoma.Some scholars think that the expression of MMP-2and MMP-9dependents onthe phosphorylation of ERK in the ERK signaling pathway in fibrosarcomacells HT1080. We can inhibit the expression of MMP-2and MMP-9byinhibiting the phosphorylation of ERK. In this experiment, MEK is animportant member in ERK signaling pathway.
In this experiment, by investigating Bufalin with different concentrationroles in esophageal carcinoma cell line in vitro (TE-13), we observe the effectof Bufalin on the expression of MMP-2, MMP-9, MEK1/2and its activatedform (P-MEK1/2). And to further explore the interaction between MEKsignaling pathway and matrix metalloproteinases (MMPS) in esophagealcancer. It may provide experimental evidence for whether MEK signalingpathway is a new target for treating esophageal carcinoma.
Methods: After dealing the esophageal cancer cell lines TE-13withBufalin of different concentration, we detected the expression of MMP-2andMMP-9by transcriptase-polymerase chain reaction RT-PCR and theexpression of MEK1/2and P-MEK1/2using Western-blot andImmunocytochemistry methods. And further more to detect the expression ofdrug effects. Statistical analysis used SPSS13.0software, date usesingle-factor analysis of variance.
Results：
1RT-PCR results showed that in esophageal cancer cells TE-13, theexpression level of MMP-2mRNA gradually decreased with the Bufalinconcentration increasing and100nmol/L was the lowest. The relative expression level of MMP-2mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019, compared with0.772±0.010of control group. Differences werestatistically significant (P＜0.05).
The expression level of MMP-9mRNA also gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of MMP-9mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017, compared with0.783±0.013of control group. Differences werestatistically significant (P＜0.05). The results was consistent withMMP-2mRNA.
2Western-blot results showed that the expression level of MEK1/2proteinhad no significant change with the Bufalin concentration increasing. Therelative expression level of MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006, compared with0.724±0.005of control group. The differencebetween the groups was not statistically significant (P＞0.05).
The expression level of P-MEK1/2protein gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of P-MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020, compared with0.710±0.006of control group. Differences werestatistically significant (P＜0.05). It explain Bufalin can inhibit the activationof MEK1/2(P-MEK1/2), and the inhibition was dose-dependent.
3The Immunocytochemistry method showed that the positive expressionrate of MEK1/2had no significant change with the Bufalin concentrationincreasing. The difference between the groups was not statistically significant(P＞0.05).
With the concentration of Bufalin increased, the number of positive cellsgradually reduced, the density of brown-yellow particles also graduallyreduced. Differences were statistically significant (P＜0.05).
Conclusion:
1In esophageal cancer cells TE-13, the expression of MEK1/2protein hadno siginificant change with the Bufalin concentration increasing, showingthat it can not be inhibited by Bufalin.
2MMP-2、MMP-9mRNA and P-MEK1/2protein expression in esophagealcancer cells TE-13can be effectively inhibited by Bufalin. And theexpression level gradually decreased with the Bufalin concentrationincreasing, showing dose-dependent. This study indicates the treatingmechanism of Bufalin may be related with down-regulation ofMMP-2,MMP-9mRNA and P-MEK1/2protein.
3In esophageal cancer, the expression between MMP-2、MMP-9and P-MEK1/2have a positive correlation. Thus, their combination may becomea new target for esophageal cancer treatment.
引文
1Meng Z, Yang P, Shen Y, et al. Pilot study of huachansu in patients withhepatocellular carcinoma, nonsmall-cell lung cancer, or pancreaticcancer [J]. Cancer,2009,115:5309-5318
2Xu R, Chen X, Chen L, et al. The mechanisms of inhibitory effect ofbufalin on human leukemia cells [J]. Zhonghua Xue Ye Xue Za Zhi,2000,21(7):359-361
3张宁,凌昌全.蟾毒灵诱导肿瘤细胞凋亡机制的研究进展[J].现代肿瘤医学,2009,17(1):126-128
4Johnson GL, Lapadat R, Mitogen-activated protein kinase pathwaysmediated by ERK, JNK and p38protein kinases [J]. Science,2002,298(5600):1911-1912
5McCubrey JA, Steelman LS, ChappellWH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant, transformation and drugresistance [J]. Biochim Biophys Acta,2007,1773(8):1263-1284
6Vilalta A, Sahuquillo J, PocaMA. Matrix metaloproteinases inneurologicalbrain lesions: a new therapeutic target [J]. Rev Neurol,2010,51(2):95-107
7Kawazoe N,Watabe M, Masuda Y, et al. Tiam1is involved in theregulation of bufalin-induced apoptosis in human leukemia cells [J].Oncogene,1999,18(15):2413-2421
8Takai N, Ueda T, Nishida M, et al. Bufalin induces growth inhibition,cellcycle arrest and apoptosis in human endometrial and ovarian cancercells[J]. Int J Mol Med,2008,21(5):637-643
9leiner DE, Stetler-Stevenson WG. Matrix metalloproteinases andmetastasis[J]. Cancer Chemother Pharmacol,1999,43Suppl: S42-S51.
10Lu HJ, Guo CB, Jin XQ, et al. The role of matrix metalloproteinaseexpression in lung cancer metastasis [J].Chin Clin Oncol,2006,11(7):486-490
11Janow ska Wieczorek A, M arquezL A, Mat suz aki A, et al. Expressionof matrix metalloproteinases(MMP-2and-9) and tissue inhibit or ofmetalloproteinases (TIMP-1and-2) in acute myelogenous leukemiablasts:comparison with normal bonemarrow cells[J]. BrJ Haem at ol,1999,105:402-411
12Janow ska Wieezorek A, Marquez LA, Doh row s ky A, et al. DifierentialMMP and TIMP production by human marrow and peripheral bloodCD34+celL in response to ehem okines[J]. Exp H emat ol,2000,28:1274-1285
13Saw icki G, Mat suz aki A, Janow ska Wieczorek A. Expression of theactive form of MMP-2on the surface of leukemie cells accounts for theirin vitro invasion [J]. Cancer Res C lin Oncol,1998,124:245-252
14Zucker S, Lysik RM, Zarrabi MH, et al. M(r)92,000type IV collagenaseis in creased in plasma of pat ients with colon cancer and breastcancer[J]. Cancer Rse,1993,53(1):140-146
15Maemura K, Shiraishi N, Sakagami K, et a1. Proliferative effects ofgamma-aminobutyric acid on the gastric cancer cell line are associatedwith extracellular signal-regulated kinase1/2activation[J]. J GastroenterolHepatol,2009,24(4):688-696
16Chang F, S teelman LS, Lee JT, et al. Signal transduction mediated by theRas/Raf/MEK/ERK pathway from cytokine receptors to transcriptionfactors: potential targeting for therapeutic intervention [J]. Leuk emia,2003,17:1263-1293
17Fiddes RJ, Janes PW, Sivertsen SP, et al. Inhibition of the MAP kinasecascade blocks heregulin2induced cell cycle progression in T-47D humanbreast cancer [J]. Oncogene,1998,16(21):2803-2813
18Nakata H, Wang SL, Chung DC, et al. Oncogenic ras induces gastrin geneexpression in colon cancer [J]. Gastroenterology1998,115(5):1144-1153
19Ding Q, Wang Q, Evers BM. Alterations of MAPK activities associatedwith intestinal cell differentiation [J]. Biochem Biophys Res Commun,2001,284(2):282-288
20Sebolt-Leopold JS, Dudley DT, Herrera R, et al.Blockade of the MAPkinase pathway suppresses growth of colon tumors in vivo [J].Nat Med,1999,5:810-816
21Zhang L, Nakaya K, Yoshida T, et al. Biochem Biophys ResCommun,1991,178(2):686-693
22Jing Y, Ohizumi H, Kawazoe N, et al. Jpn J Cancer Res,1994,85(6):645-651
23Masuda Y, Kawazoe N, Nakajo S,et al. Leuk Res,1995,19(8):549-556
24王江宁. ERK1/2和MMP-9蛋白在胆管癌组织中的表达及其临床意义[D].中国医科大学学报,2007
25Maeda-Yamamoto M, Suzuki N, Sawai Y, et al.Association of suppressionof extra cellular signal-regulated kinase phosphorylation byepigallocatechin gallate with the reduction of matrix metalloproteinaseactivities in human fibrosarcoma HT1080cells [J]. Agric Food Chem,2003,51(7):1858-1863
1张丰,李楠. ERK/MAPK信号传导途径在乳腺肿瘤治疗中的意义[J].中国肿瘤生物治疗杂志，2007(14):497-500
2Maemura K, Shiraishi N, Sakagami K, et a1. Proliferative effects ofgamma-aminobutyric acid on the gastric cancer cell line are associatedwith extracellular signal-regulated kinase1/2activation [J].J GastroenterolHepatol,2009,24(4):688-696
3Almog T, Naor Z. Mitogen activated protein kinases (MAPKs) asrugulators of spermatogenesis and spermatozoa functions [J]. Mol CellEndocrinol,2008,282(122):39-44
4Qi MS, Elion EA. MAP kinase pathways [J]. J Cell Sci,2005,118(16):3569-3572
5俞华芳，樊晓明，蒋淼，等． P38MAPK抑制剂对小鼠急性水肿型胰腺炎的作用[J].中国临床医学，2008，15(1):82-83
6McCubrey JA, Steelman LS, ChappellWH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant, transformation and drugresistance [J]. Biochim Biophys Acta,2007,1773(8):1263-1284
7Cobb MH. MAP kinase pathways [J]. Prog Biophys Mol Biol,199971(3-4):479-500
8Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specificmessages from ubiquitous messengers [J]. Mol Cell Biol,1999,19(14):2435-2444
9Dhanasekaran N, Premkumar Reddy E. Signaling by dual specificitykinases [J]. Oncogene,1998,17(11Reviews):1447-1455
10Seth A, Gonzalez FA, Gupta S, et al. Signal transduction within thenucleus by mitogen-activated protein kinase [J]. J Biol Chem,1992,267(34):24796-24804
11Greenwood JM, Dragunow M. M3muscarinic receptors promote cellsurvival through activation of the extracellular regulated kinase (ERKl／2)pathway [J]. Eur J Phanllacol,20l0,640(1-3):38-45
12Lorenz K, Schmitt JP, Vidal M, Lohse MJ. Cardiac hypertrophy: targetingRaf／MEK／ERKl／2-signaling [J].Int J Biochem Cell Biol,2009,41(12):2351-2355
13Schuringa J J, Jonk L J, Dokter W H, et al. Interleukin-6-induced STAT3transactivation and Ser727phosphorylation involves Vav, Rac-1and thekinase SEK-1/MKK-4as signal transduction components [J].Biochem J,2000,347:89-96
14Ding Q, Wang Q, Evers BM.Alterations of MAPK activities associatedwith intestinal cell differentiation [J]. Biochem Biophys Res Commun,2001,284:282-288
15Maemura K,Shiraishi N,Sakagami K,et al.Proliferative effects ofgammaaminobutyric acid on the gastric cancer cell line are associated withextracellular signal-regulated kinase1/2activation [J]. J GastroenterolHepatol,2009,24:688-696
16王殊，王杉，祝学光等。细胞外信号调节激酶及其上游激酶在人乳腺癌发生发展中的意义[J].中华外科杂志,2002,40(3):171-174
17Chin R, Earnest-Silveira L, Koeberlein B, et al. Modulation ofMAPK pathways and cell cycle by replicating hepatitis B virus:factors contributing to hepatocarcinogenesis [J]. J Hepatol,2007,47(3):325-337
18Looby E, Abdel-Latif MM, Athié-Morales V, et al. Deoxycholate inducesCOX-2expression via Erk1/2-,p38-MAPK and AP-1-dependentmechanisms in esophageal cancer cells [J]. BMC Cancer,2009,9:190
19Caja L, Sancho P, Bertran E, et al. Overactivation of the MEK/ERKpathway in liver tumor cells confers resistance to TGF-{beta}-induced celldeath through impairing upregulation of the NADPH oxidase NOX4[J].Cancer Res,2009,69(19):7595-7602
20Chen YJ, Tsai YM, Kuo CD, et al. Norcantharidin is a small-moleculesynthetic compound with antiangiogenes is effect [J]. Life Sci,2009,85(17-18):642-651
21Komi Y, Sogabe Y, Ishibashi N, et al. Acyclic retinoid inhibitsangiogenesis by suppressing the MAPK pathway [J]. Lab Invest,2010,90(1):52-60
22梁璇，丁新民，霍艳英． ERK1/2-Sp l信号通路对肺癌细胞VEGF的调控作用[J].癌变·畸变·突变，2007，19(4):280-284
23Cassano A, Bagala C, Battelli C, et al.Expression of vascular endothelialgrowth factor,mitogen-activated protein kinase and p53in humancolorectal cancer [J]. Anticancer Res,2002,22(4):2179-2184
24Zhang Yu-hua, WEI Wei, XU Hao, et al.Inducing effects of hepatocytegrowth factor on the expression of vascular endothelial growth factor inhuman colorectal carcinoma cells through MEK and PI3K signalingpathways [J]. Chin Med J,2007,120(9):743-748
25Vilalta A, Sahuquillo J, PocaMA. Matrix metaloproteinases inneurologicalbrain lesions: a new therapeutic target [J]. Rev Neurol,2010,51(2):95-107
26Lai KC, Huang AC, Hsu SC, et al. Benzyl isothiocyanate (BITC) inhibitsmigration and invasion of human coloncancer HT29cells by inhibitingmatrix metalloproteinase-2/-9and urokinase plasminogen (uPA) throughPKC and MAPK signaling pathway [J]. J Agric Food Chem,2010,58(5):2935-2942
27Weng CJ, Chau CF, Hsieh YS, et al. Lucidenic acid inhibits PMA-inducedinvasion of human hepatoma cells through inactivating MAPK/ERK signaltransduction pathway and reducing binding activities of NF-kappaB andAP-1[J]. Carcinogenesis,2008,29(1):147-156
28Endo H, Watanabe T, Sugioka Y, et al. Activation of two distinct MAPKpathways governs constitutive expression of matrix metalloproteinase-1inhuman pancreatic cancer cell lines[J]. Int J Oncol,2009,35(6):1237-1245
29Maeda-Yamamoto M, Suzuki N, Sawai Y, et al.Association of suppressionof extra cellular signal-regulated kinase phosphorylation byepigallocatechin gallate with the reduction of matrix metalloproteinaseactivities in human fibrosarcoma HT1080cells [J]. J Agric Food Chem,2003,51(7):1858-1863
30Steinmetz R, Wagoner HA, Zeng P, et al.Mechanisms Regulating theConstitutive Act ivation of the Extracellular Signal-Regulated Kinase(ERK) Signaling Pathway in Ovarian Cancer and the Effect ofRibonucleic Acid Interference for ERK1/2on Cancer Cell Proliferation[J]. Molecular Endocrinology,2004,18(10):2570-2582
31Yao Q, Luo JR, Chen JH, et al. Expression and activation of MAPKpathway signaling molecules in human breast cancer cell lines [J].Chin JCell Mol Immunol,2004,20(3):3282-3330
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34尤嘉琮,汪宏斌,杨宗伟,等.高转移倾向乳腺癌细胞中P-ERK促进增殖和迁移作用的信号转导途径[J].中国生物化学与分子生物学报,2006,22(12):1007-1013
35Chang F, S teelman LS, Lee JT, et al. Signal transduction mediated by theRas/Raf/MEK/ERK pathway from cytokine receptors to transcriptionfactors: potential targeting for therapeutic intervention [J]. Leuk emia,2003,17:1263-1293
36Fiddes RJ, Janes PW, Sivertsen SP, et al. Inhibition of the MAP kinasecascade blocks heregulin2induced cell cycle progression in T-47D humanbreast cancer [J]. Oncogene,1998,16(21):2803-2813
37Nakata H, Wang SL, Chung DC, et al. Oncogenic ras induces gastrin geneexpression in colon cancer [J]. Gastroenterology,1998,115(5):1144-1153
38Ding Q, Wang Q, Evers BM. Alterations of MAPK activities associatedwith intestinal cell differentiation [J]. Biochem Biophys Res Commun,2001,284(2):282-288
39Sebolt-Leopold JS, Dudley DT, Herrera R, et al.Blockade of the MAkinase pathway suppresses growth of colon tumors in vivo [J]. Nat Med,1999,5:810-816
40Kim TI, Jin SH, Kang EH, et al. The role of mitogen-activated proteinkinases and their relationship with NF-kappaB and PPARgamma inindomethacin-Induced apoptosis of colon cancer cells [J]. Ann N Y AcadSci,2002,973:241-245
2Xu R, Chen X, Chen L, et al. The mechanisms of inhibitory effect ofbufalin on human leukemia cells [J]. Zhonghua Xue Ye Xue Za Zhi,2000,21(7):359-361
3张宁,凌昌全.蟾毒灵诱导肿瘤细胞凋亡机制的研究进展[J].现代肿瘤医学,2009,17(1):126-128
4Johnson GL, Lapadat R, Mitogen-activated protein kinase pathwaysmediated by ERK, JNK and p38protein kinases [J]. Science,2002,298(5600):1911-1912
5McCubrey JA, Steelman LS, ChappellWH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant, transformation and drugresistance [J]. Biochim Biophys Acta,2007,1773(8):1263-1284
6Vilalta A, Sahuquillo J, PocaMA. Matrix metaloproteinases inneurologicalbrain lesions: a new therapeutic target [J]. Rev Neurol,2010,51(2):95-107
7Kawazoe N,Watabe M, Masuda Y, et al. Tiam1is involved in theregulation of bufalin-induced apoptosis in human leukemia cells [J].Oncogene,1999,18(15):2413-2421
8Takai N, Ueda T, Nishida M, et al. Bufalin induces growth inhibition,cellcycle arrest and apoptosis in human endometrial and ovarian cancercells[J]. Int J Mol Med,2008,21(5):637-643
9leiner DE, Stetler-Stevenson WG. Matrix metalloproteinases andmetastasis[J]. Cancer Chemother Pharmacol,1999,43Suppl: S42-S51.
10Lu HJ, Guo CB, Jin XQ, et al. The role of matrix metalloproteinaseexpression in lung cancer metastasis [J].Chin Clin Oncol,2006,11(7):486-490
11Janow ska Wieczorek A, M arquezL A, Mat suz aki A, et al. Expressionof matrix metalloproteinases(MMP-2and-9) and tissue inhibit or ofmetalloproteinases (TIMP-1and-2) in acute myelogenous leukemiablasts:comparison with normal bonemarrow cells[J]. BrJ Haem at ol,1999,105:402-411
12Janow ska Wieezorek A, Marquez LA, Doh row s ky A, et al. DifierentialMMP and TIMP production by human marrow and peripheral bloodCD34+celL in response to ehem okines[J]. Exp H emat ol,2000,28:1274-1285
13Saw icki G, Mat suz aki A, Janow ska Wieczorek A. Expression of theactive form of MMP-2on the surface of leukemie cells accounts for theirin vitro invasion [J]. Cancer Res C lin Oncol,1998,124:245-252
14Zucker S, Lysik RM, Zarrabi MH, et al. M(r)92,000type IV collagenaseis in creased in plasma of pat ients with colon cancer and breastcancer[J]. Cancer Rse,1993,53(1):140-146
15Maemura K, Shiraishi N, Sakagami K, et a1. Proliferative effects ofgamma-aminobutyric acid on the gastric cancer cell line are associatedwith extracellular signal-regulated kinase1/2activation[J]. J GastroenterolHepatol,2009,24(4):688-696
16Chang F, S teelman LS, Lee JT, et al. Signal transduction mediated by theRas/Raf/MEK/ERK pathway from cytokine receptors to transcriptionfactors: potential targeting for therapeutic intervention [J]. Leuk emia,2003,17:1263-1293
17Fiddes RJ, Janes PW, Sivertsen SP, et al. Inhibition of the MAP kinasecascade blocks heregulin2induced cell cycle progression in T-47D humanbreast cancer [J]. Oncogene,1998,16(21):2803-2813
18Nakata H, Wang SL, Chung DC, et al. Oncogenic ras induces gastrin geneexpression in colon cancer [J]. Gastroenterology1998,115(5):1144-1153
19Ding Q, Wang Q, Evers BM. Alterations of MAPK activities associatedwith intestinal cell differentiation [J]. Biochem Biophys Res Commun,2001,284(2):282-288
20Sebolt-Leopold JS, Dudley DT, Herrera R, et al.Blockade of the MAPkinase pathway suppresses growth of colon tumors in vivo [J].Nat Med,1999,5:810-816
21Zhang L, Nakaya K, Yoshida T, et al. Biochem Biophys ResCommun,1991,178(2):686-693
22Jing Y, Ohizumi H, Kawazoe N, et al. Jpn J Cancer Res,1994,85(6):645-651
23Masuda Y, Kawazoe N, Nakajo S,et al. Leuk Res,1995,19(8):549-556
24王江宁. ERK1/2和MMP-9蛋白在胆管癌组织中的表达及其临床意义[D].中国医科大学学报,2007
25Maeda-Yamamoto M, Suzuki N, Sawai Y, et al.Association of suppressionof extra cellular signal-regulated kinase phosphorylation byepigallocatechin gallate with the reduction of matrix metalloproteinaseactivities in human fibrosarcoma HT1080cells [J]. Agric Food Chem,2003,51(7):1858-1863
1张丰,李楠. ERK/MAPK信号传导途径在乳腺肿瘤治疗中的意义[J].中国肿瘤生物治疗杂志，2007(14):497-500
2Maemura K, Shiraishi N, Sakagami K, et a1. Proliferative effects ofgamma-aminobutyric acid on the gastric cancer cell line are associatedwith extracellular signal-regulated kinase1/2activation [J].J GastroenterolHepatol,2009,24(4):688-696
3Almog T, Naor Z. Mitogen activated protein kinases (MAPKs) asrugulators of spermatogenesis and spermatozoa functions [J]. Mol CellEndocrinol,2008,282(122):39-44
4Qi MS, Elion EA. MAP kinase pathways [J]. J Cell Sci,2005,118(16):3569-3572
5俞华芳，樊晓明，蒋淼，等． P38MAPK抑制剂对小鼠急性水肿型胰腺炎的作用[J].中国临床医学，2008，15(1):82-83
6McCubrey JA, Steelman LS, ChappellWH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant, transformation and drugresistance [J]. Biochim Biophys Acta,2007,1773(8):1263-1284
7Cobb MH. MAP kinase pathways [J]. Prog Biophys Mol Biol,199971(3-4):479-500
8Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specificmessages from ubiquitous messengers [J]. Mol Cell Biol,1999,19(14):2435-2444
9Dhanasekaran N, Premkumar Reddy E. Signaling by dual specificitykinases [J]. Oncogene,1998,17(11Reviews):1447-1455
10Seth A, Gonzalez FA, Gupta S, et al. Signal transduction within thenucleus by mitogen-activated protein kinase [J]. J Biol Chem,1992,267(34):24796-24804
11Greenwood JM, Dragunow M. M3muscarinic receptors promote cellsurvival through activation of the extracellular regulated kinase (ERKl／2)pathway [J]. Eur J Phanllacol,20l0,640(1-3):38-45
12Lorenz K, Schmitt JP, Vidal M, Lohse MJ. Cardiac hypertrophy: targetingRaf／MEK／ERKl／2-signaling [J].Int J Biochem Cell Biol,2009,41(12):2351-2355
13Schuringa J J, Jonk L J, Dokter W H, et al. Interleukin-6-induced STAT3transactivation and Ser727phosphorylation involves Vav, Rac-1and thekinase SEK-1/MKK-4as signal transduction components [J].Biochem J,2000,347:89-96
14Ding Q, Wang Q, Evers BM.Alterations of MAPK activities associatedwith intestinal cell differentiation [J]. Biochem Biophys Res Commun,2001,284:282-288
15Maemura K,Shiraishi N,Sakagami K,et al.Proliferative effects ofgammaaminobutyric acid on the gastric cancer cell line are associated withextracellular signal-regulated kinase1/2activation [J]. J GastroenterolHepatol,2009,24:688-696
16王殊，王杉，祝学光等。细胞外信号调节激酶及其上游激酶在人乳腺癌发生发展中的意义[J].中华外科杂志,2002,40(3):171-174
17Chin R, Earnest-Silveira L, Koeberlein B, et al. Modulation ofMAPK pathways and cell cycle by replicating hepatitis B virus:factors contributing to hepatocarcinogenesis [J]. J Hepatol,2007,47(3):325-337
18Looby E, Abdel-Latif MM, Athié-Morales V, et al. Deoxycholate inducesCOX-2expression via Erk1/2-,p38-MAPK and AP-1-dependentmechanisms in esophageal cancer cells [J]. BMC Cancer,2009,9:190
19Caja L, Sancho P, Bertran E, et al. Overactivation of the MEK/ERKpathway in liver tumor cells confers resistance to TGF-{beta}-induced celldeath through impairing upregulation of the NADPH oxidase NOX4[J].Cancer Res,2009,69(19):7595-7602
20Chen YJ, Tsai YM, Kuo CD, et al. Norcantharidin is a small-moleculesynthetic compound with antiangiogenes is effect [J]. Life Sci,2009,85(17-18):642-651
21Komi Y, Sogabe Y, Ishibashi N, et al. Acyclic retinoid inhibitsangiogenesis by suppressing the MAPK pathway [J]. Lab Invest,2010,90(1):52-60
22梁璇，丁新民，霍艳英． ERK1/2-Sp l信号通路对肺癌细胞VEGF的调控作用[J].癌变·畸变·突变，2007，19(4):280-284
23Cassano A, Bagala C, Battelli C, et al.Expression of vascular endothelialgrowth factor,mitogen-activated protein kinase and p53in humancolorectal cancer [J]. Anticancer Res,2002,22(4):2179-2184
24Zhang Yu-hua, WEI Wei, XU Hao, et al.Inducing effects of hepatocytegrowth factor on the expression of vascular endothelial growth factor inhuman colorectal carcinoma cells through MEK and PI3K signalingpathways [J]. Chin Med J,2007,120(9):743-748
25Vilalta A, Sahuquillo J, PocaMA. Matrix metaloproteinases inneurologicalbrain lesions: a new therapeutic target [J]. Rev Neurol,2010,51(2):95-107
26Lai KC, Huang AC, Hsu SC, et al. Benzyl isothiocyanate (BITC) inhibitsmigration and invasion of human coloncancer HT29cells by inhibitingmatrix metalloproteinase-2/-9and urokinase plasminogen (uPA) throughPKC and MAPK signaling pathway [J]. J Agric Food Chem,2010,58(5):2935-2942
27Weng CJ, Chau CF, Hsieh YS, et al. Lucidenic acid inhibits PMA-inducedinvasion of human hepatoma cells through inactivating MAPK/ERK signaltransduction pathway and reducing binding activities of NF-kappaB andAP-1[J]. Carcinogenesis,2008,29(1):147-156
28Endo H, Watanabe T, Sugioka Y, et al. Activation of two distinct MAPKpathways governs constitutive expression of matrix metalloproteinase-1inhuman pancreatic cancer cell lines[J]. Int J Oncol,2009,35(6):1237-1245
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