公开(公告)号：EP2998406A1

公开(公告)日：2016-03-23

申请号：EP14797327.5

申请日：2014-05-09

申请人： Medicinal Bioconvergence Research Center

发明人： LEE, Jung Weon ,  KIM, Sunghoon ,  LEE, Mi-Sook

IPC分类号： C12Q1/04 ,  C12N5/09 ,  C12M1/34

CPC分类号： G01N33/574 ,  C12N5/0693 ,  C12N2501/727 ,  C12N2503/02 ,  C12N2533/54 ,  G01N33/5011 ,  G01N33/5023 ,  G01N33/5026 ,  G01N33/5029 ,  G01N33/57415 ,  G01N2333/4703 ,  G01N2333/4704 ,  G01N2333/4706 ,  G01N2440/14 ,  G01N2500/10

摘要： The present invention relates to a method for monitoring migration, invasion, and metastasis of cancer cells by observing the shape of cancer cells cultured in a three-dimensional environment and measuring the activity, expression, and changes in expression sites of proteins associated with invadopodia formation and metastasis, and the degradation of an extracellular matrix; and to a method for screening a cancer metastasis inhibitor. More specifically, it was verified that the reduction in c-Jun phosphorylation induced the increase in snail1 and the decrease in cortactin expression in the cells cultured in a three-dimensional environment and the expression regulation relations between the proteins were identical to those in breast cancer tissues obtained from patients. In addition, it was verified that, when breast cancer cells in a three-dimensional collagen gel environment were treated with a JNK inhibitor, the shape of the cells became longer; the contact region of the cells and the extracellular matrix became flattened and thinner; the migration of cancer cells was decreased; and the changes in protein expression was observed, such as the increase in TGFβ1 expression, the increases in smad2 and smad3 expression and activity, the increase in snail1 expression, the decrease in cortactin expression, and the resulting decrease in invadopodia formation. In addition, in a three-dimensional collagen gel environment, MT1-MMP besides the cortactin can be used as a marker of invadopodia, and it was verified that the inhibition of JNK led to the decrease in cortactin expression and the increase in snail1 expression, badly influenced the site and role of MT1-MMP to inhibit the formation of invadopodia, and inhibited the degrading activity of a collagen gel substrate. Thus, the present invention can be used as a method for monitoring migration, invasion, metastasis, and the degree of metastasis of cancer cells and a method for screening a cancer metastasis inhibitor, and will be useful as one of screening methods capable of creating low-cost, high-efficient added value at the time of pre-clinical tests required for drug development.

摘要翻译： 本发明涉及一种用于通过观察癌症细胞在三维环境中培养的形状和测量的活性，表达监控迁移，侵袭，和癌细胞的转移，并且在蛋白质的表达位点的变化与树突状伪足的形成相关 和转移，以及细胞外基质的降解; 并用于筛选癌转移抑制剂的方法。 更具体地，它什么验证确实诱导SNAIL1的增加和在三维环境和蛋白质之间的表达调控关系中培养的细胞在皮层蛋白表达的减少在c-Jun的磷酸化的降低的那些相同。在乳腺癌 从患者的组织中。 此外，验证是什么，当乳腺癌细胞在三维胶原凝胶环境用JNK抑制剂治疗，所述细胞的形状wurde长; 细胞和细胞外基质wurde的接触区域变平，更薄; 癌细胞其中减少的迁移; 并且，观察到的，：如增加TGF²1表达，在Smad2和Smad3的表达和活性的增加，表达SNAIL1的增加，在皮层蛋白表达的减少，并且在树突状伪足的形成所产生的下降的变化的蛋白质表达。 此外，在三维胶原蛋白凝胶的环境中，除了皮层蛋白MT1-MMP可以用作树突状伪足的标志物，对于可以确认确实JNK的抑制导致皮层蛋白表达的减少和在SNAIL1表达的增加， 严重影响MT1-MMP的位点和作用，以抑制树突状伪足的形成，和抑制胶原蛋白凝胶基质的降解活性。 因此，本发明可以被用作用于监测迁移，侵入，转移和癌细胞的转移和用于筛选癌症转移抑制剂的方法的程度的方法，并且将是有用的作为能够产生低的筛选方法之一 -cost，在药物开发所需的临床前试验的时间高效的附加值。

公开(公告)号：EP3006941A1

公开(公告)日：2016-04-13

申请号：EP14797157.6

申请日：2014-05-13

申请人： Medicinal Bioconvergence Research Center

发明人： LEE, Jung Weon ,  KIM, Sunghoon ,  NAM, Seo-Hee ,  KIM, Dae Gyu

IPC分类号： G01N33/574 ,  C12Q1/68 ,  C12M1/34 ,  C12M3/00

CPC分类号： G01N33/573 ,  C12Q1/6886 ,  C12Q2600/112 ,  C12Q2600/136 ,  C12Q2600/158 ,  G01N33/5011 ,  G01N33/574 ,  G01N33/57419 ,  G01N2333/4704 ,  G01N2333/9015 ,  G01N2500/00 ,  G01N2500/04 ,  G01N2500/10

摘要： The present invention relates to a method for scanning a cancer metastasis inhibitor by analyzing the activity of lysyl-tRNA synthetase (KRS) in a cancer cell line cultured in a three-dimensional collagen gel environment, and to a method for monitoring the dissemination of cancer cells from aggregated cancer cells, and the epithelial-mesenchymal transition, migration, invasion, and metastasis of cancer cells. Specifically, it was verified that, in the case where a cell line or a spheroidically aggregated cell line, in which KRS has been regulated to be expressed or unexpressed, was constructed by using various colorectal cancer cells including HCT116 cell line and then cultured in a two-dimensional environment, the incomplete epithelial-to-mesenchymal transition phenotype (incomplete ECM phenotype) was induced in the cell line inhibiting KRS expression, and the inhibition of KRS expression inhibited cell-extracellular matrix (ECM) adhesion and cell-ECM signaling activity. In addition, it was verified that, in the case where the constructed spheroid cell line was cultured in an aqueous environment or a three-dimensional collage gel culture environment, the inhibition of KRS expression induced cells into mesenchymal cells but failed to reach the disintegration of cell-cell adhesion; inhibited the cell-ECM adhesion and the related signaling activity, causing the inhibition of the dissemination of cells from spheroid cells cultured in a three-dimensional collagen gel culture environment; and failed to induce the dissemination of cells through TGFβ1 present in the cellular microenvironment. Thus, the present invention can be used as a method for screening a cancer metastasis inhibitor and a method for monitoring the migration, invasion and metastasis of cancer cells, and will be useful as one of the screening methods capable of creating low-cost, high-efficient added value at the time of pre-clinical tests required for drug development.

摘要翻译： 本发明涉及一种用于通过在三维胶原凝胶环境培养的癌细胞系分析赖氨酰-tRNA合成酶（KRS）的活性扫描癌转移抑制剂，以及用于监控癌症的传播的方法 细胞从聚集的癌细胞，和上皮 - 间充质转换，迁移，侵入，和癌细胞的转移。 具体而言，什么证实，在的情况下待表达的细胞系或一个spheroidically聚集细胞系，其中KRS已调节或不表达什么通过使用各种结直肠癌细胞，包括HCT116细胞系，然后在培养的构筑 二维环境下，不完全的上皮 - 间充质在细胞系中抑制KRS表达从而诱发转变表型（不完全的ECM表型），以及KRS表达的抑制抑制细胞 - 细胞外基质（ECM）的粘附和细胞-ECM信号活动 ， 此外，它什么证实，在的情况下，其在wässrige环境培养在所构造的旋转椭圆体细胞系或三维拼贴凝胶培养环境，KRS表达诱导细胞的抑制成间充质细胞，但未能达到的崩解 粘附的细胞 - 细胞; 抑制细胞-ECM粘附和相关的信号转导活性，导致从在三维胶原蛋白凝胶中培养环境中培养球体细胞的细胞传播的抑制; 并不能诱导细胞通过TGF²1本传播中细胞微环境。 因此，本发明可以被用作用于筛选癌转移抑制剂以及用于监测癌症细胞的迁移，侵袭和转移的方法的方法，并且将是有用的作为能够产生低成本的，高的筛选方法之一 在效率高达用于药物开发所需的前期临床试验的时间增值。