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Development of anti-cancer drug targeted on cancer suppression gene products

以抑癌基因产物为靶点的抗癌药物的开发

基本信息

批准号：
11557085
负责人：
TAGUCHI Tetsuya
金额：
$ 3.39万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557085/
关键词：
MCF-7 NF2 calpeptin Sensitivity Test CD-DST method CE Docetaxel Docetaxel 乳癌

项目摘要

Human breast cancer cell line.MCF-7, typical ER positive cell line, was subcultured and tested the following.Determination of NF2 protein in MCF-7 cell.NF2 protein in human breast cancer cell was identified by wetern blotting method. The anti-NF2 polyclonal antibody which commercially available was used for confirming existence of NF 2 protein. The NF2 protein which identified was about 78 kDa, the same molecular weight as reported before.Identification of NF2 protein expression by immunohistochemical staining in breast cancer tissue.Using anti-NF2 polyclonal antibody, immunohistochemical staining for the breast cancer tissue was performed. As a result, 25% of the cases were strongly NF2 positive. In cases of strongly positive staining, there were a high ratio of advanced disease, in contrast, in cases of negative staining, there were a lot of stage 1 disease.NF2 resolution by calpain, inhibition of decomposition by calpeptin.Same as the previous experiment, various density of calpeptin, which is the inhibitor of calpain, and other calpain inhibitors were added to the culture medium of MCF-7 cells and cell growth was observed. By western blotting, activation of the calpain was inhibited in dose dependent manner by calpeptin and by other calpain inhibitors. Inhibition of NF2 protein decomposition was also identified.Prediction of chemotherapeutic response by CD-DST method in human breast cancers.Preparing for the development of new anti-cancer drug, Collagen Gel Droplet Embedded Culture-Drug Sensitivity Test(CD-DST)in clinically available breast cancer tissues was pefarmed using highly effective drug, Cyclophosphamide+Epirubicin(CE)or Docetaxel, for breast cancer. As a result, concordance between CD-DST and clinical response of CE and DOC was 85.7% and 96.4%, respectively. It might be useful to perform CD-DST for the new drug under development.

人乳腺癌细胞系MCF-7是典型的ER阳性细胞系，传代培养并检测MCF-7细胞中NF2蛋白的测定。采用免疫印迹法鉴定人乳腺癌细胞中NF2蛋白。使用市售的抗NF2多克隆抗体来确认NF 2蛋白的存在。鉴定的NF2蛋白分子量约为78 kDa，与之前报道的分子量相同。通过免疫组化染色鉴定乳腺癌组织中NF2蛋白的表达。使用抗NF2多克隆抗体，对乳腺癌组织进行免疫组化染色。结果，25% 的病例呈 NF2 强阳性。在强阳性染色的情况下，晚期疾病的比例很高，相反，在阴性染色的情况下，有很多1期疾病。通过calpain分解NF2，通过calpeptin抑制分解。与之前的实验相同，在MCF-7细胞的培养基中添加不同浓度的calpeptin（calpain的抑制剂）和其他calpain抑制剂，观察细胞生长。通过蛋白质印迹，钙蛋白酶的活化被钙肽素和其他钙蛋白酶抑制剂以剂量依赖性方式抑制。还发现了对NF2蛋白分解的抑制作用。通过CD-DST方法预测人类乳腺癌的化疗反应。为开发新的抗癌药物做准备，胶原凝胶液滴嵌入培养-药物敏感性试验（CD-DST）在临床可用的乳腺癌组织中使用高效药物环磷酰胺+表柔比星（CE）或多西他赛进行，用于乳腺癌。结果，CD-DST 与 CE 和 DOC 临床反应之间的一致性分别为 85.7% 和 96.4%。对正在开发的新药进行 CD-DST 可能会有用。