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Investigation of oral cancer development by using apoptosis and its application for clinical evaluation

利用细胞凋亡研究口腔癌发生发展及其在临床评价中的应用

基本信息

批准号：
12671836
负责人：
OHBA Takeshi
金额：
$ 2.11万
依托单位：
Kyushu Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671836/
关键词：
Anti-canccer drug Apoptosis Egr-1 NORs Oral carcinoma Protein phosphatase Protein phosphatase Fas antigen nucleolin anti-cancerdrug

项目摘要

Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCKN cells were minimally sensitive to this reagent. Peplomycin caused apoptosis SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 5 μM and 50 μM, respecnvely, as determined by phase-contrast microscopy, WST-1 cell viability assay, Hoechst 33342 staining, and DNA ladder formation. These results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin.We examined the expression of Egr-1 in human squamous carcinoma cell line SCCKN cells treated with okada1C acid. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. Suppression of Egr-1 protein expression okadaic acid-treated cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. Treatment of SCCKN cells with okadaic acid suppressed the cell viability of SCCKN cells and induced apoptosis in these cells.The expression and cytolocalization of protein phosphatase type 1 δ isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Anti-nucleolin antibody interacted with the 100- and 95-kDa proteins present in the whole cell lysate. The 100-kDa protein was detected in nuclear and nucleolar fractions. The 95-kDa protein was detected in cytosolic and nucleoplasmic fractions. Protein phosphatase 1δ and nucleolin were co-localized and interact with each other in nucleolus in MG63 and Saos-2 cells revealed bv immunofluorescent and immunoprecipitation method.

口腔鳞癌细胞株SSCKN对博莱霉素高度敏感，而SCCKN细胞对博莱霉素最低敏感。倒置显微镜、WST-1细胞存活率测定、Hoechst 33342染色和DNA梯带形成实验表明，培普霉素诱导SCCKN和SCCTF细胞凋亡呈剂量依赖性，5μM和50μM的作用最强。这些结果表明培普霉素诱导口腔鳞癌细胞株的凋亡依赖于这些细胞对博莱霉素的敏感性。我们检测了Okada1C酸处理的人鳞癌细胞株SCCKN细胞中Egr-1的表达。Western印迹分析表明，Egr-1在SCCKN细胞中有较强的表达，冈田酸降低了这些细胞中Egr-1蛋白的表达。通过RT-RCR方法确定，冈田酸处理的细胞中Egr-1蛋白表达的抑制源于对Egr-1mRNA水平的抑制。凝胶迁移率改变分析表明，冈田酸处理SCCKN细胞抑制了Egr-1与地高辛标记的Egr-1共识寡核苷酸探针的结合。冈田酸对人成骨细胞系MG63和SAOS-2细胞中蛋白磷酸酶1亚型δ亚型和核仁蛋白的表达及细胞定位进行了研究。抗核仁素抗体与存在于整个细胞裂解物中的100和95 kDa蛋白相互作用。在细胞核和核仁中检测到100 kDa的蛋白。在胞浆和核质中检测到95 kDa的蛋白。免疫荧光法和免疫沉淀法显示蛋白磷酸酶1、δ和核仁在细胞核内共定位并相互作用。