Laboratory of Molecular and Chemical Biology, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Talanta. 2018 Mar 1;179:845-852. doi: 10.1016/j.talanta.2017.12.010. Epub 2017 Dec 5.
Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1.
Pi 类谷胱甘肽 S-转移酶 (GSTP1) 在各种癌细胞和前瘤组织中高度表达，在这些组织中，它参与凋亡抵抗或几种抗肿瘤化疗药物的代谢。因此，GSTP1 是恶性和前恶性细胞的标志物，是可视化和药物开发的有前途的靶点。在这里，我们证明荧光素二乙酸酯 (FDA)，一种用于生命染色的荧光探针，在各种胞质 GST 中，人 GSTP1 (hGSTP1) 的酯酶活性选择性地激活荧光。在过表达 hGSTP1 的 MCF7 细胞中外源性观察到 FDA 对 GST 抑制剂的荧光激活，但在过表达 hGSTA1 或 hGSTM1 的细胞中没有观察到。在几种内源性表达 GSTP1 的癌细胞系中也观察到对抑制剂敏感的荧光激活，表明 GSTP1 参与了这些细胞中的 FDA 酯解。在所检查的 FDA 衍生物中，发现 FOMe-Ac，荧光素 O-甲基醚的乙酰酯，是 GSTP1 活性以及羧酸酯酶活性的潜在报告物。由于 GSTP1 在各种类型的癌细胞中比其正常对应物表达更高，因此，提高荧光底物对 GSTP1 的酯解活性而不是羧酸酯酶的选择性，应该会导致开发用于检测 GSTP1 过表达癌细胞和研究 GSTP1 生物学功能的工具。
