Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application

• Published in: Talanta (Oxford) 2018-03, Vol.179, p.845-852
• Main Authors: Fujikawa, Yuuta, Nampo, Taiki, Mori, Masaya, Kikkawa, Manami, Inoue, Hideshi
• Format: Article
• Language: English
• Subjects: Biomarkers, Tumor - antagonists & inhibitors; Biomarkers, Tumor - chemistry; Fluoresceins - chemistry; Fluorescent Dyes - chemistry; Fluorescent probe; Glutathione; Glutathione - chemistry; Glutathione S-Transferase pi - antagonists & inhibitors; Glutathione S-Transferase pi - chemistry; Glutathione Transferase - chemistry; GSTP1; HeLa Cells; Humans; MCF-7 Cells; Oxadiazoles - chemistry; Spectrometry, Fluorescence; Substrate Specificity
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2017.12.010

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. We report the application of fluorescein diacetate (FDA) and monoacetate derivative (FOMe-Ac) to detection/visualization of human glutathione S-transferase P1 (hGSTP1) based on its esterolytic activity. [Display omitted] •FDA is a good substrate with selectivity for hGSTP1 among cytosolic GSTs.•FDA is applicable to visualization of GSTP1 activity in living cells.•FOMe-Ac is fluorogenic substrate for GSTP1 applicable to kinetic analysis.