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An ultrasensitive fluorogenic probe for revealing the role of glutathione in chemotherapy resistance.

ABSTRACT: Unveiling the detailed roles of glutathione (GSH) in chemoresistance necessitates a reliable assay for its detection in intact live specimens. Herein, by taking advantage of the susceptibility of electron-poor C_sp² -S_sufinyl bond to GSH nucleophilic attack, we developed a naphthalimide-sulfoxide based fluorogenic probe (Na-8) applicable for tracking endogenous GSH fluctuation in live cells. Na-8 features a high degree of sensitivity towards GSH as demonstrated by its utmost 2200-fold fluorogenic response. As a proof of concept, Na-8 has been applied to image GSH in liver cancer HepG2 cells with the normal L02 cell counterparts serving as a control, and elevated GSH level was observed in HepG2 in contrast to L02. Further experiments showed that this elevated GSH level was involved in doxorubicin-resistance but not in cisplatin-resistance. Noteworthy, monitoring GSH change in HepG2 and L02 cells in response to doxorubicin treatment revealed that while normal cells showed a burst of GSH in adaption to doxorubicin treatment, no significant change was detected in HepG2 cells, suggesting that HepG2 cells have been preconditioned by their intrinsic oxidative stress which confers drug-resistance. Given the observed sensitivity and spatiotemporal resolution, Na-8 should hold promise for future study on the detailed roles of GSH in drug-resistance.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC5853925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually are carried out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 x 10(4) M(-1)s(-1). The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation, and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of beta-NADPH (beta-nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half-maximum (fwhm) for the internal standard were 2.5%, 2.0%, and 1.0%, respectively, with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and approximately 10(-18) mol, respectively. The Michaelis constants (K(m)) for GSSG and beta-NADPH were found to be 40 +/- 11 and 4.4 +/- 0.6 muM, respectively, comparable with those obtained from UV/vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation, and detection for continuous GSH determinations.

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An ultrasensitive fluorogenic probe for revealing the role of glutathione in chemotherapy resistance.

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