Dataset Information

Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells.

ABSTRACT: Glutathione (GSH), the constituent of the redox buffer system, is a scavenger of reactive oxygen species (ROS), and its ratio to oxidized glutathione (GSSG) is a key indicator of oxidative stress in the cell. Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy characterized by aberrant levels of reduced and oxidized GSH due to oxidative stress. Therefore, the real-time, dynamic, and highly sensitive detection of GSH/GSSG in AML cells is of great interest for the clinical diagnosis and treatment of leukemia. The application of genetically encoded sensors to monitor GSH/GSSG levels in AML cells is not explored, and the underlying mechanism of how the drugs affect GSH/GSSG dynamics remains unclear. In this study, we developed subcellular compartment-specific sensors to monitor GSH/GSSG combined with high-resolution fluorescence microscopy that provides insights into basal GSH/GSSG levels in the cytosol, mitochondria, nucleus, and endoplasmic reticulum of AML cells, in a decreasing order, revealing substantial heterogeneity of GSH/GSSG level dynamics in different subcellular compartments. Further, we investigated the response of GSH/GSSG ratio in AML cells caused by Prussian blue and Fe₃O₄ nanoparticles, separately and in combination with cytarabine, pointing to steep gradients. Moreover, cytarabine and doxorubicin downregulated the GSH/GSSG levels in different subcellular compartments. Similarly, live-cell imaging showed a compartment-specific decrease in response to various drugs, such as CB-839, parthenolide (PTL), and piperlongumine (PLM). The enzymatic activity assay revealed the mechanism underlying fluctuations in GSH/GSSG levels in different subcellular compartments mediated by these drugs in the GSH metabolic pathway, suggesting some potential therapeutic targets in AML cells.

SUBMITTER: Abbas G

PROVIDER: S-EPMC9909732 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells.

Abbas Ghulam G Cui Mengmeng M Wang Dianbing D Li Min M Zhang Xian-En XE

Analytical chemistry 20230126 5

Glutathione (GSH), the constituent of the redox buffer system, is a scavenger of reactive oxygen species (ROS), and its ratio to oxidized glutathione (GSSG) is a key indicator of oxidative stress in the cell. Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy characterized by aberrant levels of reduced and oxidized GSH due to oxidative stress. Therefore, the real-time, dynamic, and highly sensitive detection of GSH/GSSG in AML cells is of great interest for the clinical ...[more]

PMID: 36701391

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Project description:BackgroundEpithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments.Materials and methodsPlasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin.ResultsExposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients.ConclusionsOur study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.

Dataset Information

Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells.

Publications

Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells.

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