Thiol profiling in cancer cell lines by HPLC-mass spectrometry

Liu, Xiaoguang; Koppula, Pranavi; Olszewski, Kellen L.; Gan, Boyi

doi:10.1016/j.xpro.2021.100977

Cited by 3 publications

(3 citation statements)

References 13 publications

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“…To accurately quantify the intracellular levels of various thiol species, we used an improved extraction and sample derivatization method to prevent thiols’ oxidation before analysis, as recently described 48 . Cells cultured in 6-well plates were quickly rinsed with 1 mL of pre-cold PBS and then extracted in 500 µL of pre-chilled extraction buffer containing 40% acetonitrile, 40% methanol, and 20% water with the addition of 1 mM EDTA and 100 mM formic acid; the addition of EDTA prevents potential oxidation induced by metal ions, while formic acid prevents the formation of the highly reactive thiolate anion.…”

Section: Methodsmentioning

confidence: 99%

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells

et al. 2023

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The cystine transporter solute carrier family 7 member 11 (SLC7A11; also called xCT) protects cancer cells from oxidative stress and is overexpressed in many cancers. Here we report a surprising finding that, whereas moderate overexpression of SLC7A11 is beneficial for cancer cells treated with H2O2, a common oxidative stress inducer, its high overexpression dramatically increases H2O2-induced cell death. Mechanistically, high cystine uptake in cancer cells with high overexpression of SLC7A11 in combination with H2O2 treatment results in toxic buildup of intracellular cystine and other disulfide molecules, NADPH depletion, redox system collapse, and rapid cell death (likely disulfidptosis). We further show that high overexpression of SLC7A11 promotes tumor growth but suppresses tumor metastasis, likely because metastasizing cancer cells with high expression of SLC7A11 are particularly susceptible to oxidative stress. Our findings reveal that SLC7A11 expression level dictates cancer cells’ sensitivity to oxidative stress and suggests a context-dependent role for SLC7A11 in tumor biology.

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Section: Methodsmentioning

confidence: 99%

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells

et al. 2023

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“…Subsequently, the media was replaced with culture media containing CB839 (1 μM), ERA (3 μM), DOX (0.2 μM) and CIS (5μM) and incubated for 24hrs at 37°C with 5% CO2. Following a 24hrs incubation, the cells underwent a wash two times with PBS, and 1ml of extraction solution (40% Methanol, 40% Acetonitrile, 20% DI water, 100mM Formic acid, and 1mM EDTA) was introduced to the cells (26). The extracted cell and solution were gathered using a cell scraper and transferred to tubes on ice.…”

Section: Methodsmentioning

confidence: 99%

Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate export and glutaminase

Choi,

Gupta,

Hensley

et al. 2023

Preprint

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In triple-negative breast cancer (TNBC) that relies on catabolism of amino acid glutamine, glutaminase (GLS) converts glutamine to glutamate, which facilitates glutathione synthesis by mediating the enrichment of intracellular cystine via xCT antiporter activity. To overcome chemo resistant TNBC, we have tested a strategy of disrupting cellular redox balance by inhibition of GLS and xCT by CB839 and Erastin, respectively. Key findings of our study include: 1. Dual metabolic inhibition (CB839+Erastin) led to significant increases of cellular superoxide level in both parent and chemo resistant TNBC cells, but superoxide level was distinctly lower in resistant cells. 2. Dual metabolic inhibition combined with doxorubicin or cisplatin induced significant apoptosis in TNBC cells and is associated with high degrees of GSH depletion.In vivo, dual metabolic inhibition plus cisplatin led to significant growth delay of chemo resistant human TNBC xenografts. 3. Ferroptosis is induced by doxorubicin (DOX) but not by cisplatin or paclitaxel. Addition of dual metabolic inhibition to DOX chemotherapy significantly enhanced ferroptotic cell death. 4. Significant changes in cellular metabolites concentration preceded transcriptome changes revealed by single cell RNA sequencing, underscoring the potential of capturing early changes in metabolites as pharmacodynamic markers of metabolic inhibitors. Here we demonstrated that 4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) PET detected xCT blockade by Erastin or its analog in mice bearing human TNBC xenografts. In summary, our study provides compelling evidence for the therapeutic benefit and feasibility of non-invasive monitoring of dual metabolic blockade as a translational strategy to sensitize chemo resistant TNBC to cytotoxic chemotherapy.

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“…Chemical derivatization is frequently used as a strategy for increasing ion yields, improving chromatographic performance, and facilitating structure identification in gas chromatography tandem-mass spectrometry and liquid chromatography-mass spectrometry (LC)-MS analysis. On-tissue chemical tagging of unionizable target analytes with easily ionized moieties is an alternative means of enhancing ionization efficiency, sensitivity, and specificity in MSI experiments. − On-tissue chemical derivatization (OTCD)-MSI methods targeting fatty acids, steroid hormones, thiol peptides, amino acids, neurotransmitters, and drugs have been developed, providing important tools that are used to determine the temporal and spatial fate of these metabolites in pathological processes. − For example, Wu et al developed an electrospray-assisted OTCD method using 2-pyridinemethanamine as the derivatization reagent and mapped the distribution of endogenous fatty acids in brain tissue using OTCD-MALDI-MSI. Guo et al developed a laser-assisted tissue transfer (LATT) technique to improve the tissue derivatization of small molecules and imaged 69 derivatized metabolites, including neurotransmitters, amino acids, dipeptides, and steroids, in the rat brain using LATT derivatization with 4-hydroxy-3-methoxycinnamaldehyde and Girard’s reagent T (GT).…”

Section: Introductionmentioning

confidence: 99%

Mapping of Fatty Aldehydes in the Diabetic Rat Brain Using On-Tissue Chemical Derivatization and Air-Flow-Assisted Desorption Electrospray Ionization-Mass Spectrometry Imaging

et al. 2022

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Fatty aldehydes (FALs) are involved in various biological processes, and their abnormal metabolism is related to the occurrence and development of neurological diseases. Because of their low ionization efficiency, methods for in situ detection and mass spectrometry imaging (MSI) analysis of FALs remain underreported. On-tissue chemical tagging of hardly ionizable target analytes with easily ionized moieties can improve ionization efficiency and detection sensitivity in MSI experiments. In this study, an on-tissue chemical derivatization-air-flow-assisted desorption electrospray ionization-MSI method was developed to visualize FALs in the rat brain. The method showed high sensitivity and specificity, allowing the use of in situ high-resolution MS 3 to identify FALs. The methodology was applied to investigate the region-specific distribution of FALs in the brains of control and diabetic encephalopathy (DE) rats. In DE rats, FALs were found to be significantly enriched in various brain regions, especially in the cerebral cortex, hippocampus, and amygdala. Thus, increased FAL levels and oxidative stress occurred in a region-dependent manner, which may contribute to cognitive function deficits in DE. In summary, we provide a novel method for the in situ detection of FALs in biological tissues as well as new insights into the potential pathogenesis of DE.

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Thiol profiling in cancer cell lines by HPLC-mass spectrometry

Cited by 3 publications

References 13 publications

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells

Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate export and glutaminase

Mapping of Fatty Aldehydes in the Diabetic Rat Brain Using On-Tissue Chemical Derivatization and Air-Flow-Assisted Desorption Electrospray Ionization-Mass Spectrometry Imaging

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