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L-Buthionine Sulfoximine Detection and Quantification in Polyurea Dendrimer Nanoformulations.

ABSTRACT: l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug delivery systems and l-BSO nanoformulations are envisaged as potential chemotherapeutics. The absorption of l-BSO at a low wavelength limits its detection by conventional analytical tools. A simple and sensitive method for l-BSO detection and quantification is now reported. In this study, l-BSO was encapsulated in a folate-targeted generation four polyurea dendrimer (PURE_G4-FA₂) and its release profile was followed for 24 h at pH 7.4 and 37 °C. The protocol uses in situ l-BSO derivatization, by the formation of a catechol-derived orto-quinone, followed by visible detection of the derivative at 503 nm. The structure of the studied l-BSO derivative was assessed by NMR spectroscopy.

SUBMITTER: Mota P

PROVIDER: S-EPMC6749587 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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l-Buthionine Sulfoximine Detection and Quantification in Polyurea Dendrimer Nanoformulations.

Mota Pedro P Pires Rita F RF Serpa Jacinta J Bonifácio Vasco D B VDB

Molecules (Basel, Switzerland) 20190827 17

l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug delivery systems and l-BSO nanoformulations are envisaged as potential chemotherapeutics. The absorption of l-BSO at a low wavelength limits its detection by conventional analytical tools. A simple and sensitive method for l-BSO detection and quantification is now reported. In this study, l-BSO was encapsulated in a folate-targete ...[more]

PMID: 31461931

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Project description:Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.Estrogen deprived MCF-7:2A cells were treated with 1 nM 17beta-estradiol (E2), 100 microM BSO, or 1 nM E2 + 100 microM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model.Exposure of MCF-7:2A cells to 1 nM E2 plus 100 microM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E2 significantly reduced tumor growth of MCF-7:2A cells.Our data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.

Project description:BackgroundHydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.Materials and methodsAnti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.ResultsNon-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.Conclusion/significanceBSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.

Dataset Information

L-Buthionine Sulfoximine Detection and Quantification in Polyurea Dendrimer Nanoformulations.

Publications

l-Buthionine Sulfoximine Detection and Quantification in Polyurea Dendrimer Nanoformulations.

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