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Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine.

ABSTRACT: Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation and poly-ADP ribose polymerase (PARP) cleavage were performed to measure the induction of apoptosis after nifurtimox treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. Tumor cells were then treated with combinations of nifurtimox and BSO and evaluated for viability using MTT assays. All neural tumor cell lines were sensitive to nifurtimox, and IC50 values ranged from approximately 20 to 210??M. Nifurtimox treatment inhibited ERK phosphorylation and induced apoptosis in tumor cells. Furthermore, the combination of nifurtimox and BSO demonstrated significant synergistic efficacy in all tested cell lines. Additional preclinical and clinical studies of the combination of nifurtimox and BSO in patients with neural tumors are warranted.

SUBMITTER: Du M

PROVIDER: S-EPMC4901277 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine.

Du Michael M Zhang Linna L Scorsone Kathleen A KA Woodfield Sarah E SE Zage Peter E PE

Scientific reports 20160610

Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation a ...[more]

PMID: 27282514

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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.

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:Neoantigens play a crucial role in cancer immunotherapy. However, the effectiveness and safety of neoantigen-based immunotherapies in patients with colorectal cancer (CRC), particularly in the Chinese population, have not been well studied. This study explored the feasibility and effectiveness of neoantigens in the treatment of CRC. Whole-exome sequencing (WES) and transcriptome sequencing were used to identify somatic mutations, RNA expression, and human leukocyte antigen (HLA) alleles. Neoantigen candidates were predicted, and immunogenicity was assessed. The neoantigens TSHZ3-L523P, RARA-R83H, TP53-R248W, EYA2-V333I, and NRAS-G12D from Patient 4 (PW4); TASP1-P161L, RAP1GAP-S215R, MOSPD1-V63I, and NAV2-D1973N from Patient 10 (PW10); and HAVCR2-F39V, SEC11A-R11L, SMPDL3B-T452M, LRFN3-R118Q, and ULK1-S248L from Patient 11 (HLA-A0201+PW11) induced a heightened neoantigen-reactive T cell (NRT) response as compared with the controls in peripheral blood lymphocytes (PBLs) isolated from patients with CRC. In addition, we identified neoantigen-containing peptides SEC11A-R11L and ULK1-S248L from HLA-A0201+PW11, which more effectively elicited specific CTL responses than the corresponding native peptides in PBLs isolated from HLA-A0201+PW11 as well as in HLA-A2.1/Kb transgenic mice. Importantly, adoptive transfer of NRTs induced by vaccination with two mutant peptides could effectively inhibit tumor growth in tumor-bearing mouse models. These data indicate that neoantigen-containing peptides with high immunogenicity represent promising candidates for peptide-mediated personalized therapy.Abbreviations: CRC: colorectal cancer; DCs: dendritic cells; ELISPOT: enzyme-linked immunosorbent spot; E:T: effector:target; HLA: human leukocyte antigen; MHC: major histocompatibility complex; Mut: mutant type; NGS: next-generation sequencing; NRTs: neoantigen-reactive T cells; PBMCs: peripheral blood mononuclear cells; STR: short tandem repeat; PBLs: peripheral blood lymphocytes; PBS: phosphate-buffered saline; PD-1: programmed cell death protein 1; TILs: tumor-infiltrating lymphocytes; RNA-seq: RNA sequencing; Tg: transgenic; TMGs: tandem minigenes; WES: whole-exome sequencing; WT: wild-type.

Dataset Information

Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine.

Publications

Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine.

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