ABSTRACT
The current study investigated the cytotoxic effect of 3-(5-chloro-2-hydroxybenzylideneamino)-2-(5-chloro-2-hydroxyphenyl)-2,3-dihydroquinazolin-41(H)-one (A) and 3-(5-nitro-2-hydroxybenzylideneamino)-2-(5-nitro-2-hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (B) on MCF-7, MDA-MB-231, MCF-10A and WRL-68 cells. The mechanism involved in apoptosis was assessed to evaluate the possible pathways induced by compound A and B. MTT assay results using A and B showed significant inhibition of MCF-7 cell viability, with IC50 values of 3. 27 ± 0.171 and 4.36 ± 0.219 µg/mL, respectively, after a 72 hour treatment period. Compound A and B did not demonstrate significant cytotoxic effects towards MDA-MB-231, WRL-68 and MCF-10A cells. Acute toxicity tests also revealed an absence of toxic effects on mice. Fluorescent microscopic studies confirmed distinct morphological changes (membrane blebbing and chromosome condensation) corresponding to typical apoptotic features in treated MCF-7 cells. Using Cellomics High Content Screening (HCS), we found that compound A and B could trigger the release of cytochrome c from mitochondria to the cytosol. The release of cytochrome c activated the expression of caspases-9 and then stimulated downstream executioner caspase-3/7. In addition, caspase-8 showed remarkable activity, followed by inhibition of NF-κB activation in A-and B-treated MCF-7 cells. The results indicated that A and B could induce apoptosis via a mechanism that involves either extrinsic or intrinsic pathways.
Subject(s)
Hydrazones/chemical synthesis , Quinazolinones/chemistry , Schiff Bases/chemical synthesis , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 7/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Cytochromes c/metabolism , Humans , Hydrazones/chemistry , Hydrazones/toxicity , MCF-7 Cells , Magnetic Resonance Spectroscopy , Membrane Potential, Mitochondrial/drug effects , Microscopy, Fluorescence , Mitochondria/metabolism , Molecular Conformation , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Quinazolinones/chemical synthesis , Quinazolinones/toxicity , Reactive Oxygen Species/metabolism , Schiff Bases/chemistry , Schiff Bases/toxicityABSTRACT
BACKGROUND: Wiskott-Aldrich syndrome protein (WASP) links T-cell receptor (TCR) signaling to the actin cytoskeleton. WASP is normally protected from degradation by the Ca(++)-dependent protease calpain and by the proteasome because of its interaction with the WASP-interacting protein. OBJECTIVE: We investigated whether WASP is degraded after TCR ligation and whether its degradation downregulates F-actin assembly caused by TCR ligation. METHODS: Primary T cells, Jurkat T cells, and transfected 293T cells were used in immunoprecipitation experiments. Intracellular F-actin content was measured in splenic T cells from wild-type, WASP-deficient, and c-Casitas B-lineage lymphoma (Cbl)-b-deficient mice by using flow cytometry. Calpeptin and MG-132 were used to inhibit calpain and the proteasome, respectively. RESULTS: A fraction of WASP in T cells was degraded by calpain and by the ubiquitin-proteasome pathway after TCR ligation. The Cbl-b and c-Cbl E3 ubiquitin ligases associated with WASP after TCR signaling and caused its ubiquitination. Inhibition of calpain and lack of Cbl-b resulted in a significantly more sustained increase in F-actin content after TCR ligation in wild-type T cells but not in WASP-deficient T cells. CONCLUSION: TCR ligation causes WASP to be degraded by calpain and to be ubiquitinated by Cbl family E3 ligases, which targets it for destruction by the proteasome. WASP degradation might provide a mechanism for regulating WASP-dependent TCR-driven assembly of F-actin.
Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Calpain/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Receptors, Antigen, T-Cell/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Animals , Calpain/antagonists & inhibitors , Cell Line , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Down-Regulation , Leupeptins/pharmacology , Mice , Mice, Knockout , T-Lymphocytes/metabolism , Ubiquitination , Wiskott-Aldrich Syndrome Protein/geneticsABSTRACT
Wiskott-Aldrich syndrome (WAS) is caused by mutations in the gene encoding WAS protein (WASP ). Recently, somatic mosaicism caused by reversions or second-site mutations has been reported in some inherited disorders including WAS. In this article, we describe somatic mosaicism in a 15-year-old WAS patient due to a second-hit mutation in the initiation codon. The patient originally had a single-base deletion (c.11delG; p.G4fsX40) in the WAS (WASP) gene, which resulted in a frameshift and abrogated protein expression. Subsequently, a fraction of T and natural killer (NK) cells expressed a smaller WASP, which binds to its cellular partner WASP-interacting protein (WIP). The T and NK cells were found to have an additional mutation in the initiation codon (c.1A>T; p.M1_P5del). The results strongly suggest that the smaller WASP is translated from the second ATG downstream of the original mutation, and not only T cells but also NK cells carrying the second mutation acquired a growth advantage over WASP negative counterparts. To our knowledge, this is the first report describing somatic mosaicism due to a second-site mutation in the initiation codon of any inherited disorders.
