Nα-Tosyl-l-phenylalanine Chloromethyl Ketone Induces Caspase-dependent Apoptosis in Transformed Human B Cell Lines with Transcriptional Down-regulation of Anti-apoptotic HS1-associated Protein X-1

Abstract

was originally synthesized as a specific inhibitor of chymotrypsin-like serine proteases (1) and has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported. Hence, TPCK was shown to prevent apoptosis in a cell death stimulus-dependent manner in some model systems (2-4), and a recent study also suggested that TPCK may diminish apoptosis by direct and nonspecific inhibition of active caspases (5). On the other hand, TPCK was also reported to potentiate apoptosis induced by certain apoptotic stimuli, and this was suggested to occur, at least in some cases, through activation of cell cycle checkpoints via inhibition of the proteasome (3, 6). TPCK alone may also induce apoptosis. TPCK thus caused a reduction in activity of the transcription factor, NF-B, in a murine B lymphoma cell line resulting in a decrease in c-myc and activation of apoptosis (7,8). Subsequent studies have shown that TPCK can induce apoptosis in a human acute monocytic leukemia cell line through activation of mitochondria-dependent apoptosis signaling (4, 9). Studies in transformed human T cells also indicated that TPCK can induce apoptosis through inhibition of constitutive NF-B activation, and it was proposed that TPCK might affect sulfhydryl groups on proteins involved in regulating cell survival and NF-B acti- 4 The abbreviations used are: TPCK, N ␣ -tosyl-L-phenylalanine chloromethyl ketone; z, N-benzyloxycarbonyl; fmk, fluoromethyl ketone; AIF, apoptosisinducing factor; cIAP1, cellular inhibitor of apoptosis protein-1; cIAP2, cellular inhibitor of apoptosis protein-2; AMC, 7-amino-4-methylcoumarin; DHE, dihydroethidium; ⌬ m , mitochondrial transmembrane potential; EBV, Epstein-Barr virus; HAX-1, HS1-associated protein X-1; IB, inhibitor of NF-B; NAC, N-acetylcysteine; NF-B, nuclear factor B; PARP, poly(ADPribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species; SCN, severe congenital neutropenia; TMRE, tetramethylrhodamine ethyl ester; XIAP, X chromosome-linked inhibitor of apoptosis protein.

A Novel Cinnamide YLT26 Induces Breast Cancer Cells ApoptosisviaROS-Mitochondrial Apoptotic Pathwayin Vitroand Inhibits Lung Metastasisin Vivo

“…Disruption of the mitochondrial membrane potential is known to induce release of cytochrome c from mitochondria into the cytosol, where cytochrome c leads to the generation of caspase-9 and activates downstream caspases, which ultimately induces the cell to undergo apoptosis [27]. In our experiment, a loss of ∆Ψm in 4T1 cells was also observed after YLT26 treatment.…”

Caspase-3 triggers a TPCK-sensitive protease pathway leading to degradation of the BH3-only protein puma

“…Nevertheless, TPCK can also inhibit some cysteine proteases including at some extent even caspases [38,39]. Contrasting reports, in some cases from the same team, have shown that, depending on the cell type, dose and the nature of co-signals, TPCK may either partially inhibit caspase activation and activity or promote caspase activation and apoptosis [38][39][40][41][42]. Nevertheless, in our experimental conditions, the degradation of Puma induced by TRAIL or anisomycin in Jurkat or BL41 cells was prevented by adding TPCK to the medium, without inhibiting the activation (as assessed by monitoring the disappearance of the proform caspase) and activity (as assessed by monitoring cleavage of the caspase-3 substrate PARP-1) of caspase-3 in these cells.…”

Requirement of Apoptotic Protease-Activating Factor-1 for Bortezomib-Induced Apoptosis but Not for Fas-Mediated Apoptosis in Human Leukemic Cells

“…Cell lysates were combined with the fluorogenic, caspase-3-specific substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) in a standard reaction buffer (Jitkaew et al, 2009), and real-time measurements of enzyme-catalyzed release of AMC were obtained using a Tecan Infinite F200 plate reader (Tecan Group Ltd, Männedorf, Switzerland) operating with Magellan version 6.5 software (Tecan Group Ltd, Männedorf, Switzerland). Fluorescence values were converted to picomoles, and the maximum rate of AMC release (picomoles per minute) was estimated for each sample.…”

“…Loss of mitochondrial membrane potential was detected using tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes, Eugene, OR) (Jitkaew et al, 2009). In brief, 20 minutes prior to harvesting the cells, 1 ml (TMRE 25 nM) was added to the cell cultures.…”