A selective and rapid cell-permeable inhibitor of human caspase-3

ABSTRACTThe individual roles and overlapping functionalities the twelve human caspases have during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permit its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.

Cytolethal Distending Toxin Induces Caspase-Dependent and -Independent Cell Death in MOLT-4 Cells

ABSTRACT Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

An Antiviral Peptide Inhibitor That Is Active against Picornavirus 2A Proteinases but Not Cellular Caspases

ABSTRACT The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2A pro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2A pro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2A pro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2A pro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2A pro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2A pro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2A pro , suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.

Molecular Mechanism of Matrine-Induced Apoptosis in Leukemia K562 Cells

Matrine, a low toxic alkaloid purified from the Chinese herb Kushen, has been reported to induce apoptosis in leukemia K562 cells. In this study, the mechanism underling this apoptotic event was investigated. Treatment of K562 cells with matrine resulted in inhibition of cell survival more significantly than treatment of non-cancer fibroblast NIH3T3 cells. When K562 cells were incubated with matrine in higher than 0.2 mg/ml doses for 48 hours, the apoptotic cells were increased and both poly (ADP-ribose) polymerase (PARP) and caspase-3 were cleaved in a dose dependent manner. General caspase inhibitor (z-VAD-fmk) or caspase-3 inhibitor (z-DEVD-fmk) almost completely suppressed matrine-induced apoptosis. In addition, matrine increased proapoptotic protein bax and caused the release of cytochrome C. Taken together, the results suggest that matrine induces a cytochrome C-mediated, caspase-dependent apoptosis.

Anti-Thymocyte Globulin (ATG) Induces Apoptosis in Myeloma Cells: A Basis for Myeloma Sero-Therapy?.

Allogeneic stem cell transplantation is a potentially curative therapy for patients with multiple myeloma. Polyclonal ATG is included in conditioning regimens to enhance engraftment and reduce the risk of severe graft-versus-host disease. Since ATG has been reported to induce depletion of T-, B- and dendritic cells, we sought to investigate its cytotoxic activity on myeloma cells. Complement-mediated and complement-independent activity of ATG-Fresenius was investigated on 4 myeloma cell lines (RPMI-8226, U266, KMS-12-BM and EJM) and bone marrow samples from 6 myeloma patients. Cytotoxicity was determined by staining with annexin V-FITC and 7AAD followed by flow cytometry. ATG at clinically relevant concentrations induced up to 100% and 85% complement-dependent killing of myeloma cell lines and primary myeloma samples respectively. In the absence of complement ATG still induced up to 50% and 80% apoptosis in myeloma cell lines and primary myeloma samples respectively. Preincubation of myeloma cells with a general caspase inhibitor (ZVAD-fmk) abrogated ATG-induced complement-independent cell death. Absorption assays indicate that ATG induced cytotoxicity is mediated by specific antibodies and antigens whose further elucidation may pave the way for antibody-based myeloma therapy.

Mild sustained and intermittent hypoxia induce apoptosis in PC-12 cells via different mechanisms

Episodic hypoxia, a characteristic feature of obstructive sleep apnea, induces cellular changes and apoptosis in brain regions associated with neurocognitive function. To investigate whether mild, intermittent hypoxia would induce more extensive neuronal damage than would a similar degree of sustained hypoxia, rat pheochromocytoma PC-12 neuronal cells were subjected to either sustained (5% O2) or intermittent (alternating 5% O2 35 min, 21% O2 25 min) hypoxia for 2 or 4 days. Quantitative assessment of apoptosis showed that while mild sustained hypoxia did not significantly increase cell apoptosis at 2 days (1.31 ± 0.29-fold, n = 8; P = NS), a significant increase in apoptosis occurred after 4 days (2.25 ± 0.4-fold, n = 8; P < 0.002), without increased caspase activation. Furthermore, caspase inhibition with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) did not modify sustained hypoxia-induced apoptosis. In contrast, mild, intermittent hypoxia induced significant increases in apoptosis at 2 days (3.72 ± 1.43-fold, n = 8; P < 0.03) and at 4 days (4.57 ± 0.82-fold, n = 8; P < 0.001) that was associated with enhanced caspase activity and attenuated by Z-VAD-FMK pretreatment. We conclude that intermittent hypoxia induces an earlier and more extensive apoptotic response than sustained hypoxia and that this response is at least partially dependent on caspase-mediated pathways. In contrast, caspases do not seem to play a role in sustained hypoxia-induced apoptosis. These findings suggest that different signaling pathways are involved in sustained and intermittent hypoxia-induced cell injury and may contribute to the understanding of differential brain susceptibility to sustained and intermittent hypoxia.

The oncostatin M receptor/gp130 ligand murine oncostatin M induces apoptosis in adrenocortical Y-1 tumor cells

The effects of murine oncostatin M (mOSM) are specifically mediated by the heterodimeric oncostatin M receptor (OSMR)/gp130 receptor complex. In the current study we demonstrate that murine adrenocortical Y-1 tumor cells express the OSMR/gp130 complex. Incubation of Y-1 cells with 1 and 10 ng/ml mOSM induces cell death due to specific induction of apoptosis. Western blot analysis of Y-1 cells incubated with mOSM for 24 h revealed caspase-3 cleavage and poly(ADP-ribase) polymerase (PARP) cleavage. In a proliferation assay system, incubation of Y-1 cells with 0.01, 0.1, 1 and 10 ng/ml mOSM for 24 h resulted in a decrease in cell numbers to 99+/-2%, 84+/-9%, 50+/-7% and 43+/-5% respectively of untreated control (defined as 100%). Pretreatment of Y-1 cells with the Jak2 inhibitor AG490 (100 microM) rescued Y-1 cells from OSM-induced (10 ng/ml) cell death. Similarly, pretreatment of Y-1 cells with the general caspase inhibitor Z-VAD-FMK (42 microM) rescued Y-1 cells from OSM-induced (10 ng/ml) cell death. In summary, we show that adrenocortical Y-1 tumor cells express the OSMR/gp130 complex and that mOSM induces the Jak-STAT signaling cascade in these cells. Murine OSM in a dose-dependent manner induces apoptosis in adrenocortical Y-1 tumor cells. Apoptosis was demonstrated by caspase-3 cleavage and PARP cleavage. Rescue of Y-1 cells from mOSM-induced apoptosis by the Jak2 inhibitor, AG490, and the general caspase inhibitor, Z-VAD-FMK, demonstrates Jak activation and subsequent caspase activation to be essential for mOSM-induced apoptosis in adrenocortical Y-1 tumor cells. The putative role of OSM as an immunotherapeutic agent in human adrenocortical cancer remains to be elucidated.

Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells

Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The μ-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal α-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of μ-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells.

The Herpes Simplex Virus 1 US3 Protein Kinase Blocks Caspase-Dependent Double Cleavage and Activation of the Proapoptotic Protein BAD

ABSTRACT An earlier report showed that the US3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the US3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the US3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the US3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the US3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the US3 protein kinase. (iv) The US3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M r of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of US3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.