Dose‐dependent effects of the caspase inhibitor Q‐VD‐OPh on different apoptosis‐related processes

Abstract

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 µM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 µM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 µM Q-VD-OPh while 10 µM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.

Decitabine and SAHA-Induced Apoptosis Is Accompanied by Survivin Downregulation and Potentiated by ATRA in p53-Deficient Cells

“…Similar relationship between ROS presence and the extent of apoptosis was documented using pan-caspase inhibitors z-VAD-fmk or Q-VD-OPh (Figure 4(b)). Q-VD-OPh is known to inhibit caspase activity of HL-60 cells already at 2  μ M, while higher concentration of Q-VD-OPh was reported to prevent PARP fragmentation or loss of cellular adhesiveness [49]. In our experiments, 30  μ M Q-VD-OPh completely cancelled DAC + SAHA + ATRA-induced apoptosis and simultaneously almost fully abolished ROS generation.…”

Eliminating Encephalitogenic T Cells without Undermining Protective Immunity

“…When the effects of etoposide are paired with Bcl-2 family disequilibrium it suggests a specific mechanism for the selective clearance of encephalitogenic T cells that we observe in vivo . To test if apoptosis or necroptosis is the primary mechanism of etoposide-mediated cell death in encephalitogenic T cell we utilized the pan-caspase inhibitor Q-VD-OPH to prevent apoptosis (48, 49), and the RIP-1 inhibitor necrostatin to prevent necroptosis (50). We found that when encephalitogenic CD4 + T cells were treated with etoposide in the presence of Q-VD-OPH cell death decreased from the level of etoposide only to the level of vehicle treated cells (Figure 8H).…”

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

“…We analyzed the effect of caspase-4 and general caspase inhibition on in vitro network formation as an indicator of the relevance of the selected signaling signature for in vivo vessel formation. To avoid toxic side effects, concentrations of caspase-4 and pan-caspase inhibitors were titrated based on previous studies [18], [19], [23] (Figure S6). Untreated UC-derived ECFCs capable of forming vascular-like networks covering the matrix area after 24 hours were used as a positive control (Figure 4A).…”

“…Our data also established the as of yet unknown role of the inflammatory caspase-4 in regulating neo-vasculogenesis. We used small molecule caspase-4 (Z-FMK-LEVD) and, as a positive control, pan-caspase (Q-VD-OPh) inhibitors that irreversibly block caspases without cyto-toxicity effect [18], [19], [23], thus verifying the drugability of caspases as targets in this experimental therapeutic application. Whether caspase-4 plays a progenitor cell-specific role in apoptosis-induced proliferation during tissue regeneration remains to be studied [38].…”