Induction of apoptosis by a calpain stimulator, ONO-3403

Abstract

The effects of a synthetic serine proteese inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 ~g/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 ~g/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified ~-calpain at a concentration higher than 100 ~g/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of ~-calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.

Calpain and calpastatin regulate neutrophil apoptosis

“…The antisense-related apoptosis could be blocked by calpain inhibitors, consistent with the hypothesis that a decreased level of calpastatin led to increased calpain activity, which resulted in apoptosis. This corroborates the finding by other investigators that a pharmacologic compound able to activate calpain in vitro was sufficient to cause apoptosis in NIH/3T3 cells (Hiwasa, 1996). This is also the first direct indication that calpastatin depletion could be a sufficient physiologic stimulus to promote calpain-dependent apoptosis.…”

Enhancement of chemosensitivity toward peplomycin by calpastatin-stabilized NF-κB p65 in esophageal carcinoma cells: possible involvement of Fas/Fas-L synergism

“…The cells were washed three times with phosphate-buffered saline (PBS) and lysed in 0.5% Nonidet P-40, 20 mM TrisHCl (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, 50 µM antipain, 50 µM pepstatin A and 50 µM ALLN 32 for 10 min at 4 • C. 33 The cell lysate was centrifuged at 13,000 × g for 10 min and the supernatant was used as cytoplasmic cell extract. The pellet was used as the nuclear fraction.…”

Phytocannabinoids, ∆⁹-tetrahydrocannabinol and cannabidiol, as human calpain-1 (CAPN1) activators

“…The mechanisms by which ∆ 9 -THC and CBD stimulate the activity of calpain-1 currently remain unclear. In structural comparisons with ONO-3403, a direct activator of calpain-1 (Hiwasa, 1996), no similarities were observed between ∆ 9 -THC/ CBD and ONO-3403. Since the whole crystal structure of human calpain-1 together with calcium has not yet been elucidated, we cannot provide rational evidence for the activation mechanism(s) used by these two cannabinoids.…”

“…Calpains may be activated via at least two mechanisms: (i) chemicals initially stimulate cellular signaling, which leads to the activation of calpain enzymes (i.e., possibly indirect activation), and (ii) the chemically-based direct activation of calpains. When compared with the proposed mechanism (i), chemicals involved in mechanism (ii) are very limited; e.g., ONO-3403, a synthetic serine protease inhibitor, has been shown to stimulate the activity of purified calpain-1 at a concentration higher than 180 μM (Hiwasa, 1996). A previous study indicated that the botanical extracts nabiximols (∆ 9 -THC/CBD) delayed disease progression in rat models of Huntington's disease through, at least in part, the up-regulation of calpain expression (Sagredo et al, 2011).…”