Induction of Metallothionein Synthesis by Cilostazol in Mice and in Human Cultured Neuronal Cell Lines

Abstract

Cilostazol, a quinolinone derivative, is approved by the Food and Drug Administration for the treatment of intermittent claudication.1) It has been shown that Cilostazol has an inhibitory effect on platelet aggregation and thronbosis in feline cerebral ischemia.2) Cilostazol has also been shown to significantly reduce the risk of recurrent strokes without affecting the occurrence of intracranial hemorrhage. Recently, the Cilostazol Stroke Prevention Study revealed that Cilostazol reduced the risk of secondary stroke by 41.7% as compared with a placebo 3) suggesting that Cilostazol has a specific effect on small-vessel disease, although the mechanism for this is not well known. It has been shown that Cilostazol and its metabolites reduced the intracellular reactive oxygen species (ROS) induced by LPS in human umbilical vein endothelial cells by directly scavenging hydroxyl radicals. 4)However, the anti-stroke effect of Cilostazol in vivo seems not to be due to its antioxidative effect, because its blood clearance is so rapid, and its concentration in the brain would be too low to directly scavenge ROS. Thus, Cilostazol may cause an anti-stroke effect via the induction of a putative anti-oxidative molecule in the brain.Metallothionein (MT) is a small sulfhydryl-rich protein, the level of which is elevated by various inducers of metals, hormones and cytokines. The induction of MT synthesis has been reported in various tissues, but rarely in the brain. MT is thought to be a multifunctional protein detoxifying heavy metals such as cadmium and inorganic mercury, maintaining zinc and copper homeostasis, and regulating the biosynthesis and activity of zinc-binding proteins such as zinc-dependent transcription factors. MT also has the ability to scavenge ROS. 5,6) Concerning the anti-oxidative activities of MT, a role of MT in neurodegenerative diseases has been proposed.7) Thus, in this experiment, we examined the induction of MT by Cilostazol in the mouse brain and liver, and also in human cultured neuronal cells in order to confirm the possibility that Cilostazol directly induces MT in the human brain as an anti-oxidative effecter molecule. Animals and Treatments Male C57BL/6J mice were purchased from Clea Japan Inc. (Tokyo Japan). They were housed in plastic cages at 23°C with a 12 h light/dark cycle and were given lab food and tap water ad libitum. Mice (7 weeks old) were randomly assigned to treatment groups and were injected i.p. with 0.5% Cilostazol suspended with 0.5% carboxylmethyl cellulose solution or vehicle alone. After various time intervals, the liver and brain were removed from each mouse under anesthetization with sodium pentobarbital and were then stored at Ϫ80°C for the determination of MT level. The mice received humane care throughout the experiments according to the guidelines of Tokushima Bunri University. MATERIALS AND METHODS MaterialsCell Cultures IMR-32 cells, a human neuroblastoma cell line, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.), and maintained ...

Protective effect of pretreatment with cilostazol on cytotoxicity of cadmium and arsenite in cultured vascular endothelial cells

“…It is well known that zinc protects against Cd toxicity through the induction of MT synthesis in various systems (Leber and Miya, 1976;Goering and Klaassen, 1984). Recent observations, including our reports, using human vascular endothelial cells have demonstrated that cilostazol, as well as Cd and iAs III , induces MT synthesis (Suzuki et al, 2007;Fujiwara et al, 2009Fujiwara et al, , 2010Fujiwara et al, and 2011. The present study also showed that cilostazol induced MT mRNA and protein levels in bovine aortic endothelial cells.…”

“…Recently, several studies have demonstrated that cilostazol induces MT synthesis in not only vascular endothelial cells but also mouse tissues, such as the brain and liver, and cultured human cells, such as IMR32 neuroblastoma cells, coronary artery smooth muscle cells and colon carcinoma Caco-2 cells (Wakida et al, 2006;Suzuki et al, 2007;Fujiwara et al, 2009). These findings suggest that cilostazol non-specifically induces MT synthesis in various tissues and cell types.…”

Peculiarities of the Molecular Structure and Functions of Metallothioneins in the Central Nervous System

“…Carey et al [28] also found induction of the MT synthesis upon the action of a lipopolysaccharide that can be released in abortions and upon the action of teratogenic factors. The metabolism of MTs is very sensitive to humoral/hormonal regulation realized via the action of glucocorticoids, catecholamines, glucagon, steroids, interleukin-6, angiotensin-2, 2-butyl cAMP and phorbol ester, as wee as of stressor factors (proteins of an acute stress phase), and other mediators [29][30][31][32][33][34][35].…”

Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals

“…Several antibodies to MT-3 have been reported for immunohistochemical staining (Uchida et al, 1991;Yanagitani et al, 1999;Pang and Ru, 2005), but an ELISA method which is able to determine the MT-3 concentration in clinical and/or experimental specimens has not been reported yet. Therefore, we have developed an ELISA method which may serve as an effective tool for MT-3 research and diagnostic purpose, together with mRNA quantification by RT-PCR (Tsuji et al, 1992;Pang and Ru, 2005;Suzuki et al, 2007;Hozumi et al, 2008;Honda et al, 2010). Most of the MT-3 research has been reported only by mRNA expressions without quantitative protein analysis.…”

“…For the detection of MT-3, mRNA measurement by RT-PCR (Erickson et al, 1997;Somji et al, 2004;Suzuki et al, 2007;Haung et al, 2001) and an immunohistochemical staining method using specific antibodies (Uchida et al, 1991;Yanagitani et al, 1999;Pang and Ru, 2005) have been reported. However, a quantitative Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals determination by RIA or ELISA has not been reported yet.…”