Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells.

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5-STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31-PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.

In vivo assay of the potential gadolinium-induced toxicity for sensory hair cells using a zebrafish animal model.

Recently, intratympanic injection of gadolinium-based contrast agent (GdC) is growing in use to visualize the endolymphatic hydrops. Although GdC has been quite safely used over 20 years through intravenous injection, the biological influence of GdC on sensory hair cells needs to be thoroughly assessed for wider clinical application of it through intratympanic injection. In this in vivo experimental study, the summated number of sensory hair cells (SO1, SO2, O1 and OC1 neuromasts) showed a steep decrease in the group exposed to 10% and 20% GdC (35.7 ± 7.3, 15.09 ± 10.82, respectively, P < .01) compared with the control group (47.18 ± 2.30). An increase in apoptosis was also observed in the group exposed to 20% gadolinium (7.20 ± 5.56), as compared with the control group (0.08 ± 0.72) or the group exposed to 10% gadolinium (3.48 ± 3.32). A significant reduction in the viable cytoplasmic mitochondria was observed in embryos exposed to 20% GdC (369 ± 124 µm2 , P = .01) as compared with control embryos (447 ± 118 µm2 ) or embryos exposed to 10% GdC (420 ± 108 µm2 ). GdC administration did not impact peripheral neural structures. GdC caused a significant reduction in sensory hair cell counts in response to high concentrations along with increased apoptosis and mitochondrial damage. However, it may not be likely that GdC will lead to hair cell toxicity, as the estimated concentration in the inner ear after clinically tried intratympanic injection is far more diluted than the non-toxic concentration (0.625%) that was tested in this study.

In vivo and in vitro anti-inflammatory activities of alpha-linolenic acid isolated from Actinidia polygama fruits.

The fruit of Actinidia polygama (AP) has long been used as a folk medicine in Korea for the treatment of pain, rheumatoid arthritis and inflammation. In the present study, bioassay-guided fractionation of AP led to the separation and identification of a polyunsaturated fatty acid, alpha-linolenic acid (ALA), which was found to show anti-inflammatory activity. The anti-inflammatory effects of ALA, using acetic acid or carrageenan-induced inflammation models, were investi gated in mice or rats, respectively. ALA significantly inhibited the acetic acid-induced vascular permeability in a dose dependent manner (34.2 and 37.7% inhibition at doses of 5 and 10 mg/ kg, respectively). ALA also significantly reduced a rat paw edema induced by a single treatment of carrageenan. To investigate the mechanism of the anti-inflammatory action of ALA, the effects of ALA on lipopolysaccharide (LPS)-induced responses in the murine mac rophages cell line, RAW 264.7, were examined. Exposure of LPS-stimulated cells to ALA inhibited the accumulation of nitrite and prostaglandin E2 (PGE2) in the culture medium. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were markedly inhibited by ALA in a dose dependent manner. These results suggest that the anti-inflammatory activity of ALA might be due to the suppression of the expressions of iNOS and COX-2 mRNA.

Role of nicotinic acetylcholine receptors in the regulation of kainic acid-induced hippocampal cell death in mice.

Kainic acid (KA) is a well-known excitatory, neurotoxic substance. In mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the CA3 pyramidal neurons. In the present study, the possible role of nicotinic acetylcholine receptors (nAchRs) in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. Methyllycaconitine (MC; nAchRs antagonist, 20 microg) attenuated KA-induced CA3 pyramidal cell death. KA increased immunoreactivities (IRs) of phorylated extracellular signal-regulated kinase (p-ERK; at 30 min), p-CaMK II (at 30 min), c-Fos (at 2 h), c-Jun (at 2 h), glial fibrillary acidic protein (GFAP at 1 day), and the complement receptor type 3 (OX-42; at 1 day) in hippocampal area. MC attenuated selectively KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. Our results suggest that p-CaMK II may play as an important regulator responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Reactive astrocytes, which was meant by GFAP IR, and activated microglia, which was meant by OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA-induced excitotoxicity. Furthermore, it is implicated that niconitic receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Antinociceptive profiles of platycodin D in the mouse.

Platycodin D (PD), one of several triterpene saponins, was isolated from roots of Platycodon grandiflorum. We previously reported that intracerebroventricular (i.c.v.) administration of PD showed an antinociceptive effect as measured by the tail-flick assay. However, its exact role in the regulation of antinociception in the various types of pain models has not yet been characterized. Thus, we attempted to find antinociceptive profiles of PD in various pain models. PD administered intraperitoneally (i.p.), i.c.v. or intrathecally (i.t.) showed antinociceptive effects in dose-dependent manners as measured by the tail-flick, writhing and formalin tests. In the tail-flick test, PD at the low doses reached the peak after 15 minutes and returned to the control level after 60 minutes. However, higher doses of PD showed a strong antinociception at least for 1 hour. PD administered i.t. showed stronger antinociception than that induced by i.c.v. administration PD in both tail-flick and writhing tests. In the formalin test, PD administered i.p., i.c.v. or i.t. showed antinociceptive effects during both the first (direct nociceptive stimulation) and second (late inflammatory) phases. Pretreatment with naltrexone i.p., i.c.v. or i.t. did not affect PD-induced inhibition of the tail-flick response. Our results suggest that PD shows a strong antinociceptive effect on the tail-flick, writhing and formalin tests, acting on central nervous system. However, PD-induced antinociception may not be mediated by the opioid receptors.

Regulation of c-fos and c-jun gene expression by lipopolysaccharide and cytokines in primary cultured astrocytes: effect of PKA and PKC pathways.

The effects of lipopolysaccharide (LPS) and several cytokines on the c-fos and c-jun mRNA expression were examined in primary cultured astrocytes. Either LPS (500 ng/mL) or interferon-gamma (IFN-gamma; 5 ng/mL) alone increased the level of c-fos mRNA (1 h). However, tumor necrosis factor-alpha (TNF-alpha; 10 ng/mL) or interleukin-1beta (IL-1beta; 5 ng/mL) alone showed no significant induction of the level of c-fos mRNA. TNF-alpha showed a potentiating effect in the regulation of LPS-induced c-fos mRNA expression, whereas LPS showed an inhibitory action against IFN-gamma-induced c-fos mRNA expression. LPS, but not TNF-alpha, IL-1beta and IFN-gamma, increased the level of c-jun mRNA (1 h). TNF-alpha and IFN-gamma showed an inhibitory action against LPS-induced c-jun mRNA expression. Both phorbol 12-myristate 13-acetate (PMA; 2.5 mM) and forskolin (FSK; 5 mM) increased the c-fos and c-jun mRNA expressions. In addition, the level of c-fos mRNA was expressed in an antagonistic manner when LPS was combined with PMA. When LPS was co-treated with either PMA or FSK, it showed an additive interaction for the induction of c-jun mRNA expression. Our results suggest that LPS and cytokines may be actively involved in the regulation of c-fos and c-jun mRNA expressions in primary cultured astrocytes. Moreover, both the PKA and PKC pathways may regulate the LPS-induced c-fos and c-jun mRNA expressions in different ways.

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice.

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Effect of ginsenoside Rd on nitric oxide system induced by lipopolysaccharide plus TNF-alpha in C6 rat glioma cells.

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-alpha (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun B and Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun B and Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun B and Fra-1 in C6 glioma cells.

Cycloheximide inhibits neurotoxic responses induced by kainic acid in mice.

In the present study, we examined the effect of cycloheximide on various pharmacological responses induced by kainic acid (KA) administered intracerebroventricularly (i.c.v.) in mice. In a passive avoidance test, a 20-min cycloheximide (200mg/kg, i.p.) pretreatment prevented the memory impairment induced by KA. The morphological damage induced by KA (0.1microg) in the hippocampus was markedly concentrated in the CA3 pyramidal neurons and cycloheximide effectively prevented the KA-induced pyramidal cell death in CA3 hippocampal region. In immunohistochemical study, KA dramatically increased the phosphorylation of extracellular signal-regulated protein kinase (p-ERK), c-Jun N-terminal kinase 1 (p-JNK1), and calcium/calmodulin-dependent protein kinase II (p-CaMK II). Cycloheximide attenuated the increased p-ERK, p-JNK1, and p-CaMK II levels induced by KA. Furthermore, cycloheximide inhibited the increased c-Fos and c-Jun protein expression levels induced by KA in the hippocampus. The activation of microglia was detected in KA-induced CA3 cell death region by immunostaining with a monoclonal antibody against the OX-42. Cycloheximide inhibited KA-induced increase of OX-42 immunoreactivity. Our results suggest that the increased expression of the c-Fos, c-Jun, and phosphorylation of ERK, JNK1, and CaMK II proteins may play important roles in the memory impairment and the cell death in CA3 region of the hippocampus induced by i.c.v. KA administration in mice. Furthermore, the activated microglia may be related to phagocytosis of degenerated neuronal elements induced by KA.

Antinociceptive mechanisms of orally administered decursinol in the mouse.

Antinociceptive profiles of decursinol were examined in ICR mice. Decursinol administered orally (from 5 to 200 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured by the tail-flick and hot-plate tests. In addition, decursinol attenuated dose-dependently the writhing numbers in the acetic acid-induced writhing test. Moreover, the cumulative response time of nociceptive behaviors induced by an intraplantar formalin injection was reduced by decursinol treatment during the both 1st and 2nd phases in a dose-dependent manner. Furthermore, the cumulative nociceptive response time for intrathecal (i.t.) injection of TNF-alpha (100 pg), IL-1 beta (100 pg), IFN-gamma (100 pg), substance P (0.7 microg) or glutamate (20 microg) was dose-dependently diminished by decursinol. Intraperitoneal (i.p.) pretreatment with yohimbine, methysergide, cyproheptadine, ranitidine, or 3,7-dimethyl-1-propargylxanthine (DMPX) attenuated inhibition of the tail-flick response induced by decursinol. However, naloxone, thioperamide, or 1,3-dipropyl-8-(2-amino-4-chloro-phenyl)-xanthine (PACPX) did not affect inhibition of the tail-flick response induced by decursinol. Our results suggests that decursinol shows an antinociceptive property in various pain models. Furthermore, antinociception of decursinol may be mediated by noradrenergic, serotonergic, adenosine A(2), histamine H(1) and H(2) receptors.

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice.

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.