Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.     

Cepharanthine triggers apoptosis in a human hepatocellular carcinoma cell line (HuH-7) through the activation of JNK1/2 and the downregulation of Akt

Cepharanthine (CEP), a biscoclaurine alkaloid, has been reported to induce cell death, however, the molecular mechanism of this phenomenon remains unclear. We herein report that CEP induced apoptosis in HuH-7 cells through nuclear fragmentation, DNA ladder formation, cytochrome c release, caspase-3 activation and poly-(ADP-ribose)-polymerase cleavage. CEP triggered the generation of reactive oxygen intermediates, the activation of mitogen activated protein kinase (MAPK) p38, JNK1/2 and p44/42, and the downregulation of protein kinase B/Akt. Antioxidants and SP600125, an inhibitor of JNK1/2, but not inhibitors of p38 MAPK and MEK1/2, significantly prevented cell death, thus implying that reactive oxygen species and JNK1/2 play crucial roles in the CEP-induced apoptosis of HuH-7 cells.     

A Switch in the Dynamics of Intra-Platelet VEGF-A from Cancer to the Later Phase of Liver Regeneration after Partial Hepatectomy in Humans

Background

Liver regeneration (LR) involves an early inductive phase characterized by the proliferation of hepatocytes, and a delayed angiogenic phase distinguished by the expansion of non-parenchymal compartment. The interest in understanding the mechanism of LR has lately shifted from the proliferation and growth of parenchymal cells to vascular remodeling during LR. Angiogenesis accompanied by LR exerts a pivotal role to accomplish the process. Vascular endothelial growth factor (VEGF) has been elucidated as the most dynamic regulator of angiogenesis. From this perspective, platelet derived/Intra-platelet (IP) VEGF-A should be associated with LR.

Material and Methods

Thirty-seven patients diagnosed with hepatocellular carcinoma and undergoing partial hepatectomy (PH) were enrolled in the study. Serum and IP VEGF-A was monitored preoperatively and at four weeks of PH. Liver volumetry was determined on computer models derived from computed tomography (CT) scan.

Results

Serum and IP VEGF-A was significantly elevated at four weeks of PH. Preoperative IP VEGF-A was higher in patients with advanced cancer and vascular invasion. Postoperative IP VEGF-A was higher after major liver resection. There was a statistically significant correlation between postoperative IP VEGF-A and the future remnant liver volume. Moreover, the soluble vascular endothelial growth factor receptor-1 (sVEGFR1) was distinctly down-regulated suggesting a fine-tuned angiogenesis at the later phase of LR.

Conclusion

IP VEGF-A is overexpressed during later phase of LR suggesting its implications in inducing angiogenesis during LR.     

2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture oftobacco (Nicotiana tabacum L.) was established using mediumcontaining 30 g/liter of sucrose and 1.5 µM 2,4-D. The2,4-D-sustained photomixotrophic line was able to show rapidregreening in the light after bleaching in the dark and characterizedwith a much slower and longer growth cycle than a heterotrophicline derived from the same original callus (cell doubling timeof 100 h vs. 40 h and duration of logarithmic phase of 17 daysvs. 7 days). The photomixotrophic line took up sucrose morerapidly than the heterotrophic line and accumulated starch duringthe early logarithmic phase when it showed a maximum photosyntheticcapacity on a chlorophyll basis (6.3µmol O₂/min/mg Chl).Chlorophyll content and photosynthetic capacity on a per cellbasis and on a cell fresh weight basis, on the other hand, decreasedduring this phase and reincreased later to reach maximum levels(310 µg Chl/g fr wt; 1.4 µmol O₂/min/g fr wt) whenthe line exhibited the highest activities of dark respiration(1.0 µmol; O₂/min/g fr wt) and cell division (mitoticindex of 3.0%). These characteristics of the photomixotrophicline were lost if it was grown in the dark to become non-chlorophyllous.Although net O₂ evolution could not be detected in the photomixotrophicline throughout the growth cycle when assayed under suboptimumlight intensity, reaccumulation of starch and a marked increasein cell fresh weight upon addition of minerals, vitamins and2,4-D without sucrose at the late logarithmic phase indicatedthe development of photosynthetic activity under the cultureconditions. ¹The investigations reported were included in the thesis submittedto the Graduate School, Faculty of Agriculture, Kobe University,in partial fulfillment of the requirement for M. Agr. degree. (Received May 30, 1988; Accepted October 5, 1988)     

Endocannabinoid, anandamide in gingival tissue regulates the periodontal inflammation through NF-kappaB pathway inhibition

Anandamide (AEA) exhibits anti-inflammatory effects. However, its role in the periodontal field remains unknown. Here, we found that gingival crevicular fluid contained a detectable level of AEA. The cannabinoid receptors CB1 and CB2 were expressed by human gingival fibroblasts (HGFs), and markedly upregulated under pathological conditions. AEA significantly reduced the production of pro-inflammatory mediators (IL-6, IL-8 and MCP-1) induced by Porphyromonas gingivalis LPS in HGFs, and this effect was attenuated by AM251 and SR144528, selective antagonists of CB1 and CB2, respectively. Moreover, AEA completely blocked LPS-triggered NF-kappaB activation, implying that AEA may regulate hyperinflammatory reactions in periodontitis.     

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.     

Quantitative analysis of biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) strains from patients with orthopaedic device-related infections

Biofilms play a pivotal role in medical device-related infections. However, epidemiological analysis of biofilm formation and genotyping among clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with orthopaedic infections has rarely been reported. A total of 168 MRSA strains were examined: 23 strains from patients with device-related infection (the device group); 55 from patients with device-non-related infection (the nondevice group); and 90 from asymptomatic nasal carriers (the colonization group). Pulsed-field gel electrophoresis analysis and five genotyping methods including agr typing were performed. Biofilm formation was quantified using a microtitre plate assay. The device group had a significantly higher incidence of agr-2 than the colonization group (78.3% vs. 34.4%, P=0.001). The biofilm index of the agr-2 (0.523 ± 0.572) strains was significantly higher than those of agr-1 (0.260 ± 0.418, P<0.0001) and agr-3 (0.379 ± 0.557, P=0.045). The prevalence of strong biofilm formers in the device group (43.5%) was significantly higher than that in the nondevice group (12.7%, P=0.003) and the colonization group (20.0%, P=0.020). agr-2 MRSA strains may be more likely to cause orthopaedic device infection because of their strong biofilm formation ability.     

ER signaling is activated to protect human HaCaT keratinocytes from ER stress induced by environmental doses of UVB

Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-Lα, an upstream signaling molecule of ER stress, decreased at 1-4 h after 10 mJ/cm² irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm² UVB at 4 h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes.