p66Shc--a longevity redox protein in human prostate cancer progression and metastasis : p66Shc in cancer progression and metastasis.

p66Shc, a 66 kDa proto-oncogene Src homologous-collagen homologue (Shc) adaptor protein, is classically known in mediating receptor tyrosine kinase signaling and recently identified as a sensor to oxidative stress-induced apoptosis and as a longevity protein in mammals. The expression of p66Shc is decreased in mice and increased in human fibroblasts upon aging and in aging-related diseases, including prostate cancer. p66Shc protein level correlates with the proliferation of several carcinoma cells and can be regulated by steroid hormones. Recent advances point that p66Shc protein plays a role in mediating cross-talk between steroid hormones and redox signals by serving as a common convergence point in signaling pathways on cell proliferation and apoptosis. This article first reviews the unique function of p66Shc protein in regulating oxidative stress-induced apoptosis. Subsequently, we discuss its novel role in androgen-regulated prostate cancer cell proliferation and metastasis and the mechanism by which it mediates androgen action via the redox signaling pathway. The data together indicate that p66Shc might be a useful biomarker for the prognosis of prostate cancer and serve as an effective target for its cancer treatment

Disruption of Immune Homeostasis in Human Dendritic Cells via Regulation of Autophagy and Apoptosis by

As fundamental processes of immune homeostasis, autophagy, and apoptosis must be maintained to mitigate risk of chronic inflammation and autoimmune diseases. Periodontitis is a chronic inflammatory disease characterized by oral microbial dysbiosis, and dysregulation of dendritic cell (DC) and T cell responses. The aim of this study was to elucidate the underlying mechanisms by which the oral microb

Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis

Periodontitis (PD) is a common dysbiotic inflammatory disease that leads to local bone deterioration and tooth loss. PD patients experience low-grade bacteremias with oral microbes implicated in the risk of heart disease, cancer, and kidney failure. Although Th17 effectors are vital to fighting infection, functional imbalance of Th17 effectors and regulatory T cells (Tregs) promote inflammatory diseases. In this study, we investigated, in a small pilot randomized clinical trial, whether expansion of inflammatory blood myeloid dendritic cells (DCs) and conversion of Tregs to Th17 cells could be modulated with antibiotics (AB) as part of initial therapy in PD patients. PD patients were randomly assigned to either 7 d of peroral metronidazole/amoxicillin AB treatment or no AB, along with standard care debridement and chlorhexidine mouthwash. 16s ribosomal RNA analysis of keystone pathoge

Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation.

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the \u27keystone\u27 pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + 

Steroids Up-Regulate p66Shc Longevity Protein in Growth Regulation by Inhibiting Its Ubiquitination

p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway

Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells.

Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD

Glycosylation inhibitors block thrombin-induced sRBC adhesion to HUVECs.

<p><b>A</b>. Thrombin induces sRBC adhesion to HUVECs. sRBCs were prepared from two sickle cell model mice (SCD 1 & SCD 2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099363#pone.0099363-Ryan1" target="_blank">[26]</a>. HUVECs were treated with 1 U/mL thrombin for 5 minutes after which sRBCs were added. The level of sRBC adhesion to untreated HUVECs was set to 100%. **, p<0.0001 <b>B</b>. Glycosylation inhibitors decrease thrombin-induced sRBC adhesion to HUVECs. HUVECs were pretreated with glycosylation inhibitors for 48 hours and P-selectin expression was stimulated by 1 U/mL thrombin. sRBCs were then added to HUVECs for adhesion assays. The levels of sRBC adhesion to HUVECs that were not treated with glycosylation inhibitor but stimulated with thrombin were set to 100%. **, p<0.0001 <b>C</b>. Representative microscopic images (x10 magnification) showing sRBC adhesion to HUVECs. Note that both tunicamycin and castanospermine inhibit sRBC adhesion to thrombin-activated HUVECs.</p

Comparison of sRBC adhesion inhibition by glycosylation inhibitors and P-selectin monoclonal antibody.

<p>HUVECs that were cultured as described in Materials and Methods were pretreated with saline or glycosylation inhibitors for 48 hours and stimulated with 1/mL thrombin. sRBCs were isolated from SCD patients and added to HUVECs. To examine the inhibition of sRBC adhesion, P-selectin monoclonal antibody was added to HUVECs prior to the addition of sRBCs. To inactivate P-selectin monoclonal antibody, the antibody was premixed with blocking peptide. Lanes: 1, HUVECs stimulated with 1U/mL thrombin; 2, HUVECs pretreated with 5 µg/mL tunicamycin; 3, HUVECs pretreated with 1 mM castanospermine; 4, HUVECs added with P-selectin monoclonal antibody; 5, HUVECs added with P-selectin monoclonal antibody premixed with blocking peptide; 6, HUVECs pretreated with 5 µg/mL tunicamycin and further added with P-selectin monoclonal antibody. **, p<0.0001; ^##, p<0.01 (vs lanes 2 & 4).</p

Glycosylation inhibitors block hypoxia-induced P-selectin expression in HUVECs.

<p>P-selectin expression in HUVECs exposed to 1% O₂ was examined by Western blotting (A) and immunofluorescence (B). <b>A</b>. Western blotting analysis of P-selectin expression in HUVECs exposed to hypoxia. HUVECs were cultured in the absence or presence of 2.5 or 5.0 µg/mL tunicamycin or 0.5 to 1.0 mM castanospermine, treated with 1 U/mL thrombin, and then were exposed to 1% O₂ for 1 hour. Cell lysates (30 µg) prepared from HUVECs were analyzed by Western blotting as described in Materials and Methods. β-Actin was as the loading control. The ratio of the P-selectin protein level to that of β-actin was set to 100%. Note that this experiment focused on the expression of the 140 kDa band in response to hypoxia. **, p<0.0001 <b>B</b>. P-selectin expression on HUVECs by immunohistochemistry. HUVECs were permeabilized and fixed as described in Materials and Methods. Note that expression of P-selectin (green) was induced by exposure to 1-hour hypoxia (1% O₂), but by pretreatment of 5 µg/mL tunicamycin.</p

Molecular effects of glycosylation inhibitors on P-selectin mRNA expression (A) and the proteasomal degradation of P-selectin (B) on HUVECs.

<p><b>A</b>. Tunicamycin has no effect on P-selectin mRNA levels. HUVECs were incubated with 2.5 or 5 µg/mL tunicamycin for 48 hours before 1 U/mL thrombin was added. Total RNAs were prepared and P-selectin mRNA levels were determined by real time-PCR analysis by using 18S rRNA as an internal control. The P-selectin mRNA level of untreated HUVECs was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with 2.5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin; 5, HUVECs pretreated with 5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin. **, p<0.0001. <b>B</b>. Effects of tunicamycin on proteasomal degradation of P-selectin. HUVECs were pretreated for 1 hour with or without 1 µM MG132, treated for 48 hours with no, 2.5, or 5 µg/mL tunicamycin, and then treated with or without 1 U/mL thrombin. Cell lysates (30 µg) prepared from HUVECs were analyzed by Western blotting as described in Materials and Methods. β-Actin was used for the loading control. The ratio of the P-selectin protein level to that of β-actin was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with MG132 and then stimulated with thrombin; 5, HUVECs pretreated with MG132, then with 5 µg/mL tunicamycin, and stimulated with thrombin. Experiments were repeated three times that gave similar results and a representative blot is shown. **, p<0.0001 <b>C</b>. Effects of glycosylation inhibitors on AKT phosphorylation. HUVECs were cultured for 48 hours in the presence or absence of tunicamycin or castanospermine at various concentrations followed by activation with 1 U/mL thrombin. Western blot analysis was performed to determine the phosphorylation status of AKT (Ser473 and Thr308). β-Actin was the loading control. <b>D</b>. AKT inhibitor suppresses CD62P expression on HUVECs. Flow cytometric analysis of P-selectin (CD62P) expression on HUVECs treated with the AKT inhibitor LY-294002 for 1 hour and subsequently stimulated with 1 U/mL thrombin. CD59-FITC was used as the positive marker for ECs. Flow cytometry analyses were repeated three times and a representative result was shown.</p