Myung Kwan Han

Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin in mice.

Cytokines produced by immune cells infiltrating pancreatic islets have been incriminated as important mediators of β-cell destruction in insulin-dependent diabetes mellitus. In non insulin-dependent diabetes, cytokines are also associated with impaired β-cell function in high glucose condition. By the screening of various natural products blocking β-cell destruction, we have recently found that epigallocatechin gallate (EGCG) can prevent thein vitro destruction of RINm5F cell, an insulinoma cell line, that is induced by cytokines. In that study we suggested that EGCG could prevent cytokine-induced β-cell destruction by down-regulation of nitric oxide synthase (NOS) through inhibition of NF-κB activation. Here, to verify thein vivo antidiabetogenic effect of EGCG, we examined the possibility that EGCG could also prevent the experimental autoimmune diabetes induced by the treatment of multiple low doses of streptozotocin (MLD-STZ), which is recognized as an inducer of type I autoimmune diabetes. Administration of EGCG (100 mg/day/kg for 10 days) during the MLD-STZ induction of diabetes reduced the increase of blood glucose levels caused by MLD-STZ.Ex vivo analysis of β-islets showed that EGCG downregulates the MLD-STZ-induced expression of inducible NOS (iNOS). In addition, morphological examination showed that EGCG treatment ameliorated the decrease of islet mass induced by MLD-STZ. In combination these results suggest that EGCG could prevent the onset of MLD-STZ-induced diabetes by protecting pancreatic islets. Our results therefore revealed the possible therapeutic value of EGCG for the prevention of diabetes mellitus progression.

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.

Involvement of sirtuin 1 in airway inflammation and hyperresponsiveness of allergic airway disease

BACKGROUND Bronchial asthma is a chronic inflammatory disorder of the airways characterized by increased expression of multiple inflammatory genes. Acetylation of histones by histone acetyltransferases is associated with increased gene transcription, whereas hypoacetylation induced by histone deacetylases is associated with suppression of gene expression. Sirtuin 1 (SIRT1) is a member of the silent information regulator 2 family that belongs to class III histone deacetylase. OBJECTIVE This study aimed to investigate the role of SIRT1 and the related molecular mechanisms in the pathogenesis of allergic airway disease. METHODS By using a murine model of ovalbumin (OVA)-induced allergic airway disease and murine tracheal epithelial cells, this study investigated the involvement of SIRT1 and its signaling networks in allergic airway inflammation and hyperresponsiveness. RESULTS In this study with mice after inhalation of OVA, the increased levels of SIRT1, hypoxia-inducible factor 1alpha (HIF-1alpha), and vascular endothelial growth factor protein in the lungs after OVA inhalation were decreased substantially by the administration of a SIRT1 inhibitor, sirtinol. We also showed that the administration of sirtinol reduced significantly the increased numbers of inflammatory cells of the airways; airway hyperresponsiveness; increased levels of IL-4, IL-5, and IL-13; and increased vascular permeability in the lungs after OVA inhalation. In addition, we have found that inhibition of SIRT1 reduced OVA-induced upregulation of HIF-1alpha in airway epithelial cells. CONCLUSIONS These results indicate that inhibition of SIRT1 might attenuate antigen-induced airway inflammation and hyperresponsiveness through the modulation of vascular endothelial growth factor expression mediated by HIF-1alpha in mice.

SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells.

As the increased acetylation of p65 is linked to nuclear factor-κB (NF-κB) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-κB p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-κB during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-κB p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-κB through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

Function of NAD glycohydrolase in ADP-ribose uptake from NAD by human erythrocytes

The function of the ectoenzyme NAD glycohydrolase (NADase) in ADP-ribose uptake from extracellular NAD was studied in human erythrocytes that express relatively high NADase activity (adult erythrocytes) and erythrocytes expressing very low activity (newborn erythrocytes). The rates of ADP-ribose uptake from NAD in human erythrocytes were correlated with their NADase activities. In contrast, there was no significant difference in the rates of ADP-ribose uptake among these cells when incubated with ADP-ribose. These results indicate that ecto-NADase may have a role as supplier of ADP-ribose for its uptake into the cells and that the cleavage of NAD by NADase is necessary for the ADP-ribose uptake by human erythrocytes. From ADP-ribose uptake studies at 37 degrees C a Km of 0.7 +/- 0.05 microM and a Vmax of 2.04 +/- 0.1 pmol/min per microliter cell water was found for the uptake of [3H]ADP-ribose. The thiol-reactive reagents p-chloromercuribenzene sulfonic acid and N-ethylmaleimide inhibited the uptake ADP-ribose with IC50 values of 50 +/- 4 and 750 +/- 25 mM, respectively. Since efflux of [3H]ADP-ribose was negligible, the ADP-ribose transport system appears to be unidirectional. The unidirectionality was supported by the evidence that transported ADP-ribose was rapidly degraded to AMP which is impermeable to the membrane.

Immunohistochemical localization of NAD glycohydrolase in human and rabbit tissues.

NAD glycohydrolase (NADase) is present in many organisms from bacteria to mammals. In any given organism, this enzyme is ubiquitous in many tissues. However, its precise localization and its physiological significance have not been defined. We have determined the distribution of NADase in normal human and rabbit tissues by immunoblotting and immunohistochemistry, using a polyclonal antibody raised in goats. Immunoblot analyses revealed that NADase was highly expressed in the heart, lung, stomach, and liver tissues of the rabbit. From immunohistochemical studies of NADase, high concentrations in both human and rabbit tissues were found in hepatocytes and sinusoidal lining cells, sinus histiocytes of the lymph node, spleen and thymus, glomerular capillary endothelial cells of the kidney, cardiac muscle, endothelium of blood vessles, and erythrocytes.

Glycosylphosphatidylinositol-anchored NAD glycohydrolase is released from peritoneal macrophages activated by interferon-gamma and lipopolysaccharide.

We have previously shown that an ectoenzyme, NAD glycohydrolase (NADase) could be solubilized by treatment with bacterial phosphatidylinositol phospholipase C (PIPLC). However, it is unknown whether endogenous PIPLC can cleave this ectoenzyme. In this study, we used mouse peritoneal exudate macrophages which have been known to have relatively high activity of NADase. The results show that release of ecto‐NADase was markedly increased when mouse peritoneal macrophages were costimulated with interferon‐γ (IFN‐γ) and bacterial lipopolysaccharide (LPS), compared to unstimulated cells. This increase was preceded by markedly enhanced activity of endogenous glycosylphosphatidy‐linositol phospholipase C (GPIPLC). The cross‐reacting determinant (CRD) of the glycosylphosphatidylinositol anchor in released NADase from activated macrophages was detected by immunoblotting with anti‐CRD antibody. Taken together, ecto‐NADase is released from peritoneal exudate macrophages during IFN‐γ/LPS‐induced activation and endogenous GPIPLC is involved in the NADase release from the activated macrophages. J. Leukoc. Biol. 56: 792–796; 1994.

hESC Expansion and Stemness Are Independent of Connexin Forty-Three-Mediated Intercellular Communication between hESCs and hASC Feeder Cells

Background Human embryonic stem cells (hESCs) are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC) is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins) is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported. Methodology/Principal Findings This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs) influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth), and stemness, as assessed by alkaline phosphatase (AP), OCT4 (POU5F1-Human gene Nomenclature Database), SOX2, and NANOG expression. Conclusions/Significance These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.

Cancer related gene alterations can be detected with next-generation sequencing analysis of bile in diffusely infiltrating type cholangiocarcinoma

Genome-wide association study in diffusely infiltrating type cholangiocarcinoma (CC) can be limited due to the difficulty of obtaining tumor tissue. We aimed to evaluate the genomic alterations of diffusely infiltrating type CC using next-generation sequencing (NGS) of bile and to compare the variations with those of mass-forming type CC. A total of 24 bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP) and 17 surgically obtained tumor tissue samples were evaluated. Buffy coat and normal tissue samples were used as controls for a somatic mutation analysis. After extraction of genomic DNA, NGS analysis was performed for 48 cancer related genes. There were 27 men and 14 women with a mean age of 65.0±11.8years. The amount of extracted genomic DNA from 3cm(3) of bile was 66.0±84.7μg and revealed a high depth of sequencing coverage. All of the patients had genomic variations, with an average number of 19.4±2.8 and 22.3±3.3 alterations per patient from the bile and tumor tissue, respectively. After filtering process, damaging SNPs (8 sites for each type of CC) were predicted by analyzing tools, and their target genes showed relevant differences between the diffusely infiltrating and mass-forming type CC. Finally, in somatic mutation analysis, tumor-normal paired 14 tissue and 6 bile samples were analyzed, genomic alterations of EGFR, FGFR1, ABL1, PIK3CA, and CDKN2A gene were seen in the diffusely infiltrating type CC, and TP53, KRAS, APC, GNA11, ERBB4, ATM, SMAD4, BRAF, and IDH1 were altered in the mass-forming type CC group. STK11, GNAQ, RB1, KDR, and SMO genes were revealed in both groups. The NGS analysis was feasible with bile sample and diffusely infiltrating type CC revealed genetic differences compared with mass-forming type CC. Genome-wide association study could be performed using bile sample in the patients with CC undergoing ERCP and a different genetic approach for accurate diagnosis, pathogenesis study, and targeted therapy will be needed in diffusely infiltrating type CC.