Bon Hong Min

SIRT1 promotes DNA repair activity and deacetylation of Ku70

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.

Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity and postprandial blood glucose

The present study examined the inhibitory effect of aqueous extract from the gall of Rhus chinensis (AEGRC) on alpha-glucosidase activity, an enzyme responsible for digestion of carbohydrate to monosaccharides in the process of intestinal absorption. AEGRC inhibited Bacillus alpha-glucosidase acitvity with an IC(50) of 0.9 micro g/ml. Its inhibition on alpha-glucosidase was determined to be noncompetitive and reversible when the enzyme-substrate mixture was simultaneously treated with AEGRC as an inhibitor. In addition, when it was orally administered to rats with sucrose (2g/kg), AEGRC (250-1000mg/kg) significantly suppressed the increase of blood glucose levels after sucrose loading in a dose dependent manner. These results suggest that AEGRC might exert anti-diabetic effect by suppressing carbohydrate absorption from intestine, and thereby reducing the postprandial increase of blood glucose.

Renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase.

Recently, pegylated arginine deiminase (ADI; EC 3.5.3.6) has been used to treat the patients with hepatocellular carcinoma or melanoma, in which the level of argininosuccinate synthetase (ASS) activity is low or undetectable. The efficacy of its antitumor activity largely depends on the level of intracellular ASS, which enables tumor cells to recycle citrulline to arginine. Thus, we examined the expression levels of ASS in various cancer cells and found that it is low in renal cell carcinoma (RCC) cells, rendering the cells highly sensitive to arginine deprivation by ADI treatment. Immunohistochemical analysis revealed that in biopsy specimens from RCC patients (n = 98), the expression of ASS is highly demonstrated in the epithelium of normal proximal tubule but not seen in tumor cells. Furthermore, RCC cells treated with ADI showed remarkable growth retardation in a dose dependent manner. ADI also exerted in vivo antiproliferative effect on the allografted renal cell carcinoma (RENCA) tumor cells and prolonged the survival of tumor‐bearing mice. Histological examination of the tumors revealed that tumor angiogenesis and vascular endothelial growth factor (VEGF) expression were significantly diminished by ADI administration. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of RCC in ways of inhibitions of arginine availability and neovascularization.

Activation of nestin-positive duct stem (NPDS) cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells.

Stem cells in adult pancreas and their specific marker are poorly characterized. We hypothesized that pancreatic stem cells could evolve from the duct system in response to neogenic stimulation and may transiently express nestin during tissue regeneration. After partial pancreatectomy (Px), we found extensive formation of ductules consisting of nestin‐positive epithelial cells with higher replicating ability in the neogenic foci, particularly at day 3 after Px. Nestin was highly expressed in the earlier stages of ductule morphogenesis and then regressed as the cells evolved toward differentiated pancreatic cell types. The neogenic ductules were isolated for the culture of nestin‐positive duct stem cells. These nestin‐positive duct cells were numerous and displayed extensive self‐replication in the duct cell explants after 2–3 days of culture, thus depicted as nestin‐positive duct stem (NPDS) cells. As seen in the tissue of neogenic foci, NPDS cells were negative for cytokeratin‐20 and vimentin, the marker for duct epithelial and mesenchymal cells, respectively. Endocrine cells, mostly insulin cells, were present in the explants at day 2 as single cells or as small clusters adjacent to the NPDS cells, and formed islet‐like masses at day 3 of culture, suggesting islet cell differentiation from NPDS cells. In addition, insulin secretion from these beta cells responded to glucose stimulation. We found transient up‐regulation of PDX‐1 expression by reverse transcriptase‐polymerase chain reaction at day 3 after Px in pancreatic tissue. Higher expression of PDX‐1 was seen in the culture of neogenic ductules than that of ducts isolated from the sham‐operated pancreas. In particular, a subpopulation of nestin‐positive cells in the duct cell explants formed from the neogenic ductules expressed PDX‐1 in their nuclei. Taken together, this information suggests that NPDS cells could be generated from adult pancreas by neogenic motivations and they may differentiate into insulin‐secreting cells. Developmental Dynamics 230:1–11, 2004.

Arginine deiminase: a potential inhibitor of angiogenesis and tumour growth

Hydrolysis of plasma arginine to citrulline by arginine deiminase (ADI) was recently shown to suppress lipopolysaccharide-induced nitric oxide (NO) synthesis. Since arginine is the precursor of NO, and the latter modulates angiogenesis, we explored whether ADI treatment significantly affected tube-like (capillary) formation of human umbilical vein endothelial cells. Inhibition occurred in a dose-dependent manner, both in the chorioallantoic membrane and the murine Matrigel plug assay. Inhibition of angiogenesis by ADI was reversed when a surplus of exogenous arginine was provided, indicating that its antiangiogenic effect is primarily due to arginine depletion, although other pathways of interference are not entirely excluded. Arginine deiminase is also shown to be as a potent inhibitor of tumour growth in vitro as in vivo, being effective at nanogram quantities per millilitre in CHO and HeLa cells. Thus, it could be highly beneficial in cancer therapy because of its two-pronged attack as both an antiproliferative and an antiangiogenic agent.

Protective Role of Clusterin/Apolipoprotein J Against Neointimal Hyperplasia via Antiproliferative Effect on Vascular Smooth Muscle Cells and Cytoprotective Effect on Endothelial Cells

Objective—Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action. Methods and Results—Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-&agr;–stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21cip1/waf1 but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-&agr;–induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-&agr;. Conclusion—These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.

Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages

Most solid tumor tissues possess a significant population of macrophages, which are known to be closely linked with tumor progression and metastasis. Clusterin has been reported to be overexpressed in various tumors and to have a tumor‐promoting role. As clusterin induction and macrophage infiltration occur concurrently at the tumor site, it raises a possibility that clusterin may regulate the function of macrophages via facilitating ECM remodeling. Here, we demonstrate for the first time the expression of MMP‐9 by clusterin in human primary monocytes as well as human and murine macrophage cell lines, THP‐1, and Raw264.7. MMP‐9 expression was accompanied by increased enzymatic activity, as revealed by gelatin zymography. The MMP‐9 activity promoted by clusterin was found to be dependent on the activation of ERK1/2 and PI3K/Akt but not p38 or JNK pathways. Inhibition of PI3K activity did not affect the activation of ERK1/2 and vice versa, indicating that the two pathways were independently operated to stimulate MMP‐9 activity. Moreover, clusterin facilitated nuclear translocation of NF‐κB p65 along with IκB‐α degradation and phosphorylation, which was critical for MMP‐9 expression. As NF‐κB is a central regulator of inflammation, clusterin may provide a molecular link between inflammation and cancer via up‐regulating NF‐κB and MMP‐9. Collectively, these data highlight a novel role of clusterin as a stimulator for MMP‐9 expression in macrophages, which may contribute to the tissue reorganization by serving as a modulator for ECM degradation.

Protective effect of clusterin from oxidative stress-induced apoptosis in human retinal pigment epithelial cells.

PURPOSE Oxidative stress to retinal pigment epithelial (RPE) cells is thought to play a critical role in the pathogenesis of age-related macular degeneration (AMD). This study was conducted to investigate whether clusterin protects human RPE cells from ROS-induced apoptosis through a PI3K/Akt survival pathway. METHODS The preventive effect of clusterin on reactive oxygen species (ROS) production and RPE cell death induced by hydrogen peroxide was determined in ARPE-19 cells. The ability of clusterin to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and DAPI staining. Furthermore, the protective effect of clusterin via the PI3K/Akt pathway was determined by Western blot analysis. RESULTS Clusterin prevented ARPE-19 cells from H(2)O(2)-induced cell death and ROS production. H(2)O(2)-induced oxidative stress increased caspase-3 activity, which was significantly inhibited by clusterin, as determined by the abrogation of apoptotic bodies. Interestingly, clusterin induced Akt phosphorylation in human RPE cells under oxidative stress, which contributed to cell viability in ARPE-19 cells. This cell survival by clusterin was blocked by a PI3K inhibitor. CONCLUSIONS Clusterin may play a protective role in responding to the local redox environment of human RPE cells, which contributes to the cell survival via the PI3K/Akt pathway. Therefore, clusterin could be considered for the preventive approach to AMD.

Effect of CYP3A5*3 genotype on the pharmacokinetics and pharmacodynamics of amlodipine in healthy Korean subjects

1,4‐Dihydropyridine calcium channel blockers, including amlodipine, are mainly metabolized by cytochrome P450 (CYP) 3A. We investigated the effect of CYP3A5*3 genotype on the pharmacokinetics and pharmacodynamics of amlodipine in healthy Korean male subjects.

Clusterin expression during regeneration of pancreatic islet cells in streptozotocin-induced diabetic rats

Abstract.Aims/hypothesis: Beta-cell regeneration has been reported after islet injury in an animal model for diabetes. Recently, we showed up-regulation of clusterin after islet injury and suggested that clusterin might be involved in cytoprotection and in the regeneration of islet cells. The aim of this study was to investigate the correlation of clusterin expression with islet regeneration and its effect on islet cell replication. Methods: Streptozotocin was administrated to rats to induce various types of diabetes. Islet regeneration and clusterin expression were examined after islet injuries. Clusterin cDNA was transfected to MIN6 cells and their proliferation activity was measured by a [3H]thymidine-incorporation assay. Results: A diabetogenic dose of streptozotocin injected in rats provoked an immediate degeneration of beta cells. In this model, islets showed increased clusterin expression with extensive proliferation of alpha cells but showed poor beta-cell replication. A subdiabetogenic dose of streptozotocin, however, led to the proliferation of beta cells with clusterin up-regulation. In streptozotocin-treated neonatal rats, up-regulation of clusterin was noted during beta-cell proliferation. In all experimental models, clusterin was expressed in alpha cells in close correlation with islet cell proliferation, higher transcription of insulin mRNA and MAPKs activation. Cell replication was increased by 31 % in the MIN6 cells transfected by the clusterin cDNA. Conclusion/interpretation: Up-regulation of clusterin in alpha cells might induce beta-cell proliferation and thus restore their population after islet injury. We suggest that clusterin could be considered as a growth factor-like molecule stimulating islet-cell proliferation by paracrine action. [Diabetologia (2001) 44: 2192–2202]