Ki-Hoan Nam

Methoxychlor and triclosan stimulates ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an estrogen receptor-dependent pathway.

Methoxychlor and triclosan are emergent or suspected endocrine-disrupting chemicals (EDCs). Methoxychlor [MXC; 1,1,1-trichlor-2,2-bis (4-methoxyphenyl) ethane] is an organochlorine pesticide that has been primarily used since dichlorodiphenyltrichloroethane (DDT) was banned. In addition, triclosan (TCS) is used as a common component of soaps, deodorants, toothpastes, and other hygiene products at concentrations up to 0.3%. In the present study, the potential impact of MXC and TCS on ovarian cancer cell growth and underlying mechanism(s) was examined following their treatments in BG-1 ovarian cancer cells. As results, MXC and TCS induced BG-1 cell growth via regulating cyclin D1, p21 and Bax genes related with cell cycle and apoptosis. A methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay confirmed that the proliferation of BG-1 ovarian cancer cells was stimulated by MXC (10(-6), 10(-7), 10(-8), and 10(-9)M) or TCS (10(-6), 10(-7), 10(-8), and 10(-9)M). Treatment of BG-1 cells with MXC or TCS resulted in the upregulation of cyclin D1 and downregulation of p21 and Bax transcriptions. In addition, the protein level of cyclin D1 was increased by MXC or TCS while p21 and Bax protein levels appeared to be reduced in these cells. Furthermore, MXC- or TCS-induced alterations of these genes were reversed in the presence of ICI 182,780 (10(-7)M), suggesting that the changes in these gene expressions may be regulated by an ER-dependent signaling pathway. In conclusion, the results of our investigation indicate that two potential EDCs, MXC and TCS, may stimulate ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an ER-dependent pathway.

Trichostatin A Exacerbates Atherosclerosis in Low Density Lipoprotein Receptor–Deficient Mice

Objective—Histone acetylation has been shown to be involved in expression of a restricted set of cellular genes including various proinflammatory molecules. We aimed to investigate the relationship between histone acetylation and atherosclerosis. Methods and Results—In low-density lipoprotein (LDL) receptor-deficient (Ldlr−/−) mice fed an atherogenic diet for 4 or 8 weeks, trichostatin A (TSA), a specific histone deacetylase inhibitor, exacerbated atherosclerosis without alteration on plasma lipid profiles. When we assayed the effects of TSA on expressions of oxidized LDL (oxLDL) receptors on RAW264.7 macrophage, we found that TSA increased CD36 mRNA and protein, as well as cell surface expression of CD36. TSA also increased acetylation at the CD36 promoter region. The uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate (Dil)-labeled oxLDL was enhanced in RAW264.7 macrophage by TSA. Furthermore, TSA treatment increased CD36 mRNA expression in aorta, and SRA, tumor necrosis factor (TNF)-&agr;, and vascular cell adhesion molecule-1 (VCAM-1) were also elevated, whereas IL-6 and IL-1&bgr; expressions were decreased. Conclusions—Our findings suggest that histone acetylation could play some role in atherogenesis by modulating expressions of oxLDL receptor and some proatherogenic genes. Therefore, our results indicate that increased histone acetylation may affect the progress of atherosclerosis.

Dose-dependent efficacy of ALS-human mesenchymal stem cells transplantation into cisterna magna in SOD1-G93A ALS mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron loss. Although the underlying cause of the disease remains unclear, a variety of pathogenic mechanisms have been proposed. Despite promising preclinical studies showing the modification of the disease progression, most trials have failed to demonstrate any significant improvement in outcome. Stem cell therapy therefore has been proposed as an alternative therapy for ALS. In this study, we evaluated the dose-dependent effects of human bone marrow mesenchymal stem cells (hMSCs) obtained from an ALS patient (ALS-hMSCs) on SOD1 mice via intrathecal injection and showed its practicality for hMSCs. We transplanted different doses (1x10(4), 2x10(5), and 1x10(6)) of ALS-hMSCs into the cisterna magna and performed clinical observations including symptom onset, survival time, and locomotor performance using the rotarod test. Nissl staining was performed to evaluate motor neurons in lumbar spinal cord sections at 109 days, and transplanted cells were evaluated by immuno-fluorescence staining at the end stage. A cell dose of 1x10(6) cells significantly prolonged life span and delayed the decline of motor performance. At this dose, the average number of motor neurons was significantly higher than those of the untreated and 1x10(4) cell treated groups. Most injected hMSCs distributed in the ventricular system and subarachnoid space, while some migrated into the brain and spinal cord. These data suggest that intrathecal injection with an optimized cell number could be a potential route for stem cell therapy in ALS patients.

Vitamin D3 Upregulated Protein 1 Suppresses TNF-α–Induced NF-κB Activation in Hepatocarcinogenesis

Vitamin D3 upregulated protein 1 (VDUP1) is a candidate tumor suppressor, the expression of which is dramatically reduced in various tumor tissues. In this study, we found that VDUP1 expression is suppressed during human hepatic carcinogenesis, and mice lacking VDUP1 are much more susceptible to diethylnitrosamine-induced hepatocarcinogenesis compared with wild type mice. VDUP1-deficient tumors proliferated significantly more than wild type tumors and had corresponding changes in the expression of key cell cycle regulatory proteins. In addition, the hepatomitogen-induced response was associated with a considerable increase in the release of TNF-α and subsequent enhancement of NF-κB activation in VDUP1-deficient mice. When cells were treated with TNF-α, the VDUP1 level was markedly reduced, concomitant with elevated NF-κB activation. Furthermore, the overexpression of VDUP1 resulted in the robust suppression of TNF-α–activated NF-κB activity via association with HDAC1 and HDAC3. These results indicate that VDUP1 negatively regulates hepatocarcinogenesis by suppressing TNF-α–induced NF-κB activation.

Progression of breast cancer cells was enhanced by endocrine-disrupting chemicals, triclosan and octylphenol, via an estrogen receptor-dependent signaling pathway in cellular and mouse xenograft models.

In the present study, we determined whether two endocrine-disrupting chemicals (EDCs), triclosan (TCS) and octylphenol (OP), are able to alter the expression of two cell cycle regulators, cyclin D1 and p21, in both in vitro and mouse breast cancer models. In addition, we determined whether the stimulatory effects of OP or TCS on breast cancer progression may be associated with an estrogen receptor (ER)-mediated signaling pathway. Altered expressions of cyclin D1 and p21 were observed in MCF-7 human breast cancer cells treated with TCS and OP, which is linked to the G1/S transition of cell cycle, leading to cell proliferation. In a xenograft mouse model, breast tumor masses were established following exposure to TCS and OP for 8 weeks. In these animals, the tumor cells with BrdU-positive nuclei were increased by treatment with 17β-estradiol (E2), OP, and TCS compared to that of a control (corn oil), suggesting that TCS and OP increase DNA synthesis during the S phase in tumor cells. Increased level of cyclin D1 protein by TCS and OP was also observed in vivo, implying that the effects of these EDCs possessing estrogenic activity alter the expression of genes related to cancer progression. It was of interest that the effects of TCS and OP were reversed by ICI 182,780, an ER antagonist, indicating that EDC-induced activities are mediated by an ER-dependent signaling pathway. Taken together, these results suggest that TCS and OP may promote breast cancer progression, via an ER-mediated signaling cascade.

Bisphenol A and Nonylphenol Have the Potential to Stimulate the Migration of Ovarian Cancer Cells by Inducing Epithelial–Mesenchymal Transition via an Estrogen Receptor Dependent Pathway

Epithelial-mesenchymal transition (EMT) is an important process appearing in embryo development and tumor migration or progression, which is influenced by 17β-estradiol (E2). Bisphenol A (BPA) and nonylphenol (NP) are suspected as endocrine disrupting chemicals (EDCs) because they can exert estrogenic properties. In this study, we examined whether E2, BPA, and NP can lead to the EMT process in BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To confirm the effect of E2, BPA, and NP, BG-1 cells were cultured under treatment with E2, BPA, or NP, and the alteration of EMT markers such as vimentin was examined at mRNA levels by using real-time and reverse-transcription (RT)-PCR. The expressions of snail, slug, and vimentin were enhanced by the treatment of E2, BPA, or NP compared to a control (DMSO). In protein levels, vimentin protein was increased by E2 and two EDCs, while E-cadherin was decreased. In addition, the expression of snail protein was enhanced by the treatment of E2 and the two EDCs in comparison with that of the control. Since EMT response in cancer cells can affect metastasis, we also performed a scratch assay and Western blot assay to show the migration ability caused by E2, BPA, or NP. Consequently, E2, BPA, and NP enhanced the migration capability of BG-1 cells and increased the expression of MMP-9 protein. Furthermore, to examine whether EMT and migration of BG-1 cancer cells are induced by BPA or NP via the ER dependent pathway, we cotreated the cells with ER-antagonist, ICI 182,780, in the presence of E2, BPA, or NP. As a result, the expressions of E-cadherin, vimentin, snail, and slug were reversed following treatment with an ER antagonist. Moreover, we confirmed that ICI 182,780 reduced the migration ability of BPA and NP to the control level. Taken together, these results indicate that BPA and NP, the potential EDCs, may have the ability to influence ovarian cancer metastasis via regulating EMT markers and migration in ER-expressing BG-1 ovarian cancer cells.

Inhibition of Adenylyl Cyclase Type 5 Prevents l-DOPA-Induced Dyskinesia in an Animal Model of Parkinson's Disease

The dopamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA) is widely used as a therapeutic choice for the treatment of patients with Parkinsons disease. However, the long-term use of l-DOPA leads to the development of debilitating involuntary movements, called l-DOPA-induced dyskinesia (LID). The cAMP/protein kinase A (PKA) signaling in the striatum is known to play a role in LID. However, from among the nine known adenylyl cyclases (ACs) present in the striatum, the AC that mediates LID remains unknown. To address this issue, we prepared an animal model with unilateral 6-hydroxydopamine lesions in the substantia nigra in wild-type and AC5-knock-out (KO) mice, and examined behavioral responses to short-term or long-term treatment with l-DOPA. Compared with the behavioral responses of wild-type mice, LID was profoundly reduced in AC5-KO mice. The behavioral protection of long-term treatment with l-DOPA in AC5-KO mice was preceded by a decrease in the phosphorylation levels of PKA substrates ERK (extracellular signal-regulated kinase) 1/2, MSK1 (mitogen- and stress-activated protein kinase 1), and histone H3, levels of which were all increased in the lesioned striatum of wild-type mice. Consistently, FosB/ΔFosB expression, which was induced by long-term l-DOPA treatment in the lesioned striatum, was also decreased in AC5-KO mice. Moreover, suppression of AC5 in the dorsal striatum with lentivirus-shRNA-AC5 was sufficient to attenuate LID, suggesting that the AC5-regulated signaling cascade in the striatum mediates LID. These results identify the AC5/cAMP system in the dorsal striatum as a therapeutic target for the treatment of LID in patients with Parkinsons disease.

Age-related telomere length dynamics in peripheral blood mononuclear cells of healthy cynomolgus monkeys measured by Flow FISH

Telomere length is a good biomarker to study the cellular senescence as well as aging of an organism, because it regulates the replicative capacity of vertebrate somatic cells. To demonstrate age‐related telomere length dynamics in the peripheral blood mononuclear cells (PBMC) of the cynomolgus monkey, we introduced a novel method of measuring telomere length by fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH). A highly significant correlation was observed between the intensity of telomere‐specific fluorescence by Flow FISH and telomere length by Southern blot analysis (R = 0·923, n = 22). The intensity of telomere fluorescence in PBMC significantly decreased with age in 55 monkeys aged from 0 to 34 years and this decrease corresponded to the loss of 62·7 base pairs per year (R = − 0·52, P < 0·00004). We also analysed the expression of naive cell‐associated markers, CD28, CD62L and CD45RA/CD62L in T lymphocytes of 47 cynomolgus monkeys. An age‐related increase in the CD28− subset was observed in CD8+ T lymphocytes in monkeys less than 11 years old and in CD4+ T lymphocytes in monkeys over 23 years old, respectively. The percentage of CD62L+ subsets was significantly decreased with age in both CD4+ (R = − 0·55) and CD8+ T lymphocytes (R = − 0·73). From the comparison of telomere length among PBMC, CD62L+ and CD62L− T lymphocytes, it was clearly evident that loss of naive subsets results in the shortening of telomere length in vivo. These results show that this method can be applicable to studying the turnover and precursor‐progeny of PBMC in cynomolgus monkeys as an animal model of aging.

Vitamin D3 up-regulated protein 1 deficiency accelerates liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS Liver regeneration is a complicated process involving a variety of interacting factors. Vitamin D3 up-regulated protein 1 (VDUP1) is a potent growth suppressor that, upon over-expression, inhibits tumor cell proliferation and cell-cycle progression. Here, we investigated the function of VDUP1 in liver regeneration following hepatectomy in mice. METHODS Liver regeneration after 70% partial hepatectomy (PH) was compared in VDUP1 knockout (KO) and wild-type (WT) mice, and the activities of proliferative- and cell-cycle-related signaling pathways were measured. RESULTS Compared with WT mice, liver recovery was significantly accelerated in VDUP1 KO mice during the first day after PH, in association with increased DNA synthesis. Consistent with this observation, the expression levels of key cell-cycle regulatory proteins, including cyclin D, cyclin E, cyclin-dependent kinase 4 (CDK4), p21, and p27, were markedly altered in the livers of VDUP1 KO mice. Induction of growth factors and activation of proliferative signaling pathway components including extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, glycogen synthase kinase 3β (GSK3β), mammalian target of rapamycin (mTOR), and p70S6 kinase (p70(S6K)), occurred much earlier and to a greater extent in VDUP1 KO mouse livers. In addition, primary hepatocytes isolated from VDUP1 KO mice displayed increased activation of ERK1/2 and Akt in response to HGF and TGF-α. CONCLUSIONS Our results reveal an important role for VDUP1 in the regulation of proliferative signaling during liver regeneration. Altered activation of genes involved in ERK1/2 and Akt signaling pathways may explain the accelerated growth responses seen in VDUP1 KO mice.

AGE-DEPENDENT REMODELING OF PERIPHERAL BLOOD CD4+ CD8+ T LYMPHOCYTES IN CYNOMOLGUS MONKEYS

Recently, we have found in adult cynomolgus monkeys that substantial peripheral blood CD4+ CD8+ double-positive (DP) T lymphocytes exhibit a resting memory phenotype and increase in proportion with age. In this study, we investigated whether phenotypic changes occur in the course of the increase in proportion of the DP T cells. The results obtained from 195 clinically healthy monkeys aged from 1 month to 31 years showed that the CD29hi and CD28 subpopulation in the DP T subset increased in proportion with age and that the increase reached a plateau at six years old for the CD29hi subpopulation and at eleven years old for the CD28 one, respectively. The phenotypic alteration preceded the abrupt increase in proportion of the DP T cells and was able to be classified into four phases on the basis of the qualitative and quantitative alteration.