Hsing-Chun Kuo

Homocysteine induces smooth muscle cell proliferation through differential regulation of cyclins A and D1 expression

The mechanism of homocysteine‐induced cell proliferation in human vascular smooth muscle cells (SMCs) remains unclear. We investigated the molecular mechanisms by which homocysteine affects the expression of cyclins A and D1 in human umbilical artery SMCs (HUASMCs). Homocysteine treatment induced proliferation of HUASMCs and increased the expression levels of cyclins A and D1. Knocking down either cyclin A or cyclin D1 by small interfering RNA (siRNA) inhibited homocysteine‐induced cell proliferation. Furthermore, treatment with extracellular signal‐related kinase (ERK) inhibitor (PD98059) and dominant negative Ras (RasN17) abolished homocysteine‐induced cyclin A expression; and treatment with phosphatidylinositol 3‐kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine‐induced cyclin D1 expression. Homocysteine also induced transient phosphorylation of ERK, Akt, and p70 ribosomal S6 kinase (p70S6K). Neutralizing antibody and siRNA for β1 integrin blocked cell proliferation, expression of cyclins A and D1, and phosphorylation of ERK and Akt. In conclusion, homocysteine‐induced differential activation of Ras/ERK and PI3K/Akt/p70S6K signaling pathways and consequent expression of cyclins A and D1 are dependent on β1 integrin. Homocysteine may accelerate progression of atherosclerotic lesions by promoting SMC proliferation. J. Cell. Physiol. 226: 1017–1026, 2011.

Proteomic analysis of the effects of baicalein on colorectal cancer cells.

Baicalein is the flavonoids with multiple pharmacological activities. The aim of our study was to investigate the effects of baicalein on colorectal cancer (CRC) and to recognize the targets of baicalein treatment. To better understand baicaleins target, proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS‐PAGE) to elucidate proteins differential display. Results from this study investigate that baicalein treatment of CRC cells results in reduced cell proliferation. As a result, differential protein displays between baicalein‐treated and untreated CRC were determined and validated. There were 11 differentially expressed proteins between baicalein‐treated and untreated CRC. Furthermore, we demonstrate that baicalein inhibits cancer cell proliferation and reduced reactive oxygen species (ROS) by up‐regulating the levels of peroxiredoxin‐6 (PRDX6). Knockdown of PRDX6 in baicalein‐treated CRC cells by specific small interfering RNA resulted in ROS production and proliferation, opposite of the baicalein treatment scenario as indicated by cell cycle distribution. These results illustrate that baicalein up‐regulates the expression of PRDX6, which attenuates the generation of ROS and inhibits the growth of CRC cells, whereas baicalein treatment have no effect on normal epithelial cells.

Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells

BackgroundStromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells.ResultsHuman gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity.ConclusionsResistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer.

Correlation among subcortical white matter lesions, intelligence and CTG repeat expansion in classic myotonic dystrophy type 1

Objectives –  To analyze the correlation among intelligence, brain magnetic resonance images (MRI) and genotype in classic myotonic dystrophy type 1 (DM1) patients.

Protective effects of Hericium erinaceus mycelium and its isolated erinacine A against ischemia-injury-induced neuronal cell death via the inhibition of iNOS/p38 MAPK and nitrotyrosine.

Hericium erinaceus, an edible mushroom, has been demonstrated to potentiate the effects of numerous biological activities. The aim of this study was to investigate whether H. erinaceus mycelium could act as an anti-inflammatory agent to bring about neuroprotection using a model of global ischemic stroke and the mechanisms involved. Rats were treated with H. erinaceus mycelium and its isolated diterpenoid derivative, erinacine A, after ischemia reperfusion brain injuries caused by the occlusion of the two common carotid arteries. The production of inflammatory cytokines in serum and the infracted volume of the brain were measured. The proteins from the stroke animal model (SAM) were evaluated to determine the effect of H. erinaceus mycelium. H. erinaceus mycelium reduced the total infarcted volumes by 22% and 44% at a concentration of 50 and 300 mg/kg, respectively, compared to the SAM group. The levels of acute inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor á, were all reduced by erinacine A. Levels of nitrotyrosine-containing proteins, phosphorylation of p38 MAPK and CCAAT enhancer-binding protein (C/EBP) and homologous protein (CHOP) expression were attenuated by erinacine A. Moreover, the modulation of ischemia injury factors present in the SAM model by erinacine A seemed to result in the suppression of reactive nitrogen species and the downregulation of inducible NO synthase (iNOS), p38 MAPK and CHOP. These findings confirm the nerve-growth properties of Hericium erinaceus mycelium, which include the prevention of ischemic injury to neurons; this protective effect seems to be involved in the in vivo activity of iNOS, p38 MAPK and CHOP.

Stromal cell‐derived factor‐1/CXC receptor 4 and β1 integrin interaction regulates urokinase‐type plasminogen activator expression in human colorectal cancer cells

The stromal cell‐derived factor‐1 (SDF‐1)/CXC receptor 4 (CXCR4) axis has been shown to play a role in colorectal cancer progression. In addition, the protease urokinase‐type plasminogen activator (uPA) is an important factor in tumor cell invasion and metastasis. However, the mechanism by which SDF‐1 mediates uPA expression in human colorectal cancer cells remains unknown. We investigated the molecular mechanism governing the interaction between SDF‐1 stimulation and uPA expression in three human colon cancer cell lines (DLD‐1, SW48, and COLO 205). We found that SDF‐1 stimulation led to an increase in the expression and secretion of uPA in these cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of p38 mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K)/Akt pathways are critical for SDF‐1‐induced uPA expression. Analysis of transcription factor binding using ELISA and chromatin immunoprecipitation assays revealed that SDF‐1 increased Sp1‐ and AP‐1‐DNA‐binding activities in DLD‐1 cells. Inhibition of Sp1 and AP‐1 activation blocked the SDF‐1‐induced expression and activity of the uPA promoter. The effect of SDF‐1 on DLD‐1 signaling and uPA expression was mediated by the CXCR4/β1 integrin axis. In summary, our findings elucidate the mechanisms of SDF‐1/CXCR4 downstream signaling and provide insights into the function of SDF‐1 in colon cancer cells. J. Cell. Physiol. 227: 1114–1122, 2012.

Hericium erinaceus mycelium and its isolated erinacine A protection from MPTP-induced neurotoxicity through the ER stress, triggering an apoptosis cascade

BackgroundHericium erinaceus is an edible mushroom; its various pharmacological effects which have been investigated. This study aimed to demonstrate whether efficacy of oral administration of H. erinaceus mycelium (HEM) and its isolated diterpenoid derivative, erinacine A, can act as an anti-neuroinflammatory agent to bring about neuroprotection using an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson’s disease, which results in motor disturbances, in addition to elucidating the mechanisms involved.MethodsMice were treated with and without HEM or erinacine A, after MPTP injection for brain injuries by the degeneration of dopaminergic nigrostriatal neurons. The efficacy of oral administration of HEM improved MPTP-induced loss of tyrosine hydroxylase positive neurons and brain impairment in the substantia nigra pars compacta as measured by brain histological examination.ResultsTreatment with HEM reduced MPTP-induced dopaminergic cell loss, apoptotic cell death induced by oxidative stress, as well as the level of glutathione, nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE). Furthermore, HEM reversed MPTP-associated motor deficits, as revealed by the analysis of rotarod assessment. Our results demonstrated that erinacine A decreases the impairment of MPP-induced neuronal cell cytotoxicity and apoptosis, which were accompanied by ER stress-sustained activation of the IRE1α/TRAF2, JNK1/2 and p38 MAPK pathways, the expression of C/EBP homologous protein (CHOP), IKB-β and NF-κB, as well as Fas and Bax.ConclusionThese physiological and brain histological changes provide HEM neuron-protective insights into the progression of Parkinson’s disease, and this protective effect seems to exist both in vivo and in vitro.

Identification of an association between genomic hypomethylation of FCGR2A and susceptibility to Kawasaki disease and intravenous immunoglobulin resistance by DNA methylation array.

Kawasaki disease (KD) is characterized by systemic vasculitis, and it is the most common acquired heart disease in children. However, the etiology and immunopathogenesis of KD are still unclear. A genome‐wide association study (GWAS) identified polymorphisms in CD40, BLK, and FCGR2A as the susceptibility genes for KD. No epigenetic array studies of KD have previously been published. This study was undertaken to investigate differences in DNA methylation in patients with KD as compared to controls.

Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker.

Magnolol protects neurons against ischemia injury via the downregulation of p38/MAPK, CHOP and nitrotyrosine.

Magnolol is isolated from the herb Magnolia officinalis, which has been demonstrated to exert pharmacological effects. Our aim was to investigate whether magnolol is able to act as an anti-inflammatory agent that brings about neuroprotection using a global ischemic stroke model and to determine the mechanisms involved. Rats were treated with and without magnolol after ischemia reperfusion brain injury by occlusion of the two common carotid arteries. The inflammatory cytokine production in serum and the volume of infarction in the brain were measured. The proteins present in the brains obtained from the stroke animal model (SAM) and control animal groups with and without magnolol treatment were compared. Magnolol reduces the total infarcted volume by 15% and 30% at dosages of 10 and 30mg/kg, respectively, compared to the untreated SAM group. The levels of acute inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 were attenuated by magnolol. Magnolol was also able to suppress the production of nitrotyrosine, 4-hydroxy-2-nonenal (4-HNE), inducible NO synthase (iNOS), various phosphorylated p38 mitogen-activated protein kinases and various C/EBP homologues. Furthermore, this modulation of ischemia injury factors in the SAM model group treated with magnolol seems to result from a suppression of reactive oxygen species production and the upregulation of p-Akt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings confirm the anti-oxidative properties of magnolol, including the inhibition of ischemic injury to neurons; this protective effect seems to involve changes in the in vivo activity of Akt, GSK3β and NF-κB.