Publications by authors named "Yiu-Kay Lai"

60 Publications

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice.

Pharmacol Res 2020 11 5;161:105183. Epub 2020 Sep 5.

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan. Electronic address:

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5-100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC > 1 μM) with cross-resistance to gefitinib (IC > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes - such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) - are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC = 12-123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.
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http://dx.doi.org/10.1016/j.phrs.2020.105183DOI Listing
November 2020

Identification of anti-HBV activities in Andr. using GRP78 as a drug target on Herbochip.

Chin Med 2017 24;12:11. Epub 2017 Apr 24.

Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

Background: Herbochip technology is a high throughput drug screening platform in a reverse screening manner, in which potential chemical leads in herbal extracts are immobilized and drug target proteins can be used as probes for screening process [BMC Complementary and Alternative Medicine (2015) 15:146]. While herbal medicines represent an ideal reservoir for drug screenings, here a molecular chaperone GRP78 is demonstrated to serve as a potential target for antiviral drug discovery.

Methods: We cloned and expressed a truncated but fully functional form of human GRP78 (hGRP78) and used it as a probe for anti-HBV drug screening on herbochips. In vitro cytotoxicity and in vitro anti-HBV activity of the herbal extracts were evaluated by MTT and ELISA assays, respectively. Finally, anti-HBV activity was confirmed by in vivo assay using DHBV DNA levels in DHBV-infected ducklings as a model.

Results: Primary screenings using GRP78 on 40 herbochips revealed 11 positives. Four of the positives, namely , , and were subjected to subsequent assays. None of the above extracts was cytotoxic to AML12 cells, but extract (PCE) was found to be cytotoxic to HepG2 2.2.15 cells. Both PCE and extract (PSE) suppressed secretion of HBsAg and HBeAg in HepG2 2.2.15 cells. The anti-HBV activity of PSE was further confirmed in vivo.

Conclusion: We have demonstrated that GRP78 is a valid probe for anti-HBV drug screening on herbochips. We have also shown that PSE, while being non-cytotoxic, possesses in vitro and in vivo anti-HBV activities. Taken together, our data suggest that PSE may be a potential anti-HBV agent for therapeutic use.
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http://dx.doi.org/10.1186/s13020-017-0132-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402670PMC
April 2017

Discovery, structure-activity relationship studies, and anti-nociceptive effects of N-(1,2,3,4-tetrahydro-1-isoquinolinylmethyl)benzamides as novel opioid receptor agonists.

Eur J Med Chem 2017 Jan 20;126:202-217. Epub 2016 Sep 20.

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County, 35053, Taiwan. Electronic address:

μ-Opioid receptor (MOR) agonists are analgesics used clinically for the treatment of moderate to severe pain, but their use is associated with severe adverse effects such as respiratory depression, constipation, tolerance, dependence, and rewarding effects. In this study, we identified N-({2-[(4-bromo-2-trifluoromethoxyphenyl)sulfonyl]-1,2,3,4-tetrahydro-1-isoquinolinyl}methyl)cyclohexanecarboxamide (1) as a novel opioid receptor agonist by high-throughput screening. Structural modifications made to 1 to improve potency and blood-brain-barrier (BBB) penetration resulted in compounds 45 and 46. Compound 45 was a potent MOR/KOR (κ-opioid receptor) agonist, and compound 46 was a potent MOR and medium KOR agonist. Both 45 and 46 demonstrated a significant anti-nociceptive effect in a tail-flick test performed in wild type (WT) B6 mice. The ED value of 46 was 1.059 mg/kg, and the brain concentrations of 45 and 46 were 7424 and 11696 ng/g, respectively. Accordingly, compounds 45 and 46 are proposed for lead optimization and in vivo disease-related pain studies.
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http://dx.doi.org/10.1016/j.ejmech.2016.09.003DOI Listing
January 2017

Identification of anti-inflammatory fractions of Geranium wilfordii using tumor necrosis factor-alpha as a drug target on Herbochip® - an array-based high throughput screening platform.

BMC Complement Altern Med 2015 May 12;15:146. Epub 2015 May 12.

Yunnan Baiyao-Herbcopoeia Laboratory Inc, 51 Xi-Ba Road, Kunming, Yunnan, China.

Background: Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays.

Methods: A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vitro model.

Results: The in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity.

Conclusion: We have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.
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http://dx.doi.org/10.1186/s12906-015-0665-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443519PMC
May 2015

Rutin potentiates insulin receptor kinase to enhance insulin-dependent glucose transporter 4 translocation.

Mol Nutr Food Res 2014 Jun 24;58(6):1168-76. Epub 2014 Feb 24.

Institute of Biotechnology, National Dong-Hwa University, Hualien, 97401, Taiwan; Institute of Biotechnology & Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan.

Scope: We investigated whether rutin, a flavonoid isolated from Toona sinensis Roem, has the ability to enhance insulin-dependent receptor kinase (IRK) activity and glucose transporter 4 (GLUT4) translocation in differentiated myotubes. We also tested the effects of rutin treatment in insulin-resistant mice using an oral glucose tolerance test (OGTT).

Methods And Results: Rutin potentiated insulin receptor kinase (IRK) phosphorylation when IRK autophosphorylation was triggered by insulin in differentiated myotubes. Co-treatment of cells with rutin and insulin attenuated S961-mediated inhibition of insulin-dependent GLUT4 translocation. In S961-treated C57BL/6 mice, an in vivo model of insulin resistance and type 2 diabetes, rutin treatment showed a normoglycemic effect in the OGTT.

Conclusion: This study shows evidence that rutin may serve as a potential agent for glycemic control through enhancement of IRK activity, thereby inducing the insulin signaling pathway causing increased GLUT4 translocation and increased glucose uptake.
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http://dx.doi.org/10.1002/mnfr.201300691DOI Listing
June 2014

Long-term Aβ exposure augments mCa2+-independent mROS-mediated depletion of cardiolipin for the shift of a lethal transient mitochondrial permeability transition to its permanent mode in NARP cybrids: a protective targeting of melatonin.

J Pineal Res 2013 Jan;54(1):107-25

Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

Mitochondrial dysfunction is a hallmark of amyloid β-peptide (Aβ)-induced neurodegeneration of Alzheimer's disease (AD). This study investigated whether mtDNA T8993G mutation-induced complex V inhibition, clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), is a potential risk factor for AD and the pathological link for long-term exposure of Aβ-induced mitochondrial toxicity and apoptosis in NARP cybrids. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy and NARP cybrids harboring 98% mutant genes along with its parental 143B osteosarcoma cells, we demonstrated that Aβ-augmented mitochondrial Ca(2+) (mCa(2+))-independent mitochondrial reactive oxygen species (mROS) formation for a cardiolipin (CL, a major mitochondrial protective phospholipid)-dependent lethal modulation of the mitochondrial permeability transition (MPT). Aβ augmented not only the amount but also the propagation rate of mROS-induced mROS formation to significantly depolarize mitochondrial membrane potential (∆Ψ(m)) and reduce mCa(2+) stress. Aβ-augmented mROS oxidized and depleted CL, thereby enhances mitochondrial fission and movement retardation, which promoted the NARP-augmented lethal transient-MPT (t-MPT) to switch to its irreversible mode of permanent-MPT (p-MPT). Interestingly, melatonin, a multiple mitochondrial protector, markedly reduced Aβ-augmented mROS formation and therefore significantly reduced mROS-mediated depolarization of ∆Ψ(m), fission of mitochondria and retardation of mitochondrial movement to stabilize CL and hence the MPT. In the presence of melatonin, Aβ-promoted p-MPT was reversed to a protective t-MPT, which preserved ∆Ψ(m) and lowered elevated mCa(2+) to sublethal levels for an enhanced mCa(2+)-dependent O(2) consumption. Thus, melatonin may potentially rescue AD patients associated with NARP symptoms.
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http://dx.doi.org/10.1111/jpi.12004DOI Listing
January 2013

Involvement of calcium-mediated reactive oxygen species in inductive GRP78 expression by geldanamycin in 9L rat brain tumor cells.

Int J Mol Sci 2013 Sep 18;14(9):19169-85. Epub 2013 Sep 18.

Department of Bioresources, Da-Yeh University, Changhua 515, Taiwan.

Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.
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http://dx.doi.org/10.3390/ijms140919169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794827PMC
September 2013

Aqueous extracts of Cordyceps militaris (Ascomycetes) lower the levels of plasma glucose by activating the cholinergic nerve in streptozotocin-induced diabetic rats.

Int J Med Mushrooms 2013 ;15(3):277-86

College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

In our previous research, Cordyceps militaris (CM) had a hypoglycemic effect in normal rats. In this study we wanted to elucidate whether CM also had an effect on diabetic rats. Twelve rats with streptozotocin-induced diabetes were separated randomly into 2 groups. First, aqueous extracts of CM 10 mg/kg (CM group) or saline (control group) was fed to the rats; then the plasma glucose levels were assayed. Second, the signaling proteins IRS-1 and GLUT-4 collected from the muscle were detected. Finally, another 2 groups of rats were injected with atropine 0.1 mg/kg intraperitoneally just before the CM/saline feeding, and the assays mentioned above were repeated. Blood glucose decreased 7.2% in the CM group but only 1.5% in the control group (P < 0.05). The IRS-1 signal was 2.9-fold higher than actin in the CM group but only 0.8-fold higher in the control group (P < 0.005). In GLUT-4 signal, the difference was 1.7- vs. 0.6-fold, respectively, compared with actin (P < 0.05). However, atropine injection made CM-induced hypoglycemia or elevation of IRS-1 and GLUT-4 not significant. In conclusion, CM had a hypoglycemic effect in diabetic rats and atropine blocked it. Therefore, the cholinergic activation also was considered to be involved in the hypoglycemic effect of CM in rats with streptozotocin-induced diabetes.
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http://dx.doi.org/10.1615/intjmedmushr.v15.i3.50DOI Listing
July 2013

The critical role of ECM proteins within the human MSC niche in endothelial differentiation.

Biomaterials 2013 Jun 13;34(17):4223-34. Epub 2013 Mar 13.

Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

Interactions between blood vessels and osteoblasts-bone-forming cells-are critical for successful bone development. We therefore investigated the endothelial differentiation capacity of mesenchymal stem cells (MSCs) derived from bone tissue. We found that fetal pre-osteoblast and adult trabecular bone-derived (TB) MSCs express similar surface markers as bone marrow (BM) MSCs and can differentiate into adipocytes, osteoblasts, and chondrocytes. However, when cultured in extracellular matrix (ECM) and endothelial differentiation conditions, bone-derived MSCs (B-MSCs) more readily form tubular structures and uptake acetylated low-density lipoproteins, fulfilling the functional criteria for endothelial cells (ECs). Moreover, addition of B-MSCs but not other cells significantly enhanced vessel formation in the in vivo chick chorioallantoic membrane assay. Mechanistically, this appears to be due to the upregulation of the endothelial transcription factor forkhead box protein C2 (FOXC2) and its downstream gene αvβ3 integrin/CD61in B-MSCs but not BMMSCs by laminin, a component protein of the ECM. Our findings not only reveal discrepant differentiation capacity for various tissue-specific MSCs, but also highlight the critical role of the niche-in this case, the ECM and its component proteins-in determining lineage commitment of stem cells.
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http://dx.doi.org/10.1016/j.biomaterials.2013.02.062DOI Listing
June 2013

In silico prediction and in vitro characterization of multifunctional human RNase3.

Biomed Res Int 2013 17;2013:170398. Epub 2013 Jan 17.

Institute of Molecular and Cellular Biology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan.

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, (34)RWRCK(38) (HBR1), (75)RSRFR(79) (HBR2), and (101)RPGRR(105) (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.
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http://dx.doi.org/10.1155/2013/170398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581242PMC
September 2013

The role of RhoA kinase inhibition in human placenta-derived multipotent cells on neural phenotype and cell survival.

Biomaterials 2013 Apr 12;34(13):3223-30. Epub 2013 Feb 12.

Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

Current advances in stem cell biology have brought much hope for therapy of neuro-degenerative diseases. However, neural stem cells (NSCs) are rare adult stem cells, and the use of non-NSCs requires efficient and high-yielding lineage-specific differentiation prior to transplantation for efficacy. We report on the efficient differentiation of placental-derived multipotent cells (PDMCs) into a neural phenotype with use of Y-27632, a clinically compliant small molecular inhibitor of Rho kinase (ROCK) which is a major mediator of cytoskeleton dynamics. Y-27632 does not induce differentiation of PDMC toward the mesodermal lineages of adipogenesis and osteogenesis, but rather a neural-like morphology, with rapid development of cell extensions and processes within 24 h. Compared with conventional neurogenic differentiation agents, Y-27632 induces a higher percentage of neural-like cells in PDMCs without arresting proliferation or cell cycle dynamics. Y-27632-treated PDMCs express several neural lineage genes at the RNA and protein level, including nestin, MAP2, and GFAP. The effect of the ROCK inhibitor is cell-specific to PDMCs, and is mainly mediated through the ROCK2 isoform and its downstream target, myosin II. Our data suggest that ROCK inhibition and cytoskeletal rearrangement may allow for induction of a neural phenotype in PDMCs without compromising cell survival.
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http://dx.doi.org/10.1016/j.biomaterials.2012.12.034DOI Listing
April 2013

Phosphate limitation induces the intergeneric inhibition of Pseudomonas aeruginosa by Serratia marcescens isolated from paper machines.

FEMS Microbiol Ecol 2013 Jun 11;84(3):577-87. Epub 2013 Mar 11.

Institute of Microbiology, University of Freiburg, Freiburg, Germany.

Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria-Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini-Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation.
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http://dx.doi.org/10.1111/1574-6941.12086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717176PMC
June 2013

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection.

BMC Biotechnol 2012 Nov 7;12:83. Epub 2012 Nov 7.

Institute of Biotechnology, Department of Life Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan, R.O.C.

Background: There are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated.

Results: In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.

Conclusion: The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.
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http://dx.doi.org/10.1186/1472-6750-12-83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505465PMC
November 2012

Extracts of Cordyceps militaris lower blood glucose via the stimulation of cholinergic activation and insulin secretion in normal rats.

Phytother Res 2012 Aug 4;26(8):1173-7. Epub 2012 Jan 4.

College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

Previous studies have shown that Cordyceps militaris (CM) has a hypoglycemic effect, but the actual mechanism remains unclear. This study explored the hypoglycemic mechanism of aqueous extracts of CM in normal Wistar rats. First, the optimal dose of CM for lowering plasma glucose and insulin secretion was tested. Further, atropine and hemicholinium-3 (HC-3) were injected and a western blot was used to investigate insulin signaling. It was found that 10 mg/kg CM extracts had a stronger hypoglycemic effect than a higher dose (100 mg/kg); therefore, a dose of 10 mg/kg was used in subsequent experiments. In normal rats, CM extracts decreased plasma glucose by 21.0% and induced additional insulin secretion by 54.5% after 30 min. When atropine or HC-3 was injected, CM induced a hypoglycemic effect, but the enhancement of insulin secretion was blocked. By western blotting, significant increases in the insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) were observed after CM feeding. However, the elevation of these signaling proteins was abolished by atropine or HC-3. Taken together, these findings indicate that CM can lower plasma glucose via the stimulation of insulin secretion and cholinergic activation involved in the hypoglycemic mechanism of normal Wistar rats.
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http://dx.doi.org/10.1002/ptr.3709DOI Listing
August 2012

Putative tumor metastasis-associated genes in human gastric cancer.

Int J Oncol 2012 Sep 31;41(3):1068-84. Epub 2012 May 31.

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.

Gastric cancer is one of the leading causes of cancer mortality and its malignancy, resulting from disseminated cancer cells of diffuse type, is clinically manifested as metastases to the liver and peritoneum. The aim of the present study was to identify putative tumor metastasis-associated genes in human gastric cancer cells of diffuse type. An MKN45 cell line constitutively expressing green fluorescent protein (MKN45-GFP) was established and selected using the Transwell® system for invasive sublines MKN45-GFP-4, MKN45-GFP-10 and MKN45-GFP-12. MKN45-GFP-10 and MKN45-GFP-12 are highly invasive compared to the others. The mRNA levels were measured with cDNA microarrays and correlated with their invasion abilities in these sublines. Many of the genes identified with a positive or negative correlation are associated with angiogenesis, cell cycle, cytoskeleton and cell motility, protease and cell adhesion, as well as cellular signal transduction. In particular, novel genes without known functions were also noted. RT-PCR and western blot analyses were applied to verify the expression of selective genes. Following orthotopical intraperitoneal implantation, MKN45-GFP-12 demonstrated significantly higher in vivo tumor malignancies than parental MKN45-GFP in ascites induction and liver -invasion in mice. We have identified putative gastric tumor metastasis-associated, as well as novel genes. These genes and their protein products are to be further explored for their functional roles associated with tumor metastasis. The molecular profiles of these identified genes, gene transcripts and proteins in the patient specimens are likely to be useful biomarkers for diagnostic, therapeutic and/or prognostics. Most importantly, they may be used as molecular targets for the discovery of antitumor drugs against human gastric cancer metastasis.
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http://dx.doi.org/10.3892/ijo.2012.1502DOI Listing
September 2012

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells.

World J Gastroenterol 2012 May;18(17):2035-42

Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, Zhejiang Province, China.

Aim: To investigate the role of transforming growth factor (TGF)-β-inducible early gene 1 (TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma (HCC) cells.

Methods: Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium (MTT) assay. The expression changes of Smad2, Smad3, Smad4, Smad7, TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line (MIHA), a TGF-β-sensitive hepatoma cell line (Hep3B) and two TGF-β-insensitive hepatoma cell lines (HepG2 and Bel7404) were examined. SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined. Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines (Bel7404 and HepG2). MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis, respectively. The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis, and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.

Results: TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line, Hep3B, but not in the resistant cell lines. The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1, whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines, which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1. Our data further suggested that stathmin was a direct target of TIEG1, as stathmin was significantly downregulated by TIEG1 overexpression, and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.

Conclusion: Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.
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http://dx.doi.org/10.3748/wjg.v18.i17.2035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342601PMC
May 2012

The clinical performance of the EGV1 self-monitoring blood glucose system.

Clin Chim Acta 2012 Jul 14;413(13-14):1039-44. Epub 2012 Jan 14.

Wei-Gong Memorial Hospital, Miaoli, Taiwan.

Background: The novel technique of blood volume detection can improve the reliability and accuracy of a self-monitoring blood glucose system. Self-management of diabetes can be improved, and the glycemic range can be efficiently controlled.

Methods: A total of 153 patients with diabetes mellitus participated in the clinical study. The accuracy, blood volume detection, interference, and altitude effect of the EGV1 self-monitoring blood glucose system were evaluated and compared among the fingerstick, alternative site testing, and venous blood.

Results: The EGV1 self-monitoring blood glucose system with fingertip demonstrated an excellent correlation with venous blood (linear regression analysis: slope=1.01, intercept=-0.8972 mg/dl, r(2)=0.96), and with other brands of glucose systems (linear regression analysis: slope=0.99, intercept=+3.5632 mg/dl, r(2)=0.94). The Clarke error grid analysis indicated that the results of fingertip and alternative sites were in the acceptable zones, A and B. The system required 0.6 ul of a blood sample to obtain an accurate reading, and was unaffected by several interferents and altitude.

Conclusions: The EGV1 self-monitoring blood glucose system using various blood samples demonstrated acceptable accuracy and reliability compared to the laboratory reference and other self-monitoring blood glucose systems.
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http://dx.doi.org/10.1016/j.cca.2011.12.012DOI Listing
July 2012

7,7''-Dimethoxyagastisflavone-induced apoptotic or autophagic cell death in different cancer cells.

Phytother Res 2012 Apr 14;26(4):528-34. Epub 2011 Sep 14.

Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

7,7''-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from the needles of Taxus × media cv. Hicksii, was evaluated for its antiproliferative and antineoplastic effects in three human cancer cell lines. Interestingly, DMGF caused cell death via different pathways in different cancer cells. DMGF induced apoptosis, activated caspase-3 activity and changed the mitochondrial membrane potential in HT-29 human colon cancer cells. However, the apoptotic pathway is not the major pathway involved in DMGF-induced cell death in A549 human lung cancer cells and HepG2 human hepatoma cells. Treatment with 3-MA, an inhibitor of autophagy, significantly decreased DMGF-induced cell death in HepG2 and A549 cells, but did not affect DMGF-induced cell death in HT-29 cells. Following DMGF treatment, the HepG2 cells increased expression of LC3B-II, a marker used to monitor autophagy in cells. Thus, DMGF induced apoptotic cell death in HT-29 cells, triggered both apoptotic and autophagic death in A549 cells and induced autophagic cell death in HepG2 cells.
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http://dx.doi.org/10.1002/ptr.3583DOI Listing
April 2012

mtDNA T8993G mutation-induced mitochondrial complex V inhibition augments cardiolipin-dependent alterations in mitochondrial dynamics during oxidative, Ca(2+), and lipid insults in NARP cybrids: a potential therapeutic target for melatonin.

J Pineal Res 2012 Jan 4;52(1):93-106. Epub 2011 Aug 4.

Department of Neurology, Kee-Lung Medical Center, Chang Gung Memorial Hospital, Kee-Lung, Taiwan.

Mitochondrial dynamics including morphological fission and mitochondrial movement are essential to normal mitochondrial and cellular physiology. This study investigated how mtDNA T8993G (NARP)-induced inhibition of mitochondrial complex V altered mitochondrial dynamics in association with a protective mitochondrial phospholipid, cardiolipin (CL), as a potential therapeutic target. NARP cybrids harboring 98% of mtDNA T8993G genes and its parental osteosarcoma 143B cells were studied for comparison, and protection provided by melatonin, a potent mitochondrial protector, was explored. We demonstrate for the first time that NARP mutation significantly enhances apoptotic death as a result of three distinct lethal mitochondrial apoptotic insults including oxidative, Ca(2+), and lipid stress. In addition, NARP significantly augmented pathological depletion of CL. NARP-augmented depletion of CL results in enhanced retardation of mitochondrial movement and fission and later swelling of mitochondria during all insults. These results suggest that CL is a common and crucial pathological target for mitochondrial apoptotic insults. Furthermore, CL possibly plays a central role in regulating mitochondrial dynamics that are associated with NARP-augmented mitochondrial pathologies. Intriguingly, melatonin, by differentially preserving CL during various stresses (oxidation > Ca(2+) > lipid), rescues differentially CL-altered mitochondrial dynamics and cell death (oxidation > Ca(2+) > lipid). Thus, melatonin, in addition to being a mitochondrial antioxidant to antagonize mitochondrial oxidative stress, a mitochondrial permeability transition modulator to antagonize mitochondrial Ca(2+) stress, may stabilize directly CL to prevent its oxidization and/or depletion and, therefore, exerts great potential in rescuing CL-dependent mitochondrial dynamics-associated mitochondrial pathologies for treatment of NARP-induced pathologies and diseases.
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http://dx.doi.org/10.1111/j.1600-079X.2011.00923.xDOI Listing
January 2012

Proteomic profiling between CNE-2 and its strongly metastatic subclone S-18 and functional characterization of HSP27 in metastasis of nasopharyngeal carcinoma.

Proteomics 2011 Jul;11(14):2911-20

Surgical Department of Head and Neck, The Third Affiliated Hospital, Kunming Medical University, Kunming, Yunnan, P. R. China.

Metastasis to secondary sites remains the leading cause of nasopharyngeal carcinoma (NPC)-associated death. In order to identify the candidate protein(s) responsible for the differential metastatic capacity, the protein expression profiling between NPC cell line CNE-2 and its highly metastatic subclone S-18 were compared by 2-DE. In total, 18 spots were differentially expressed between these two cell lines. Among all, seven proteins were identified with further MS analysis. Western blotting further validated upregulation of HSP27 and ezrin, and downregulation of valosin containing protein and keratin 18 in S-18. Moreover, the knockdown of HSP27 was found to significantly decrease the invasive ability of S-18. On the other hand, overexpression of HSP27 in NP460 cells, which generated little endogenous HSP27 and less invasive, was noted to gain enhanced metastatic capability. Real-time PCR confirmed that the transcriptional levels of NF-κB and MMP9, MMP11 were downregulated after inhibition of HSP27 in S-18, which implicated that HSP27 enhanced the metastatic property of NPC cells probably via the NF-κB-mediated activation of MMPs. The findings in this work provided us a platform for further elucidating the underlying mechanisms of NPC metastasis and demonstrated that HSP27 would be a valid target for anti-cancer drug development.
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http://dx.doi.org/10.1002/pmic.201000483DOI Listing
July 2011

Recurrent insertion of 5'-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs.

RNA 2011 Jul 25;17(7):1321-35. Epub 2011 May 25.

Centre de Génétique Moléculaire du C.N.R.S., 91190 Gif-sur-Yvette, France.

A survey of sequence databases revealed 10 instances of subgroup IIB1 mitochondrial ribosomal introns with 1 to 33 additional nucleotides inserted between the 5' exon and the consensus sequence at the intron 5' end. These 10 introns depart further from the IIB1 consensus in their predicted domain VI structure: In contrast to its basal helix and distal GNRA terminal loop, the middle part of domain VI is highly variable and lacks the bulging A that serves as the branchpoint in lariat formation. In vitro experiments using two closely related IIB1 members inserted at the same ribosomal RNA site in the basidiomycete fungi Grifola frondosa and Pycnoporellus fulgens revealed that both ribozymes are capable of efficient self-splicing. However, whereas the Grifola intron was excised predominantly as a lariat, the Pycnoporellus intron, which possesses six additional nucleotides at the 5' end, yielded only linear products, consistent with its predicted domain VI structure. Strikingly, all of the introns with 5' terminal insertions lack the EBS2 exon-binding site. Moreover, several of them are part of the small subset of group II introns that encode potentially functional homing endonucleases of the LAGLIDADG family rather than reverse transcriptases. Such coincidences suggest causal relationships between the shift to DNA-based mobility, the loss of one of the two ribozyme sites for binding the 5' exon, and the exclusive use of hydrolysis to initiate splicing.
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http://dx.doi.org/10.1261/rna.2655911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138568PMC
July 2011

Lentivirus-mediated RNA interference targeting Bax inhibitor-1 suppresses ex vivo cell proliferation and in vivo tumor growth of nasopharyngeal carcinoma.

Hum Gene Ther 2011 Oct 5;22(10):1201-8. Epub 2011 May 5.

Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang, Guangdong, China.

Bax inhibitor-1 (Bi-1), an anti-apoptotic protein that belongs to the Bcl-2 family, plays an important role in the mitochondrial apoptosis pathway to suppress Bax-induced apoptosis. In several human cancers, including nasopharyngeal carcinoma, its expression was found to be increased; however, up-regulated expression of this protein has been linked to increased cell proliferations. In this study, we down-regulated the gene expression of Bi-1 in nasopharyngeal carcinoma cells by using a lentivirus transfection system packed with short hairpin RNA targeting Bi-1 and used an in vivo model to assess its efficacy as a target in human gene therapy. The data indicated that human malignant nasopharyngeal carcinoma cells, CNE-1 and SUNE-1, transfected with lentiviral short hairpin RNA targeting Bi-1 grew more slowly and showed a higher degree of apoptosis. Moreover, the tumorigenicity of CNE-1 was significantly suppressed when inoculated mice were intratumorically injected with the same vector. Taken together, these data lead us to conclude that Bi-1 plays a crucial role in CNE-1 tumorigenesis and that Bi-1 may be a novel therapeutic target for nasopharyngeal carcinoma.
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http://dx.doi.org/10.1089/hum.2010.178DOI Listing
October 2011

Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy.

Analyst 2011 May 29;136(10):2111-8. Epub 2011 Mar 29.

Division of Medical Engineering Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan ROC.

Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFα) with TNFα antibody (anti-TNFα) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 ± 4.9 μm(2) s(-1) and 48.96 ± 2.52 μm(2) s(-1) for Alexa488-TNFα and Atto647N-anti-TNFα were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFα and Atto647N-anti-TNFα were approximately 4.89 ± 0.24 nm and 9.99 ± 0.52 nm, respectively, which agrees with the values of 5.20 ± 1.23 nm and 9.28 ± 0.86 nm for the native TNFα and the anti-TNFα as determined from dynamic light scattering measurements. For the binding kinetics, association (k(on)) and dissociation (k(off)) rate constants were (1.13 ± 0.08) × 10(4) M(-1) s(-1) and (1.53 ± 0.19) × 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 °C was (1.36 ± 0.10) × 10(-7) M. We believe this is the first report on the binding kinetics for TNFα-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response.
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http://dx.doi.org/10.1039/c0an00995dDOI Listing
May 2011

Enhanced chemotherapy of cancer using pH-sensitive mesoporous silica nanoparticles to antagonize P-glycoprotein-mediated drug resistance.

Mol Cancer Ther 2011 May 16;10(5):761-9. Epub 2011 Mar 16.

Division of Medical Engineering Research, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan.

Multidrug resistance (MDR) is the major clinical obstacle in the management of cancer by chemotherapy. Overexpression of ATP-dependent efflux transporter P-glycoprotein (PGP) is a key factor contributing to multidrug resistance of cancer cells. The purpose of the present study was to use the endosomal pH-sensitive MSN (mesoporous silica nanoparticles; MSN-Hydrazone-Dox) for controlled release of doxorubicin (Dox) in an attempt to overcome the PGP-mediated MDR. In vitro cell culture studies indicate that uptake of MSN-Hydrazone-Dox by the human uterine sarcoma MES-SA/Dox-resistant tumor (MES-SA/Dx-5) cell occurs through endocytosis, thus bypassing the efflux pump resistance. This improves the efficacy of the drug and leads to significant cytotoxicity and DNA fragmentation evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA laddering assays. In vivo studies show that the intratumor injection of MSN-Hydrazone-Dox induces significant apoptosis of MES-SA/Dox-resistant cancer cells. This is validated by active caspase-3 immunohistochemical analysis. However, MSN-Hydrazone, without doxorubicin conjugation, cannot induce apoptosis in vitro and in vivo. In conclusion, both in vitro and in vivo studies show that MSN could serve as an efficient nanocarrier entering cell avidly via endocytosis, thus bypassing the PGP efflux pump to compromise the PGP-mediated MDR. MSN-Hydrazone-Dox could further respond to endosomal acidic pH to release doxorubicin in a sustained manner. Besides the cell study, this is the first report that successfully shows the therapeutic efficacy of using MSN against MDR cancer in vivo.
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http://dx.doi.org/10.1158/1535-7163.MCT-10-0884DOI Listing
May 2011

Targeting S100P inhibits colon cancer growth and metastasis by Lentivirus-mediated RNA interference and proteomic analysis.

Mol Med 2011 9;17(7-8):709-16. Epub 2011 Feb 9.

Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, China.

S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and β-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis.
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http://dx.doi.org/10.2119/molmed.2011.00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146612PMC
December 2011

Staphylococcus aureus hijacks a skin commensal to intensify its virulence: immunization targeting β-hemolysin and CAMP factor.

J Invest Dermatol 2011 Feb 18;131(2):401-9. Epub 2010 Nov 18.

Division of Dermatology, Department of Medicine, University of California, San Diego, San Diego, California 92121, USA.

The need for a new anti-Staphylococcus aureus therapy that can effectively cripple bacterial infection, neutralize secretory virulence factors, and lower the risk of creating bacterial resistance is undisputed. Here, we propose what is, to our knowledge, a previously unreported infectious mechanism by which S. aureus may commandeer Propionibacterium acnes, a key member of the human skin microbiome, to spread its invasion and highlight two secretory virulence factors (S. aureus β-hemolysin and P. acnes CAMP (Christie, Atkins, Munch-Peterson) factor) as potential molecular targets for immunotherapy against S. aureus infection. Our data demonstrate that the hemolysis and cytolysis by S. aureus were noticeably augmented when S. aureus was grown with P. acnes. The augmentation was significantly abrogated when the P. acnes CAMP factor was neutralized or β-hemolysin of S. aureus was mutated. In addition, the hemolysis and cytolysis of recombinant β-hemolysin were markedly enhanced by recombinant CAMP factor. Furthermore, P. acnes exacerbated S. aureus-induced skin lesions in vivo. The combination of CAMP factor neutralization and β-hemolysin immunization cooperatively suppressed the skin lesions caused by coinfection of P. acnes and S. aureus. These observations suggest a previously unreported immunotherapy targeting the interaction of S. aureus with a skin commensal.
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http://dx.doi.org/10.1038/jid.2010.319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057116PMC
February 2011

The differences of eosinophil- and neutrophil-related inflammation in elderly allergic and non-allergic chronic obstructive pulmonary disease.

J Asthma 2010 Nov;47(9):1040-4

Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.

Background: Chronic obstructive pulmonary disease (COPD) is a common disease in the elderly population and is characterized by airway inflammation. Whether it is a progressive condition resulting from allergic inflammation or a distinct condition involving a pathogen-induced reaction remains unclear.

Objectives: To determine the role of allergic inflammation in the pathogenesis of elderly COPD.

Methods: A total of 63 elderly adults (21 mite-allergic COPD patients, 29 non-allergic COPD patients, and 13 normal controls) were recruited in this study. The serum-specific IgE for mites, level of interleukin-5 (IL-5), IL-8, leptin, adiponectin, regulated upon activation normal T cell expressed and secreted (RANTES), growth-related oncogene-α (GRO-α), vitamin E, and glutathione (GSH) were determined.

Results: The serum levels of GRO-α in patients with COPD were higher in comparison to normal controls (105.8 ± 32.7 vs. 7.5 ± 7.5 pg/mL, p= .021). Compared to patients with non-allergic COPD, patients with mite allergies had a higher serum level of IL-8 (63.2 ± 12.6 vs. 35.0 ± 8.2 pg/mL, p= .022). Although both IL-5 and RANTES levels were increased in COPD patients, there were no significant differences between allergic and non-allergic COPD. There were also no differences in serum levels of leptin, adiponectin, vitamin E, and GSH between COPD patients and normal controls.

Conclusions: The increased serum levels of GRO-α indicate that it may have potential as a candidate biomarker for elderly COPD patients. There was no difference of eosinophils-related chemokines in allergic and non-allergic COPD. These results indicated that both adipokines and eosinophil-related chemokines only play trivial roles in the pathogenesis of COPD.
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http://dx.doi.org/10.1080/02770903.2010.491145DOI Listing
November 2010

A fusion protein with the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A) is an antagonist of angiogenesis in cancer treatment: Simultaneous blocking of VEGF receptor-1 and 2.

Cancer Biol Ther 2010 Nov 1;10(9):865-73. Epub 2010 Nov 1.

Department of Orthopedics, Hualien Armed Forces Hospital, Hualien, Taiwan, ROC.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that signals through VEGFR-1 and VEGFR-2, which are expressed preferentially in proliferating endothelial cells. Thus, simultaneous blockage of both VEGF receptors may provide a more efficient therapeutic response in cancer treatment. We created a recombinant fusion protein (RBDV-IgG1 Fc), which is composed of the receptor binding domain of human VEGF-A (residues 8-109) and the Fc region of human IgG1 immunoglobulin. The recombinant protein can bind to both mouse VEGFR-1 and VEGFR-2 to decrease VEGF-induced proliferation and tube formation of endothelial cells in vitro. In this study, the RBDV-IgG1 Fc fusion protein reduced the effects of proliferation, migration and tube formation induced by VEGF in murine endothelial cells in vitro. In vivo tumor therapy with RBDV-IgG1 Fc resulted in tumor inhibition by reducing angiogenesis. Pathological evidence also shows that RBDV-IgG1 Fc can seriously damage vessels, causing the death of tumor cells. These findings suggest that this chimeric protein has potential as an angiogenesis antagonist in tumor therapy.
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http://dx.doi.org/10.4161/cbt.10.9.13230DOI Listing
November 2010

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway.

Biochem Biophys Res Commun 2010 Jun 24;397(2):283-9. Epub 2010 May 24.

Department of Biological Science and Technology, National Chiao-Tung University, Hsin-Chu, Taiwan.

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCbeta2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCbeta2/Ca2+ signal transduction in endothelial cells.
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http://dx.doi.org/10.1016/j.bbrc.2010.05.101DOI Listing
June 2010

TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

BMC Cell Biol 2010 Jan 20;11. Epub 2010 Jan 20.

Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
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http://dx.doi.org/10.1186/1471-2121-11-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2819994PMC
January 2010