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Search Results: 1 - 10 of 24217 matches for " Hui Chua "
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GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing (siRNA) in senescent human diploid fibroblasts  [PDF]
Suzana Makpol, Azalina Zainuddin, Kien Hui Chua2
American Journal of Molecular Biology (AJMB) , 2012, DOI: 10.4236/ajmb.2012.24041
Abstract: Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.
Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells
Azizah Ugusman, Zaiton Zakaria, Chua Hui, Nor Megat Mohd Nordin
BMC Complementary and Alternative Medicine , 2011, DOI: 10.1186/1472-6882-11-31
Abstract: HUVECs were divided into four groups:- control; treatment with 180 μM hydrogen peroxide (H2O2); treatment with 150 μg/mL AEPS and concomitant treatment with AEPS and H2O2 for 24 hours. Total RNA was extracted from all the groups of HUVEC using TRI reagent. Subsequently, qPCR was carried out to determine the mRNA expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx. The specificity of the reactions was verified using melting curve analysis and agarose gel electrophoresis.When stimulated with H2O2, HUVECs expressed higher level of ICAM-1 (1.3-fold) and Nox4 (1.2-fold) mRNA expression. However, AEPS treatment led to a reduction in the mRNA expression of ICAM-1 (p < 0.01) and Nox4 (p < 0.05) in the H2O2-induced HUVECs. AEPS also upregulated the mRNA expression of SOD1 (p < 0.05), CAT (p < 0.01) and GPx (p < 0.05) in oxidative stress-induced HUVECs. There was no significant change in the mRNA expression of VCAM-1 and E-selectin.The expressional suppression of ICAM-1 and Nox4 and induction of antioxidant enzymes might be an important component of the vascular protective effect of AEPS.Atherosclerosis has been recognized as a chronic inflammatory disease and oxidative stress plays a pivotal role in its initiation and progression [1]. Endothelial dysfunction is considered to be an early marker for atherosclerosis [2]. Evidence suggests that increased production of reactive oxygen species (ROS) and vascular inflammation play important roles in endothelial dysfunction.Vascular disorders, through over expression of adhesion molecules and cytokines are involved in the development of atherosclerosis. Endothelial cells in human atherosclerotic lesions have increased cell adhesion molecules expression such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) [3,4]. The adhesion of monocytes to the arterial wall and their subsequent infiltration and differentiation into macrophages are th
Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts
Suzana Makpol,Norhazira Abdul Rahim,Chua Kien Hui,Wan Zurinah Wan Ngah
Oxidative Medicine and Cellular Longevity , 2012, DOI: 10.1155/2012/785743
Abstract: In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (<0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (<0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (<0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.
Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts
Suzana Makpol,Lina Wati Durani,Kien Hui Chua,Yasmin Anum Mohd Yusof,Wan Zurinah Wan Ngah
Journal of Biomedicine and Biotechnology , 2011, DOI: 10.1155/2011/506171
Abstract: This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.
Characterization of Novel and Uncharacterized p53 SNPs in the Chinese Population – Intron 2 SNP Co-Segregates with the Common Codon 72 Polymorphism
Beng Hooi Phang,Hui Wan Chua,Huihua Li,Yeh Ching Linn,Kanaga Sabapathy
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015320
Abstract: Multiple single nucleotide polymorphisms (SNPs) have been identified in the tumor suppressor gene p53, though the relevance of many of them is unclear. Some of them are also differentially distributed in various ethnic populations, suggesting selective functionality. We have therefore sequenced all exons and flanking regions of p53 from the Singaporean Chinese population and report here the characterization of some novel and uncharacterized SNPs - four in intron 1 (nucleotide positions 8759/10361/10506/11130), three in intron 3 (11968/11969/11974) and two in the 3′UTR (19168/19514). Allelic frequencies were determined for all these and some known SNPs, and were compared in a limited scale to leukemia and lung cancer patient samples. Intron 2 (11827) and 7 (14181/14201) SNPs were found to have a high minor allele frequency of between 26–47%, in contrast to the lower frequencies found in the US population, but similar in trend to the codon 72 polymorphism (SNP12139) that shows a distribution pattern correlative with latitude. Several of the SNPs were linked, such as those in introns 1, 3 and 7. Most interestingly, we noticed the co-segregation of the intron 2 and the codon 72 SNPs, the latter which has been shown to be expressed in an allele-specific manner, suggesting possible regulatory cross-talk. Association analysis indicated that the T/G alleles in both the co-segregating intron 7 SNPs and a 4tagSNP haplotype was strongly associated increased susceptibility to lung cancer in non-smoker females [OR: 1.97 (1.32, 3.394)]. These data together demonstrate high SNP diversity in p53 gene between different populations, highlighting ethnicity-based differences, and their association with cancer risk.
Inhibition of Hypoxia-Inducible Factor-1α (HIF-1α) Protein Synthesis by DNA Damage Inducing Agents
Jessica Jie Wei Lou,Yee Liu Chua,Eng Hui Chew,Jie Gao,Martin Bushell,Thilo Hagen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010522
Abstract: Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that is composed of a hypoxia-inducible α subunit (HIF-1α and HIF-2α) and a constitutively expressed β subunit (HIF-1β). HIF mediates the adaptation of cells and tissues to low oxygen concentrations. It also plays an important role in tumorigenesis and constitutes an important therapeutic target in anti-tumor therapy. We have screened a number of reported HIF inhibitors for their effects on HIF-transcriptional activity and found that the DNA damage inducing agents camptothecin and mitomycin C produced the most robust effects. Camptothecin is a reported inhibitor of HIF-1α translation, while mitomycin C has been reported to induce p53-dependent HIF-1α degradation. In this study we demonstrate that the inhibitory effect of mitomycin C on HIF-1α protein expression is not dependent on p53 and protein degradation, but also involves HIF-1α translational regulation. Initiation of a DNA damage response with the small molecule p53 activator NSC-652287 (RITA) has been reported to inhibit HIF-1α protein synthesis by increasing the phosphorylation of eIF2α. However, we show here that even when eIF2α phosphorylation is prevented, the DNA damage inducing drugs mitomycin C, camptothecin and NSC-652287 still inhibit HIF-1α protein synthesis to the same extent. The inhibitory effects of camptothecin on HIF-1α expression but not that of mitomycin C and NSC-652287 were dependent on cyclin-dependent kinase activity. In conclusion, specific types of DNA damage can bring about selective inhibition of HIF-1α protein synthesis. Further characterization of the involved mechanisms may reveal important novel therapeutic targets.
Effects of edible bird's nest (EBN) on cultured rabbit corneal keratocytes
Fadhilah Zainal Abidin, Chua Hui, Ng Luan, Elvy Suhana Mohd Ramli, Lee Hun, Norzana Abd Ghafar
BMC Complementary and Alternative Medicine , 2011, DOI: 10.1186/1472-6882-11-94
Abstract: Corneal keratocytes from six New Zealand White Rabbits were isolated and cultured until Passage 1. The proliferative effects of EBN on corneal keratocytes were determined by MTT assay in serum-containing medium (FDS) and serum-free medium (FD). Keratocytes phenotypical changes were morphologically assessed and gene expression of aldehyde dehydrogenase (ALDH), collagen type 1 and lumican were determined through RT-PCR.The highest cell proliferation was observed when both media were supplemented with 0.05% and 0.1% EBN. Cell proliferation was also consistently higher in FDS compared to FD. Both phase contrast micrographs and gene expression analysis confirmed the corneal keratocytes retained their phenotypes with the addition of EBN.These results suggested that low concentration of EBN could synergistically induce cell proliferation, especially in serum-containing medium. This could be a novel breakthrough as both cell proliferation and functional maintenance are important during corneal wound healing. The in vitro test is considered as a crucial first step for nutri-pharmaceutical formation of EBN-based eye drops before in vivo application.The cornea comprises of three distinct layers: the epithelium, the stroma and the endothelium. Each layer provides specific properties which are crucial to the optimal functionality of the cornea in normal vision while acting as a protective barrier from external environment [1]. The corneal stroma makes up 90% of the corneal volume and is filled with keratocytes bound by extracellular matrix which forms the structural backbone of the cornea [2,3]. Keratocytes are mesenchymal-derived cells of the corneal stroma responsible for the synthesis and maintenance of the extracellular matrix (ECM) components [4]. Normal, quiescent keratocytes residue between collagen lamellae of the corneal stroma as a sparse population of flattened-cells [5], connecting to one other through a network of extensive processes [6]. The keratocytes have low ce
Effect of MDM2 SNP309 and p53 codon 72 polymorphisms on lung cancer risk and survival among non-smoking Chinese women in Singapore
Hui Chua, Daniel Ng, Serena Choo, San Lum, Huihua Li, Li Soh, Kanaga Sabapathy, Adeline Seow
BMC Cancer , 2010, DOI: 10.1186/1471-2407-10-88
Abstract: We therefore examined the role of the SNPs in the p53 pathway (p53 codon 72 and MDM2 SNP309) on lung cancer risk and prognosis of a life-time non-smoking female Chinese population, in a hospital-based case-control study of 123 cases and 159 age-matched controls, by PCR analysis.Our findings reveal that the risk of lung cancer among individuals with the MDM2 SNP309 TT genotype was 2.1 (95% CI 1.01-4.36) relative to the GG genotype, contrary to initial expectations that the GG genotype with elevated MDM2 levels will increase cancer risk. Those who had this genotype in combination with the p53 Pro allele had a risk of 2.5 (95% CI 1.2-5.0). There was however no effect of either polymorphism on age at diagnosis of lung cancer or on overall survival.The results thus demonstrate that the MDM2 SNP309 TT rather than the GG genotype is associated with increased risk of lung cancer in this population, suggesting that other mechanisms independent of increased MDM2 levels can influence cancer susceptibility.The TP53 tumour suppressor pathway plays a critical role in cell cycle regulation and apoptosis in many cancers, including lung carcinomas [1], and variation in the genes that regulate this pathway may exert an important influence on tumour development, and hence, cancer risk. Recent interest has focused on the murine double minute-2 protein (MDM2), a nuclear phospoprotein that inhibits p53 activity by promoting its degradation [2]. A single nucleotide polymorphism (SNP309) in the MDM2 promoter has been found to influence transcription of this gene via a greater affinity for the SP1 transcription factor, and hence, individuals with the GG genotype have higher MDM2 levels leading to attenuation of the p53 pathway [3,4]. This has been especially so in the case of females, due to the involvement of the MDM2 SNP in the estrogen receptor signaling pathway [3].Several epidemiologic studies have evaluated this association with varying results. No overall association between MDM2 SNP
Weak expression of cyclooxygenase-2 is associated with poorer outcome in endemic nasopharyngeal carcinoma: analysis of data from randomized trial between radiation alone versus concurrent chemo-radiation (SQNP-01)
Susan Loong, Jacqueline Hwang, Hui Li, Joseph Wee, Swee Yap, Melvin Chua, Kam Fong, Terence Tan
Radiation Oncology , 2009, DOI: 10.1186/1748-717x-4-23
Abstract: 58 out of 88 patients from this institution had samples available for analysis. COX-2 expression levels were stratified by immunohistochemistry, into negligible, weak, moderate and strong, and correlated with overall and disease specific survivals.58% had negligible or weak COX-2 expression, while 14% and 28% had moderate and strong expression respectively. Weak COX-2 expression conferred a poorer median overall survival, 1.3 years for weak versus 6.3 years for negligible, 7.8 years, strong and not reached for moderate. There was a similar trend for disease specific survival.Contrary to literature published on other malignancies, our findings seemed to indicate that over-expression of COX-2 confer a better prognosis in patients with endemic NPC. Larger studies are required to conclusively determine the significance of COX-2 expression in these patients.Nasopharyngeal carcinoma (NPC) is the sixth most common male cancer in Singapore. The current standard of care for locally advanced NPC is concurrent chemo-radiation, which is associated with increased acute and long term morbidities [1,2]. Increasing effort has been directed toward developing molecular targeted therapies for the treatment of NPC with increasing interest in cyclooxygenase-2 (COX-2) inhibitors.COX-2 is a 68 kDA enzyme that catalyses the conversion of arachidonic acid to prostaglandins. Over-expression of COX-2 has been found in a variety of malignancies, both gastrointestinal (colon, oesophagus, stomach, pancreas) as well as outside the gastrointestinal tract (lung, breast, bladder and cervix), and shown to correlate with poorer outcomes [3-6].We hereby describe a retrospective analysis of 58 samples from patients, diagnosed with endemic NPC, who had previously been randomized into a trial of radiotherapy (RT) alone versus concurrent chemo-radiation (CRT) [7]. The aims of the study were to determine the expression level of COX-2 in our cohort of patients and to correlate this with known prognostic fact
Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts
Makpol, Suzana;Zainuddin, Azalina;Chua, Kien Hui;Yusof, Yasmin Anum Mohd;Ngah, Wan Zurinah Wan;
Clinics , 2012, DOI: 10.6061/clinics/2012(02)08
Abstract: objective: human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. the beneficial effects of vitamin e in aging have been established, but studies to determine the mechanisms of these effects are ongoing. this study determined the molecular mechanism of γ-tocotrienol, a vitamin e homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. methods: primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. the expression levels of eln, col1a1, mmp1, ccnd1, rb1, and il6 genes were determined using the quantitative real-time polymerase chain reaction. cell cycle profiles were determined using a facscalibur flow cytometer. results: the cell cycle was arrested in the g0/g1 phase, and the percentage of cells in s phase decreased with senescence. ccnd1, rb1, mmp1, and il6 were upregulated in senescent fibroblasts. a similar upregulation was not observed in young cells. incubation with γ-tocotrienol decreased ccnd1 and rb1 expression in senescent fibroblasts, decreased cell populations in the g0/g1 phase and increased cell populations in the g2/m phase. γ-tocotrienol treatment also upregulated eln and col1a1 and downregulated mmp1 and il6 expression in young and senescent fibroblasts. conclusion: γ-tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.
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