Human cDNA Microarray Research Articles

Gene expression profiling of HepG2 cells after treatment with black tea polyphenols

This study aimed to determine the effects of black tea polyphenols on gene expression in hepatocellular cancer cells. The total RNA from HepG2 hepatocellular cancer cells treated with black tea polyphenols was subjected to Human 14K cDNA microarray analysis. Real-time PCR and Western blot analysis were conducted to verify microarray data. Black tea polyphenols treatment at the dose of 20 mg/L, 40 mg/L or 80 mg/L for one to three days inhibited the growth of HepG2 cells in a dose and time dependent manner. A total of 48 genes showed more than two-fold change after black tea polyphenols treatment, including 17 upregulated genes and 31 downregulated genes, and they were involved in the regulation of cell growth, cell cycle, apoptosis, signaling, angiogenesis, tumor invasion and metastasis. Real-time PCR analysis of the selected genes showed that their mRNA expression changes were consistent with the microarray data. In addition, Western blot analysis of the selected genes showed that their protein expression changes were consistent with mRNA expression. In conclusion, gene expression profiles provide comprehensive molecular mechanisms by which black tea polyphenols exerts growth inhibition effects on cancer cells. The novel molecular targets identified in this study may be further exploited as therapeutic strategies for hepatocellular cancer.

A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer.

Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

Evaluation by microarray of the potential safety of Sarracenia purpurea L. (Sarraceniaceae) a traditional medicine used by the Cree of Eeyou Istchee.

The purpose of this study was to assess safety of the traditional antidiabetic extracts of either S. purpurea or its lead active principle, morroniside at the transcriptional level. The overarching objective was to profile and validate transcriptional changes in the cytochrome P450 family of genes, in response to treatment with S. purpurea ethanolic extract or its lead active, morroniside. Transcriptional activity was profiled using a 19K human cDNA microarray in C2BBe1 cells, clone of Caco-2 intestinal cells, which are a model of first-pass metabolism (1, 2). Cells were treated with S. purpurea extract for 4 and 24 hrs, as well as the pure compound morroniside for 4 hrs, to determine their effects. No evidence of cytochrome P450 transcriptome regulation or of transcriptional activation of other diabetes relevant mRNA was detected after rigorous quantitative-PCR validation of microarray results. Our data do not support a transcriptional mechanism of action for either S. purpurea extract or its lead active, morroniside. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Mitochondria-focused gene expression profile reveals common pathways and CPT1B dysregulation in both rodent stress model and human subjects with PTSD.

Posttraumatic stress disorder (PTSD), a trauma-related mental disorder, is associated with mitochondrial dysfunction in the brain. However, the biologic approach to identifying the mitochondria-focused genes underlying the pathogenesis of PTSD is still in its infancy. Previous research, using a human mitochondria-focused cDNA microarray (hMitChip3) found dysregulated mitochondria-focused genes present in postmortem brains of PTSD patients, indicating that those genes might be PTSD-related biomarkers. To further test this idea, this research examines profiles of mitochondria-focused gene expression in the stressed-rodent model (inescapable tail shock in rats), which shows characteristics of PTSD-like behaviors and also in the blood of subjects with PTSD. This study found that 34 mitochondria-focused genes being upregulated in stressed-rat amygdala. Ten common pathways, including fatty acid metabolism and peroxisome proliferator-activated receptors (PPAR) pathways were dysregulated in the amygdala of the stressed rats. Carnitine palmitoyltransferase 1B (CPT1B), an enzyme in the fatty acid metabolism and PPAR pathways, was significantly over-expressed in the amygdala (P<0.007) and in the blood (P<0.01) of stressed rats compared with non-stressed controls. In human subjects with (n=28) or without PTSD (n=31), significant over-expression of CPT1B in PTSD was also observed in the two common dysregulated pathways: fatty acid metabolism (P=0.0027, false discovery rate (FDR)=0.043) and PPAR (P=0.006, FDR=0.08). Quantitative real-time polymerase chain reaction validated the microarray findings and the CPT1B result. These findings indicate that blood can be used as a specimen in the search for PTSD biomarkers in fatty acid metabolism and PPAR pathways, and, in addition, that CPT1B may contribute to the pathology of PTSD.

Abstract 393: Genome-wide molecular characterization of CIMP positive stage III colon cancer using cDNA microarray analysis

Abstract Background: The CpG island methylator phenotype (CIMP) is a distinctive subtype of colon cancers, which leads to the loss of tumor suppressor gene functions by epigenetic regulation. However, molecular characteristics of CIMP+ cancers are not fully understood. Here, we aimed to elucidate the different patterns of gene expression according to the CIMP status. Method: 158 patients with stage III colon cancer were enrolled for the analysis of CIMP status. Pyrosequencing was used to examine the methylation status of 7 CpG island loci (p14, p16, MINT1, MINT2, MINT31, hMLH1, and WNT5a) in DNA extracted from formalin-fixed paraffin-enbedded specimens. Gene expression analyses of the same samples were also performed using a human cDNA microarray to identify molecular signatures. Result: Among the 158 patients, 31 (19.3%) were CIMP+. Microarray results revealed distinctive gene expression profiles between CIMP+ and CIMP- cancers. Among 57 genes which showed significant changes between CIMP+ and CIMP- cancers, Axin2, the canonical Wnt suppressor, displayed the strongest correlation with the CIMP status (p=0.007). Axin2 was downregulated in CIMP+ cancers compared to CIMP- cancers (p=0.005). In terms of disease-free survival (DFS), tumors with low Axin2 expression showed poor prognosis compared to tumors with high Axin2 expression in microsatellite stable (MSS) patients (p=0.038), while there were no significant changes in DFS of microsatellite instable (MSI) patients (p=0.32). These findings indicate the clinical importance of epigenetic silencing of Axin2 and the subsequent activation of Wnt signaling pathway in MSS colon cancer patients. Conclusion: Axin2 is downregulated in CIMP+ stage III colon cancer patients and the consequent decreased expression of Axin2 is a poor prognostic factor for the DFS in MSS patients. Citation Format: Hongjae Jeon, Se Hyun Kim, Kyu Hyun Park, Hei-Cheul Jeung, Nam Kyu Kim, Minkyu Jung, Kang Young Lee, Chan Hee Park, Sun Young Rha, Hyun Cheol Chung, Joong Bae Ahn. Genome-wide molecular characterization of CIMP positive stage III colon cancer using cDNA microarray analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 393. doi:10.1158/1538-7445.AM2014-393

Differential Expression of Genes in Cells Cultured from Juxtacanalicular Trabecular Meshwork and Schlemm's Canal

The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues. A set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and β-spectrin was assessed by immunofluorescence. Schlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM). We found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.

Differential expression of leukocyte β2 integrin signal transduction-associated genes in patients with symptomatic pulmonary embolism

Whole human genome oligo microarrays were employed to systematically investigate the differential expression characteristics of associated mRNAs, which were found in the signal transduction pathway of β2 integrins in peripheral blood mononuclear cells (PBMCs) between patients with symptomatic pulmonary embolism (PE) and controls. A total of 20cases of PE patients and twenty gender‑ and age‑matched controls were recruited for the study. Human cDNA microarray analysis was used to detect the differences in mRNA expression between the two groups and a random variance model corrected t‑test was used to analyze the statistical data. A total of 80 associated mRNAs were detected. The mRNA expression of chemokines, ligands, inside‑out and outside‑in signaling pathway‑associated proteins were upregulated significantly in the PE group, compared with the controls. In five subunit‑associated mRNAs, the mRNA expression of ITGAL, ITGAM, ITGAX and ITGB2, which encode for the subunits of αL, αM, αX and β2, were upregulated in the PE group and the differences, with the exception of ITGB2, were statistically significant (P<0.05). The mRNA expression of ITGAD was downregulated; however, there was no significant difference (P>0.05). The expression of Fgr mRNA was significantly downregulated (P<0.01). Thus, in PE patients, bilateral signal transduction pathways of β2 integrins in neutrophils and monocytes were activated, enhancing innate immunity.

Abstract B110: Characterization of the role of TRAF2 and TNFRSF14 in breast cancer

Abstract Human breast cancer patients display significant diversity in terms of their survival, recurrence, metastasis as well as response to treatment. Our group recruited a multi-centre prospective cohort of over 1900 women with ANN breast cancer and followed them clinically. Tumors from 135 women in this group were selected for gene expression analysis on human cDNA microarrays to examine the molecular alterations in ANN patients that could be predictive of recurrence. From genes that demonstrated a statistically significant difference in expression in tumors from patients who have experienced an early recurrence versus who have remained disease free for over 10 years, TRAF2 (tumor necrosis factor (TNF) receptor-associated factor 2) was found to be highly expressed in early recurrence. TRAF2 is involved in immune system cytokine signaling pathways and interacts with many receptors in the TNF receptor superfamily and mediates the survival effects of these receptors including NF-κB, LTβR, JNK and MAPKs kinases. TRAF2 has been reported to be overexpressed or constitutively phosphorylated in some cancers, including lymphoma and pancreatic cancers. The tumor necrosis factor receptor TNFRSF14, also called LIGHTR or HVEM is located on the short arm of chromosome 1p36. Deletion of 1p36 is especially frequent in breast tumors and is associated with progression and lymph node metastasis, poor prognosis, higher rate of recurrence, larger tumor size, and DNA aneuploidy7. Cheung and al. (2010) have described somatic TNFRSF14 mutations in Follicular Lymphoma (FL) which are associated with worse prognosis. They identified 46 cases (18.3%) with non-synonymous mutations affecting TNFRSF14 in a cohort of 251 FL patients. The significance of such acquired mutations on the structure-function relationship of TNFRSF14 is still unknown, but it appears that TNFRSF14 can be considered as a potential candidate gene that might contribute to FL development. TNFRSF14 belongs to the TNF-receptor superfamily and contains multiple copies of a cysteine-rich motif in the extracellular region that is known to provide the ligand recognition motifs. Activation of TNF-receptor family members is induced by their cognate, trivalent, TNF-related ligand, clustering multiple receptors, which in turn recruits the cytosolic TNF receptor-associated factors (TRAFs) adaptors leading to serine kinase activation of the NFκB system. TNFRSF14 associated with TRAF2 and other members of the TRAF family and activates JNK as well as NF κB and AP-1, which control expression of multiple immune, inflammatory and acute phase action genes in response to infection or cellular stress. The short cytoplasmic regions are frequently capable of stimulating the transcription factors activator protein 1 (AP1) and NFKB1. This particular region of the TNFRSF14 gene interacts with members of the TNFR-associated factor (TRAF) family and mediates the signal transduction pathways that activate the immune response. Since TNFRSF14 interacts with TRAF2 and/or other TRAFs to mediate the NF-κB activation and TRAF2 is highly expressed in breast cancer early recurrence, we hypothesized that altered TRAF2 and TNFRSF14 could contribute to recurrence in ANN breast cancer. Single-strand conformation polymorphism (SSCP) and DNA Sequencing were performed to detect any small alteration in PCR-amplified product from 130 ANN breast cancer tumor samples and 48 germline breast cancer tumor samples. Known polymorphisms were excluded and direct sequencing revealed one new coding SNP (Pro262Leu; C&amp;gt;T) located at exon 8. The genotypes CC were observed in 177 of the 178 samples in total. Only one ANN breast cancer tumor sample (936A) revealed a heterozygote genotype (CT). According to algorithms ‘SIFT’ and PolyPhen-2, the Pro262Leu amino acid exchange alters the secondary structure of TNFRSF14 and is predicted to affect the function of the protein. Due to the impact of TNFRSF14 mutations associated with poor prognosis during carcinogenesis, this variant may play a role in the therapy outcome in breast cancer. Quantitative Real-time RT-PCR was performed to quantify TRAF2 expression in a subset of cell lines and tumor samples. Current studies include immunohistochemistry of tissue microarrays containing 888 ANN tumors to detect TRAF2 protein levels and localization and investigation of the association of TRAF2 expression with clinical outcome. In addition, the characterization of the importance of alterations in TRAF2 gene and its associated pathways is being performed using a combination of bioinformatics, to identify potential. Furthermore, the TNFRSF14 Pro262Leu_C&amp;gt;T SNP is being analysed using TaqMan allelic discrimination to investigate a putative association of this variant with breast cancer risk or recurrence. These studies could lead to the discovery of new prognostic markers that may improve our understanding of the molecular pathways responsible for breast cancer development and progression and identify novel targets for breast cancer therapeutics. Citation Format: Sandrine Tchatchou, Lucine Bosnoyan-Collins, Dushanthi Pinnaduwage, Shelley B. Bull, Irene L. Andrulis. Characterization of the role of TRAF2 and TNFRSF14 in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B110.

Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy

Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios≥2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD.

Characteristics of the complement system gene expression deficiency in patients with symptomatic pulmonary embolism

IntroductionPulmonary embolism (PE) is a disease with a high mortality and morbidity rate, and the pathogenesis of PE remains still unclear. We aimed to investigate the gene expression differences of the complement system in peripheral blood mononuclear cells (PBMCs) from patients with symptomatic PE and controls. MethodsTwenty cases of PE patients and twenty sex and age matched controls were recruited into the study. Human cDNA microarray analysis was used to detect the gene expression difference of the complement system between the two groups. Results1). Expression of twenty-one genes encoding complement components was detected. In PE patients, expression of the genes encoding C1qα, C1qβ, C4b, C5 and Factor P was significantly greater (P<0.05) than controls, while C6, C7, C9, mannose-binding lectin (MBL) and mannan-binding lectin serine peptidase 1 (MASP1) mRNAs were lower (P<0.05) than controls. 2). Expression of seven genes encoding complement receptors was examined. In PE patients, CR1, integrin αM, integrin αX and C5aR mRNAs were significantly up-regulated (P<0.01) compared with controls. 3). Seven genes encoding complement regulators were examined. The mRNA expression of CD59 and CD55 was significantly up-regulated (P<0.05), whereas Factor I mRNA was significantly down-regulated (P<0.05) in PE patients than controls. ConclusionsIn PE patients, the mRNA expressions of complement components, receptors and regulators were unbalanced, suggesting dysfunction and/or deficiency of the complement system, which leads to decreased function of MAC-induced cell lysis in PE patients finally.

Gene expression levels of cytokines in peripheral blood mononuclear cells from patients with pulmonary embolism

The aim of this study was to investigate the differential gene expression of cytokines in peripheral blood mononuclear cells (PBMCs) from patients with pulmonary embolism (PE) and controls. Twenty patients with PE and twenty control patients matched for gender and age with the PE group were recruited into the study. Human cDNA microarray analysis was used to detect differences in the expression of cytokine-associated genes between the two groups. In PE patients, the expression levels of the genes encoding IFNα5, IFNα6, IFNα8, IFNα14, IFNκ, IFNω1, IFNε1 and IFNγ were significantly lower compared with controls (P<0.05). The expression levels of the genes encoding IL1α, IL2, IL3, IL9, IL13, IL17β, IL19, IL22, IL23α, IL24, IL25 and IL31 were significantly lower (P<0.05), while IL10 and IL28A mRNA expression levels were higher in PE patients compared with controls (P<0.05). In PE patients, Cxcl1, Cxcl2, Cxcl6, Cxcl13 and Cxcl14 mRNAs were significantly upregulated (P<0.05), however, Cxcl10 mRNA was significantly downregulated (P<0.01). In PE patients, the mRNA expression levels of TNF superfamily members 1, 9 and 13, and TNF receptor superfamily members 1A, 1B, 9, 10B, 10C, 10D and 19L, were significantly upregulated (P<0.05), whereas TNF receptor superfamily members 11B, 19 and 25 were significantly downregulated compared with controls (P<0.05). The mRNA expression levels of granulocyte-macrophage colony‑stimulating factor, granulocyte colony-stimulating factor, erythropoietin, thrombopoietin and mast cell growth factor were significantly lower in PE patients compared with controls (P<0.05). In PE patients, the mRNA expression levels of a variety of cytokines were imbalanced and cellular immune function was downregulated compared with controls. Thus, PE patients may be more susceptible to infections caused by viruses, intracellular bacteria and parasites.

MRNA expression of interleukins and Th1/Th2 imbalance in patients with pulmonary embolism

Few studies have investigated the changes of Th1- and Th2-type cytokines in pulmonary embolism (PE) patients. In this study, the gene expression of interleukins and the balance of Th1- and Th2-type cytokines in the peripheral blood mononuclear cells (PBMCs) of PE patients and controls were investigated. A total of 20 PE patients and 20gender- and age-matched controls were included in the study. Human cDNA microarray analysis was used to detect the differences in cytokine gene expression between the two groups and a random variance model corrected t-test was used to analyze the statistical data. In comparison with the controls, 12genes were found to be downregulated, specifically IL1A, IL9, IL17B, IL19, IL23A, IL25 (p<0.05), IL2, IL3, IL13, IL22, IL24 and IL31 (p<0.01), and 2genes were found to be upregulated, specifically IL10 and IL28A, in the PE patients. The expression levels of IFN-γ and IL2 mRNA in the PE patients were significantly lower than those in the control group (p<0.01), while the IL20 mRNA expression levels were significantly upregulated (p<0.01). We conclude that there are significant differences in interleukin gene expression between the PE patients and the control group. A shift of the Th1/Th2 balance comprising enhanced Th2 activity and reduced Th1 activity in the PE patients is also demonstrated.

N-Butyl Benzyl Phthalate Promotes Breast Cancer Progression by Inducing Expression of Lymphoid Enhancer Factor 1

Environmental hormones play important roles in regulating the expression of genes involved in cell proliferation, drug resistance, and breast cancer risk; however, their precise role in human breast cancer cells during cancer progression remains unclear. To elucidate the effect of the most widely used industrial phthalate, n-butyl benzyl phthalate (BBP), on cancer progression, we evaluated the results of BBP treatment using a whole human genome cDNA microarray and MetaCore software and selected candidate genes whose expression was changed by more than ten-fold by BBP compared with controls to analyze the signaling pathways in human breast cancer initiating cells (R2d). A total of 473 genes were upregulated, and 468 were downregulated. Most of these genes are involved in proliferation, epithelial-mesenchymal transition, and angiogenesis signaling. BBP induced the viability, invasion and migration, and tube formation in vitro, and Matrigel plug angiogenesis in vivo of R2d and MCF-7. Furthermore, the viability and invasion and migration of these cell lines following BBP treatment was reduced by transfection with a small interfering RNA targeting the mRNA for lymphoid enhancer-binding factor 1; notably, the altered expression of this gene consistently differentiated tumors expressing genes involved in proliferation, epithelial-mesenchymal transition, and angiogenesis. These findings contribute to our understanding of the molecular impact of the environmental hormone BBP and suggest possible strategies for preventing and treating human breast cancer.

A novel fumarate hydratase-deficient HLRCC kidney cancer cell line, UOK268: a model of the Warburg effect in cancer

The role of energy deregulation and altered/adapted metabolism in tumor cells is an increasingly important issue in understanding cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of fumarate hydratase (FH), followed by somatic loss of the remaining wild-type allele and known to be a highly metastatic and lethal malignancy compared to other RCCs. The intrinsic loss of normal tricarboxylic acid (TCA) cycle presumably aids tumorigenesis due to the necessary metabolic alterations required and the enforced dependence on glycolysis derived energy, mimicking the Warburg effect. Thus, there is considerable utility in establishing a preclinical cell model from these tumors to study energy metabolism deregulation, as well as developing new targeted therapeutic approaches for TCA cycle enzyme-deficient cancers. Here, we describe a new immortalized cell line, UOK268, derived from a patient's primary HLRCC-associated kidney cancer. This represents the first primary renal cell line to model TCA cycle gene loss and provides a perfect partner cell line to our previously described metastasis-derived HLRCC-associated cell line, UOK262. We identified a novel germline FH missense mutation, p.His192Asp, and the subsequent loss of heterozygosity in UOK268. The UOK268 cell line expressed mutant FH protein, which localized to the mitochondria, but with loss of almost all catalytic activity. The UOK268 cells had severely compromised oxidative phosphorylation and increased glycolytic flux. Ingenuity pathways analysis of human mitochondria-focused cDNA microarray (hMitChip3) gene chip data confirmed the altered mRNA expression patterns of genes involved in several important pathways, such as lipid metabolism, apoptosis, and energy production/glycolysis. UOK268 provides a unique model of a primary cell line demonstrating an enforced, irreversible Warburg effect and, combined with UOK262, provides a unique invitro preclinical model for studying the bioenergetics of the Warburg effect in human cancer.

Identification of cellular genes showing differential expression associated with hepatitis B virus infection

To investigate the impact of hepatitis B virus (HBV) infection on cellular gene expression, by conducting both in vitro and in vivo studies. Knockdown of HBV was targeted by stable expression of short hairpin RNA (shRNA) in huH-1 cells. Cellular gene expression was compared using a human 30K cDNA microarray in the cells and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) (qRT-PCR) in the cells, hepatocellular carcinoma (HCC) and surrounding non-cancerous liver tissues (SL). The expressions of HBsAg and HBx protein were markedly suppressed in the cells and in HBx transgenic mouse liver, respectively, after introduction of shRNA. Of the 30K genes studied, 135 and 103 genes were identified as being down- and up-regulated, respectively, by at least twofold in the knockdown cells. Functional annotation revealed that 85 and 62 genes were classified into four up-regulated and five down-regulated functional categories, respectively. When gene expression levels were compared between HCC and SL, eight candidate genes that were confirmed to be up- or down-regulated in the knockdown cells by both microarray and qRT-PCR analyses were not expressed as expected from HBV reduction in HCC, but had similar expression patterns in HBV- and hepatitis C virus-associated cases. In contrast, among the eight genes, only APM2 was constantly repressed in HBV non-associated tissues irrespective of HCC or SL. The signature of cellular gene expression should provide new information regarding the pathophysiological mechanisms of persistent hepatitis and hepatocarcinogenesis that are associated with HBV infection.

Hesperidin Displays Relevant Role in the Nutrigenomic Effect of Orange Juice on Blood Leukocytes in Human Volunteers: A Randomized Controlled Cross-Over Study

BackgroundWe previously showed, in healthy, middle-aged, moderately overweight men, that orange juice decreases diastolic blood pressure and significantly improves postprandial microvascular endothelial reactivity and that hesperidin could be causally linked to the observed beneficial effect of orange juice. The objective was to determine the effect of chronic consumption of orange juice on the gene expression profile of leukocytes in healthy volunteers and to assess to what extent hesperidin is involved in the effect of orange juice.Methodology/Principal FindingsVolunteers were included in a randomized, controlled, crossover study. Throughout three 4-week periods, volunteers consumed daily: 500 ml orange juice, 500 ml control drink plus hesperidin or 500 ml control drink and placebo. Blood samplings were performed on 10 overnight-fasted subjects after the 4-week treatment period. Global gene expression profiles were determined using human whole genome cDNA microarrays. Both orange juice and hesperidin consumption significantly affected leukocyte gene expression. Orange juice consumption induced changes in expression of, 3,422 genes, while hesperidin intake modulated the expression of 1,819 genes. Between the orange juice and hesperidin consumption groups, 1,582 regulated genes were in common. Many of these genes are implicated in chemotaxis, adhesion, infiltration and lipid transport, which is suggestive of lower recruitment and infiltration of circulating cells to vascular wall and lower lipid accumulation.ConclusionsThis study shows that regular consumption of orange juice for 4 weeks alters leukocyte gene expression to an anti-inflammatory and anti-atherogenic profile, and hesperidin displays a relevant role in the genomic effect of this beverage.Trial RegistrationClinicalTrials.gov NCT 00983086

Transcriptional profiling of human skin fibroblast cell line Hs27 induced by herbal formula Astragali Radix and Rehmanniae Radix

Ethnopharmacological relevanceThe herbs Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. Aim of the studyChinese herbal formulus comprising Astragali Radix (AR) and Rehmanniae Radix (RR) have been shown to improve the healing of diabetic foot ulcer through enhancing the viability of primary fibroblasts in diabetic patients suffering insulin resistance. Our previous study demonstrated that the herbal formula NF3 comprising of AR and RR in the ratio of 2:1 was effective in promoting wound healing in diabetic rats, and in vitro data indicated that the wound healing effects of NF3 might be due to the regulation and coordination of inflammation, angiogenesis and tissue regeneration. However, the underlying molecular mechanism has not been well investigated. In this study, we investigated the cellular and molecular effects of the herbal formula NF3 on human skin fibroblast cells. Materials and methodsHuman skin fibroblast cells Hs27 were treated with NF3 ranging from 0 to 8mg/ml for 24h, and the cells without NF3 treatment were used as control. Cell proliferation assay and cell cycle analysis were performed. Transcriptional profiles of Hs27 cells upon NF3 treatment were acquired by using a human cDNA microarray containing 10,000 genes, and the signaling pathways differentially regulated by NF3 were identified and analyzed. ResultsNF3 promoted Hs27 cell proliferation and cell cycle progression. Microarray analysis revealed that 116 genes were differentially expressed upon NF3 treatment. Functional analysis of the genes indicated that NF3 mainly activated Wnt and angiogenesis related pathways, which are directly related to cell proliferation, angiogenesis, extracellular matrix (ECM) formation and inflammation during the process of wound healing. ConclusionThis study provides insight into the molecular mechanism of how the herbal formula Astragali Radix and Rehmanniae Radix may serve as potential therapeutics for wound healing.

The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray

While the autophagic process is mainly regulated at the post-translational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy.lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.

Expression profiling of estrogen responsive genes on bisphenol A, 4-nonylphenol and 17β-estradiol treatment using in house cDNA microarray

Bisphenol A (BPA) and nonylphenol (NP) are thought to mimic estrogens in their action, and are called endocrine disrupting chemicals. These phenolic chemicals are used in a number of commercial products and have been reported to be weakly estrogenic in previous studies. Their estrogenic activities are mainly dependent on their binding affinity for the estrogen receptors in vitro and in vivo. To identify genes elicited by BPA and NP, we carried out a microarray analysis in MCF-7 cells treated with BPA and NP using human c-DNA microarray including 416 endocrine system related genes. At the minimum foldchange criteria of 1.5, 14 and 29 genes were identified showing significant changes in gene expression resulting from BPA and NP, respectively. Especially, 2 genes were repressed and 6 genes were induced by BPA and NP as 17β-estradiol (E2). To validate the gene expression profiles identified from microarray analyses, the expression patterns of 4 representative genes, Selenoprotein P, plasma, 1 (SEPP1), Matrix Gla protein (MGP), H2A histone family, member X (H2AFX) and breast cancer 1, early onset (BRCA1) were examined by real time RT-PCR. We further examined the expression patterns of phthalic chemicals with estrogenic activity by comparing with those of E2 or phenolic chemicals to evaluate that alterations of these estrogen responsive genes may act as useful biomarkers to the endocrine disrupting chemicals with estrogenic activity.