Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 18 de 18
Filter
1.
Carcinogenesis ; 42(7): 995-1007, 2021 07 16.
Article in English | MEDLINE | ID: mdl-34089582

ABSTRACT

MicroRNAs, as a group of post-transcriptional regulators, regulate multiple pathological processes including metastasis during tumor development. Here, we demonstrated the metastasis-suppressive function of microRNA (miR)-338-5p in esophageal squamous cell carcinoma (ESCC). Overexpression of miR-338-5p had inhibitory effect on invasive ability of ESCC cells and extracellular matrix degradation, whereas silencing miR-338-5p had opposite effects. Mechanistically, miR-338-5p directly targeted the 3' untranslated regions of hepatocellular growth factor receptor cMet (cMET) and epidermal growth factor receptor (EGFR). As a result, miR-338-5p inhibited the downstream signaling cascades of cMET and EGFR and repressed cMET- and EGFR-mediated ESCC cell invasion. Re-expression of cMET or EGFR in miR-338-5p overexpressing ESCC cells was sufficient to derepress ESCC cell invasion both in vitro and in vivo. We further showed that such manipulation downregulated the expression and secretion of matrix metalloproteinases 2 and 9, which resulted in impaired extracellular matrix degradation and cell invasion. Most importantly, systemic delivery of miR-338-5p mimic significantly inhibited metastasis of ESCC cells in nude mice. Taken together, our results uncovered a previously unknown mechanism through which miR-338-5p suppresses ESCC invasion and metastasis by regulating cMET/EGFR-matrix metalloproteinase 2/9 axis and highlighted the potential significance of miR-338-5p-based therapy in treating patients with metastatic ESCC.


Subject(s)
Esophageal Neoplasms/prevention & control , Esophageal Squamous Cell Carcinoma/prevention & control , Gene Expression Regulation, Neoplastic , Lung Neoplasms/prevention & control , MicroRNAs/genetics , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Apoptosis , Cell Proliferation , ErbB Receptors/antagonists & inhibitors , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/pathology , Humans , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Mice , Mice, Nude , MicroRNAs/administration & dosage , Neoplasm Invasiveness , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
BMC Cancer ; 17(1): 409, 2017 Jun 10.
Article in English | MEDLINE | ID: mdl-28601093

ABSTRACT

BACKGROUND: Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach. METHODS: The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections. RESULTS: We identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman's correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Heat-Shock Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Liver Neoplasms/metabolism , Signal Transduction , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Female , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Hep G2 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Male , Middle Aged , Progranulins , Protein Binding
3.
Oncotarget ; 8(24): 38811-38824, 2017 Jun 13.
Article in English | MEDLINE | ID: mdl-28415588

ABSTRACT

Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-dependent activation of Smad2/3 and Smad4 in hepatocellular carcinoma (HCC) CSCs and in HCC primary tumors. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing stemness gene expression, and increasing chemoresistance. Cyclin D1 interacts with and activates Smad2/3 and Smad4 to result in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. The cyclin D1-dependent activation of Smad2/3 and Smad4 is also found in HCC patients and predicts disease progression. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-mediated activation of Smad2/3 and Smad4 is an important regulatory mechanism in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequently significant chemosensitization, which could be an effective strategy to target CSCs.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cyclin D1/metabolism , Drug Resistance, Neoplasm , Neoplastic Stem Cells/pathology , Smad2 Protein/metabolism , Smad4 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Cell Differentiation/drug effects , Cell Proliferation , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, SCID , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Prognosis , Signal Transduction/drug effects , Smad2 Protein/antagonists & inhibitors , Smad4 Protein/antagonists & inhibitors , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Lung Cancer ; 85(2): 282-92, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24976335

ABSTRACT

BACKGROUND: Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines. METHODS: LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed. RESULTS: Fifty resected lung AD were included (M:F=23:27, smokers:non-smokers=19:31, EGFR mutant:wild-type=21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR=0.217; p=0.003; Overall survival RR=0.238; p=0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels. CONCLUSIONS: LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors.


Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/mortality , ErbB Receptors/genetics , Gene Expression , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Mutation , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/surgery , Adenocarcinoma of Lung , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , DNA Mutational Analysis , ErbB Receptors/metabolism , Female , Gene Knockdown Techniques , Gene Silencing , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/surgery , MAP Kinase Signaling System , Male , Middle Aged , Neoplasm Recurrence, Local , Neoplasm Staging , Prognosis , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Risk Factors , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism
5.
Mol Cell Proteomics ; 12(11): 3379-87, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23924514

ABSTRACT

MicroRNAs (miRNAs) regulate gene expression through translational repression and RNA degradation. Recently developed high-throughput proteomic methods measure gene expression changes at protein level and therefore can reveal the direct effects of miRNAs' translational repression. Here, we present a web server, ProteoMirExpress, that integrates proteomic and mRNA expression data together to infer miRNA-centered regulatory networks. With both types of high-throughput data from the users, ProteoMirExpress is able to discover not only miRNA targets that have decreased mRNA, but also subgroups of targets with suppressed proteins whose mRNAs are not significantly changed or with decreased mRNA whose proteins are not significantly changed, which are usually ignored by most current methods. Furthermore, both direct and indirect targets of miRNAs can be detected. Therefore, ProteoMirExpress provides more comprehensive miRNA-centered regulatory networks. We used several published data to assess the quality of our inferred networks and prove the value of our server. ProteoMirExpress is available online, with free access to academic users.


Subject(s)
MicroRNAs/genetics , MicroRNAs/metabolism , Proteome/genetics , Proteome/metabolism , Proteomics/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Gene Expression Profiling/statistics & numerical data , Gene Regulatory Networks , HeLa Cells , High-Throughput Nucleotide Sequencing/methods , High-Throughput Nucleotide Sequencing/statistics & numerical data , Humans , Proteomics/statistics & numerical data , Software
6.
Int J Cancer ; 131(8): 1930-40, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22261816

ABSTRACT

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.


Subject(s)
Apoptosis/drug effects , Epstein-Barr Virus Infections/pathology , Herpesvirus 4, Human/drug effects , Hydroxamic Acids/pharmacology , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/pathology , Virus Replication/drug effects , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Carcinoma , Cell Proliferation/drug effects , Epstein-Barr Virus Infections/drug therapy , Epstein-Barr Virus Infections/virology , Female , Flow Cytometry , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Mice, Nude , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/virology , Poly(ADP-ribose) Polymerases/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Vorinostat
7.
Mol Carcinog ; 51(12): 963-72, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22006341

ABSTRACT

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-ß-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not p-STAT3 (Y705) and p-STAT3 (S727), in the nucleus of HK-1 cells. Pre-treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl-xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p-STAT3 in the poorly differentiated C666-1 cells. Pharmacological inhibition of STAT3 or Bcl-2/xL resulted in a decrease in endoreduplication of C666-1 cells. Taken together, the expression of p-STAT5 and p-STAT3 was upregulated in 2ME2-induced endoreduplicating HK-1 and C666-1 cells, respectively. Combination of 2ME2 with Bcl-2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.


Subject(s)
Endoreduplication/drug effects , Estradiol/analogs & derivatives , Nasopharyngeal Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/physiology , STAT3 Transcription Factor/physiology , STAT5 Transcription Factor/physiology , bcl-X Protein/physiology , 2-Methoxyestradiol , Base Sequence , Blotting, Western , Carcinoma , Cell Line, Tumor , Estradiol/pharmacology , Flow Cytometry , Humans , Nasopharyngeal Carcinoma , RNA, Small Interfering
8.
Am J Pathol ; 176(4): 1828-40, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20110407

ABSTRACT

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33 degrees C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy.


Subject(s)
Conjunctiva/virology , Influenza A Virus, H1N1 Subtype/metabolism , Influenza, Human/virology , Respiratory System/virology , Alveolar Epithelial Cells/metabolism , Animals , Bronchi/cytology , Cytokines/metabolism , Dogs , Epithelial Cells/cytology , Humans , Orthomyxoviridae/metabolism , Pandemics , Seasons , Species Specificity
9.
Cancer Epidemiol Biomarkers Prev ; 18(10): 2709-16, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19815638

ABSTRACT

PURPOSE: Nasopharyngeal carcinoma is an epithelial malignancy with a remarkable racial and geographic distribution. Previous cytogenetic studies have shown nasopharyngeal carcinoma to be characterized by gross genomic aberrations. However, identification of susceptible gene loci in advanced nasopharyngeal carcinoma has been poorly discussed. EXPERIMENTAL DESIGN: A genome-wide survey of gene copy number changes was initiated with two nasopharyngeal carcinoma cell lines by array-based comparative genomic hybridization analysis. These alterations were confirmed by a parallel analysis with the data from the gene expression microarray and were validated by quantitative PCR. Clinical association of the defined target genes was analyzed by fluorescence in situ hybridization on 48 metastatic tumors. RESULTS: A high percentage of genes were consistently altered in dosage and expression levels with gain on 3q26.2-q26.32 and losses on 3p12.3-p14.2 and 9p21.3-p23. Six candidate genes, GPR160 (3q26.2-q27), SKIL (3q26), ADAMTS9 (3p14.2-p14.3), LRIG1 (3p14), MPDZ (9p22-p24), and ADFP (9p22.1) were validated by quantitative PCR. Fluorescence in situ hybridization studies revealed amplification of GPR160 (in 25% of cases) and SKIL (33%); and deletion of ADAMTS9 (30%), LRIG1 (35%), MPDZ (15%), and ADFP (15%). Clinical association analyses indicated a poor survival rate with genetic alterations at the defined 3p deletion (P = 0.0012) and the 3q amplification regions (P = 0.0114). CONCLUSION: The combined microarray technologies suggested novel candidate oncogenes, amplification of GPR160 and SKIL at 3q26.2-q26.32, and deletion of tumor suppressor genes ADAMTS9 and LRIG1 at 3p12.3-p14.2. Altered expression of these genes may be responsible for malignant progression and could be used as potential markers for nasopharyngeal carcinoma.


Subject(s)
Chromosomes, Human, Pair 3 , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/pathology , Cell Line, Tumor , Gene Amplification , Gene Dosage , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genome, Human , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Oligonucleotide Array Sequence Analysis , Prognosis
10.
PLoS One ; 3(8): e2990, 2008 Aug 20.
Article in English | MEDLINE | ID: mdl-18714356

ABSTRACT

BACKGROUND: Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. METHODOLOGY/PRINCIPAL FINDINGS: Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing. CONCLUSIONS/SIGNIFICANCE: Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.


Subject(s)
Carcinoma/genetics , Cell Adhesion Molecules/genetics , DNA Methylation , Epigenesis, Genetic/genetics , Gene Silencing , Genes, Tumor Suppressor , Lymphoma/genetics , Nerve Tissue Proteins/genetics , Alternative Splicing , Chromosomes, Human, Pair 11 , Female , GPI-Linked Proteins , Genetic Variation , Humans , Loss of Heterozygosity , Male , Nasopharyngeal Neoplasms/genetics , Reference Values , Transcription, Genetic
11.
Carcinogenesis ; 29(9): 1742-50, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18632752

ABSTRACT

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.


Subject(s)
Apoptosis/drug effects , Drug Resistance, Neoplasm , Dual Specificity Phosphatase 6/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Oxidative Stress/physiology , Animals , Apoptosis/physiology , Blotting, Western , Cell Adhesion/physiology , Cell Proliferation/drug effects , Cisplatin/pharmacology , Disease Progression , Dual Specificity Phosphatase 6/antagonists & inhibitors , Dual Specificity Phosphatase 6/genetics , Female , Humans , Hydrogen Peroxide/metabolism , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Ovarian Neoplasms/genetics , Phosphorylation , Promoter Regions, Genetic , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Tumor Cells, Cultured , Ubiquitin/metabolism
12.
J Cell Biochem ; 104(5): 1625-35, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18320592

ABSTRACT

Esophageal cancer (EC) persists to be a leading cancer-related death in northern China. Clinical outcome of EC is the most dismal among many types of digestive tumors because EC at early stage is asymptomatic. The current study used 2-DE-based proteomics to identify differentially expressed proteins between esophageal cancer cell lines and immortal cell line. Fifteen proteins were identified with differences of more than five folds, comprising the down-regulation of annexin A2, histone deacetylase 10 isoform beta and protein disulfide-isomerase ER-60 precursor, and the up-regulation of heat shock 70 kDa protein 9B precursor, solute carrier family 44 Member 3, heterogeneous nuclear ribonucleoprotein L (hnRNP L), eukaryotic translation initiation factor 4A isoform 2, triosephosphate isomerase1 (TPI), peroxiredoxin1 (PRX1), forminotransferase cyclodeaminase form (FTCD), fibrinogen gamma-A chain precursor, kinesin-like DNA binding protein, lamin A/C, cyclophilin A (CypA), and transcription factor MTSG1. Expression pattern of annexin A2 was verified by Western blotting, immunocytochemistry and immunohistochemistry analysis. The implication of these protein alterations correlated to the esophageal malignant transformation is discussed.


Subject(s)
Carcinoma, Squamous Cell/chemistry , Cell Transformation, Neoplastic/chemistry , Esophageal Neoplasms/chemistry , Neoplasm Proteins/analysis , Proteomics , Annexins/metabolism , Blotting, Western , Carcinoma, Squamous Cell/pathology , Cell Adhesion , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Esophageal Neoplasms/pathology , Humans , Immunohistochemistry
13.
J Mol Neurosci ; 32(2): 160-7, 2007.
Article in English | MEDLINE | ID: mdl-17873300

ABSTRACT

Telomerase, a specialized reverse transcriptase that maintains telomere during cell division, is commonly associated with cell proliferation. Increasing evidence suggests that telomerase may bear functions other than telomere elongation. We investigated whether telomerase is expressed in the continuously growing goldfish retina. Telomeric repeat amplification protocol (TRAP) assay reveals telomerase activity in goldfish retina. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot show that telomerase catalytic subunit (TERT) is expressed at both mRNA and protein levels. Localization of TERT by immunohistochemistry indicates prominent expression of TERT in the outer nuclear layer, the inner nuclear layer, and, in a small population of cells, in the ganglion cell layer. Coexpression of TERT with proliferative cell nuclear antigen (PCNA) immunoreactivity is found in rod progenitor cells. These results suggest the role of telomerase in vertebrate central nervous system (CNS) other than telomere maintenance, such as regulation of cell cycle progression and maintenance of retinal cell phenotypes.


Subject(s)
Retina/enzymology , Telomerase/genetics , Amino Acid Sequence , Animals , Blotting, Western , Consensus Sequence , Gene Amplification , Goldfish/genetics , Molecular Sequence Data , Protein Biosynthesis , Repetitive Sequences, Amino Acid , Reverse Transcriptase Polymerase Chain Reaction
14.
Lab Invest ; 87(7): 644-50, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17384664

ABSTRACT

Aberrant activation of the wingless-type- (Wnt)-signaling pathway is common in many cancers including nasopharyngeal (NPC) and esophageal squamous cell (ESCC) carcinomas, both prevalent in Southern China and Southeast Asia. However, the molecular mechanism leading to this abnormality is still obscure. Wnt inhibitory factor-1 (WIF1) is a secreted antagonist of the Wnt pathway, and is recently shown to be inactivated by epigenetic mechanism in some tumors. Here, we examined whether WIF1 is also inactivated epigenetically in NPC and ESCC. With semiquantitative reverse transcription-PCR and methylation-specific PCR, we detected WIF1 downregulation or silencing in 6/6 of NPC and 12/19 of ESCC cell lines, which is well correlated with its methylation status. Methylation was further confirmed by high-resolution bisulfite genomic sequencing. Methylation was also frequently observed in a large collection of primary tumors of NPC (85%, 55/65) and ESCC (27%, 25/92), with WIF1 expressed and unmethylated in normal NPC and esophageal cell lines and normal tissues. Treatment of 5-aza-2'-deoxycytidine demethylated WIF1 and induced its expression in NPC and ESCC cell lines, highlighting a direct role of epigenetic inactivation. Ectopic expression of WIF1 in NPC and ESCC tumor cells resulted in significant inhibition of tumor cell colony formation, similar to TP53, and also significant downregulation of beta-catenin protein level in NPC cells. Thus, WIF1 functions as a tumor suppressor for both NPC and ESCC through suppressing the Wnt-signaling pathway, but is frequently silenced by epigenetic mechanism in a tumor-specific way. Our study indicates that epigenetic inactivation of WIF1 contributes to the aberrant activation of Wnt pathway and is involved in the pathogenesis of both tumors. WIF1 methylation could also serve as a specific biomarker for these tumors.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carcinoma/genetics , DNA Methylation , Esophageal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Nasopharyngeal Neoplasms/genetics , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/drug effects , Adaptor Proteins, Signal Transducing/genetics , Animals , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biomarkers, Tumor , Carcinoma/metabolism , Carcinoma/pathology , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Methylation/drug effects , Decitabine , Down-Regulation , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Gene Silencing , Humans , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Promoter Regions, Genetic/genetics , Repressor Proteins/drug effects , Repressor Proteins/genetics , Tumor Suppressor Proteins/drug effects , Tumor Suppressor Proteins/genetics , Wnt1 Protein/genetics , Wnt1 Protein/metabolism
15.
Mol Hum Reprod ; 12(7): 451-60, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16772430

ABSTRACT

Placental trophoblast cells are unique endocrine cells that play vital roles during the processes of embryonic implantation and placentation. However, research into the function of human trophoblast has been largely restrained mainly due to a lack of adequate cell models. A normal placenta-origin cytotrophoblast cell line (NPC) was previously established by our group, but these cells showed replicating senescence after 50 population doublings (PDs). In this study, the human telomerase catalytic subunit gene (htert) was transferred into B6 strain of NPC cells, and strains with reconstituted telomerase activity (B6Tert) were established. It was shown that B6Tert-1 cells produce various biomarkers of normal extravillous cytotrophoblasts during the early weeks of gestation. Meanwhile, the cell invasiveness was inhibited by transforming growth factor beta (TGFbeta). However, their ability to form syncytium was relatively low when stimulated with fetal calf serum (FCS). The cells maintained normal cell growth properties and failed to elicit tumours in nude mice. They proliferated continuously with no signs of senescence until the final count at 210 PDs. The growth rate of B6Tert-1 cells was increased when compared with the parental cells, which results, at least partly, from facilitating release of the G1/S checkpoint during the cell-cycle regulation. This is the first report of immortalizing human normal cytotrophoblast (CTB) cells by activation of telomerase activity. The cells will provide an ideal in vitro model for the study of human extravillous trophoblast (EVT) functions and consequently the mechanisms of embryonic implantation and placentation.


Subject(s)
DNA-Binding Proteins/genetics , Telomerase/genetics , Telomere/metabolism , Trophoblasts/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Line , DNA-Binding Proteins/metabolism , Female , Humans , Immunohistochemistry , Karyotyping , Keratins/analysis , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Phase-Contrast/methods , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/metabolism , Telomere/genetics , Transfection , Trophoblasts/cytology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vimentin/analysis
16.
Int J Oncol ; 28(3): 767-73, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16465383

ABSTRACT

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.


Subject(s)
Carcinoma, Squamous Cell/pathology , DNA Methylation , Esophageal Neoplasms/pathology , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins/genetics , Carcinoma, Squamous Cell/epidemiology , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , China/epidemiology , Chromosomes, Human, Pair 3/genetics , Esophageal Neoplasms/epidemiology , Esophageal Neoplasms/genetics , Genes, Tumor Suppressor , Humans , Incidence , Japan/epidemiology , Neoplasm Staging , Survival Analysis
17.
Int J Cancer ; 118(10): 2461-9, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16353136

ABSTRACT

An analysis of gene expression profiles obtained from cervical cancers was performed to find those genes most aberrantly expressed. Total RNA was prepared from 29 samples of cervical squamous cell carcinoma and 18 control samples, and hybridized to Affymetrix oligonucleotide microarrays with probe sets complementary to over 20,000 transcripts. Unsupervised hierarchical clustering of the expression data readily distinguished normal cervix from cancer. Supervised analysis of gene expression data identified 98 and 139 genes that exhibited >2-fold upregulation and >2-fold downregulation, respectively, in cervical cancer compared to normal cervix. Several of the genes that were differentially regulated included SPP1 (Osteopontin), CDKN2A (p16), RPL39L, Clorf1, MAL, p11, ARS and NICE-1. These were validated by quantitative RT-PCR on an independent set of cancer and control specimens. Gene Ontology analysis showed that the list of differentially expressed genes included ones that were involved in multiple biological processes, including cell proliferation, cell cycle and protein catabolism. Immunohistochemical staining of cancer specimens further confirmed differential expression of SPP1 in cervical cancer cells vs. nontumor cells. In addition, 2 genes, CTGF and RGS1 were found to be upregulated in late stage cancer compared to early stage cancer, suggesting that they might be involved in cancer progression. The pathway analysis of expression data showed that the SPP1, VEGF, CDC2 and CKS2 genes were coordinately differentially regulated between cancer and normal. The present study is promising and provides potential new insights into the extent of expression differences underlying the development and progression of cervical squamous cell cancer. This study has also revealed several genes that may be highly attractive candidate molecular markers/targets for cervical cancer diagnosis, prognosis and therapy.


Subject(s)
Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Uterine Cervical Neoplasms/genetics , Case-Control Studies , Diagnosis, Differential , Female , Genes, Neoplasm , Genetic Markers , Hong Kong , Humans , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/therapy
18.
Cancer ; 106(1): 146-55, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16329133

ABSTRACT

BACKGROUND: Lung cancer is a prevalent cancer with a poor prognosis. To develop a useful in vitro cell model, a cell line of lung squamous cell carcinoma (SCC-35) was established. METHODS: The SCC-35 cell was characterized by comparative genomic hybridization (CGH) and spectral karyotyping (SKY). Chromosome microdissection, fluorescence in situ hybridization (FISH), and Southern and Northern blots analyses were used to study target genes. RESULTS: Two amplicons were found at chromosomes 7p12 and 11q13. Amplification and overexpression of epidermal growth factor receptor (EGFR) at 7p12 and fibroblast growth factor 3 (FGF3) at 11q13 were found. To understand the correlation between these two genes in nonsmall cell lung carcinoma (NSCLC) more comprehensively, overexpression of FGF3 and EGFR was investigated by immunohistochemistry with a tissue microarray containing 406 NSCLC samples. Cytoplasmic overexpression of FGF3 and EGFR was detected in 61% and 69% NSCLC cases, respectively. More interestingly, a significant correlation between overexpression of FGF3 and EGFR was found in NSCLC. CONCLUSION: These results suggest that co-overexpression of FGF3 and EGFR may play an important role in the pathogenesis of lung carcinoma.


Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Squamous Cell/metabolism , ErbB Receptors/metabolism , Fibroblast Growth Factor 3/metabolism , Lung Neoplasms/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 7/genetics , ErbB Receptors/genetics , Fibroblast Growth Factor 3/genetics , Humans , Lung Neoplasms/genetics , Male , Middle Aged , Spectral Karyotyping , Telomerase/genetics , Telomerase/metabolism , Tissue Array Analysis
SELECTION OF CITATIONS
SEARCH DETAIL
...