Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Clin Exp Metastasis ; 32(3): 255-65, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25647720

ABSTRACT

Metastasis, responsible for most deaths from breast cancer (BC), is a multistep process leading to cancer cell spread. Extracellular matrix (ECM)-related adhesion and apoptosis resistance play pivotal role in metastasis. Ras suppressor-1 (RSU-1) localizes to cell-ECM adhesions and binds to pro-survival adhesion protein PINCH-1. Little is known about the role of RSU-1 in BC. In the present study, we investigated the role of RSU-1 in BC metastasis using two BC cell lines that differ in terms of their metastatic potential and a set of 32 human BC samples from patients with or without lymph node metastasis. We show that RSU-1 is upregulated in the aggressive MDA-MB-231 cells compared to MCF-7 and that its silencing by siRNA leads to upregulation of PINCH-1, induction of proliferation and reduction of apoptosis through downregulation of the pro-apoptotic gene p53-upregulated-modulator-of-apoptosis (PUMA). Our findings in the cell lines were further validated in the human BC tissues where normal adjacent tissues were used as controls. We demonstrate for the first time, that RSU-1 expression is upregulated in metastatic BC samples and downregulated in non-metastatic while it is negatively correlated with PINCH-1 and positively correlated with PUMA expression, suggesting that a pro-apoptotic mechanism is in place in metastatic BC samples and identifying RSU-1 as a potentially interesting molecule that needs to be evaluated further as a novel BC metastasis biomarker.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenocarcinoma, Mucinous/secondary , Apoptosis Regulatory Proteins/metabolism , Apoptosis , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/secondary , Carcinoma, Lobular/secondary , LIM Domain Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenocarcinoma, Mucinous/genetics , Adenocarcinoma, Mucinous/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins/genetics , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Lobular/genetics , Carcinoma, Lobular/metabolism , Cell Proliferation , Female , Humans , LIM Domain Proteins/genetics , Lymphatic Metastasis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Tumor Cells, Cultured
2.
Cell Oncol (Dordr) ; 38(2): 155-64, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25539992

ABSTRACT

PURPOSE: Tamoxifen is a major treatment modality for estrogen receptor positive breast cancer, but the occurrence of resistance remains a problem. Recently, obesity-related leptin has been found to interfere with tamoxifen in breast cancer MCF-7 cells. In the present study we investigated the effect of leptin on three tamoxifen-treated breast cancer cell types (i.e., MDA-MB-231, MCF-7 and MCF-7/HER2). METHODS: The effect of tamoxifen/leptin treatment was evaluated using a MTT cell viability assay. mRNA expression was assessed by real time PCR and protein expression by Western blotting. WWOX, Survivin and BCL2 gene promoter activities were evaluated by chromatin immunoprecipitation. RESULTS: Cell viability assays revealed that estrogen receptor negative MDA-MB-231 cells were resistant, that estrogen receptor positive MCF-7 cells were sensitive and that MCF-7/HER2 cells were relatively resistant to tamoxifen, while leptin co-administration 'rescued' MCF-7 and, especially, MCF-7/HER2 cells from the anti-proliferative effect of tamoxifen. The cell lines also exhibited a different phosphorylation status of STAT3, a transcription factor that is activated by the obesity related leptin receptor b (Ob-Rb). Most importantly, chromatin immunoprecipitation assays revealed differential STAT3 binding to the anti-apoptotic BCL2 and pro-apoptotic WWOX gene promoters in MCF-7 and MCF-7/HER2 cells, leading to concomitant modifications of its mRNA/protein expression levels, thus providing a selective advantage to HER2 over-expressing MCF-7/HER2 cells after treatment with tamoxifen and tamoxifen plus leptin. CONCLUSIONS: Our study provides novel evidence indicating that synergy between the leptin/Ob-Rb/STAT3 signalling pathway and the HER2 receptor protects tamoxifen-treated HER2 over-expressing cells from the inhibitory effect of tamoxifen through differential regulation of apoptosis-related genes.


Subject(s)
Apoptosis/drug effects , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm/drug effects , Leptin/metabolism , Receptor, ErbB-2/metabolism , Apoptosis/physiology , Blotting, Western , Cell Line, Tumor , Chromatin Immunoprecipitation , Drug Resistance, Neoplasm/physiology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leptin/pharmacology , Real-Time Polymerase Chain Reaction , STAT3 Transcription Factor/metabolism , Selective Estrogen Receptor Modulators/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Tamoxifen/pharmacology
3.
Biochem Biophys Res Commun ; 430(2): 494-9, 2013 Jan 11.
Article in English | MEDLINE | ID: mdl-23237804

ABSTRACT

Osteoarthritis (OA) is a debilitating disease of the joints characterized by cartilage degradation but to date there is no available pharmacological treatment to inhibit disease progression neither is there any available biomarker to predict its development. In the present study, we examined the expression level and possible involvement of novel cell-ECM adhesion-related molecules such as Iintegrin Linked Kinase (ILK), PINCH, parvin, Mig-2 and Migfilin in OA pathogenesis using primary human articular chondrocytes from healthy individuals and OA patients. Our findings show that only ILK and Migfilin were upregulated in OA compared to the normal chondrocytes. Interestingly, Migfilin silencing in OA chondrocytes rather exacerbated than ameliorated the osteoarthritic phenotype, as it resulted in even higher levels of catabolic and hypertrophic markers while at the same time induced reduction in ECM molecules such as aggrecan. Furthermore, we also provide a link between Migfilin and ß-catenin activation in OA chondrocytes, showing Migfilin to be inversely correlated with ß-catenin. Thus, the present study emphasizes for the first time to our knowledge the role of Migfilin in OA and highlights the importance of cell-ECM adhesion proteins in OA pathogenesis.


Subject(s)
Cell Adhesion Molecules/metabolism , Chondrocytes/metabolism , Cytoskeletal Proteins/metabolism , Osteoarthritis/metabolism , beta Catenin/biosynthesis , Cell Adhesion Molecules/genetics , Cells, Cultured , Chondrocytes/pathology , Cytoskeletal Proteins/genetics , Disease Progression , Extracellular Matrix/metabolism , Gene Silencing , Humans , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 13/genetics , Osteoarthritis/pathology , Phenotype , RNA, Messenger/biosynthesis , Up-Regulation
4.
Int J Radiat Biol ; 87(6): 609-21, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21627565

ABSTRACT

PURPOSE: Telomerase activity (TA), frequently observed in cancer, compensates for telomere shortening thus preventing cell senescence and conferring resistance to therapy. In the present study, we investigated the expression of human telomerase reverse transcriptase (hTERT) and TA and their regulation, as well as apoptotic rates and correlation with the presence of human epidermal growth factor receptor 2 (HER2), in irradiated tumour-derived breast cancer cells. MATERIALS AND METHODS: In 50 breast cancer tissue samples hTERT mRNA expression and TA were correlated with cell features (HER2, Estrogen and Progesterone Receptor status). Cells from six samples were then irradiated with 10 and 20 Gy; apoptotic rates were measured by flow cytometry, hTERT mRNA expression by real-time polymerase chain reaction and TA by telomeric repeat amplification protocol assay, at 24-144 h post-irradiation. Chromatin immunoprecipitation was performed to investigate hTERT and cellular-myelocytomatosis (c-myc) promoters' activity. HER2 gene knockdown was performed using small interfering RNA technology. RESULTS: hTERT/TA were found increased only in irradiated HER2-positive cells, which were found to be more radioresistant, while HER2 knockdown led to hTERT/TA downregulation. HER2 was found to mediate hTERT expression through activation of Nuclear Factor-kappa B (NF-κB) and c-myc. CONCLUSIONS: The present study suggests that following irradiation, HER2 receptor activates hTERT/telomerase, increasing the breast cancer cells' survival potential, through sequential induction of transcription factors NF-κΒ and c-myc.


Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , NF-kappa B/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Receptor, ErbB-2/metabolism , Telomerase/metabolism , Apoptosis , Chromatin Immunoprecipitation , Female , Humans , Phenotype , RNA, Messenger/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Telomere/metabolism , Time Factors
5.
J Cell Mol Med ; 15(7): 1542-50, 2011 Jul.
Article in English | MEDLINE | ID: mdl-20716114

ABSTRACT

Radiotherapy is an important treatment modality against cancer resulting in apoptosis and inhibition of cell growth. Survivin is an important cancer biomarker conferring to tumour cells increased survival potential by inhibiting apoptosis. In the present study, we investigated the implication of breast cancer cells features, as hormone receptors and p53 status, in the radio-resistance of breast cancer cells and in the regulation of survivin's expression by nuclear factor (NF)-κB and c-myc. Six breast cancer cell lines Michigan Cancer Foundation (MCF-7), MCF-7/Human Epidermal Growth Factor Receptor (HER)2, M. D. Anderson - Metastatic Breast (MDA-MB-231), SK-BR-3, BT-474 and Human Breast Lactating (HBL-100) were irradiated and cell viability as well as cell cycle distribution were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Survivin mRNA and protein levels were evaluated by real time PCR and Western blot analysis. Survivin and HER2 gene knockdown was performed with siRNA technology and investigation of transcription factors binding to survivin and c-myc gene promoters was assessed by chromatin immunoprecipitation. Student's t-test and F-statistics were used for statistical evaluation. Our results demonstrated that only HER2(+) breast cancer cells up-regulated survivin upon irradiation, whereas HER2 knockdown in HER2(+) cells led to survivin's down-regulation. Survivin and especially HER2 knockdown abolished the observed G2/M cell cycle checkpoint and reduced the radio-resistance of HER2 overexpressing breast cancer cells. Additionally, HER2 was found to regulate survivin's expression through NF-κB and c-myc transcription factors. This study revealed the significance of HER2 in the radio-resistance of HER2(+) breast cancer cells through induction of transcription factors NF-κB and c-myc, leading to activation of survivin, a downstream target oncogene preventing apoptosis.


Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Gene Expression Regulation, Neoplastic , Inhibitor of Apoptosis Proteins/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Receptor, ErbB-2/metabolism , Cell Line, Tumor/radiation effects , Female , Gene Knockdown Techniques , Humans , Inhibitor of Apoptosis Proteins/genetics , NF-kappa B/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Receptor, ErbB-2/genetics , Survivin , Transcription Factors/metabolism
6.
Int J Oncol ; 35(5): 1141-9, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19787269

ABSTRACT

Cancer cell characteristics may play a pivotal role in the response to therapy by activating or deactivating different molecular pathways. In the present study, we investigated the implication of breast cancer cell features, such as HER2 and p53 in the activation of telomerase upon exposure to ionizing radiation. Telomerase is among the most important cancer biomarkers, conferring to tumor cells unlimited proliferative capacity, increased survival potential and resistance to several types of cellular stress. We investigated possible mechanisms regulating telomerase in six irradiated breast cancer cell lines (MCF-7, MCF-7/HER2, MDA-MB-231, SK-BR-3, BT-474 and HBL-100) differing in their HER2, p53 and ERalpha status. hTERT mRNA expression was evaluated by real-time PCR and telomerase activity by the TRAP assay. HER2, c-myc, p53 and p21 protein levels were evaluated by Western blotting. Silencing of hTERT and HER2 was achieved by small interfering RNA technology. Chromatin immunoprecipitation was used to evaluate H3 histone acetylation status, as well as myc/mad/max and p53 transcription factors interaction with the hTERT promoter. Our results showed for the first time, that only HER2-positive cells, independently of their p53 status, upregulated hTERT/telomerase, while knockdown of hTERT increased radio-sensitivity. Knockdown of HER2 also led to increased radio-sensitivity and downregulation of hTERT/telomerase. We also demonstrated that c-myc and mad1 regulate hTERT expression in all irradiated breast cancer cells. We conclude, for the first time, that HER2 phenotype upregulates hTERT through c-myc activation and confers radio-resistance to breast cancer cells.


Subject(s)
Breast Neoplasms/genetics , Gene Expression/radiation effects , Radiation Tolerance/genetics , Receptor, ErbB-2/genetics , Telomerase/biosynthesis , Tumor Suppressor Protein p53/genetics , Biomarkers, Tumor/radiation effects , Blotting, Western , Breast Neoplasms/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/radiation effects , Cell Line, Tumor , Chromatin Immunoprecipitation , Female , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/radiation effects , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/radiation effects , RNA, Small Interfering , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/radiation effects , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/genetics , Telomerase/radiation effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/radiation effects , Up-Regulation
SELECTION OF CITATIONS
SEARCH DETAIL
...