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1.
Cell Signal ; 34: 76-85, 2017 06.
Article in English | MEDLINE | ID: mdl-28302567

ABSTRACT

Breast cancer is the most frequent malignancy among women worldwide. We have described the expression of α2-adrenoceptors in breast cancer cell lines, associated with increased cell proliferation and tumor growth. A mitogenic autocrine/paracrine loop of prolactin (Prl) has been described in breast cancer cells. We hypothesized that the α2-adrenergic enhancement of proliferation could be mediated, at least in part, by this Prl loop. In both T47D and MCF-7 cell lines, the incubation with the α2-adrenergic agonist dexmedetomidine significantly increased Prl release into the culture medium (measured by the Nb2 bioassay), this effect being reversed by the α2-adrenergic antagonist rauwolscine. No change in Prl receptors (PrlR) was observed by RT-qPCR in these cell lines. In IBH-6 cells a decrease in Prl secretion was observed at the lower dexmedetomidine concentration. The signaling pathways involved in ovine Prl (oPrl) and dexmedetomidine action were also assessed. Both compounds significantly activated STAT5 and ERK in all three cell lines. In T47D and MCF-7 cell lines also AKT was activated by both Prl and dexmedetomidine. We therefore describe the STAT5 phosphorylation by an α2-adrenergic agonist, dexmedetomidine. In T47D cells, the α2-adrenergic stimulation of cell proliferation is probably mediated, at least in part, by the Prl autocrine/paracrine loop, because this effect is abrogated by the specific PrlR antagonist Δ1-9-G129R-hPrl. The implication of Prl loop describes a novel mechanism of action of this GPCR.


Subject(s)
Adrenergic alpha-2 Receptor Agonists/pharmacology , Dexmedetomidine/pharmacology , Prolactin/pharmacology , Signal Transduction/drug effects , Animals , Autocrine Communication/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cattle , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , MCF-7 Cells , Microscopy, Fluorescence , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , STAT5 Transcription Factor/metabolism
2.
Oncotarget ; 7(37): 60133-60154, 2016 09 13.
Article in English | MEDLINE | ID: mdl-27507057

ABSTRACT

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits.Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/ß-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1.Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.


Subject(s)
Breast Neoplasms/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Glypicans/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Glypicans/metabolism , Humans , MCF-7 Cells , Mice, Nude , RNA Interference , Transplantation, Heterologous , Tumor Burden/genetics , Zinc Finger E-box-Binding Homeobox 1/genetics , Zinc Finger E-box-Binding Homeobox 1/metabolism
3.
J Cell Physiol ; 222(3): 596-605, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19957299

ABSTRACT

Epithelial cadherin (E-cadherin) is a 120 kDa cell-cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as beta-catenin. Loss of E-cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down-regulation of E-cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH-6 and IBH-4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E-cadherin and related proteins in IBH-6 and IBH-4 cell lines was evaluated. In IBH-6 and IBH-4 cell extracts, only an 89 kDa E-cadherin form (Ecad89) was detected, which is truncated at the C-terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E-cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH-6 and IBH-4 cells showed an intracellular scattered E-cadherin localization. beta-catenin accompanied E-cadherin localization, and actin stress fibers were identified in both cell types. E-cadherin mRNA levels were remarkably low in IBH-6 and IBH-4 cells. The E-cadherin mRNA and genomic sequence encoding exons 14-16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up-regulation of Twist mRNA was found in both cell lines. In conclusion, IBH-6 and IBH-4 breast cancer cells show down-regulation of E-cadherin expression with aberrant protein localization, and up-regulation of Twist; these features can be related to their invasive/metastatic characteristics.


Subject(s)
Breast Neoplasms/metabolism , Cadherins/metabolism , Carcinoma, Ductal, Breast/metabolism , Actins/metabolism , Antigens, CD , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cadherins/genetics , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Cell Line, Tumor , Exons , Female , Gene Expression Regulation, Neoplastic , Humans , Ion Channels , Membrane Glycoproteins/metabolism , Microfilament Proteins , Microscopy, Fluorescence , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stress Fibers/metabolism , Twist-Related Protein 1/metabolism , beta Catenin/metabolism
4.
J Steroid Biochem Mol Biol ; 110(1-2): 170-5, 2008 May.
Article in English | MEDLINE | ID: mdl-18486470

ABSTRACT

Catecholestrogens are estrogen metabolites formed by hydroxylation of 17beta-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via alpha(2)-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E(2)), 2-hydroxy-estrone (2-OH-E(1)) and 4-hydroxy-estrone (4-OH-E(1)) competed for [(3)H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E(2)) did not. The contribution of alpha(2)-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific alpha(2)-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E(2). The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the alpha(2)-adrenoceptors and ERs for 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1) was mixed, whereas for 4-OH-E(2), the only receptor implied was an ER. In MDA-MB-231 cells (ER-alpha negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that alpha(2)-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1).


Subject(s)
Cell Proliferation/drug effects , Estrogens, Catechol/pharmacology , Receptors, Adrenergic, alpha-2/physiology , Adrenergic alpha-2 Receptor Antagonists , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Estradiol/analogs & derivatives , Estradiol/pharmacology , Fulvestrant , Humans , Protein Binding/drug effects , Receptors, Adrenergic, alpha-2/metabolism , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/metabolism , Receptors, Estrogen/physiology , Yohimbine/pharmacology
5.
J Immunol ; 174(11): 7022-32, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15905545

ABSTRACT

Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.


Subject(s)
Cathepsins/physiology , Cysteine Endopeptidases/physiology , Extracellular Matrix Proteins/biosynthesis , Lymphoid Tissue/immunology , Lymphoid Tissue/metabolism , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cathepsin L , Cathepsins/deficiency , Cathepsins/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Movement/genetics , Cell Movement/immunology , Cell Proliferation , Cysteine Endopeptidases/deficiency , Cysteine Endopeptidases/genetics , Down-Regulation/genetics , Extracellular Matrix Proteins/antagonists & inhibitors , Glycoproteins/biosynthesis , Integrin alpha5/biosynthesis , Integrin alpha6/biosynthesis , Integrin beta1/biosynthesis , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocyte Count , Lymphoid Tissue/cytology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Mutant Strains , Thymus Gland/metabolism , Up-Regulation/genetics
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