Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro

Overexpression of the small heat shock protein Hsp27 has been shown by us to inhibit the in vitro proliferation rate and to delay tumor development of a human melanoma cell line (A375) in nude mice. We hypothesized that Hsp27 may influence the neoplastic phenotype. In the present study Hsp27 transfectants from this cell line were analyzed for various cellular aspects associated with the metastatic process. We found that Hsp27-overexpressing clones exhibited an altered cellular morphology as compared with control transfected cells. The Hsp27-positive cells tended to develop an epithelial-like phenotype growing in clusters and were characterized by a loss of transcytoplasmic stressfibers. In parallel, Hsp27-expressing cells lost the ability to form colonies in soft agar. The invasive potential was studied in vitro by the use of a reconstituted extracellular matrix-coated filter (Matrigel). Compared with controls, Hsp27-overexpressing cells showed decreased cell invasiveness through Matrigel. A correlation between invasion and activation of matrix metalloproteinases (MMPs) has been shown in several cell models. Secretion of MMPs (MMP-2 and MMP-9) was studied by gelatin-substrate zymogram analysis, as well as by a sensitive gelatinase activity assay. The Hsp27-transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 as compared with the control transfected cells. Integrins are adhesion receptors and function in cell invasion by mediating cell movement on matrix molecules and by regulating the expression of MMPs. Both fluorescence-activated cell sorter analysis and immunofluorescence analysis revealed a loss of alpha(v)beta3 integrin in Hsp27-transfected cell colonies. Our results demonstrate that Hsp27 overexpression has a profound impact on several parameters regulating the invasive and metastatic potential of melanoma cells in vitro.     

Reversal of melanocytic malignancy by keratinocytes is an E-cadherin-mediated process overriding beta-catenin signaling

Loss of E-cadherin in melanoma cells frees them from keratinocytes-mediated proliferation and phenotypic control, which can be restored by forced E-cadherin expression. In this study, E-cadherin and its derivatives were introduced into metastatic melanoma line 1205Lu. E-cadherin and E-cadherin-alpha-catenin fusion protein were functional in mediating cell adhesion, downregulating MCAM(4) in coculture, and inhibiting proliferation regardless of beta-catenin expression levels and activation status. In contrast, cytoplasmic domain-deleted (E-cadDeltaCYT) derivative was not able to reverse malignancy. The results indicate that E-cadherin-mediated cell adhesion is required for keratinocyte-mediated control of melanocytic cells, which can override proliferative activity of beta-catenin.     

MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes

Molecular and Cellular Biochemistry - We investigated for the first time the expression of melanoma cell adhesion molecule (MCAM) and its involvement in the differentiation of 3T3-L1 fibroblasts to...     

The effects of patchouli alcohol and combination with cisplatin on proliferation,apoptosis and migration in B16F10 melanoma cells

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G₀/G₁ phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

Connexin 32 down-regulates the fibrinolytic factors in metastatic renal cell carcinoma cells

Fibrinolytic factors have an important role in tumor progression through the degradation of extracellular matrix. The increased levels of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR) and type-1 PA inhibitor (PAI-1) are reported in human renal cell carcinoma (RCC). Connexin (Cx) gene, a member of gap junction, is known to act as a tumor suppressor gene. We have reported that Cx32 improves malignant phenotypes of metastatic RCC cells via the inhibition of Src-dependent signaling. In this study, we examined the effect of expression of Cx32 gene on the production of uPA, uPAR and PAI-1, and on the induction of PAI-1 stimulated by hypoxia in a human metastatic RCC cell line, Caki-1 cells. Cx32 expression decreased both mRNA level and production of PAI-1, uPA and uPAR in Caki-1 cells. Cx32 also decreased hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha mRNA level. PP1, a Src inhibitor, significantly decreased PAI-1, uPA, uPAR and HIF-alpha mRNA levels in Caki-1 cells. Furthermore, Cx32 suppressed the induction of HIF-2alpha protein in Caki-1 cells under hypoxia. PAI-1 mRNA level in Cx32-transfected Caki-1 cells was lower than that of mock transfectant under hypoxic conditions. These results suggest that Cx32 might reduce PAI-1, uPA and uPAR production in metastatic RCC cells via the inhibition of Src-dependent induction of HIF-1alpha and HIF-2alpha gene expression and that Cx32 might suppress hypoxia-inducible gene expression under hypoxic conditions.     

CA125/MUC16 interacts with Src family kinases,and over-expression of its C-terminal fragment in human epithelial cancer cells reduces cell–cell adhesion

MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and β-catenin at the cell–cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca²⁺-dependent cell–cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/β-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell–cell adhesion of epithelial cancer cells.     

Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.     

Down-regulation of MUC1 in cancer cells inhibits cell migration by promoting E-cadherin/catenin complex formation

MUC1, a tumor associated glycoprotein, is over-expressed in most cancers and can promote proliferation and metastasis. The objective of this research was to study the role of MUC1 in cancer metastasis and its potential mechanism. Pancreatic (PANC1) and breast (MCF-7) cancer cells with stable 'knockdown' of MUC1 expression were created using RNA interference. beta-Catenin and E-cadherin protein expression were upregulated in PANC1 and MCF-7 cells with decreased MUC1 expression. Downregulation of MUC1 expression also induced beta-catenin relocation from the nucleus to the cytoplasm, increased E-cadherin/beta-catenin complex formation and E-cadherin membrane localization in PANC1 cells. PANC1 cells with 'knockdown' MUC1 expression had decreased in vitro cell invasion. This study suggested that MUC1 may affect cancer cell migration by increasing E-cadherin/beta-catenin complex formation and restoring E-cadherin membrane localization.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.