Lens placode modulates extracellular matrix formation during early eye development.

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

Downregulation of metallothionein 2A reduces migration, invasion and proliferation activities in human squamous cell carcinoma cells.

BACKGROUND: The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma. METHODS: Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays. RESULTS: CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α. CONCLUSIONS: MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

Laminin-derived peptide C16 regulates Tks expression and reactive oxygen species generation in human prostate cancer cells.

Laminin peptides influence cancer biology. We investigated the role of a laminin-derived peptide C16 regulating invadopodia molecules in human prostate cancer cells (DU145). C16 augmented invadopodia activity of DU145 cells, and stimulated expression Tks4, Tks5, cortactin, and membrane-type matrix metalloproteinase 1. Reactive oxygen species generation is also related to invadopodia formation. This prompted us to address whether C16 would induce reactive oxygen species generation in DU145 cells. Quantitative fluorescence and flow cytometry showed that the peptide C16 increased reactive oxygen species in DU145 cells. Furthermore, significant colocalization between Tks5 and reactive oxygen species was observed in C16-treated cells. Results suggested that the peptide C16 increased Tks5 and reactive oxygen species in prostate cancer cells. The role of C16 increasing Tks and reactive oxygen species are novel findings on invadopodia activity.

ADAMTS-1 disrupts HGF/c-MET signaling and HGF-stimulated cellular processes in fibrosarcoma.

Extracellular matrix (ECM) serves as a reservoir for biologically active factors, such as growth factors and proteases that influence the tumor cell behavior. ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a secreted protease that has the ability to modify the ECM during physiological and pathological processes. Here, we analyzed the role played by ADAMTS-1 regulating HGF and TGF-ß1 activities in the high-grade fibrosarcoma cell line (HT1080). We generated HT1080 and HEK293T cells overexpressing ADAMTS-1. HT1080 cells overexpressing ADAMTS-1 (HT1080-MPA) exhibited a significant decrease in cell proliferation and migration velocity, both in presence of HGF. We obtained similar results with ADAMTS-1-enriched conditioned medium from other cell type. However, ADAMTS-1 overexpression failed to affect TGF-ß1 activity associated with HT1080 cell proliferation and migration velocity. Immunoblotting showed that ADAMTS-1 overexpression disturbs c-Met activation upon HGF stimulation. Downstream ERK1/2 and FAK signaling pathways are also influenced by this protease. Additionally, ADAMTS-1 decreased the size of the fibrosarcospheres, both under normal conditions and in the presence of HGF. Likewise, in presence of HGF, ADAMTS-1 overexpression in HT1080 disrupted microtumors formation in vivo. These microtumors, including individual cells, presented characteristics of non-invasive lesions (rounded morphology). Our results suggest that ADAMTS-1 is involved in regulating HGF-related functions on fibrosarcoma cells. This protease may then represent an endogenous mechanism in controlling the bioavailability of different growth factors that have a direct influence on tumor cell behavior.

The laminin-derived peptide C16 regulates GPNMB expression and function in breast cancer.

Breast cancer is an important public health problem, and its progression may be related to the extracellular matrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECM proteins regulating tumor biology. The laminin-derived peptide C16 regulates different properties of tumor cells. Here we analyzed C16-induced differential gene expression in MDA-MB-231 breast cancer cells. MCF-10A normal-like breast cells served as control. Among different cancer-related genes, C16 induced overexpression of GPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non-metastatic B), involved with malignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene and protein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancer cells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highly aggressive MDA-MB-231 cancer cells. We addressed regulatory mechanisms involved in C16-mediated increase of GPNMB protein levels in MDA-MB-231 cells, and observed that C16 stimulates ß1 integrin and Src phosphorylation. Furthermore, Src inhibition decreases peptide-induced GPNMB expression levels. To contextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer human tissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed in breast cancer compared to normal tissue. We concluded that laminin-derived peptide C16 regulates gene and protein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion of highly aggressive MDA-MB-231 cells, probably through Src signaling. GPNMB presented increased expression in breast cancer in vivo compared to normal breast tissue.

Role of HIF-1α and CASPASE-3 in cystogenesis of odontogenic cysts and tumors.

OBJECTIVES: Odontogenic cysts and tumors are the most relevant lesions that affect the gnathic bones. These lesions have in common the formation of cystic areas and this common feature may suggest involvement of similar mechanisms. The hypoxia inducible factor 1 alpha (HIF-1α), a responsive protein to hypoxia and caspase-3, an irreversible apoptosis marker, may contribute to cyst formation. Thus, this study aimed to investigate the immunoexpression of these proteins in odontogenic cysts and tumors. MATERIAL AND METHODS: Twenty cases of ameloblastoma, keratocystic odontogenic tumor (KOT) (n = 20), radicular cyst (RC) (n = 18), dentigerous cyst (DC) (n = 11), calcifying cystic odontogenic tumor (n = 8), and dental follicle (DF) (n = 10) were used to investigate HIF-1α and caspase-3 expression in sequential serial cuts by immunohistochemistry. RESULTS: HIF-1α was overexpressed in RC, DC, and ameloblastoma when compared with DF. The basal and sometimes the lower suprabasal layer showed no or very low expression in DC, KOT, and ameloblastoma, the last also showing strong expression in solid epithelial areas and initial cystic formation regions. Caspase-3 was found to be overexpressed in all lesions, with the highest expression in odontogenic cysts compared to tumors. HIF-1α and caspase-3 were localized in similar areas of the same lesions, especially in the epithelium surrounding cystic formations. CONCLUSIONS: This study showed distinct immunoexpression of HIF-1α and caspase-3 in odontogenic cyst and tumors, with higher expression observed in odontogenic cysts. CLINICAL RELEVANCE: These findings suggest a possible correlation between hypoxia, apoptosis, and cystogenesis, leading to understand the mechanisms responsible to cystic formation in odontogenic lesions.

ß2-Adrenoceptor is involved in connective tissue remodeling in regenerating muscles by decreasing the activity of MMP-9.

We investigated the role of ß2-adrenoceptors in the connective tissue remodeling of regenerating muscles from ß2-adrenoceptor knockout (ß2KO) mice. Tibialis anterior muscles from ß2KO mice were cryolesioned and analyzed after 3, 10, and 21 days. Regenerating muscles from ß2KO mice showed a significant increase in the area density of the connective tissue and in the amount of collagen at 10 days compared with wild-type (WT) mice. A greater increase occurred in the expression levels of collagen I, III, and IV in regenerating muscles from ß2KO mice evaluated at 10 days compared with WT mice; this increase continued at 21 days, except for collagen III. Matrix metalloproteinase (MMP-2) activity increased to a similar extent in regenerating muscles from both ß2KO and WT mice at 3 and 10 days. This was also the case for MMP-9 activity in regenerating muscles from both ß2KO and WT mice at 3 days; however, at 10 days post-cryolesion, this activity returned to baseline levels only in WT mice. MMP-3 activity was unaltered in regenerating muscles at 10 days. mRNA levels of tumor necrosis factor-α increased in regenerating muscles from WT and ß2KO mice at 3 days and, at 10 days post-cryolesion, returned to baseline only in WT mice. mRNA levels of interleukin-6 increased in muscles from WT mice at 3 days post-cryolesion and returned to baseline at 10 days post-cryolesion but were unchanged in ß2KO mice. Our results suggest that the ß2-adrenoceptor contributes to collagen remodeling during muscle regeneration by decreasing MMP-9 activity.

EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.

Expression of molecules related to AKT pathway as putative regulators of ameloblastoma local invasiveness.

BACKGROUND: Ameloblastoma is an odontogenic neoplasm with local invasiveness and high recurrence. We previously suggested that growth factors, matrix metalloproteinases (MMPs), and TIMPs influence ameloblastoma invasiveness (Pathol. Res. Pract., 208, 2012, 225; Oral. Surg. Oral. Med. Oral. Pathol. Oral Radiol. Endod., 111, 2011, 474). Signals generated by this molecular network would be transduced by ERK 1/2 pathway (Oral. Surg. Oral. Med. Oral. Pathol. Oral Radiol. Endod., 111, 2011, 474). Others signaling pathways may influence ameloblastoma biology. Here, we studied expression of AKT and related molecules in ameloblastoma. METHODS: Fourteen cases of solid/multicystic ameloblastomas were examined. Immunohistochemistry was carried out to detected AKT (phospho-AKT), NF-қB (phospho-NF-қB), ß-catenin, cyclin-D1, and COX-2 in ameloblastoma samples. These molecules were evaluated in neoplastic cells and stroma. RESULTS: All proteins were detected in ameloblastoma. Expression of these markers was quantified and compared. Spearman's rank test was carried out to address positive correlations between proteins (P < 0.05). Ameloblastoma had a significant positive correlation of AKT (phospho-AKT) with ß-catenin. ß-catenin correlated with Cyclin-D1 and COX-2 in neoplastic cells. AKT (phospho-AKT) correlated with ß-catenin; ß-catenin with Cyclin-D1; AKT (phospho-AKT) with NF-қB (phospho-NF-қB); and NF-қB (phospho-NF-қB) with COX-2 in stromal cells. CONCLUSIONS: Results suggest that proteins studied are present and probably involved in a functional pathway in neoplastic cells and stroma and may therefore influence the local invasiveness of ameloblastoma.

Lens Placode Modulates Extracellular Matrix Formation During Early Eye Development.

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2 , a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion.

BACKGROUND: ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. RESULTS: In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. CONCLUSIONS: ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line.

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. ß1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through ß1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour.

AIMS: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness. The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. METHODS AND RESULTS: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. CONCLUSIONS: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta1 integrin.

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and beta1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Progesterone decreases ovarian cancer cells migration and invasion.

The progression of cancer depends on the interaction between the cells and their microenvironment. Progesterone is a steroid and progestogen sex hormone produced by the corpus luteum, which is a transitory endocrine gland in female mammals and prepares the endometrium for implantation. Also, progesterone is involved in antitumorigenic process in different types of cancer. Our goal is to investigate the role of progesterone in cell invasion and migration. Ovarian cells were treated with different concentrations of progesterone. 500 nM or 1 µM progesterone decreased the migration of the cells in 24 h or less without affecting the viability. Immunoblot showed that treatment with 1 µM progesterone decreased the phosphorylated forms of Src and FAK, and the cells were less polarized. Our results suggest that progesterone interferes with migration and invasion of ovarian cells. Inhibitory experiments inferred the progesterone receptor playing a role in migration and invasion. Decreased phosphorylation of molecules involved in these processes was also found.

Expression patterns of cell adhesion molecules in mice's lung after administration of meso-2,3-dimercaptosuccinic acid-coated maghemite nanoparticles.

We studied the expression pattern of cell adhesion molecules associated to transendothelial migration of leukocytes in different lung's vascular compartments after administration of a magnetic fluid sample containing maghemite nanoparticles surface-coated with meso-2,3-dimercaptosuccinic acid. The analyses were conducted in mice 4 and 12 h after endovenous administration of the magnetic fluid in control mice. Firstly, the migratory activity of leukocytes after magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration was confirmed using broncho-alveolar lavage and light microscopy. Then, the expression of cell adhesion molecules in the lung's vascular compartments was investigated by immunofluorescence microscopy of frozen sections, using antibodies against L-selectin, P-selectin, E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1. L- and P-selectin showed similar pattern of expression in the pulmonary vasculature in animals treated with magnetic fluid and in the control group. In contrast, macrophage antigen-1 and leukocyte function associated antigen-1 were found in capillary only in animals treated with magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration. In addition, after magnetic fluid administration E-selectin was found in post-capillary sites. Our findings demonstrated that magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration exhibits modulation effects on expression patterns of E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1 in the lung's vascular compartments. These findings are very important in a strategy to reduce the potential toxicity of magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration for medical applications.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment.

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted Hs578T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta1 integrin.

We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.

Secretion of collagen I and tenascin is modulated by laminin-111 in 3D culture of human adenoid cystic carcinoma cells.

Adenoid cystic carcinoma is a frequent malignant salivary gland neoplasm with high levels of recurrence and metastasis. This neoplasm expresses prominent extracellular matrix (ECM). We are studying regulatory mechanisms underlying secretion of ECM molecules in adenoid cystic carcinoma. We have previously demonstrated that laminin modulates the phenotype of a human adenoid cystic carcinoma (CAC2) cell line. Thus, this molecule would be a good candidate to regulate secretion of ECM molecules in these cells. Here we analysed the role played by laminin-111 [formerly laminin-1; Aumailley et al. (2005). Matrix Biol. 24, 326] stimulating secretory activity of CAC2 cells. Three-dimensional cultures of cells in laminin-111 (treated) or agarose (controls) were studied by light and electron microscopy. Ultrastructural analysis of CAC2 cells grown within laminin-111 showed pseudocysts filled with secretory-like material. Cells exhibited prominent and dilated endoplasmic reticulum and coated and uncoated vesicles. Ultrastructural findings suggested that laminin-111 induced secretory activity in CAC2 cells. We further investigated this point by light microscopy, immunofluorescence and confocal microscopy. Histochemistry showed periodic acid-Schiff (PAS)-positive diastase-resistant material in CAC2 cells treated by laminin-111. This material could represent laminin-induced secretion of ECM molecules. We searched for collagen I and tenascin in CAC2 cells treated by laminin-111. Confocal microscopy and immunoblot showed that laminin-111 enhanced secretion of collagen I and tenascin in CAC2 cells. We suggest that laminin-111 modulates secretion of collagen I and tenascin in cells derived from human adenoid cystic carcinoma.