New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT.

Further improvements in Photodynamic therapy (PDT) necessitate that the dye targets more selectively tumour tissues or neovascularization than healthy cells. Different enzymes such as matrix metalloproteinases (MMPs) are overexpressed in tumour areas. Among these MMPs, gelatinases (MMP-2 and MMP-9) and its activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of many cancers such as glioblastoma multiforme (GBM), an aggressive malignant tumour of the brain. These last years, the concept of photodynamic molecular beacons (PMB) became interesting for controlling the photosensitizer's ability to generate singlet oxygen (1O2) close to target biomolecules as MMPs. We report herein novel PMBs triggered by MMP-2 and/or MMP-9 and/or MMP-14, comprising a photosensitizer and a singlet oxygen quencher linked by MMP cleavable peptide linker (H-GRIGFLRTAKGG-OH). First of all, we focused on the synthesis and the photophysical study of different derivatives photosensitizer-peptide. This preliminary work concluded on an influence of the nature and the distance from the peptide, but not of the position of the photosensitizer in these derivatives on the proteolytic enzymatic action. The nature of the quencher used (a blackberry quencher (BBQ-650) or a black hole quencher (BHQ3)) does not influence the enzymatic action. We also studied the influence of an additional PEG spacer. Finally, the synthesis, the singlet oxygen quenching efficiency and the enzymatic activation of these new MMP- cleavable-PMBs were compared.

Differential MMP-14 Targeting by Lumican-Derived Peptides Unraveled by In Silico Approach.

Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), displays anti-tumor properties through its direct interaction with MMP-14. Lumican-derived peptides, such as lumcorin (17 amino acids) or L9M (10 amino acids), are able to inhibit the proteolytic activity of MMP-14 and melanoma progression. This work aimed to visualize the interactions of lumican-derived peptides and MMP-14. Molecular modeling was used to characterize the interactions between lumican-derived peptides, such as lumcorin, L9M, and cyclic L9M (L9Mc, 12 amino acids), and MMP-14. The interaction of L9Mc with MMP-14 was preferential with the MT-Loop domain while lumcorin interacted more with the catalytic site. Key residues in the MMP-14 amino acid sequence were highlighted for the interaction between the inhibitory SLRP-derived peptides and MMP-14. In order to validate the in silico data, MMP-14 activity and migration assays were performed using murine B16F1 and human HT-144 melanoma cells. In contrast to the HT-144 melanoma cell line, L9Mc significantly inhibited the migration of B16F1 cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. L9Mc significantly inhibited the proliferation of B16F1 but not of HT-144 cells in vitro and primary melanoma tumor growth in vivo. Thus, the site of interaction between the domains of MMP-14 and lumcorin or L9Mc were different, which might explain the differences in the inhibitory effect of MMP-14 activity. Altogether, the biological assays validated the prediction of the in silico study. Possible and feasible improvements include molecular dynamics results.

LRP-1 Matricellular Receptor Involvement in Triple Negative Breast Cancer Tumor Angiogenesis.

BACKGROUND: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression. METHODS: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs' angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways. RESULTS: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs' angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-ß signaling and plasminogen/plasmin system. CONCLUSIONS: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

Contribution of the Low-Density Lipoprotein Receptor Family to Breast Cancer Progression.

The low-density lipoprotein receptor (LDLR) family comprises 14 single-transmembrane receptors sharing structural homology and common repeats. These receptors specifically recognize and internalize various extracellular ligands either alone or complexed with membrane-spanning co-receptors that are then sorted for lysosomal degradation or cell-surface recovery. As multifunctional endocytic receptors, some LDLR members from the core family were first considered as potential tumor suppressors due to their clearance activity against extracellular matrix-degrading enzymes. LDLRs are also involved in pleiotropic functions including growth factor signaling, matricellular proteins, and cell matrix adhesion turnover and chemoattraction, thereby affecting both tumor cells and their surrounding microenvironment. Therefore, their roles could appear controversial and dependent on the malignancy state. In this review, recent advances highlighting the contribution of LDLR members to breast cancer progression are discussed with focus on (1) specific expression patterns of these receptors in primary cancers or distant metastasis and (2) emerging mechanisms and signaling pathways. In addition, potential diagnosis and therapeutic options are proposed.

Crosstalk between adenosine receptor (A2A isoform) and ERalpha mediates ethanol action in MCF-7 breast cancer cells.

Alcohol consumption increases the risk of breast cancer but the underlying mechanisms are not well understood. We have shown previously that ethanol activates ER signalling pathway in a cAMP/PKA-mediated ligand-independent manner. Since the activation of A2A adenosine receptor (A2AAR) by ethanol has been reported in other cell types, here we tested if cross-talk between this Gs-coupled receptor and ERalpha could be involved in ethanol effects in breast cancer cells. Our study shows that A2AAR is expressed and functional in the hormone-dependent breast cancer cell line MCF-7. Interestingly, activation of this receptor by the selective agonist CGS21680 stimulates the transcription of progesterone receptor, a well known estrogen target gene. CGS21680 also stimulates the pEREtkLuc reporter activity in transfected MCF-7 cells, an effect antagonized by the antiestrogen ICI182,780. Moreover, CGS21680 stimulates the proliferation of MCF-7 cells similarly to E2. Finally, the A2AAR antagonist MSX-3 inhibits the ethanol-induced activation of ERalpha signalling pathway. These results demonstrate cross-talk between A2AAR and ERalpha that is involved in ethanol action. This could open new perspectives for the therapy of estrogen-dependent breast cancer.

Ethanol-induced ligand-independent activation of ERalpha mediated by cyclic AMP/PKA signaling pathway: an in vitro study on MCF-7 breast cancer cells.

Alcohol consumption is an increased risk factor for hormone-dependent breast cancer but the underlying molecular bases are unknown. Several studies suggest that ethanol could activate the estrogen signaling pathway. We have performed an in vitro study in order to investigate the molecular players involved in this phenomenon. Exposure of MCF-7 breast cancer cells to ethanol induced an increase in the mRNA level of two well known estrogen target genes: progesterone receptor (PR) and pS2. This result was confirmed by an increase in luciferase activity in pEREtkLuc-transfected MCF-7 cells exposed to ethanol. These effects, whose intensity was similar to those of E2, were observed also in steroid-free medium and were inhibited by the antiestrogen ICI 182,780. This suggested a ligand-independent activation of ERalpha that was confirmed by the absence of ERalpha proteolysis in ethanol-treated cells. Using PKA inhibitor (H89), the study of phospho-CREB by Western blot and transfection experiments with a CRE-reporter construct demonstrated that PKA was involved in ethanol-induced transcription of ERalpha target genes. Adenylyl cyclase inhibition impaired the activation of estrogen signaling pathway induced by ethanol. The results obtained in vitro, are discussed in regard to alcohol consumption and relevance to humans.

A new gallium complex inhibits tumor cell invasion and matrix metalloproteinase MMP-14 expression and activity.

In this study, we investigated the effect of [N-(5-chloro-2-hydroxyphenyl)-l-aspartato] chlorogallate (GS2), a new water soluble gallium complex, on cell invasion and on the expression and activity of matrix metalloproteinases (MMPs) in human metastatic HT-1080 fibrosarcoma and MDA-MB 231 breast carcinoma cells. The effect on cell invasion was studied using a modified Boyden chamber coated with a type-I collagen. We analyzed the effect of GS2 on MMP-2, MMP-9, and MMP-14 via zymography and enzymatic assay using high affinity fluorogenic substrates. The expression of MMP mRNA was analyzed via qRT-PCR. GS2 induced a decrease in cell invasion. A dose-dependent inhibition effect was observed on the activities of MMP-2, MMP-9, and MMP-14 with the IC50 values of 168, 82, and 20 µM, respectively. A decrease in the expression of MMP-14 mRNA was observed in both cell lines, whereas the expression of MMP-2 and MMP-9 mRNA was decreased only in the MDA-MB231 cells. Data obtained for the expression of MMP-14 mRNA were confirmed via Western blotting. In fact, MMP-14 expression was decreased in the presence of GS2. Overall, these data show that GS2 is a promising compound for anti-invasive and anticancer therapy.

Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts inhibitory activity against matrix metalloproteinases and cytokine-like effects. We previously showed that TIMP-1 reduces neurite outgrowth in mouse cortical neurons and that this cytokine-like effect depends on TIMP-1 endocytosis mediated by the low-density lipoprotein receptor-related protein-1 (LRP-1). To gain insight into the interaction between TIMP-1 and LRP-1, we considered conformational changes that occur when a ligand binds to its receptor. TIMP-1 conformational changes have been studied using biomolecular simulations, and our results provide evidence for a hinge region that is critical for the protein movement between the N- and C-terminal TIMP-1 domains. In silico mutants have been proposed on residues F12 and K47, which are located in the hinge region. Biological analyses of these mutants show that F12A or K47A mutation does not alter MMP inhibitory activity but impairs the effect of TIMP-1 on neurite outgrowth. Interestingly, these mutants bind to LRP-1 but are not endocytosed. We conclude that the intrinsic dynamics of TIMP-1 are not involved in its binding to LRP-1 but rather in the initiation of endocytosis and associated biological effects.

Ethanol stimulates the secretion of matrix metalloproteinases 2 and 9 in MCF-7 human breast cancer cells.

Alcohol consumption is a well-established risk factor for hormone-dependent breast cancer. In vitro studies performed to understand the mechanisms by which ethanol acts on breast cancer cells have shown that this compound stimulates both proliferation and migration. In the present study, we show by gelatin zymography that, when exposed to ethanol, MCF-7 human breast cancer cells display a higher amount of active metalloproteinases (MMP) 2 and 9 in their culture medium. This increase is somewhat higher than those observed in the case of 17beta-estradiol (E2) exposure. As expected, anti-estrogen ICI 182,780 inhibits the E2-induced overexpression of a well-known estrogen responsive gene, the progesterone receptor, in MCF-7 cells. ICI 182,780 also inhibits the E2-induced increase in MMP-2 and -9 secretion. Nevertheless, in the case of ethanol exposure, this ER ant-agonist was only efficient on MMP-9 secretion. In addition, although MMP-9 transcription was not sensitive to E2 or ethanol, MMP-2 transcription was stimulated in MCF-7 cells exposed to ethanol. Collectively, these results give new insights into the effects of alcohol on breast cancer cell migration, which are not due solely to an estrogen-like activity of alcohol.

Analysis of the effects of different alcohols on MCF-7 human breast cancer cells.

Alcohol consumption is known to be an increased risk factor for breast cancer, but the underlying molecular mechanisms are not well understood. We have recently shown that the exposure of MCF-7 breast cancer cells to 0.1% ethanol enhanced their proliferation and increased their content in both estrogen receptor-alpha (ERalpha) and aromatase. The aim of the present work was to determine if the effects of ethanol could be mimicked by other short-chain aliphatic alcohols such as methanol and 1-butanol. Our results show that these compounds do not stimulate MCF-7 cell proliferation. An increase in ERalpha content was observed by Western blot in methanol-treated cells, but this parameter was not affected in butanol-treated cells. Neither of these two alcohols induced an increase in aromatase mRNA level. So despite a similarity in molecular structure, these primary alcohols do not exert the same effects. Taken together, these results suggest that the increase in aromatase expression might be a key event required for the enhanced proliferation observed in the presence of ethanol.

Ethanol stimulates proliferation, ERalpha and aromatase expression in MCF-7 human breast cancer cells.

It is well documented that alcohol is associated with an increased risk factor for breast carcinogenesis although the underlying mechanisms are not clearly understood. It has been reported that in vitro, the culture of estrogen receptor (ER) expressing breast cancer cells in ethanol containing medium was associated with an increase in the proliferation rate, in the ERalpha content as well as in ER transcriptional activity. Since these changes are not observed in ER negative breast cancer cells, and since alcohol intake has been associated to an increased level of circulating estrogens, we have postulated that aromatase expression could be increased following ethanol exposure. The results of our studies show a 1.3-fold increase in cell proliferation after 6 days of culture of MCF-7 cells in the presence of 0.1% ethanol. This enhanced proliferation is confirmed by the use of clonogenic assays which show a 1.5-fold increase in clonal growth in the presence of 0.1% ethanol. No statistically significant changes were observed in the presence of higher ethanol concentration (0.3%). After a 6-day exposure to 0.1% ethanol, RT-PCR analyses reveal a 1.7-fold increase in ERalpha mRNA that was not significant, whereas western blot analyses show a significant 3.3-fold increase in ERalpha content. At the same stage, RT-PCR studies demonstrate a 2.4-fold increase in aromatase mRNA level which is confirmed at the protein level by western blots performed after immuno-precipitation of the enzyme. Taken together, these results are in agreement with the involvement of ER signalling in ethanol-induced stimulation of breast cancer cell proliferation and could help to understand why alcohol consumption is associated with breast cancer risk.

Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.

LRP-1: a checkpoint for the extracellular matrix proteolysis.

Low-density lipoprotein receptor-related protein-(LRP-1) is a large endocytic receptor that binds more than 35 ligands and exhibits signaling properties. Proteinases capable of degrading extracellular matrix (ECM), called matrix proteinases in this paper, are mainly serine proteinases: the activators of plasminogen into plasmin, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, and the members of the matrix metalloproteinase (MMP) family. LRP-1 is responsible for clearing matrix proteinases, complexed or not with inhibitors. This paper attempts to summarize some aspects on the cellular and molecular bases of endocytic and signaling functions of LRP-1 that modulate extra- and pericellular levels of matrix proteinases.

A crucial role for Lyn in TIMP-1 erythroid cell survival signalling pathway.

TIMP-1, a well-known MMP inhibitor, displays other biological activities such as cell survival, proliferation and differentiation in hematopoietic cells. In this report, we investigated the role of the Src-related kinase Lyn in TIMP-1 induced UT-7 erythroleukemic cell survival. We showed that (i) tyrosine 507 of Lyn was dephosphorylated and Lyn kinase activity enhanced by TIMP-1, (ii) Lyn silencing suppressed TIMP-1 anti-apoptotic activity and (iii) Lyn was activated upstream the JAK2/PI 3-kinase/Akt pathway. Our data suggest a novel role for Lyn in erythroid cell survival.