Vincenza Dolo

Shedding of the Matrix Metalloproteinases MMP-2, MMP-9, and MT1-MMP as Membrane Vesicle-Associated Components by Endothelial Cells

Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) originating from localized areas of the cell plasma membrane, as revealed by ultrastructural analysis. Normal and reverse zymography, Western blot, and immunogold analyses of the vesicles showed two gelatinases, MMP-2 and MMP-9, in both the active and proenzyme forms, the MT1-MMP proenzyme located on the external side of the vesicle membrane and the two inhibitors TIMP-1 and TIMP-2. Serum and the angiogenic factors, fibroblast growth factor-2 and vascular endothelial growth factor, stimulated the shedding of MMPs as vesicle components. Shedding the vesicle was rapid, as it was already completed after 4 hours. Addition of shed vesicles to human umbilical vein endothelial cells resulted in autocrine stimulation of invasion through a layer of reconstituted basement membrane (Matrigel) and cord formation on Matrigel. We conclude that endothelial cells shed MMP-containing vesicles and this may be a mechanism for regulating focalized proteolytic activity vital to invasive and morphogenic events during angiogenesis.

Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro

The inxa0vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon inxa0vivo and inxa0vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen α-Folate Receptor (α-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABAxa0I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABAxa0I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABAxa0I cells shed membrane vesicles, which were morphologically similar to those identified inxa0vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both inxa0vivo and inxa0vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABAxa0I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both inxa0vivo and inxa0vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.

Enrichment and localization of ganglioside GD3 and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.

Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens

We have compared the pattern of surface antigen expression, as detected by monoclonal antibodies (mAbs), in plasma membranes vs shed membrane vesicles of two human breast carcinoma cell lines, MCF-7 and 8701-BC. Antigen expression was detected on cells by immunofluorescence (IF) analysis, whilst, due to their small dimensions, the same technique was not applicable to vesicles. For these structures dot-blot analysis and immunoelectron microscopy (IEM) were employed. When applicable, both cell membranes and membrane vesicles were immunoprecipitated and the precipitate (IP) was analyzed by SDS-PAGE. Cells of both lines expressed HLA class I antigens, epithelial cytokeratins, β1 integrins, CEA and the glycoprotein detected by mAb 19.9, but only MCF-7 cells expressed Lewis Y, episialin and globo-H antigens and only 8701-BC cells expressed folate receptor. Membrane vesicles of both cell lines appeared to be rich in β1, α3 and α5 integrin chains, expressed HLA class I antigens and carried most of the plasma membrane antigens found in the cell membranes. Overall we have analyzed 17 antigens on the two cell lines and on their vesicles. The results obtained for cells (IF and IP) and those for vesicles (dot-blot and IP) were generally concordantly positive or concordantly negative. We obtained a total of 26 clearly concordant combinations on 34 analyses. In three cases we found discordant results, whereas in the remaining combinations we observed slight reactivity and we found difficulties in determining concordance. Discordant results concerned the expression of the following antigens: folate receptors, which were clearly expressed in 8701-BC cells but not detected by dot-blot analysis or IEM on their shed membrane vesicles; neu (c-erb-B2) receptor found in MCF-7 cell membranes but not in their vesicles; and the globo-H antigen recognized by mAb MBr1, detected at low levels on 8701-BC plasma membranes but undetectable on their membrane vesicles. Like vesicles shed in vitro by cultured cells, the vesicles shed in vivo by human breast carcinoma cells could be tagged with several antibodies against tumor-associated antigens. The vesicles shed in vivo were found in association with a fiber network. Some of the fibers had the characteristic fibrin periodicity. These data suggest that tumor markers detected in the circulation of carcinoma patients, at least in part, are carried by shed membrane vesicles. Moreover the observation that membrane vesicles carry both tumor-associated antigens and HLA class I molecules indicate that these structures could in principle present antigens to the immune system. Together with our previous demonstration that membrane vesicles shed by breast carcinoma cells contain TGF-β, these results suggest an important role for vesicles in the immunological escape of these cells. The presence in membrane vesicles of integrins, together with the previous observation that they are rich in gelatinolytic activities, also points to a possible role of these structures in the metastatic behavior of tumor cells.

Azacitidine improves antitumor effects of docetaxel and cisplatin in aggressive prostate cancer models

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells. In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2 antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were also tolerable in mice and superior to either agent alone. As DTX is the standard first-line chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest in this disease stage. Our results provide a rationale for clinical trials on combination treatments with azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.

Differential effects of PXD101 (belinostat) on androgen-dependent and androgen-independent prostate cancer models

Histone deacetylase inhibitors (HDACi) are promising epigenetic cancer chemotherapeutics rapidly approaching clinical use. In this study, we tested using in vitro and in vivo models the differential biological effects of a novel HDAC inhibitor [belinostat (PXD101)], in a wide panel of androgen-sensitive and androgen-independent tumor cells. Belinostat significantly increased acetylation of histones H3 and H4. Belinostat potently inhibited the growth of prostate cancer cell lines (IC50 range from 0.5 to 2.5 µM) with cytotoxic activity preferentially against tumor cells. This agent induced G2/M arrest and increased significantly the percentage of apoptosis mainly in androgen-sensitive tumor cells confirming its growth-inhibitory effects. The cell death mechanisms were studied in three different prostate cancer cell lines with different androgen dependence and expression of androgen receptor; LAPC-4 and 22rv1 (androgen-dependent and expressing androgen receptor) and PC3 (androgen-independent not expressing androgen receptor). Belinostat induced the expression of p21 and p27, acetylation of p53 and G2/M arrest associated with Bcl2 and Bcl-Xl downmodulation and significant reduction of survivin, IAPs and Akt/pAkt and increased caspase-8 and -9 expression/activity. Belinostat effectiveness was dependent on the androgen receptor (AR), since the stable transfection of AR greatly increased the efficacy of this HDAC inhibitor. These observations were correlated using in vivo models. We demonstrated that belinostat preferentially resulted in antitumor effect in androgen-dependent tumor cells expressing AR. Our findings provide evidence that belinostat may be a promising anticancer drug for prostate cancer expressing AR, supporting its clinical role in prostate cancer.

Shed Membrane Vesicles and Selective Localization of Gelatinases and MMP‐9/TIMP‐1 Complexes

Shedding of membrane vesicles is a vital phenomenon frequently observed in tumor cells and suggested to be involved in several aspects of tumor progression. 1 As we have recently shown, in 8701-BC breast carcinoma cells, vesicle shedding is modulated by extracellular signal; and several molecules, probably involved in tumor progression, are specifically densified on shed vesicles. 2 One of the clustered molecules is gelatinase B (MMP-9), which is present in both active and proenzyme forms. Western blot analyses of vesicles shed by 8701-BC breast carcinoma cells demonstrate that most MMP-9 molecules bound to vesicle membranes are in association with TIMP-1, as components of high M r complexes. 2 ProMMP-9/TIMP-1 complexes have been detected in vesicles shed by all other analyzed cell lines. Other proteolytic enzymes, on the contrary, were detected in vesicles shed by some cell lines and not in others. The urokinase type of plasminogen activator (uPA), while not detected in vesicles shed by 8701-BC and MCF-7 cells, is present in MDA MB-231. In vesicles shed by HT-1080 fibrosarcoma, we find not only MMP-9 and uPA, but also gelatinase A (MMP-2). 3 We observe a positive correlation between the quantity of shed vesicles, the amount of lytic enzymes they carry, and the in vitro invasive capability of the different cell lines. These findings indicate that vesicle production might represent an important mechanism by which tumor cells induce the localized degradation of the extracellular matrix and acquire metastatic capabilities. We have no evidence of which membrane vesicle components are involved in binding of proMMP-9/TIMP-1 complexes. Since Olson et al. recently reported that, in breast epithelial cells, proMMP-9 binds to α 2 (IV) chain of collagen IV, 4 we tested 8701-BC shed vesicles for the presence of this molecule. As shown by F IGURE 1, when Western blot analyses were performed in nonboiled, nonreducing conditions, antibodies against the α 2 (IV) chain of human collagen IV, recognized two components. One of them, having an electrophoretic mobility of 190 kDa, corresponds to the α 2 (IV) chain of collagen IV, the molecule identified as the proMMP-9 receptor

Differential expression and function of cadherin-like proteins in the sea urchin embryo

Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.

Extracellular vesicles in prostate cancer: new future clinical strategies?

Prostate cancer (PCa) is the most common cancer—excluding skin tumors—in men older than 50 years of age. Over time, the ability to diagnose PCa has improved considerably, mainly due to the introduction of prostate-specific antigen (PSA) in the clinical routine. However, it is important to take into account that although PSA is a highly organ-specific marker, it is not cancer-specific. This shortcoming suggests the need to find new and more specific molecular markers. Several emerging PCa biomarkers have been evaluated or are being assessed for their potential use. There is increasing interest in the prospective use of extracellular vesicles as specific markers; it is well known that the content of vesicles is dependent on their cellular origin and is strongly related to the stimulus that triggers the release of the vesicles. Consequently, the identification of a disease-specific molecule (protein, lipid or RNA) associated with vesicles could facilitate their use as novel biological markers. The present review describes several in vitro studies that demonstrate the role of vesicles in PCa progression and several in vivo studies that highlight the potential use of vesicles as PCa biomarkers.

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation

When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearance of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. The active fraction does not readily filter through Amicon XM-50 and is retarded by column chromatography on Bio-Gel P-60.