Chemical Modification for Proteolytic Stabilization of the Selective αvß3 Integrin RGDechi Peptide: in Vitro and in Vivo Activities on Malignant Melanoma Cells.

Herein, we report the synthesis and biological characterization of the new peptide ψRGDechi as the first step toward novel-targeted theranostics in melanoma. This pseudopeptide is designed from our previously reported RGDechi peptide, known to bind selectively αvß3 integrin, and differs for a modified amide bond at the main protease cleavage site. This chemical modification drastically reduces the enzymatic degradation in serum, compared to its parental peptide, resulting in an overall magnification of the biological activity on a highly expressing αvß3 human metastatic melanoma cell line. Selective inhibition of cell adhesion, wound healing, and invasion are demonstrated; near-infrared fluorescent ψRGDechi derivative is able to detect αvß3 integrin in human melanoma xenografts in a selective fashion. More, molecular docking studies confirm that ψRGDechi recognizes the receptor similarly to RGDechi. All these findings pave the way for the future employment of this novel peptide as promising targeting probe and therapeutic agent in melanoma disease.

A Combined NMR and Computational Approach to Determine the RGDechi-hCit-αv ß3 Integrin Recognition Mode in Isolated Cell Membranes.

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv ß3 and αv ß5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv ß3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv ß3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv ß3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv ß5 integrin.

RGDechi-hCit: αvß3 selective pro-apoptotic peptide as potential carrier for drug delivery into melanoma metastatic cells.

αvß3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvß3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvß3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvß3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

Cripto haploinsufficiency affects in vivo colon tumor development.

Colorectal cancer is one of the most common and aggressive cancers arising from alterations in various signaling pathways, such as the WNT, RAS-MAPK, PI3K and transforming growth factor-ß (TGF-ß) pathways. Cripto (also called Teratocarcinoma-derived growth factor), the original member of the vertebrate EGF-CFC family, plays a key role in all of these pathways and is deeply involved in early embryo development and cancer progression. The role of Cripto in colon and breast cancer, in particular, has been investigated, as it is still not clearly understood. In this article, we provide the first in vivo functional evidence of a role of Cripto in colon cancer development. We analyzed the effect of Cripto haploinsufficiency on colon tumor formation by treating Cripto heterozygous mice with the colonotropic carcinogen azoxymethane (AOM). Of note, in our model system, Cripto haploinsufficiency increased tumorigenesis. Moreover, we revealed a correlation between the differential AOM response found in wt and Cripto⁺/⁻ mice and the expression levels of glucose regulated protein-78 (Grp78), a heat shock protein required for Cripto signaling pathways. We hypothesize that the balance between Cripto and Grp78 expression levels might be crucial in cancer development and may account for the increased tumorigenesis in Cripto heterozygous mice. In summary, our results highlight the heterogeneous effect of Cripto on tumorigenesis and the consequent high level of complexity in the Cripto regulatory pathway, whose imbalance causes tumors.

Characterization of the functional properties of the neuroectoderm in mouse Cripto(-/-) embryos showing severe gastrulation defects.

During development of the mammalian embryo, there is a complex relation between formation of the mesoderm and the neuroectoderm. In mouse, for example, the role of the node and its mesendoderm derivatives in anterior neural specification is still debated. Mouse Cripto(-/-) embryos could potentially help settle this debate because they lack almost all embryonic endoderm and mesoderm, including the node and its derivatives. In the present paper, we show that Cripto(-/-) embryos can still form functional neural stem cells that are able to differentiate and maintain a neural phenotype both in vivo and in vitro. These data suggest that signals emanating from the mesoderm and endoderm might not be essential for the formation and differentiation of neural stem cells. However, we use grafting experiments to show that the Cripto(-/-) isthmus (the secondary organizer located at the midbrain-hindbrain boundary) loses its inductive ability. We further show that the Cripto(-/-)isthmus expresses lower amounts of the isthmic signalling molecule, Fgf8. Since nearby tissues remain competent to respond to exogenously added Fgf8, this reduction in Fgf8 levels in the Cripto(-/-) isthmus is the potential cause of the loss of patterning ability in graft experiments. Overall, we interpret our data to suggest that the mammalian node and primitive streak are essential for the development of the regional identities that control the specification and formation of the secondary organizers within the developing brain.

TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation.

Pax8 and TTF-1 are transcription factors involved in the morphogenesis of the thyroid gland and in the transcriptional regulation of thyroid-specific genes. Both proteins are expressed in few tissues but their simultaneous presence occurs only in the thyroid where they interact physically and functionally allowing the regulation of genes that are markers of the thyroid differentiated phenotype. TAZ is a transcriptional coactivator that regulates the activity of several transcription factors therefore playing a central role in tissue-specific transcription. The recently demonstrated physical and functional interaction between TAZ and TTF-1 in the lung raised the question of whether TAZ could be an important regulatory molecule also in the thyroid. In this study, we demonstrate the presence of TAZ in thyroid cells and the existence of an important cooperation between TAZ and the transcription factors Pax8 and TTF-1 in the modulation of thyroid gene expression. In addition, we reveal that the three proteins are co-expressed in the nucleus of differentiated thyroid cells and that TAZ interacts with both Pax8 and TTF-1, in vitro and in vivo. More importantly, we show that this interaction leads to a significant enhancement of the transcriptional activity of Pax8 and TTF-1 on the thyroglobulin promoter thus suggesting a role of TAZ in the control of genes involved in thyroid development and differentiation.

Cripto-independent Nodal signaling promotes positioning of the A-P axis in the early mouse embryo.

During early mouse development, the TGFbeta-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto(-/-) embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.

Immunolocalization of 3beta-HSD and 17beta-HSD in the testis of the spotted ray Torpedo marmorata.

Using polyclonal antibodies, we examined the localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as markers of the site of steroidogenetic activity during the spermatogenesis of Torpedo marmorata. These enzymes play a central role in the biosynthesis of steroid hormones, including androgen and oestrogen production. We demonstrated that in the spotted ray testis, Sertoli and Leydig cells, as well as spermatogonia, show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies. In particular, we demonstrated that Sertoli cells show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies in cysts containing spermatogonia and spermatozoa, while Leydig cells present a positive reaction only when they are located between cysts containing meiotic cells. This study strongly suggests that, as hypothesised in our previous study [Prisco, M., Liguoro, A., D'Onghia, B., Ricchiari, L., Andreuccetti, P., Angelini, F., 2002. Fine structure of Leydig and Sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata. Mol. Reprod. Dev. 63, 192-201.], Sertoli and Leydig cells are differently involved in the hormonal control of spermatogenesis: Sertoli cells before the beginning of meiosis and after spermiation, Leydig cells only during meiosis phase. Moreover, the present paper deals with the possibility that also spermatogonia are engaged in the production of androgen hormones, as they are characterized by the presence of 3beta-HSD and 17beta-HSD enzymes, and show the ultrastructural features of steroid hormone-producing cells.

Cadherin in developing and maturing cysts of Torpedo Marmorata Testis.

We investigated the presence of cadherins, Ca++ dependent cell-cell adhesion molecules, during the development and maturation of cysts in the testis of the spotted ray Torpedo marmorata. Using different anti-cadherin antibodies, we provide evidence by means of immunohistochemistry and immunoblotting that cadherins are involved in the interaction between Sertoli and germ cells. During the development and maturation of cysts, in fact, cadherins occur between Sertoli and germ cells when they begin to interact to build a cyst. Later on, the presence of cadherins between Sertoli and germ cells persists; furthermore, during the formation of spermatoblast, it is also evident at the level of indentations, arising from Sertoli cells and encompassing germ cells. Finally, the present findings strongly suggest that cadherins are also involved in the spermiogenesis as germ cells, when male gamete differentiation starts, are intensively stained, while, when spermiation is completed, the spermatozoa appear unlabeled.

Distribution of terminal sugar residues in the testis of the spotted ray Torpedo marmorata.

Lectins represent a class of proteins/glycoproteins binding specifically to terminal sugar residues. The present investigation aims to identify lectin-binding sites in testis of Torpedo marmorata. Using a panel of lectins coupled with fluoresceine isothiocyanate, we demonstrated that germ and somatic cells present in Torpedo testis contain glycoconjugates, whose distribution at the level of the surface, the cytoplasm and the nucleus changes during germ cell differentiation. Moreover our observations demonstrate that the germ cells undergoing apoptosis (Prisco et al., 2003a: Mol Reprod Dev 64:341-348) overexpress a residual sugar recognised by WFA lectin that can be considered a specific marker for apoptotic germ cells. Finally, our results indicate that there is a progressive increase in glycosilation during spermatogenesis, especially at the level of the acrosome in the spermatocyte-spermatid step, and that Leydig cells are differently stained in relation to the spermatogenetic cycle.

Apoptosis during spermatogenesis in the spotted ray Torpedo marmorata.

This article is a cytological and molecular investigation on the occurrence of apoptosis during spermatogenesis in Torpedo, a cartilaginous fish characterised by a typical cystic testis. Using DNA fragmentation and Bak gene expression, it demonstrated that germ cells undergo apoptosis only at the stages of spermatocyte and spermatid, and degeneration also involves Sertoli but not Leydig cells. In immature cysts, this cellular process probably occurs when the ratio of germ cells to the only Sertoli cell (SC) forming the spermatoblast changes. Apoptosis also takes place in mature cysts after sperm release to eliminate most of the SCs. Few of them, however, become cytoplasts and probably continue secreting androgens so as to control the final events of spermatogenesis, i.e., passage of spermatozoa through the ductus deferentes. Finally, the present investigation demonstrated that, in Torpedo testis, Bak mRNA is expressed during spermatogenesis, thus suggesting that the mitochondrial pathway might be active. This observation in one of the oldest vertebrate classes indicates that, in all vertebrates, the apoptotic process during spermatogenesis is conserved, contributing to testicular homeostasis.