ACS Med Chem Lett. 2015 Nov 10;7(1):28-33.
Role of a Preorganized Scaffold Presenting Four Residues of a GM-3 Lactone Mimetic on Melanoma Progression.
- 1Department of Chemistry “Ugo Schiff″, University of Florence , via della Lastruccia, 13 50019 Sesto F.no (FI), Italy.
- 2Department of Experimental and Clinical Biomedical Sciences, Biochemistry, Human Health Medical School, University of Florence , viale Morgagni 50, 50134 Firenze, Italy.
- 3Université Grenoble-Alpes, and CNRS, DCM, 38000 Grenoble, France; Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005 Paris, France.
- 4Université Grenoble-Alpes, and CNRS, DCM , 38000 Grenoble, France.
- 5Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Ecole Nationale Supérieure de Chimie de Montpellier , 8 Rue de l’Ecole Normale, 34296 Montpellier cedex 5, France.
- 6Core Research Laboratory, Istituto Toscano Tumori , viale Pieraccini, 6, 50139 Firenze, Italy.
- 7Department of Chemical and Pharmaceutical Sciences, University of Trieste , via L. Giorgieri 1, 34127 Trieste, Italy.
- 8Department of Chemistry “Ugo Schiff″, University of Florence, via della Lastruccia, 13 50019 Sesto F.no (FI), Italy; FiorGen, Polo Scientifico e Tecnologico, via Sacconi, 6 50019 Sesto F.no (FI), Italy.
Two tetravalent architectures, the glycocalix 7 and the RAFT 9, presenting four residues of a GM-3 ganglioside lactone mimetic, target the host compartment of melanoma and significantly abrogate the effect induced by cancer-associated fibroblasts (CAFs) contact + hypoxia in the motility and invasiveness of tumor cells. The data reported support the involvement of glycosphingolipids (GSLs) in hypoxia and show an interesting role played by compound 9 in targeting melanoma cells thereby interfering with melanoma progression. The unprecedented findings reported for the glycocluster 9 may contribute to the understanding of the critical and complex interactions between tumor cells and their local environment paving the way for new therapeutic agents.
KEYWORDS: Glycosides; mimetics; tumor antigen; tumor microenvironment
Cancer cells are easily compliant to environmental and genetic changes, thereby obtaining an enormous advantage during the long metastatic route, disseminated of severe environmental modifications in each step of the pathway. Moreover, tumor cells do not proliferate and progress in isolation, rather, they are dependent on support from their surrounding stromal cells, which play a compulsory role for their metastatic potential. As tumor progression proceeds, the stromal cells create a “reactive/useful stroma” that releases a variety of signals facilitating the escape of cancer cells from primary tumors to spread at distance (1). The host cells thus engage a continuous molecular cross talk with the cancer cells, secreting large amounts of factors/cytokines, which trigger invasion and metastasis. (2,3)
Glycosphingolipids (GSLs) and gangliosides in particular, are known to be involved in cell adhesion and signalling and to affect cellular phenotype. The understanding of the GSLs-mediated mechanisms in cancer progression is an attracting goal, which would reasonably lead to the identification of novel targets for anti-cancer therapy. Nonetheless, the structure complexity of many GSLs and their scarce availability reduced a real comprehension of their role in vivo. In 1980s a special attention was paid to GM-3 ganglioside, the ganglioside largely expressed on normal melanocytes and over expressed in melanoma cells with metastatic potential. On melanoma cells the GM-3 ganglioside cyclize to form the corresponding GM-3 lactone, (4,5) which has been recognized as a melanoma antigen. The GM-3 lactone is not stable under physiological conditions and this instability greatly limited a complete investigation of its role in tumor progression.
Figure 1. Structure of calix 1, mimetic 3 and glycosyl RAFTs 2 and 4.
We developed a mimetic of the GM-3 lactone, the thioether 3 (Figure 1) (6) that is structurally simpler than the endogenous GM-3 lactone and stable under physiological conditions. The mimetic 3 was manipulated and used to decorate a calix and a RAFT scaffolds to obtain the tetravalent architectures 1 and 2 (Figure 1). We also employed the GM-3 mimetic to decorate gold nanoparticles (GNPs, average diameter 6 nm) to get a globular presentation of the antigen.
We already observed the interesting effect of the GM-3 mimetic on melanoma progression (7), thus the effect of derivatives 1, 2 and GNPs were tested in microenvironment-promoted events, on melanoma cell adhesion and migration with the aim to highlight a possible role (positive or detrimental) played by the calix and RAFT scaffolds and by the spatial orientation of the mimetic residues on melanoma modulation. Human melanoma A375 cells were treated with conditioned media from CAFs (Cancer Associated Fibroblasts) in hypoxia in the presence of 1, 2 or GNPs and assayed for their ability to migrate or to invade through reconstituted matrigel barrier. As shown in Figure 2, compounds 1 and 2 have a very strong effect on both 2D migration (Figure 2, left) and invasiveness (Figure 2, right) of melanoma cells, completely abrogating the effects exerted by tumor microenvironment (CAFs + hypoxia). Compound 2 was also active in inhibiting survival in the absence of proper integrin-mediated adhesion, a peculiar ability of cancer cells, including malignant melanoma. Conversely, GNPs tested alone or in synergy, did not show any real improvement with respect to 1 and 2 in the in vitro assays performed.
Figure 2. Sub-confluent HDFs and CAFs were serum starved for 48h to obtain the corresponding CM. A375 melanoma cells were then incubated in hypoxia with CM from the above fibroblasts, in the presence or in absence of tested compounds (50 μM). Migration assay (A) Cells were allowed to migrate to 24h toward 10% serum as chemo-attractant. For each filter the number of cells in the randomly chosen field was determined. Invasiveness assay (B) Invasiveness was evaluated as for the migration assay except that matrigel has been used to represent 3D ECM. The Calix, RAFT and blank_GNPs were tested as negative controls. *** , P < 0,001 versus each correspondent controls.
The properties showed by compound 2 (i.e. inhibition of migration, invasion and induction of tumor cells apoptosis) demonstrated its wide activity towards several key features of melanoma malignancy and were confirmed by orthotopic xenographs in vivo (Figure 4). In order to improve the immunogenicity of 2, we armed the RAFT scaffold introducing in addition of the four residues of GM-3 mimetic on the upper rim a residue of Melan peptide on the lower rim. However the introduction of the peptide made the glycosylated RAFT poorly soluble in dimethyl sulphoxyde, that is, unsuitable for in vitro tests.
Figure 3 : Effect of the glycosyl-RAFT 2 and R(4Lys-1Ox) on in vivo tumor growth. A) Three groups of C57BL/6 mice (10 mice per group) were treated with 2, R(4Lys-1Ox) and PBS (as negative control) on days 0, 14, 28, 49 and 72 (doses: 400mg; all i.p.). On day 69, 10.000 B16F10 murine melanoma cells were injected subcutaneously in two lateral flanks. The volume of the tumor (20 tumors per group) was measured every 2-3 days with a caliper, and tumor volumes were calculated with the formula V=W2xLx0.5, where W and L are tumor width and length, respectively. Data are presented as mean±SEM. *, p<0.05; **, p<0.001 versus each correspondent PBS control. B) Weight of mice for each group of treatment at the end of the experiment. It shows that treatment does not affect mouse weight, suggesting little, if any, toxic effects.
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