Gomesin is a potent antimicrobial peptide (AMP) isolated from hemocytes of the spider Acanthoscurria gomesiana. The present study aimed at determining whether gomesin exerted antitumor activity in vitro and in vivo. Topical treatment of subcutaneous murine melanoma with gomesin incorporated in a cream base significantly delayed tumor growth. A direct cytotoxicity of gomesin in murine melanoma B16F10-Nex2 cells and several human tumor cell lineages was observed in vitro, with IC(50) values below 5 microM. The beta-hairpin structure of gomesin with disulfide bridges seemed essential for optimal activity. d-Gomesin was equally active. A membrane-permeabilizing activity was suggested, as gomesin bound to the cell membrane and cytoplasmic lactate dehydrogenase was detected extracellularly. At doses causing partial growth of tumor cells, gomesin allowed internalization of macromolecules (immunoglobulins), which increased the cytotoxic effect. The in vivo antitumor effect of gomesin might also involve a cytotoxic effect on endothelial cells because cultured human endothelial cells were killed in vitro at a similar concentration range. This effect represents a novel and potential use for gomesin as a topical agent against unsuccessfully treated intradermal and epithelial skin cancers. To our knowledge, this is the first report on the successful topical use of AMPs in cancer treatment.

Background: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd₂[S_(-)C², N-dmpa]₂(μ-dppe)Cl₂} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies.Methods: B16F10-Nex2 cells were treatedin vitrowith C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treatedin vitrowith C7a.Results: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineagesin vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells.Conclusions: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

Silencing of SOCS1 protein with shRNAi lentivirus (shR-SOCS1) led to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS1 silencing inhibited cell migration and invasion as well as in vitro growth by cell cycle arrest at S phase with increased cell size and nuclei. Down-regulation of SOCS1 decreased the expression of epidermal growth factor receptor, Ins-Rα, and fibroblast growth factor receptors. The present work aimed at analyzing the SOCS1 cell signaling and expression of proteins relevant to tumor development. An RNA microarray analysis of B16F10-Nex2 melanoma cells with SOCS1 silenced by shRNAi-SOCS1 was undertaken in comparison with cells transduced with the empty vector. Among 609 differentially expressed genes, c-Kit, Met and EphA3 cytokine/tyrosine-kinase (TK) receptors were down regulated. A significant decrease in the expression of TK receptors, the phosphorylation of mediators of ERK1/2 and p38 pathways and STAT3 (S727) were observed. Subcutaneous immunization with shR-SOCS1-transduced viable tumor cells rendered protection against melanoma in a syngeneic model, with decreased expression of PD-L1 and of matrix metallo-proteinases (MMPs) and CD-10 in those cells. The present work shows the role of SOCS1 in murine melanoma development and the potential of SOCS1-silenced tumor cells in raising an effective anti-melanoma immune response.

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

Background: CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d glycoproteins are related to the class I MHC molecules, and are involved in the presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT cells. In the present work we attempted to identify the endogenous lipid mediator expressed in melanoma cells inducing such immunesurveillance response and study the possibility of protecting animals challenged with tumor cells with lipid-primed DC.Results: Crude cytosolic and membrane fractions fromin vivogrowing melanoma contained iNKT-stimulating substances. Lipids were then extracted from these cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to produce IL-2. The active fraction was analyzed by electrospray ionization-mass spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally produced indicating iNKT cell activation. GM3 consistently inhibited this response. To assess the antitumor response-induced by iGb3, a cytotoxicity assayin vitrowas used with [³H]-thymidine labeled B16F10-Nex2 cells. At target/effector (iGb3-activated iNKT) cell ratio of 100^-1-100^-4tumor cell lysis was shown. The antitumor activityin vivowas tested in mice challenged i.v. with B16F10-Nex2 cells and treated with iGb3- or α-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was observed with either treatment.Conclusion: Our results show the expression of globo and isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 and its precursor, iGb4, on tumor cells may prime an effective iNKT cell-dependent antitumor response, modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.