PLP2-derived peptide Rb4 triggers PARP-1-mediated necrotic death in murine melanoma cells.

Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.

Intracellular Targeting of Poly Lactic-Co-Glycolic Acid Nanoparticles by Surface Functionalization with Peptides.

Nanoparticles (NPs) are a promising strategy for delivering drugs to specific sites because of their tunable size and surface chemistry variety. Among the availablematerials, NPs prepared with biopolymers are of particular interest because of their biocompatibility and controlled release of encapsulated drugs. Poly lactic-co-glycolic acid (PLGA) is one of the most widely used biopolymers in biomedical applications. In addition to material choice modulation of the interaction between NPs and biological systems is essential for the safety and effective use of NPs. Therefore, this work focused on evaluating different surface functionalization strategies to promote cancer cell uptake and intracellular targeting of PLGA NPs. Herein, cell-penetrating peptides (CPPs) were shown to successfully drive PLGA NPs to the mitochondria and nuclei. Furthermore, the functionalization of PLGA NPs with peptide AC-1001 H3 (GQYGNLWFAY) was proven to be useful for targeting actin filaments. The PLGA NPs cell internalization mechanism by B16F10-Nex2 cells was identified as caveolae-mediated endocytosis, which could be inhibited by the presence of methyl-ß-cyclodextrin. Notably, when peptide C (CVNHPAFAC) was used to functionalize PLGA NPs, none of the tested inhibitors could avoid cell internalization of PLGA NPs. Therefore, we suggest this peptide as a promising surface modification agent for enhancing drug delivery to cancer cells. Finally, PLGA NPs showed slow release kinetics and low cytotoxic profile, which, combined with the surface functionalization strategies addressed in this study, highlight the potential of PLGA NPs as a drug delivery platform for improving cancer therapy.

MIF inhibition as a strategy for overcoming resistance to immune checkpoint blockade therapy in melanoma.

Immune checkpoint blockade (ICB) has demonstrated an impressive outcome in patients with metastatic melanoma, yet, durable complete response; even with Ipilimumab/Nivolumab combo are under 30%. Primary and acquired resistance in response to ICB is commonly due to a tumor immune escape mechanism dictated by the tumor microenvironment (TME). Macrophage Migratory Inhibition Factor (MIF) has emerged as an immunosuppressive factor secreted in the TME. We have previously demonstrated that blockade of the MIF-CD74 signaling on macrophages and dendritic cells restored the anti-tumor immune response against melanoma. Here, we report that inhibition of the MIF-CD74 axis combined with ipilimumab could render resistant melanoma to better respond to anti-CTLA-4 treatment. We provide evidence that blocking the MIF-CD74 signaling potentiates CD8+ T-cells infiltration and drives pro-inflammatory M1 conversion of macrophages in the TME. Furthermore, MIF inhibition resulted in reprogramming the metabolic pathway by reducing lactate production, HIF-1α and PD-L1 expression in the resistant melanoma cells. Melanoma patient data extracted from the TCGA database supports the hypothesis that high MIF expression strongly correlates with poor response to ICB therapy. Our findings provide a rationale for combining anti-CTLA-4 with MIF inhibitors as a potential strategy to overcome resistance to ICB therapy in melanoma, turning a "cold" tumor into a "hot" one mediated by the activation of innate immunity and reprogramming of tumor metabolism and reduced PD-L1 expression in melanoma cells.

Intranasal Vaccine Using P10 Peptide Complexed within Chitosan Polymeric Nanoparticles as Experimental Therapy for Paracoccidioidomycosis in Murine Model.

Paracoccidioidomycosis (PCM) is a granulomatous fungal disease caused by the dimorphic fungal species of Paracoccidioides, which mainly affects the lungs. Modern strategies for the treatment and/or prevention of PCM are based on a Th1-type immune response, which is important for controlling the disease. One of the most studied candidates for a vaccine is the P10 peptide, derived from the 43 kDa glycoprotein of Paracoccidioides brasiliensis. In order to improve its immune modulatory effect, the P10 peptide was associated with a chitosan-conjugated nanoparticle. The nanoparticles presented 220 nm medium size, poly dispersion index (PDI) below 0.5, zeta potential of +20 mV and encapsulation efficiency around 90%. The nanoparticles' non-toxicity was verified by hemolytic test and cell viability using murine macrophages. The nanoparticles were stable and presented physicochemical characteristics desirable for biological applications, reducing the fungal load and the usual standard concentration of the peptide from 4 to 20 times.

Antitumor effect of chiral organotelluranes elicited in a murine melanoma model.

Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric forms of 1-[Butyl(dichloro)-λ4-tellanyl]-2-[1S-methoxyethyl]benzene (organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumor cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.

Molecular, Biological and Structural Features of VL CDR-1 Rb44 Peptide, Which Targets the Microtubule Network in Melanoma Cells.

Microtubules are important drug targets in tumor cells, owing to their role in supporting and determining the cell shape, organelle movement and cell division. The complementarity-determining regions (CDRs) of immunoglobulins have been reported to be a source of anti-tumor peptide sequences, independently of the original antibody specificity for a given antigen. We found that, the anti-Lewis B mAb light-chain CDR1 synthetic peptide Rb44, interacted with microtubules and induced depolymerization, with subsequent degradation of actin filaments, leading to depolarization of mitochondrial membrane-potential, increase of ROS, cell cycle arrest at G2/M, cleavage of caspase-9, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-2, altogether resulting in intrinsic apoptosis of melanoma cells. The in vitro inhibition of angiogenesis was also an Rb44 effect. Peritumoral injection of Rb44L1 delayed growth of subcutaneously grafted melanoma cells in a syngeneic mouse model. L1-CDRs from immunoglobulins and their interactions with tubulin-dimers were explored to interpret effects on microtubule stability. The opening motion of tubulin monomers allowed for efficient L1-CDR docking, impairment of dimer formation and microtubule dissociation. We conclude that Rb44 VL-CDR1 is a novel peptide that acts on melanoma microtubule network causing cell apoptosis in vitro and melanoma growth inhibition in vivo.

Leucurogin and melanoma therapy.

Leucurogin is an ECD disintegrin-like protein, cloned from Bothrops leucurus venom gland. This new protein, encompassing the disintegrin region of a PIII metalloproteinase, is produced by recombinant technology and its biological and functional activity was partially characterized in this study. Biological activity was characterized in vitro using human fibroblasts. Functional activity of leucurogin was analysed in vitro and in vivo with murine B16F10 Nex-2 and human melanoma BLM cells. The results show that leucurogin inhibits cellular processes dependent on collagen type I. In a competition assay with collagen, leucurogin inhibits, in a dose-dependent manner, the adhesion of fibroblast to collagen. At 10 µM leucurogin reduces adhesion (40%) and migration (70%) of hFb and inhibits migration (32%) and proliferation (65%) of BLM cells. At 2.5 µM leucurogin inhibits 80% cell proliferation of B16F10 Nex-2 melanoma cells. At 4.8 µM leucurogin inhibits, in vitro, the vascular structures formation by endothelial cells by 66%. Leucurogin, injected intraperitoneally, i.p. (5 µg/animal, two-month old C57/Bl6 male mice) on alternate days for 15 days, inhibits lung metastasis of B16F10 Nex-2 cells by 70-75%. In the treatment of human melanoma, grafted intradermally in the nude mice flank, leucurogin (7.5 µg/kg in alternate days during 17 days) inhibits tumor growth by more than 40%. Leucurogin can be considered a promising agent for melanoma treatment.

Immunomodulatory Protective Effects of Rb9 Cyclic-Peptide in a Metastatic Melanoma Setting and the Involvement of Dendritic Cells.

The cyclic VHCDR3-derived peptide (Rb9) from RebMab200 antibody, directed to a NaPi2B phosphate-transport protein, displayed anti-metastatic melanoma activity at 50-300 µg intraperitoneally injected in syngeneic mice. Immune deficient mice failed to respond to the peptide protective effect. Rb9 induced increased CD8+ T and low Foxp3+ T cell infiltration in lung metastases and high IFN-γ and low TGF-ß in lymphoid organs. The peptide co-localized with F-actin and a nuclear site in dendritic cells and specifically bound to MIF and CD74 in a dot-blot setting. Murine bone-marrow dendritic cells preincubated with Rb9 for 6 h were treated with MIF for short time periods. The modulated responses showed stimulation of CD74 and inhibition of pPI3K, pERK, and pNF-κB as compared to MIF alone. Rb9 in a melanoma-conditioned medium, stimulated the M1 type conversion in bone marrow-macrophages. Functional aspects of Rb9 in vivo were studied in therapeutic and prophylactic protocols using a melanoma metastatic model. In both protocols Rb9 exhibited a marked anti-melanoma protection. Human dendritic cells were also investigated showing increased expression of surface markers in response to Rb9 incubation. Rb9 either stimulated or slightly inhibited moDCs submitted to inhibitory (TGF-ß and IL-10) or activating (LPS) conditions, respectively. Lymphocyte proliferation was obtained with moDCs stimulated by Rb9 and tumor cell lysate. In moDCs from cancer patients Rb9 exerted immunomodulatory activities depending on their functional status. The peptide may inhibit over-stimulated cells, stimulate poorly activated and suppressed cells, or cause instead, little phenotypic and functional alterations. Recently, the interaction MIF-CD74 has been associated to PD-L1 expression and IFN-γ, suggesting a target for melanoma treatment. The effects described for Rb9 and the protection against metastatic melanoma may suggest the possibility of a peptide reagent that could be relevant when associated to modern immunotherapeutic procedures.

Peptide R18H from BRN2 Transcription Factor POU Domain Displays Antitumor Activity In Vitro and In Vivo and Induces Apoptosis in B16F10-Nex2 Cells.

BACKGROUND: BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor. OBJECTIVE: To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells. METHODS: Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment. RESULTS: Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H. CONCLUSION: Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo.

Blockade of MIF-CD74 Signalling on Macrophages and Dendritic Cells Restores the Antitumour Immune Response Against Metastatic Melanoma.

Mounting an effective immune response against cancer requires the activation of innate and adaptive immune cells. Metastatic melanoma is the most aggressive form of skin cancer. While immunotherapies have shown a remarkable success in melanoma treatment, patients develop resistance by mechanisms that include the establishment of an immune suppressive tumor microenvironment. Thus, understanding how metastatic melanoma cells suppress the immune system is vital to develop effective immunotherapies against this disease. In this study, we find that macrophages (MOs) and dendritic cells (DCs) are suppressed in metastatic melanoma and that the Ig-CDR-based peptide C36L1 is able to restore MOs and DCs' antitumorigenic and immunogenic functions and to inhibit metastatic growth in lungs. Specifically, C36L1 treatment is able to repolarize M2-like immunosuppressive MOs into M1-like antitumorigenic MOs, and increase the number of immunogenic DCs, and activated cytotoxic T cells, while reducing the number of regulatory T cells and monocytic myeloid-derived suppressor cells in metastatic lungs. Mechanistically, we find that C36L1 directly binds to the MIF receptor CD74 which is expressed on MOs and DCs, disturbing CD74 structural dynamics and inhibiting MIF signaling on these cells. Interfering with MIF-CD74 signaling on MOs and DCs leads to a decrease in the expression of immunosuppressive factors from MOs and an increase in the capacity of DCs to activate cytotoxic T cells. Our findings suggest that interfering with MIF-CD74 immunosuppressive signaling in MOs and DCs, using peptide-based immunotherapy can restore the antitumor immune response in metastatic melanoma. Our study provides the rationale for further development of peptide-based therapies to restore the antitumor immune response in metastatic melanoma.

Fungicidal activity of peptides encoded by immunoglobulin genes.

Evidence from previous works disclosed the antimicrobial, antiviral, anti-tumour and/or immunomodulatory activity exerted, through different mechanisms of action, by peptides expressed in the complementarity-determining regions or even in the constant region of antibodies, independently from their specificity and isotype. Presently, we report the selection, from available databases, of peptide sequences encoded by immunoglobulin genes for the evaluation of their potential biological activities. Synthetic peptides representing the translated products of J lambda and J heavy genes proved to act in vitro against pathogenic fungi, entering yeast cells and causing their death, and exerted a therapeutic effect in a Galleria mellonella model of infection by Candida albicans. No haemolytic, cytotoxic and genotoxic effects were observed on mammalian cells. These findings raise the hypothesis that antibodies could be the evolutionary result of the adaptive combination of gene products ancestrally devoted to innate antimicrobial immunity.

Peptide Vaccine Against Paracoccidioidomycosis.

The chapter reviews methods utilized for the isolation and characterization of a promising immunogen candidate, aiming at a human vaccine against paracoccidioidomycosis. Peptide P10 carries a T-CD4+ epitope and was identified as an internal sequence of the major diagnostic antigen known as gp43 glycoprotein. It successfully treated massive intratracheal infections by virulent Paracoccidioides brasiliensis in combination with chemotherapy.An introduction about the systemic mycosis was found essential to understand the various options that were considered to design prophylactic and therapeutic vaccine protocols using peptide P10.

Inhibition of melanoma metastasis by dual-peptide PLGA NPS.

Despite the positive results observed in vitro and in vivo, clinical trials with bioactive peptides are generally hampered by their fast degradation in the biological system. Two bioactive peptides, P20 (CSSRTMHHC) and the combined peptide C (CVNHPAFACGYGHTMYYHHYQHHL) have been identified as anticancer therapeutics. Combined peptide C consists of peptide C (CVNHPAFAC), a tumor-homing peptide, conjugated to the antiangiogenic peptide HTMYYHHYQHHL with a GYG. In this work, PLGA NPs with peptide C were applied as a dual-peptide carrier for application in cancer therapy. Peptide P20 was loaded into the NPs and combined peptide C was conjugated to the NPs surface. These NPs were evaluated as a therapeutic system to treat metastatic melanoma. In vivo assays showed that P20 encapsulation in PLGA NPs enhanced its antitumor activity. The inhibitory activity of P20-PLGANPs was similar to the activity of non-encapsulated P20 in a dose fivefold higher. The inhibitory activity was even higher when P20PLGA NPs were functionalized with combined peptide C. P20PLGAPepC NPs reduced in 28% the number of lung nodules in a syngeneic model of metastatic melanoma as compared to untreated animals. Additionally to the better tumor targeting and the in situ release of P20, it is expected that the therapeutic efficiency of the dual-peptide PLGA NPs was further enhanced by a synergistic effect between P20 and combined peptide C. Our encouraging results showed that by enabling the co-delivery of two peptides and promoting tumor targeting, PLGA NPs coupled with peptide C is a promising platform for peptide-based cancer therapy.

TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models.

Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.

AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model.

Antibody-derived peptides modulate functions of the immune system and are a source of anti-infective and antitumor substances. Recent studies have shown that they comprise amino acid sequences of immunoglobulin complementarity-determining regions, but also fragments of constant regions. VH CDR3 of murine mAb AC-1001 displays antimetastatic activities using B16F10-Nex2 murine melanoma cells in a syngeneic model. The peptide was cytotoxic in vitro in murine and human melanoma cells inducing reactive oxygen species (ROS) and apoptosis by the intrinsic pathway. Signs of autophagy were also suggested by the increased expression of LC3/LC3II and Beclin 1 and by ultrastructural evidence. AC-1001 H3 bound to both G- and F-actin and inhibited tumor cell migration. These results are important evidence of the antitumor activity of Ig CDR-derived peptides.

The Ig VH complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

Antibodies Against Glycolipids Enhance Antifungal Activity of Macrophages and Reduce Fungal Burden After Infection with Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo.

Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca(2+) together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy.

A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays antitumor activity against melanoma in vitro and in vivo.

Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.