Jorge Augusto Borin Scutti

α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model

Malignant melanoma is one the most aggressive types of cancer and its incidence has gradually increased in the last years, accounting for about 75% of skin cancer deaths. This poor prognosis results from the tumor resistance to conventional drugs mainly by deregulation of apoptotic pathways. The aim of this work was to investigate the cell death mechanism induced by α-pinene and its therapeutic application. Our results demonstrated that α-pinene was able to induce apoptosis evidenced by early disruption of the mitochondrial potential, production of reactive oxygen species, increase in caspase-3 activity, heterochromatin aggregation, DNA fragmentation and exposure of phosphatidyl serine on the cell surface. Most importantly, this molecule was very effective in the treatment of experimental metastatic melanoma reducing the number of lung tumor nodules. This is the first report on the apoptotic and antimetastatic activity of isolated α-pinene.

Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo.

Background Malignant melanoma is a deadly type of metastatic skin cancer with increased incidence over the past 30 years. Despite the advanced knowledge on the biology, immunobiology and molecular genetics of melanoma, the alternatives of treatment are limited with poor prognosis. On clinical trials, natural products and among them redox-active quinones have been tested in the attempt to control the growth of cancer cells. Recently, we isolated jacaranone from Pentacalia desiderabilis, a benzoquinone derivative that showed a broad antitumor activity and protective anti-melanoma effect in a syngeneic model. The purified substance is active at micromolar concentrations, is not hemolytic, and is not toxic in naïve mice. Methodology/Principal Findings The jacaranone antitumor activity was shown against several human cancer cell lines in vitro. Moreover, the induction of apoptosis in murine melanoma cells and jacaranone antitumor activity in vivo, in a melanoma experimental model, were also shown. Jacaranone renders antiproliferative and proapoptotic responses in tumor cells, by acting on Akt and p38 MAPK signaling pathways through generation of reactive oxygen species (ROS). The free radical scavenger N-acetyl-cysteine (NAC) was able to completely suppress cell death induced by jacaranone as it blocked Akt downregulation, p38 MAPK activation as well as upregulation of proapoptotic Bax. Notably, treatment of melanoma growing subcutaneously in mice with jacaranone significantly extended the mean survival times in a dose-dependent manner. Conclusions/Significance The results provide evidence for the mechanisms of action of jacaranone and emphasize the potential use of this quinone for the treatment of melanoma.

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.

Melanoma: Perspectives of a Vaccine Based on Peptides

Anti-tumor peptides have been identified in internal sequences of immunoglobulins, mainly the complementarity determining regions (CDRs), and also as sequences of signaling proteins. The in vitro effects of these peptides against several tumor cell lines involved different receptors and mechanisms mainly affecting the cell invasion, proliferation, cell cycle and apoptosis. Peptides also exerted in vivo protection using a melanoma subcutaneous and metastatic model, but this effect depended on the immune response. More specifically, peptides activated dendritic cells (DCs) and these cells exerted a potent anti-tumor effect. Experiments with DCs activated ex-vivo and transferred to tumor-challenged animals showed a protection identical to that of peptides alone suggesting that the latter activate DCs in vivo. A vaccine based on peptides is encouraged by the protection exerted by a signaling protein-derived peptide that caused arrest of a 200-mm3 growing subcutaneous melanoma, and by Ig-CDR readily synthesized, purified peptides that displayed anti-metastatic melanoma activities.

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Background: Pyrostegia venusta (Ker. Gawl.) Miers (Bignoniacea) is a medicinal plant from the Brazilian Cerrado used to treat leucoderma and common diseases of the respiratory system. Objective: To investigate the antitumor activity of P.venusta extracts against melanoma. Materials and Methods: The cytotoxic activity and tumor induced cell death of heptane extract (HE) from P. venusta flowers was evaluated against murine melanoma B16F10-Nex2 cells in vitro and in a syngeneic model in vivo. Results: We found that HE induced apoptosis in melanoma cells by disruption of the mitochondrial membrane potential, induction of reactive oxygen species and late apoptosis evidenced by plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, exposure of phosphatidylserine on the cell surface and activation of caspase-2,-3,-8,-9. HE was also protective against singeneyc subcutaneous melanoma HE compounds were also able to induce cell cycle arrest at G2/M phases on tumor cells. On fractionation of HE in silica gel we isolated a cytotoxic fraction that contained a mixture of saturated hydrocarbons identified by 1H NMR and GC-MS analyses. Predominant species were octacosane (C28H58-36%) and triacontane (C30H62-13%), which individually showed significant cytotoxic activity against murine melanoma B16F10-Nex2 cells in vitro and a very promising antitumor protection against subcutaneous melanoma in vivo. Conclusion: The results suggest that the components of the heptane extract, mainly octasane and triacontane, which showed antitumor properties in experimental melanoma upon regional administration, might also be therapeutic in human cancer, such as in the mostly epidermal and slowly invasive melanomas, such as acral lentiginous melanoma, as an adjuvant treatment to surgical excision.

A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

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.

Abstract 2867: A novel WT1-derived peptide induces cellular senescence and inhibits tumor growth in a human melanoma cell line and xenograft model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant melanoma is the most aggressive and deadly type of skin cancer. The Wilms tumor protein 1 (WT1) is a transcription factor that has been associated with melanoma oncogenesis and proliferation, hence could be a target of tumor control procedures. A lysine-arginine rich peptide (WT1 pTj) based on WT1 C-terminal zinc finger domain was synthesized and tested for anti-proliferative effects on human and murine melanoma cell lines (A2058 and B16F10-Nex2, respectively), including inhibition of cell growth and colony formation, cell cycle arrest and induction of cell senescence. Internalization of WT1 pTj into A2058 cells was established by confocal microscopy, using biotinylated peptide and staining by streptoavidin-FITC. An effect on p53 activity was measured by a luciferase reporter assay. Competitive ELISA assay was performed to verify whether the peptide interferes with p53-WT1 interaction. In vivo, athymic nude mice and C57Bl/6 were inoculated with A2058 and B16F10-Nex2 cells, respectively, and treated for 5 consecutive days with C-amidated WT1 pTj. Here, we show that WT1 pTj decreased, in a dose and time dependent manner, the number of melanoma viable cells. The anti-proliferative activity of WT1 pTj was linked to an irreversible G1 cell cycle arrest and induction of senescence as suggested by positive staining for beta-galactosidase. Treatment with WT1 pTj reduced the total number of soft-agar colonies by A2058 melanoma cells. The peptide entered the cell within 15 minutes and was diffusely distributed throughout the cell, including the nucleus. Moreover, WT1 pTj enhanced p53 activity and competed with WT1 protein for binding to p53. In vivo, WT1 pTj reduced the number of lung nodules in the B16F10-Nex2 syngeneic model of metastatic melanoma and prolonged survival of mice in the A2058 subcutaneous xenograft model. In conclusion, WT1 pTj peptide entered cancer cells and promoted antitumor activity in vitro and in vivo. The data support the hypothesis that WT1 pTj may prevent important interactions between WT1 protein and its partners via a zinc finger motif, inhibiting melanoma development. Therefore, WT1 pTj is a promising agent against WT1-expressing malignancies (e.g. MCF-7 but not SK-BR-3 breast cancer or normal human fibroblasts; and also HL60 acute myeloid leukemia). WT1 pTj may act also as a trojan carrier peptide for potential drug delivery. Supported by FAPESP grant 2010/51423-0 and the Brazilian National Research Council (CNPq) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2867. doi:1538-7445.AM2012-2867

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

ABSTRACT 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.