title: BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity.
abstract: Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma.
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil; Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen, The Netherlands. Electronic address: ferreira-kleber@usp.br.
authors.forename: Adilson Kleber
authors.initials: AK
authors.lastname: Ferreira
authors.affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Kerly Fernanda Mesquita
authors.initials: KF
authors.lastname: Pasqualoto
authors.affiliation: Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen, The Netherlands.
authors.forename: Frank A E
authors.initials: FA
authors.lastname: Kruyt
authors.affiliation: Laboratory of Drug Design and Development, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Fanny
authors.initials: F
authors.lastname: Palace-Berl
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Ricardo Alexandre
authors.initials: RA
authors.lastname: Azevedo
authors.affiliation: Laboratory of Cytopathology, Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Kely Medeiros
authors.initials: KM
authors.lastname: Turra
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Cecilia Pessoa
authors.initials: CP
authors.lastname: Rodrigues
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Ana Carolina Franco
authors.initials: AC
authors.lastname: Ferreira
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Maria Alejandra Clavijo
authors.initials: MA
authors.lastname: Salomón
authors.affiliation: Laboratory of Genetics, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Paulo Luiz
authors.initials: PL
authors.lastname: de Sá Junior
authors.affiliation: Experimental Oncology Section, The Federal University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Camyla Fernandes
authors.initials: CF
authors.lastname: Farias
authors.affiliation: Experimental Oncology Section, The Federal University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Carlos Rogerio
authors.initials: CR
authors.lastname: Figueiredo
authors.affiliation: Laboratory of Drug Design and Development, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Leoberto Costa
authors.initials: LC
authors.lastname: Tavares
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil; Cell and Molecular Therapy Center NUCEL-NETCEM, University of São Paulo, SP, Brazil.
authors.forename: José Alexandre Marzagão
authors.initials: JA
authors.lastname: Barbuto
authors.affiliation: Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP, Brazil. Electronic address: sdjorge@usp.br.
authors.forename: Salomão Dória
authors.initials: SD
authors.lastname: Jorge
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001583:Benzoxazoles; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects*; D019938:Cyclin D1 / Q000096:biosynthesis; D051358:Cyclin-Dependent Kinase 4 / Q000096:biosynthesis; D005434:Flow Cytometry; D006834:Hydrazines / Q000494:pharmacology*; D008546:Melanoma, Experimental / Q000276:immunology*; D051379:Mice; D008856:Microscopy, Fluorescence; D062105:Molecular Docking Simulation; D010069:Oxadiazoles / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors*
chemical_list: C000609140:BFD-22; D001583:Benzoxazoles; D006834:Hydrazines; D010069:Oxadiazoles; D019938:Cyclin D1; C014354:benzofuroxan; D048493:Proto-Oncogene Proteins B-raf; D051358:Cyclin-Dependent Kinase 4
keywords: Anti-metastatic; BFD-22; Benzofuroxan derivatives; Cell death; Melanoma