Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo.
BACKGROUND
Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model.


METHODS
B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins.


RESULTS
Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.


CONCLUSIONS
We conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.
Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. camyla.ff@gmail.com.
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Farias
Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. mari.massa@gmail.com.
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Massaoka
Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. nataliagirola@hotmail.com.
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Girola
Laboratory of Tumor Immunology, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. ricardoaazevedo@hotmail.com.
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Azevedo
Experimental Physiopathology, Faculty of Medicine, University of São Paulo, São Paulo, SP, Brazil. ferreira-kleber@usp.br.
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Ferreira
Laboratory of Tumor Immunology, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. sdjorge@gmail.com.
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Jorge
Department of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo, SP, Brazil. leoberto@usp.br.
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Tavares
Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. rogernty@hotmail.com.
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Figueiredo
Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. luiztravassos@gmail.com.
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Travassos
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D001583:Benzoxazoles / Q000737:chemistry* / Q000494:pharmacology / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D051058:Oncogene Protein v-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D010069:Oxadiazoles
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001583:Benzoxazoles; D008565:Membrane Proteins; D051058:Oncogene Protein v-akt; D011518:Proto-Oncogene Proteins; C000606775:BCL2L11 protein, human; C000606776:Bcl2l11 protein, mouse; C014354:benzofuroxan; D010069:Oxadiazoles