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

[ Vol. 19 , Issue. 3 ]

Author(s):

Fernanda F.M. da Cunha, Katia C.U. Mugnol, Filipe M. de Melo , Marta V.S.Q. Nascimento, Ricardo A. de Azevedo, Raquel T. S. Santos, Jéssica A. Magalhães, Danilo C. Miguel , Dayane B. Tada, Renato A. Mortara, Luiz R. Travassos and Denise C. Arruda*   Pages 389 - 401 ( 13 )

Abstract:


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.

Keywords:

Peptide, melanoma, apoptosis, transcription factor, R18H, Brn-2.

Affiliation:

Núcleo Integrado de Biotecnologia (NIB), Universidade de Mogi das Cruzes, UMC, Mogi das Cruzes, SP, Centro Interdisciplinar de Investigação Bioquímica (CIIB) Universidade de Mogi das Cruzes, UMC, Mogi das Cruzes, SP, Departamento de Imunologia, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, UMC, Mogi das Cruzes, SP, Unidade de Oncologia Experimental (UNONEX), Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Núcleo Integrado de Biotecnologia (NIB), Universidade de Mogi das Cruzes, UMC, Mogi das Cruzes, São Paulo, SP, Laboratório de Nanomateriais e Nanotoxicologia, Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo (UNIFESP), São José dos Campos, SP, Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Laboratório de Nanomateriais e Nanotoxicologia, Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo (UNIFESP), São José dos Campos, SP, Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Unidade de Oncologia Experimental (UNONEX), Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Núcleo Integrado de Biotecnologia (NIB), Universidade de Mogi Das Cruzes, UMC, Mogi das Cruzes, SP



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