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

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 , Denise C. Arruda* .

Journal Name: Anti-Cancer Agents in Medicinal Chemistry

Volume 19 , Issue 3 , 2019

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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.

[1]
American Cancer Society. Melanoma Skin Cancer., https://www. cancer.org/cancer/melanoma-skin-cancer/about/key-statistics.html/ (Accessed August 03, 2018).
[2]
World Health Organization. Health effects of UV radiation. http://www.who.int/uv/health/uv_health2/en/index1.html/ (Accessed August 03, 2018).
[3]
Block, K.I.; Gyllenhaal, C.; Lowe, L.; Amedei, A.; Amin, A.R.M.R.; Amin, A.; Aquilano, K.; Arbiser, J.; Arreola, A.; Arzumanyan, A.; Ashraf, S.S.; Azmi, A.S.; Benencia, F.; Bhakta, D.; Bilsland, A.; Bishayee, A.; Blain, S.W.; Block, P.B.; Boosani, C.S.; Carey, T.E.; Carnero, A.; Carotenuto, M.; Casey, S.C.; Chakrabarti, M.; Chaturvedi, R.; Chen, G.Z.; Chen, H.; Chen, S.; Chen, Y.C.; Choi, B.K.; Ciriolo, M.R.; Coley, H.M.; Collins, A.R.; Connell, M.; Crawford, S.; Curran, C.S.; Dabrosin, C.; Damia, G.; Dasgupta, S.; DeBerardinis, R.J.; Decker, W.K.; Dhawan, P.; Diehl, A.M.E.; Dong, J.T.; Dou, Q.P.; Drew, J.E.; Elkord, E.; El-Rayes, B.; Feitelson, M.A.; Felsher, D.W.; Ferguson, L.R.; Fimognari, C.; Firestone, G.L.; Frezza, C.; Fujii, H.; Fuster, M.M.; Generali, D.; Georgakilas, A.G.; Gieseler, F.; Gilbertson, M.; Green, M.F.; Grue, B.; Guha, G.; Halicka, D.; Helferich, W.G.; Heneberg, P.; Hentosh, P.; Hirschey, M.D.; Hofseth, L.J.; Holcombe, R.F.; Honoki, K.; Hsu, H.Y.; Huang, G.S.; Jensen, L.D.; Jiang, W.G.; Jones, L.W.; Karpowicz, P.A.; Keith, W.N.; Kerkar, S.P.; Khan, G.N.; Khatami, M.; Ko, Y.H.; Kucuk, O.; Kulathinal, R.J.; Kumar, N.B.; Kwon, B.S.; Le, A.; Lea, M.A.; Lee, H.Y.; Lichtor, T.; Lin, L.T.; Locasale, J.W.; Lokeshwar, B.L.; Longo, V.D.; Lyssiotis, C.A.; MacKenzie, K.L.; Malhotra, M.; Marino, M.; Martinez-Chantar, M.L.; Matheu, A.; Maxwell, C.; McDonnell, E.; Meeker, A.K.; Mehrmohamadi, M.; Mehta, K.; Michelotti, G.A.; Mohammad, R.M.; Mohammed, S.I.; Morre, D.J.; Muralidhar, V.; Muqbil, I.; Murphy, M.P.; Nagaraju, G.P.; Nahta, R.; Niccolai, E.; Nowsheen, S.; Panis, C.; Pantano, F.; Parslow, V.R.; Pawelec, G.; Pedersen, P.L.; Poore, B.; Poudyal, D.; Prakash, S.; Prince, M.; Raffaghello, L.; Rathmell, J.C.; Rathmell, W.K.; Ray, S.K.; Reichrath, J.; Rezazadeh, S.; Ribatti, D.; Ricciardiello, L.; Robey, R.B.; Rodier, F.; Rupasinghe, H.P.V.; Russo, G.L.; Ryan, E.P.; Samadi, A.K.; Sanchez-Garcia, I.; Sanders, A.J.; Santini, D.; Sarkar, M.; Sasada, T.; Saxena, N.K.; Shackelford, R.E.; Shantha-Kumara, H.M.C.; Sharma, D.; Shin, D.M.; Sidransky, D.; Siegelin, M.D.; Signori, E.; Singh, N.; Sivanand, S.; Sliva, D.; Smythe, C.; Spagnuolo, C.; Stafforini, D.M.; Stagg, J.; Subbarayan, P.R.; Sundin, T.; Talib, W.H.; Thompson, S.K.; Tran, P.T.; Ungefroren, H.; Vander-Heiden, M.G.; Venkateswaran, V.; Vinay, D.S.; Vlachostergios, P.J.; Wang, Z.; Wellen, K.E.; Whelan, R.L.; Yang, E.S.; Yang, H.; Yang, X.; Yaswen, P.; Yedjou, C.; Yin, X.; Zhu, J.; Zollo, M. Designing a broad-spectrum integrative approach for cancer prevention and treatment. Semin. Can Biol., 2015, 35, S276-S304.
[4]
Röckmann, H.; Schadendorf, D. Drug resistance in human melanoma: Mechanisms and therapeutic opportunities. Onkologie, 2003, 26, 581-587.
[5]
Soengas, M.S.; Lowe, S.W. Apoptosis and melanoma chemoresistance. Oncogene, 2003, 22(20), 3138-3151.
[6]
Sookraj, K.A.; Adler, V.; Sarafraz-Yazdi, E.; Bowne, W.B. W1961 Novel p53-derived peptide induces extensive necrosis in cancer cells. Gastroenterology, 2008, 134(4), 7743.
[7]
Fulda, S.; Wick, W.; Weller, M.; Debatin, K.M. Smac agonists sensitize for Apo2L/TRAIL-or anticancer drug-induced apoptosis and induced regression of malignant glioma in vivo. Nat. Med., 2002, 8(8), 808-815.
[8]
Polonelli, L.; Pontón, J.; Elguezabal, N.; Moragues, M.D.; Casoli, C.; Pilotti, E.; Ronzi, P.; Dobroff, A.S.; Rodrigues, E.G.; Juliano, M.A.; Maffei, D. L Antibody Complementarity-Determining Regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities. PLoS One, 2008, 3(6), 2371.
[9]
Arruda, D.C.; Santos, L.C.P.; Melo, F.M.; Pereira, F.V.; Figueiredo, C.R.; Matsuo, A.L.; Mortara, R.A.; Juliano, M.A.; Rodrigues, E.G.; Dobroff, A.S.; Polonelli, L.; Travassos, L.R. β-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma. J. Biol. Chem., 2012, 287(18), 14912-14922.
[10]
Maijaroen, S.; Jangpromma, N.; Daduang, J.; Klaynongsruang, S. KT2 and RT2 modified antimicrobial peptides derived from Crocodylus siamensis Leucrocin I show activity against human colon cancer HCT-116 cells. Environ. Toxicol. Pharmacol., 2018, 62, 164-176.
[11]
Feng, Z.; Wang, H.; Du, X.; Shi, J.; Li, J.; Xu, B. Minimal C-terminal modification boosts peptide self-assembling ability for necroptosis of cancer cells. Chem. Commun. (Camb.), 2016, 52(37), 6332-6335.
[12]
Krause, G.C.; Lima, K.G.; Dias, H.B.; Da-Silva, E.F.G.; Haute, G.V.; Basso, B.S.; Gassen, R.B.; Marczak, E.S.; Nunes, R.S.B.; De-Oliveira, J.R. Liraglutide, a glucagon-like peptide-1 analog, induce autophagy and senescence in HepG2 cells. Eur. J. Pharmacol., 2017, 15(809), 32-41.
[13]
Rabaça, A.N.; Arruda, D.C.; Figueiredo, C.R.; Massaoka, M.H.; Farias, C.F.; Tada, D.B.; Maia, V.C.; Silva, Jr, P.I.; Girola, N.; Real, F.; Mortara, R.A.; Polonelli, L.; Travassos, L.R. AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model. FEBS Open Bio, 2016, 6(9), 885-901.
[14]
Cook, A.L.; Sturm, R.A. POU domain transcription factors: BRN2 as a regulator of melanocytic growth and tumourigenesis. Pigm Cell Melanoma Res., 2008, 21(6), 611-626.
[15]
Goodall, J.; Wellbrock, C.; Dexter, T.J.; Roberts, K.; Marais, R.; Goding, C.R. The BRN2 transcription factor links activated BRAF to melanoma proliferation. Mol. Cell. Biol., 2004, 24, 2923-2931.
[16]
Wellbrock, C.; Rana, S.; Paterson, H.; Pickersgill, H.; Brummelkamp, T.; Marais, R. Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF. PLoS One, 2008, 163(7), e2734 15..
[17]
Fane, M.E.; Chhabra, Y.; Smith, A.G.; Sturm, R.A. BRN2, a POUerful driver of melanoma phenotype switching and metastasis. Pigment Cell Melanoma Res., 2019, 32, 9-24.
[18]
Goodall, J.; Carreira, S.; Denat, L.; Kobi, D.; Davidson, I.; Nuciforo, P.; Sturm, R.; Larue, L.; Goding, C.R. BRN2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells. Cancer Res., 2008, 68(19), 7788-7794.
[19]
Bonvin, E.; Falletta, P.; Shaw, H.; Delmas, V.; Goding, C.R. A phosphatidylinositol 3-kinase-Pax3 axis regulates BRN2 expression in melanoma. Mol. Cell. Biol., 2012, 32(22), 4674-4683.
[20]
Dobroff, A.S.; Rodrigues, E.G.; Moraes, J.Z.; Travassos, L.R. Protective anti-tumor monoclonal antibody recognizes a conformational epitope similar to melibiose at the surface of invasive murine melanoma cells. Hybrid. Hybridomics, 2002, 21, 321-331.
[21]
Miguel, D.C.; Flannery, A.R.; Mittra, B.; Andrews, N.W. Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence. Infect. Immun., 2013, 81(10), 3620-3626.
[22]
Oddo, A.; Hansen, P.R. Hemolytic activity of antimicrobial peptides. Methods Mol. Bio., 2017, 1548, 427-435.
[23]
Atkinson, E.A.; Barry, M.; Darmon, A.J.; Shostak, I.; Turner, P.C.; Moyer, R.W.; Bleackley, R.C. Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. J. Biol. Chem., 1998, 273(33), 21261-21266.
[24]
Dong, T.; Liao, D.; Liu, X.; Lei, X. Using small molecules to dissect non-apoptotic programmed cell death: Necroptosis, ferroptosis, and pyroptosis. Chembiochem, 2015, 16(18), 2557-2561.
[25]
Figueiredo, C.R.; Matsuo, A.L.; Pereira, F.V.; Rabaça, A.N.; Farias, C.F.; Girola, N.; Massaoka, M.H.; Azevedo, R.A.; Scutti, J.A.; Arruda, D.C.; Silva, L.P.; Rodrigues, E.G.; Lago, J.H.; Travassos, L.R.; Silva, R.M. Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons inducesapoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo. Pharmacogn. Mag., 2014, 10(Suppl. 2), S363-S376.
[26]
Negoescu, A.; Guillermet, C.; Lorimier, P.; Brambilla, E.; Labat-Moleur, F. Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed. Pharmacother., 1998, 52(6), 252-258.
[27]
Amin, H.M.; Medeiros, L.J.; Ma, Y.; Feretzaki, M.; Das, P.; Leventaki, V.; Rassidakis, G.Z.; O’Connor, S.L.; McDonnell, T.J.; Lai, R. Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma. Oncogene, 2003, 22(35), 5399-5407.
[28]
Kuypers, F.A.; Lewis, R.A.; Hua, M.; Schott, M.A.; Discher, D.; Ernst, J.D.; Lubin, B.H. Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood, 1996, 87(3), 1179-1187.
[29]
Thornberry, N.A.; Lazebnik, Y. Caspases: Enemies within. Science, 1998, 281(5381), 1312-1316.
[30]
Sinthujaroen, P.; Wanachottrakul, N.; Pinkaew, D.; Petersen, J.R.; Phongdara, A.; Sheffield-Moore, M.; Fujise, K. Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo. BBA Clin., 2014, 2, 103-111.
[31]
Chan, F.K.; Moriwaki, K.; De-Rosa, M.J. Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol. Biol., 2013, 979, 65-70.
[32]
Girola, N.; Matsuo, A.L.; Figueiredo, C.R.; Massaoka, M.H.; Farias, C.F.; Arruda, D.C.; Azevedo, R.A.; Monteiro, H.P.; Resende-Lara, P.T.; Cunha, R.L.; Polonelli, L.; Travassos, L.R. 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. Peptides, 2016, 85, 1-15.
[33]
Massaoka, M.H.; Matsuo, A.L.; Figueiredo, C.R.; Girola, N.; Faria, C.F.; Azevedo, R.A.; Travassos, L.R. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems. FEBS Open Bio, 2014, 21(4), 153-161.
[34]
Wang, X.; Qiao, Y.; Asangani, I.A.; Ateeq, B.; Poliakov, A.; Cieślik, M.; Pitchiaya, S.; Chakravarthi, B.V.; Cao, X.; Jing, X.; Wang, C.X.; Apel, I.J.; Wang, R.; Tien, J.C.; Juckette, K.M.; Yan, W.; Jiang, H.; Wang, S.; Varambally, S.; Chinnaiyan, A.M. Development of peptidomimetic inhibitors of the ERG gene fusion product in prostate cancer. Canc Cell, 2017, 31(4), 532-548.
[35]
Peixoto, P.; Liu, Y.; Depauw, S.; Hildebrand, M.P.; Boykin, D.W.; Bailly, C.; Wilson, W.D.; David-Cordonnier, M.H. Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furanbenzimidazole dication. Nucleic Acids Res., 2008, 36, 3341-3353.
[36]
Pathria, G.; Ronai, Z.A. BRN 2 Invade. Cancer Cell, 2018, 34(1), 1-3.
[37]
Renz, A.; Berdel, W.E.; Kreuter, M.; Belka, C.; Schulze-Osthoff, K.; Los, M. Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo. Blood, 2001, 98, 1542-1548.
[38]
Gobe, G.; Crane, D. Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicol. Lett., 2010, 198(1), 49-55.
[39]
Luetjens, C.M.; Kögel, D.; Reimertz, C.; Düßmann, H.; Renz, A.; Schulze-Osthoff, K.; Nieminen, A.L.; Poppe, M.; Prehn, J.H. Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis. Mol. Pharmacol., 2001, 60, 1008-1019.
[40]
Paradies, G.; Petrosillo, G.; Pistolese, M.; Ruggiero, F.M. Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage. Gene, 2002, 286, 135-141.
[41]
Vakifahmetoglu-Norberg, H.; Ouchida, A.T.; Norberg, E. The role of mitochondria in metabolism and cell death. Biochem. Biophys. Res. Commun., 2017, 482(3), 426-431.
[42]
Masse, M.; Glippa, V.; Saad, H.; Le Bloas, R.; Gauffeny, I.; Berthou, C.; Czjzek, M.; Cormier, P.; Cosson, B. An eIF4E-interacting peptide induces cell death in cancer cell lines. Cell Death Dis., 2014, 5(10)e1500
[43]
Franz, S.; Frey, B.; Sheriff, A.; Gaipl, U.S.; Beer, A.; Voll, R.E.; Kalden, J.R.; Herrmann, M. Lectins detect changes of the glycosylation status of plasma membrane constituents during late apoptosis. Cytometry A, 2006, 69, 230-239.
[44]
Sun, L.; Wang, H.; Wang, Z.; He, S.; Chen, S.; Liao, D.; Wang, L.; Yan, J.; Liu, W.; Lei, X.; Wang, X. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell, 2012, 148(1-2), 213-227.
[45]
Sun, L.; Wang, H.; Wang, Z.; He, S.; Chen, S.; Liao, D.; Wang, L.; Yan, J.; Liu, W.; Lei, X.; Wang, X. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol. Cell, 2014, 148(1), 213-227.
[46]
Asaro, R.J.; Zhu, Q.; Cabrales, P.; Carruthers, A. Do skeletal dynamics mediate sugar uptake and transport in human erythrocytes? Biophys. J., 2018, 114(6), 1440-1454.
[47]
Shamloo, A.; Mehrafrooz, B. Nanomechanics of actin filament: A molecular dynamics simulation. Cytoskeleton, 2018, 75(3), 118-130.
[48]
He, L.; Sayers, E.J.; Watson, P.; Jones, A.T. Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates. Sci. Rep., 2018, 8(1), 7318.


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VOLUME: 19
ISSUE: 3
Year: 2019
Page: [389 - 401]
Pages: 13
DOI: 10.2174/1871520618666181109164246
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