Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β -casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo , antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci . In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.
S. Riedl, D. Zwytick and K. Lohner, “Membrane-Active Host Defense Peptides—Challenges and Perspectives for the Development of Novel Anticancer Drugs,” Chemistry and Physics of Lipids, Vol. 164, No. 8, 2011, pp. 766-781. doi:10.1016/j.chemphyslip.2011.09.004
A. L. Matsuo, A. S. Tanaka, M. A. Juliano, E. G. Rodrigues and L. R. Travassos, “A Novel Melanoma-Targeting Peptide Screened by Phage Display Exhitits Antitumor Activity,” Journal of Molecular Medicine, Vol. 88, No. 12, 2010, pp. 1255-1264. doi:10.1007/s00109-010-0671-9
A. K. Ferreira, R. Meneguelo, S. C. Neto, G. O. Chierice and D. A. Maria, “Synthetic Phosphoethanolamine Induces Apoptosis through Caspase-3 Pathway by Decreasing Expression of Bax/Bad Protein and Changes Cell Cycle in Melanoma,” Journal Cancer Science & Therapy, Vol. 3, No. 3, 2011, pp. 53-59.
A. K. Ferreira, R. Meneguelo, S. C. Neto, A. Pereira, O. M. R. Filho, G. O. Chierice and D. A. Maria, “Anticancer Effects of Synthetic Phosphoethanolamine on Ehrlich Ascites Tumor: An Experimental Study,” Anticancer Research, Vol. 32, No. 1, 2012, pp. 95-104.
C. Araya and B. Lomonte, “Antitumor Effects of Cationic Synthetic Peptides Derived from Lys49 Phospholipase A2 Homologues of Snake Venoms,” Cell Biology International, Vol. 31, No. 3, 2007, pp. 263-268. doi:10.1016/j.cellbi.2006.11.007
T. Kawahara, D. Katayama and H. Otani, “Effect of β -Casein (1-28) on Proliferative Responses and Secretory Functions of Human Immunocompetent Cell Lines,” Bioscience, Biotechnology, and Biochemistry, Vol. 68, No. 10, 2004, pp. 2091-2095.
P. W. Parodi, “A Role for Milk Proteins and Their Peptides in Cancer Prevention,” Current Pharmaceutical Design, Vol. 13, No. 8, 2007, pp. 813-828. doi:10.2174/138161207780363059
O. Hallgren, S. Aits, P. Brest, L. Gustafsson, A. K. Mossberg, B. Wult and C. Svanborg, “Apoptosis and Tumor Cell Death in Response to HAMLET (Human Alpha-Lac-talbumin Made Lethal to Tumor Cells),” Advances in Experimental Medicine and Biology, Vol. 66, 2008, pp. 217- 240. doi:10.1007/978-0-387-74087-4_8
J. Fast, A. K. Mossberg, H. Nilsson, C. Svanborg, M. Akke and S. Linse, “Compact Oleic Acid in HAMLET,” FEBS Letter, Vol. 579, No. 27, 2005, pp. 6095-6100. doi:10.1016/j.febslet.2005.08.089
M. Svensson, A. Hakansson, S. Linse and C. Svanborg, “Conversion of Alpha-Lactlbumin to a Protein Inducing Apoptosis,” Proceedings of the National Academy Science of the United States America, Vol. 97, No. 8, 2000, pp. 4221-4226. doi:10.1073/pnas.97.8.4221
A. K. Mossberg, B. Wullt, L. Gustafsson, W. Mansson, E. Ljunggren and C. Svanborg, “Bladder Cancers Respond to Intravesical Installation of HAMLET (Human α-Lactalbumin Made Lethal to Tumor Cells),” International Journal of Cancer, Vol. 121, No. 6, 2007, pp. 1352-1359. doi:10.1002/ijc.22810
C. Kohler, V. Gogvadze, A. Hakansson, C. Svanborg, S. Orrenius and B. Zhivotovsky, “A Folding Variant of Human Alpha-Lactalbumin Induces Mitochondrial Permeability Transition in Isolated Mitochondrial,” European Journal of Biochemistry, Vol. 268, No. 1, 2001, pp. 186-191. doi:10.1046/j.1432-1327.2001.01870.x
C. Düringer, A. Hamiche, L. Gustafsson, H. Kimura and C. Svanborg, “HAMLET Interacts with Histones and Chromatin in Tumor Cell Nuclei,” Journal Biological Chemistry, Vol. 278, No. 43, 2003, pp. 42131-42135. doi:10.1074/jbc.M306462200
S. Permyakov, I. V. Pershikiva, A. P. Zhadan, J. Goers, A. G. Bakunts, V. N. Uversku, L. J. Berliner and E. A. Permyakov, “Conversion of Human Alpha-Lactalbumin to an Apo-Like State in the Complexes with Basic Poly-Amino Acids: Toward Understanding of the Molecular Mechanism of Antitumor Action of HAMLET,” Journal Proteome Research, Vol. 4, No. 2, 2005, pp. 564-569. doi:10.1021/pr0497778
M. Hoyer-Hansen and M. Jaattela, “Autophagy: An Emerging Target for Cancer Therapy,” Autophagy, Vol. 4, No. 5, 2008, pp. 574-580.
S. Aits, L. Gustafsson, O. Hallgren, P. Brest, M. Gustafsson, M. Trulsson, A. K. Mossberg, H. U. Simon, B. Mograbi and C. Svanborg, “HAMLET (Human Alpha-Lactalbumin Made Lethal to Tumor Cells) Triggers Autophagic Tumor Cell Death,” International Journal of Cancer, Vol. 124, No. 5, 2009, pp. 1008-1019. doi:10.1002/ijc.24076
E. A. Perpetuo, L. Juliano and I. Lebrun, “Biochemical and Pharmacological of Two Bradykinin-Potentiating Peptides Obtained from Tryptic Hydrolysis Of Casein,” Journal of Protein Chemistry, Vol. 22, No. 7-8, 2003, pp. 601-606. doi:10.1023/B:JOPC.0000008724.98339.ff
I. Lebrun, V. Carvalho, L. Juliano, M. A. Juliano and M. C. de Souza e Silva, “Effects of ‘Casoparan’, a Peptide Isolated from Casein Hydrolysates with Mastoparan-Like Properties,” Mediators Inflammation, Vol. 13, No. 4, 2004, pp. 263-268. doi:10.1080/09629350400003068
N. V. Vellarkad, K. G. Arup, R. R. Ganapathi and K. R. Roland, “Atomic Physicochemical Parameters for Three Dimensional Structure Directed Quantitative Structure-Activity Relationships. 4. Additional Parameters for Hydrophobic and Dispersive Interactions and Their Application for an Automated Superposition of Certain Naturally Occurring Nucleoside Antibiotics,” Journal Chemical Information Computer Science, Vol. 29, No. 3, 1989, pp. 163-172. doi:10.1021/ci00063a006
K. Gilles, L. Ju-Yun, W. Shaomeng and D. Mario, “Computer Automated Log P Calculations Based on an Extended Group Contribution Approach,” Journal Chemical Information Computer Science, Vol. 34, No. 4, 1994, pp. 752-781. doi:10.1021/ci00020a009
C. Ferenc, T. K. Anna, I. Panderi and D. Ferenc, “Prediction of Distribution Coefficient from Structure. 1. Estimation Method,” Journal of Pharmaceutical Sciences, Vol. 86, No. 7, 1997, pp. 865-871. doi:10.1021/js960177k
M. Asano, N. Nio and Y. Ariyoshi, “Inhibition of Prolyl Endopeptidase by Synthetic-Casein Peptides and Their Derivatives with a C-Terminal Prolinol or Prolinal,” Bioscience Biotechnology Biochemistry, Vol. 56, No. 2, 1992, pp. 976-977. doi:10.1271/bbb.56.976
J. S. Armstrong, “Mitochondria: A Target for Cancer Therapy,” British Journal of Pharmacology, Vol. 14, No. 3, 2006, pp. 7239-7248. doi:10.1038/sj.bjp.0706556
F. Schwizer, “Cationic Amphiphilic Peptides with Cancer-Selective Toxicity,” European Journal of Pharmacology, Vol. 625, No. 1, 2009, pp. 190-194. doi:10.1016/j.ejphar.2009.08.043
D. C. Pimenta and I. Lebrun, “Cryptides: Buried Secrets in Proteins,” Peptides, Vol. 28, No. 12, 2007, pp. 2403-2410.
D. J. Autelitano, A. Rajic, A. I. Smith, M. C. Berndt, L. L. Ilag and M. Vadas, “The Cryptome: A Subset of the Proteome, Comprising Cryptic Peptides with Distinct Bioactivities,” Drug Discovery Today, Vol. 11, No. 7-8, 2006, pp. 306-314.
B. Fadnes, O. Rekdal and L. Uhlin-Hansen, “The Anticancer Activity of Lytic Peptides Is Inhibited by Heparin Sulfate on the Surface of the Tumor Cells,” BMC Cancer, Vol. 15, No. 9, 2006, pp. 1-13. doi:10.1186/1471-2407-9-183
N. Papo and Y. Shai, “Host Defense Peptides as New Weapons in Cancer Treatment,” Cellular and Molecular Life Sciences, Vol. 62, No. 7-8, 2005, pp. 784-790. doi:10.1007/s00018-005-4560-2
S. K. Bhutia and T. K. Maiti, “Targeting Tumor with Peptides from Natural Sources,” Trends Biotechnology, Vol. 26, No. 4, 2008, pp. 210-217.
M. Wieczorek, H. Jenssen, J. Kindrachuk, W. R. P. Scott, M. Elliott, K. Hilpert, J. T. J. Cheng, R. E. W. Hancock and S. K. Straus, “Structural Studies of a Peptide with Immune Modulating and Direct Antimicrobial Activity,” Chemistry Biology, Vol. 17, No. 9, 2010, pp. 970-980. doi:10.1016/j.chembiol.2010.07.007
P. W. Soballe, W. L. Maloy, M. L. Myrga, L. S. Jacob and M. Herlyn, “Experimental Local Therapy of Human Melanoma with Lytic Magainin Peptides,” International Journal Cancer, Vol. 60, No. 2, 1995, pp. 280-284. doi:10.1002/ijc.2910600225
R. E. W. Hancock and H. G. Sahi, “Antimicrobial and Host-Defense Peptides as New Anti-Infective Therapeutic Strategies,” Nature Biotechnology, Vol. 24, No. 12, 2006, pp. 1551-1557. doi:10.1038/nbt1267
D. R. Pfeiffer, T. I. Gudz, A. S. Novgorodov and W. L. Ferdahl, “The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition,” Journal of Biological Chemistry, Vol. 270, No. 9, 1995, pp. 4923- 4932. doi:10.1074/jbc.270.9.4923
Y. Yamada, Y. Shinohara, T. Kakudo, S. C. S. Futaki, H. Kamiya and H. Harashima, “Mitochondrial Delivery of Mastoparan with Transferrin Liposomes Equipped with a pH-Sensitive Fusogenic Peptide for Selective Cancer Therapy,” International Journal of Pharmaceutics, Vol. 303, No. 1-2, 2005, pp. 1-7.
P. Costantini, E. Jacotot, D. Decaudin and G. Kroemer, “Mitochondrion as a Novel Target of Anticancer Chemotherapy,” Journal of National Cancer Institute, Vol. 92, No. 13, 2000, pp. 1042-1053. doi:10.1093/jnci/92.13.1042