Anti-tumor activities of the host-defense peptide hymenochirin-1B Samir Attoub a , Hama Arafat a , Milena Mechkarska b , J. Michael Conlon b, ⁎ a Department of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates b Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates abstract article info Article history: Received 8 July 2013 Received in revised form 23 October 2013 Accepted 28 October 2013 Available online 31 October 2013 Keywords: Host-defense peptide Hymenochirin Frog skin Structure–activity Anti-cancer The hymenochirins are a family of cationic, amphipathic, α-helical host-defense peptides, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Of the four hymenochirins tested, hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIVAKMV.NH 2 ) shows the greatest cytotoxic potency against non-small cell lung adenocarcinoma A549 cells (LC 50 = 2.5 ± 0.2 μM), breast adenocarcinoma MDA-MB-231 cells (LC 50 = 9.0 ± 0.3 μM), colorectal adenocarcinoma HT-29 cells (LC 50 = 9.7 ± 0.2 μM), and hepatocarcinoma HepG2 cells (LC 50 = 22.5 ± 1.4 μM) with appreciably less hemolytic activity against human erythrocytes (LC 50 = 213 ± 18 μM). Structure–activity relationships were investigated by synthesizing analogs of hymenochirin-1B in which Pro 5 , Glu 6 and Asp 9 on the hydrophilic face of the helix were replaced by one or more L-lysine or D-lysine residues. The [D9K] analog displays the greatest increase in potency against all four cell lines (up to 6 fold) but hemolytic activity also increases (LC 50 = 174 ± 12 μM). The [D9k] and [E6k,D9k] analogs retain relatively high cytotoxic potency against the tumor cells (LC 50 in the range 2.1–21 μM) but show reduced hemolytic activity (LC 50 N 300 μM). The data suggest that hymenochirin- 1B has therapeutic potential as a template to generate potent, non-toxic anti-cancer agents. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Peptides with potent antibacterial and antifungal activity and the ability to permeabilize mammalian cells play an important role in the system of innate immunity that constitutes the first-line defense against invading pathogens for both vertebrate and invertebrate species. Such peptides are multifunctional possessing immunomodulatory and chemoattractant properties as well as cytotoxic activities so that it is more informative to refer to them as host-defense peptides rather than antimicrobial peptides [1,2]. Skin secretions from many species of Anura (frogs and toads) contain host-defense peptides that, with few exceptions, are cationic (molecular charge between +2 and +6 at pH 7), contain at least 50% hydrophobic amino acids, and have the propensity to adopt an amphipathic α-helical conformation in a membrane-mimetic environment (reviewed in [3]). Although there is no single mechanism by which the peptides produce cell death, their ac- tion generally involves a non-specific perturbation of the cell membrane and insertion into the lipid bilayer leading to membrane disruption and cell lysis rather than binding to specific receptors on the cell membrane or intracellular targets [4,5]. Host-defense peptides have excited interest because of the possibil- ities for development into anti-infective drugs for use against pathogen- ic microorganisms that have developed resistance against commonly used antibiotics (reviewed in [6]). The problem of multidrug resistance in the treatment of bacterial infections is also encountered in cancer chemotherapy [7]. Because of their non-specific and destructive mech- anism of action, naturally occurring host-defense peptides show thera- peutic potential for development into anti-cancer agents in cases where the tumor is not responsive to conventional pharmaceutical interven- tion. Several cationic host-defense peptides, first identified on the basis of their antimicrobial activity, have shown selective cytotoxic ac- tivity against a range of tumor cell lines (reviewed in [8–11]). Among the naturally occurring frog skin peptides, these include ascaphin-8 from Ascaphus truei [12], magainin-2 from Xenopus laevis [13–15], peptide XT-7 from Silurana epitropicalis [12], alyteserin-2a from Alytes obstetricans [16], dermaseptins from Hylomantis lemur [17] and Phyllomedusa bicolor [18], aureins from Litoria aurea and Litoria raniformis [19], pentadactylin from Leptodactylus labyrinthicus [20], brevinin-1BYa and its dicarba derivative from Rana boylii [21], temporin-1CEa from Rana chensinensis [22], and esculentin-2CHa from Lithobates chiricahuensis [23]. These peptides are not tumor- specific in their cytotoxic action but show N 5 fold greater potency against tumor cells than against erythrocytes or fibroblasts. The hymenochirins are a family of five host-defense peptides that were first isolated from norepinephrine-stimulated skin secretions from the Congo clawed frog Hymenochirus boettgeri (Pipidae) [24]. The peptides display potent, broad-spectrum growth inhibitory activity against a range of reference strains of clinically relevant bacteria and are associated with relatively weak hemolytic activity. The present study compares the effects of hymenochirin-1B, -2B, -3B, and -4B on the viabil- ity of four well-characterized human tumor cell lines: non-small cell lung Regulatory Peptides 187 (2013) 51–56 ⁎ Corresponding author at: Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al Ain, United Arab Emirates. Tel.: +971 3 7137484; fax: +971 3 7672033. E-mail address: jmconlon@uaeu.ac.ae (J.M. Conlon). 0167-0115/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2013.10.006 Contents lists available at ScienceDirect Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep