Rhodamine B-conjugated encrypted vipericidin nonapeptide is a potent toxin to zebrafish and associated with in vitro cytotoxicity

• Published in: Biochimica et biophysica acta Vol. 1850; no. 6; pp. 1253 - 1260
• Main Authors: Wang, Liang, Chan, Judy Y.W., Rêgo, Juciane V., Chong, Cheong-Meng, Ai, Nana, Falcão, Cláudio B., Rádis-Baptista, Gandhi, Lee, Simon M.Y.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2015
• Subjects: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents - isolation & purification; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; Antineoplastic Agents - toxicity; breast neoplasms; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; calcium; Calcium Signaling - drug effects; Cathelicidins - isolation & purification; Cathelicidins - metabolism; Cathelicidins - pharmacology; Cathelicidins - toxicity; Cell Membrane - drug effects; Cell Membrane - pathology; Cell Survival - drug effects; Cell-penetrating peptide; cytoplasm; cytotoxicity; Cytotoxin; Danio rerio; Dose-Response Relationship, Drug; Encrypted peptides; enzymes; Female; fluorescence; homeostasis; Humans; Larva - drug effects; larvae; Lethal Dose 50; MCF-7 Cells; Molecular Sequence Data; neoplasm cells; Oligopeptides - isolation & purification; Oligopeptides - metabolism; Oligopeptides - pharmacology; Oligopeptides - toxicity; proteins; Rhodamine B-conjugated peptide; Rhodamines - metabolism; Rhodamines - pharmacology; Rhodamines - toxicity; Time Factors; toxins; venoms; Viper Venoms - chemistry; Vipericidins; Zebra-fish model; Zebrafish - embryology; CRAMP; Zebra-fish model; Rhodamine B-conjugated peptide; Vipericidins; Encrypted peptides; dpf; Cytotoxin; EVP; ACP; RhoB; Cell-penetrating peptide
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.02.013

Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6μM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide. [Display omitted] •Vipericidins, reptilian cathelicidins, have short in tandem cryptic peptides (EVP-50).•Rhodamine B-conjugated encrypted vipericidin nonaneptide is converted in a cytotoxin.•Zebrafish cardiovascular system is a target tissue for the action of RhoB-EVP50.•RhoB-EVP50 causes calcium imbalance and membrane dysfunction in cancer cells.