Antibacterial Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Cytotoxic Effect against MDA-MB-468 and MDA-MB-231 Breast Cancer Cell Lines
Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B, containing the RRWQWR motif, were designed, synthesized, purified, and characterized using RP-HPLC chromatography and MALDI-TOF mass spectrometry. The antibacterial activity of the designed peptides against E. coli (ATCC 11775 and 25922) and their cytotoxic effect against MDA-MB-468 and MDA-MB-231 breast cancer cell lines were evaluated. Dimeric and tetrameric peptides showed higher antibacterial activity in both
... ctivity in both bacteria strains than linear peptides. The dimeric peptide (RRWQWR) 2 K-Ahx exhibited the highest antibacterial activity against the tested bacterial strains. Furthermore, the peptides with high antibacterial activity exhibited significant cytotoxic effect against the tested breast cancer cell lines. This cytotoxic effect was fast and dependent on the peptide concentration. The tetrameric molecule containing RRWQWR motif has an optimal cytotoxic effect at a concentration of 22 µM. The evaluated dimeric and tetrameric peptides could be considered as candidates for developing new therapeutic agents against breast cancer. Polyvalence of linear sequences could be considered as a novel and versatile strategy for obtaining molecules with high anticancer activity. of life of the patient. These treatments prolong the survival time, but recurrence of the disease is frequent. Chemotherapy causes several adverse effects, such as neutropenia, nausea and vomiting, amenorrhea, alopecia, neurological toxicity, weight gain, secondary leukemia, and cardiotoxicity [2, 3]. Hormone therapy also causes adverse effects, such as hot flashes, musculoskeletal pain, fatigue, mood disturbances, nausea, vomiting, and fractures, among others  . Antimicrobial peptides (AMPs) exhibit a cytotoxic effect on cancer cell lines and antimicrobial activity because of the electrostatic interaction between the amino acid side chain and the negative charge of the membrane surface [5, 6] . AMPs are able to discriminate between neoplastic and non-neoplastic cells, interacting specifically with negatively-charged membrane components such as phosphatidylserine, sialic acid, or heparan sulfate, which differ between cancer and non-cancer cells  . AMPs are considered to be a main source of molecules that are candidates for developing new drugs based on peptides. Bovine lactoferrin (BLF) is a milk protein with great biological activity. LF exhibits antimicrobial activity against pathogenic bacteria, fungi, parasites, and viruses. Furthermore, BLF inhibits colon, esophagus, lung, and bladder carcinogenesis in rats when administered orally in the post-initiation stage. BLF has the capacity to reduce the metastatic properties of both MDA-MB-231 and MCF-7 cell lines  . The bovine lactoferricin LfcinB: 17 FKCRRWQWRMKKLGAPSITCVRRAF 41 is a 25 amino acid-peptide belonging to the N-terminal region of BLF [9-12]. It has been suggested that LfcinB is responsible for the antimicrobial activity of BLF, and LfcinB exhibits greater antimicrobial and anticancerigenic activity than BLF itself    . LfcinB contains aromatic amino acids, such as tryptophan and phenylalanine, and basic residues (arginine and lysine) that confer amphipathic properties [9, 16] . These positively-charged residues interact electrostatically with the negative charges of the bacterial cell wall lipopolysaccharide (LPS), allowing the peptide to approach the bacterial membrane [9, 16] . Thereupon, hydrophobic residues interact with the membrane lipid bilayer, causing its disruption and cell lysis [9, 16] . LfcinB exhibits a cytotoxic effect against human cancer cell lines of breast, gastric, and colorectal cancer, leukemia, fibrosarcomas, melanomas, and colon cancer      . Subcutaneous administration of LfcinB to mice inhibits lymphoma cell metastasis to the liver and lungs [17, 22] . The minimal motif (RRWQWR) exhibited cytotoxic activity in leukemia cells, and the dead cells were related to both Cathepsin B and caspase activity [17, 23, 24] . In the present paper, linear, dimeric, and tetrameric peptides were synthesized via solid-phase peptide synthesis (SPPS) and purified and characterized using reverse phase high performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Peptide antibacterial activity (against E. coli strains) and cytotoxicity against two breast cancer cell lines were evaluated. Our results indicate that peptides derived from LfcinB that exhibit antibacterial activity also exhibit a cytotoxic effect in breast cancer cell lines. Tetrameric and dimeric peptides containing the minimal motif have the greatest cytotoxic effect against both MDA-MB-468 and MDA-MB-231 breast cancer cell lines. This activity was fast and dependent on peptide concentration. Results and Discussion The aim of this study was to establish if the polyvalence or molecular restriction of LfcinB-derived peptides increases its antibacterial activity and/or cytotoxic effect in breast cancer cell lines. Therefore, linear, dimeric, tetrameric, and cyclic peptides containing sequences derived from LfcinB were designed and synthesized through SPPS, using the Fmoc/tBu strategy. Specifically, sequences LfcinB (20-25): 20 RRWQWR 25 , LfcinB (20-30): 20 RRWQWRMKKLG 30 , and [Ala 19 ]-LfcinB (17-31): 17 FKARRWQWRMKKLGA 31 were selected. In order to obtain families of each sequence, all groups contained four members, specifically (i) a linear; (ii) a dimeric; (iii) a cyclic; and (iv) a tetrameric peptide, as it is shown in Figure 1 .