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 bacteria strains than linear peptides. The dimeric peptide (RRWQWR)2K-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.

Dimeric peptides ( Figure 1B) were synthesized using the MAPs (multiple antigen peptides) methodology. The cyclic peptides ( Figure 1C) were obtained by oxidation of cysteine residues located at the sequence C and N-terminal ends. Finally, the tetrameric peptides ( Figure 1D) were obtained by the formation of an inter-disulfide bridge by the oxidation of purified dimeric precursor peptides (Table 1). All crude products were characterized using RP-HPLC and then purified by means of SPE. In all cases, the chromatographic profile of the purified products exhibited the main specie and purity was determined by RP-HPLC. MALDI-TOF-MS analysis showed that the synthesized peptides had the expected molecular weight (Table 1). As an example, Figure 2 shows the analytical results for peptide LfcinB (20)(21)(22)(23)(24)(25); the chromatographic profile of crude product (Panel A) presents a main peak (tR: 4.3 min; purity: 40%). This product was purified and characterized, the RP-HPLC analysis shows a peak with the same retention time and a purity of 92% and its MALDI-TOF MS spectrum ( Figure 2C) has a main signal at m/z 986,55 corresponding to [M + H] + . Oxidation reactions were monitored by RP-HPLC; Figure 2D presents the oxidation of a dimeric precursor, (RRWQWR)2K-Ahx-C, at reaction times of 0, 1, and 6 h, producing the tetramer (Lfcin B (20-25)4: (RRWQWR)4K2-Ahx2-C2). Dimeric peptides ( Figure 1B) were synthesized using the MAPs (multiple antigen peptides) methodology. The cyclic peptides ( Figure 1C) were obtained by oxidation of cysteine residues located at the sequence C and N-terminal ends. Finally, the tetrameric peptides ( Figure 1D) were obtained by the formation of an inter-disulfide bridge by the oxidation of purified dimeric precursor peptides (Table 1). All crude products were characterized using RP-HPLC and then purified by means of SPE. In all cases, the chromatographic profile of the purified products exhibited the main specie and purity was determined by RP-HPLC. MALDI-TOF-MS analysis showed that the synthesized peptides had the expected molecular weight (Table 1). As an example, Figure 2 shows the analytical results for peptide LfcinB (20)(21)(22)(23)(24)(25); the chromatographic profile of crude product (Panel A) presents a main peak (t R : 4.3 min; purity: 40%). This product was purified and characterized, the RP-HPLC analysis shows a peak with the same retention time and a purity of 92% and its MALDI-TOF MS spectrum ( Figure 2C) has a main signal at m/z 986,55 corresponding to [M + H] + . Oxidation reactions were monitored by RP-HPLC; Figure 2D presents the oxidation of a dimeric precursor, (RRWQWR) 2 K-Ahx-C, at reaction times of 0, 1, and 6 h, producing the tetramer (Lfcin B (20-25) 4 : (RRWQWR) 4 K 2 -Ahx 2 -C 2 ).   [15,25,26]. These results showed that dimeric and tetrameric peptides containing the RRWQWR sequence have greater antibacterial activity against E. coli strains (ATCC 11775 and ATCC 25922) than the other tested bacterial strains. In a similar way, the tetramer LfcinB (20-25)4 exhibited greater cytotoxicity against oral squamous carcinoma cell lines than its linear peptide analogue, LfcinB (20)(21)(22)(23)(24)(25) [27].
For this research, peptide activity was evaluated in two different models: (i) bacteria and (ii) breast cancer cell lines. First, the designed peptide families were tested against two E. coli strains (ATCC 11775 and 25922). It was found that dimeric and tetrameric peptides exhibit greater antibacterial activity against the evaluated strains than their linear counterpart sequences (Table 1), confirming that the polyvalence enhanced the antibacterial activity. This behavior is in accordance with the mechanism suggested for LfcinB, which involves the initial electrostatic interaction with the negative charges of the bacterial cell wall. It has been proposed that the increase of the positive charge of the molecules enables the interaction with the charged negative molecules of the bacterial surface [10][11][12][13][14]. It has been suggested that LfcinB peptides self-assemble, forming a polymeric structures as a requisite for the interaction with the bacterial surface [9,28]. On the other hand, cyclic peptides exhibit antibacterial activity similar to monomeric peptides, suggesting that molecular restriction of these amino acid sequences does not increase antibacterial activity in the evaluated strains. This indicates that the relevant properties for antibacterial activity are both positively charged and have an amphipathic sequence.
Many new therapies are currently being used for cancer treatment; among these new methods, chemotherapy based on antimicrobial peptides (AMPs) has been of great interest due to the unique advantages of this kind of molecule, such as low molecular weight, ability to specifically target tumor cells, and low toxicity in normal tissues [29]. For example, the cytotoxic effect of AMPs normally occurs at micromolar levels, and it is not accompanied by significant levels of hemolysis or toxicity to other mammalian cells. In most cases, the mechanisms underlying such activity involve disruption  [15,25,26]. These results showed that dimeric and tetrameric peptides containing the RRWQWR sequence have greater antibacterial activity against E. coli strains (ATCC 11775 and ATCC 25922) than the other tested bacterial strains. In a similar way, the tetramer LfcinB (20-25) 4 exhibited greater cytotoxicity against oral squamous carcinoma cell lines than its linear peptide analogue, LfcinB (20)(21)(22)(23)(24)(25) [27].
For this research, peptide activity was evaluated in two different models: (i) bacteria and (ii) breast cancer cell lines. First, the designed peptide families were tested against two E. coli strains (ATCC 11775 and 25922). It was found that dimeric and tetrameric peptides exhibit greater antibacterial activity against the evaluated strains than their linear counterpart sequences (Table 1), confirming that the polyvalence enhanced the antibacterial activity. This behavior is in accordance with the mechanism suggested for LfcinB, which involves the initial electrostatic interaction with the negative charges of the bacterial cell wall. It has been proposed that the increase of the positive charge of the molecules enables the interaction with the charged negative molecules of the bacterial surface [10][11][12][13][14]. It has been suggested that LfcinB peptides self-assemble, forming a polymeric structures as a requisite for the interaction with the bacterial surface [9,28]. On the other hand, cyclic peptides exhibit antibacterial activity similar to monomeric peptides, suggesting that molecular restriction of these amino acid sequences does not increase antibacterial activity in the evaluated strains. This indicates that the relevant properties for antibacterial activity are both positively charged and have an amphipathic sequence.
Many new therapies are currently being used for cancer treatment; among these new methods, chemotherapy based on antimicrobial peptides (AMPs) has been of great interest due to the unique advantages of this kind of molecule, such as low molecular weight, ability to specifically target tumor cells, and low toxicity in normal tissues [29]. For example, the cytotoxic effect of AMPs normally occurs at micromolar levels, and it is not accompanied by significant levels of hemolysis or toxicity to other mammalian cells. In most cases, the mechanisms underlying such activity involve disruption of mitochondrial or plasmatic membranes of the target tumor cells [5]. AMPs are considered to be promising molecules for developing new drugs for treating different cancer types. LfcinB is an AMP with potential for designing molecules with antibacterial and anticancer properties. In this context, it is important to identify short sequences derived from LfcinB with anticancerigenic activity, specifically against breast cancer.
Specifically, the tetrameric peptide exhibits the maximum cytotoxic effect against MDA-MB-468 cell lines at a concentration of 11 µM (50 µg/mL), indicating that these cells are very sensitive to this molecule. The dimeric peptide LfcinB (20-25) 2 (200 µg/mL) exhibited an intermediate cytotoxic effect in both tested cell lines. In a similar way, dimeric and tetrameric peptides containing the 20 RRWQWRMKKLG 30 sequence exhibited greater cytotoxic effect against both breast cancer cells lines than the linear sequence ( Figure 3B). The dimer and tetramer exhibited the maximum cytotoxic effect at a concentration of 100 µg/mL, which corresponds to 30 µM and 15 µM, respectively, and their effect is constant at higher concentrations. In this family, the cyclic peptide showed a great cytotoxic effect against MDA-MB-468 cells, the cell viability being near to zero when the peptide concentration was 200 µg/mL (107 µM). The linear peptide exhibited a minimal cytotoxic effect in both breast cancer cell lines. For dimeric and tetrameric peptides containing the 17 FKARRWQWRMKKLGA 31 sequence, a greater cytotoxic effect against the breast cancer cell lines than their linear and cyclic analogues was also found ( Figure 3C).
Molecules 2017, 22, 1641 6 of 11 17 FKARRWQWRMKKLGA 31 sequence, a greater cytotoxic effect against the breast cancer cell lines than their linear and cyclic analogues was also found ( Figure 3C).  (11-44 µM). It was found that at 30 min of treatment, the tetrameric peptide cytotoxic effect was significant, and after the first hour, cell viability was near 5% and was constant up to 4 h (Figure 4). The cytotoxic effect of the tetrameric peptide LfcinB (20)(21)(22)(23)(24)(25) 4 against MDA-MB-468 breast cancer cell lines was tested at different incubation times, ranging from 30 to 240 min, with a peptide concentration of between 50 and 200 µg/mL (11-44 µM). It was found that at 30 min of treatment, the tetrameric peptide cytotoxic effect was significant, and after the first hour, cell viability was near 5% and was constant up to 4 h (Figure 4). The results indicate that the cytotoxic effect is fast and independent of incubation time. It depends on peptide concentration, 100 µg/mL (22 µM) being the minimum concentration with maximum cytotoxic effect. This behavior was also observed for oral squamous-cell carcinoma (OSCC) cell lines, SCC15 and CAL27. When they were treated with this tetramer, its cytotoxic effect was significant after the first hour of treatment, and it was constant up to 24 h [27,30].
The peptides LfcinB (20-25)2 and LfcinB (20-25)4 exhibited a greater and faster cytotoxic effect on MDA-MB-231 cells than has been reported for LfcinB (after 18 h of incubation, 45% cell death) [17]. Similarly, dimeric and tetrameric peptides also exhibited a greater and faster cytotoxic effect than BLF and LfcinB in other cancer models: BLF and LfcinB exhibited cytotoxic activity and significantly stimulated the apoptosis of HT-29 cells. The maximum effects were observed at 12 h of treatment, and the optimal concentrations for BLF and LfcinB were 800 µg/mL and 400 µg/mL, respectively [18]. It has been reported that incubation (24 h) of human MDA-MB-435 breast carcinoma cells in the presence of LfcinB caused cell death by apoptosis [29].
Dimeric and tetrameric peptides that exhibited a cytotoxic effect against MDA-468 breast cancer cells, while exhibiting minimal cytotoxic effect in fibroblasts cells (PCS 201-012). The peptide LfcinB (20-25)4 exhibited great cytotoxic effects in MDA-468 cells at 50 µg/mL, while the cytotoxic effect was minimal in PCS-201-012 cells at the same concentration ( Figure 5). Dimeric and tetrameric peptides containing 17 FKARRWQWRMKKLGA 31 and 20 RRWQWRMKKLG 30 sequences exhibited a similar behavior at 100 µg/mL ( Figure 5). These results indicate that the cytotoxic effect of these peptides could be selective for breast cancer cells lines.
In Summary, the results indicate that polyvalence of linear sequences increases the antibacterial activity and cytotoxic effects against both oral and breast cancer cell lines. Dimeric and tetrameric peptides containing sequences shorter than LfcinB could be considered as candidates for developing new therapeutic agents against both breast and oral cancer.
It has been reported that both LFB and LfcinB has activity against different cancer types [11,24], we specifically had found that the LfcinB-derived tetramer has activity in oral cancer [30] and, herein, we show that LfcinB-derived peptides have selective cytotoxicity against breast cancer cells. These results are promissory and it is important to evaluate the cytotoxic effect in other breast cancer cell lines as well as in other normal epithelial cell lines (e.g., mammary, bladder, bronchial, corneal, prostate, and renal epithelial cell lines) to determine the spectrum of activity and the selectivity of these peptides. It is also important to establish if these peptides have antitumoral activity in animal model assays. The results indicate that the cytotoxic effect is fast and independent of incubation time. It depends on peptide concentration, 100 µg/mL (22 µM) being the minimum concentration with maximum cytotoxic effect. This behavior was also observed for oral squamous-cell carcinoma (OSCC) cell lines, SCC15 and CAL27. When they were treated with this tetramer, its cytotoxic effect was significant after the first hour of treatment, and it was constant up to 24 h [27,30].
The peptides LfcinB (20)(21)(22)(23)(24)(25) 2 and LfcinB (20-25) 4 exhibited a greater and faster cytotoxic effect on MDA-MB-231 cells than has been reported for LfcinB (after 18 h of incubation, 45% cell death) [17]. Similarly, dimeric and tetrameric peptides also exhibited a greater and faster cytotoxic effect than BLF and LfcinB in other cancer models: BLF and LfcinB exhibited cytotoxic activity and significantly stimulated the apoptosis of HT-29 cells. The maximum effects were observed at 12 h of treatment, and the optimal concentrations for BLF and LfcinB were 800 µg/mL and 400 µg/mL, respectively [18]. It has been reported that incubation (24 h) of human MDA-MB-435 breast carcinoma cells in the presence of LfcinB caused cell death by apoptosis [29].
In Summary, the results indicate that polyvalence of linear sequences increases the antibacterial activity and cytotoxic effects against both oral and breast cancer cell lines. Dimeric and tetrameric peptides containing sequences shorter than LfcinB could be considered as candidates for developing new therapeutic agents against both breast and oral cancer.
It has been reported that both LFB and LfcinB has activity against different cancer types [11,24], we specifically had found that the LfcinB-derived tetramer has activity in oral cancer [30] and, herein, we show that LfcinB-derived peptides have selective cytotoxicity against breast cancer cells. These results are promissory and it is important to evaluate the cytotoxic effect in other breast cancer cell lines as well as in other normal epithelial cell lines (e.g., mammary, bladder, bronchial, corneal, prostate, and renal epithelial cell lines) to determine the spectrum of activity and the selectivity of these peptides. It is also important to establish if these peptides have antitumoral activity in animal model assays.

Reverse Phase HPLC
RP-HPLC analysis was performed on a Merck Chromolith ® C18 (50 mm × 4.6 mm) column using an Agilent 1200 liquid chromatograph (Omaha, NE, USA) with UV-Vis detector (210 nm). For peptide analysis (1.0 mg/mL crude or purified molecule), 10 µL samples were injected and a linear gradient was applied from 5% to 70% Solvent B (0.05% TFA in ACN) in Solvent A (0.05% TFA in water) for 11.5 min at a flow rate of 2.0 mL/min at room temperature.

Peptide Purification
Molecules were purified using solid-phase extraction columns (SUPELCO LC-18 with 2.0 g resin). SPE columns were activated prior to use with 30 mL acetonitrile (containing 0.1% TFA) and equilibrated with 30 mL water (containing 0.1%TFA). Crude peptides were passed through the column, and a gradient was used for their elution. Collected fractions were analyzed using RP-HPLC (as described above). Fractions that contained pure products were lyophilized.

MALDI-TOF MS
The purified peptides were analyzed by MALDI-TOF mass spectrometry. For sample preparation a solution of peptide (1 mg/mL) was mixed with the matrix (1.0 mg/mL of 2,5-dihydroxybenzoic acid, or sinapinic acid) in a relation of 2:18 (v/v) and 1 µL was seeded on the steel target. The experiment was performed on an Ultraflex III TOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in reflectron mode, using an MTP384 polished steel target (Bruker Daltonics, Bremen, Germany), Laser: 500 shots and 25-30% power.

Antibacterial Activity Assays
The minimal inhibitory concentration (MIC) was determined using a microdilution assay [26]. In brief, bacterial strains were incubated for 18 to 24 h at 37 • C in an Muller Hinton broth (MHB) until an optical density of 0.15 to 0.30 (620 nm) was obtained. 90 µL of MHB was mixed with 90 µL of peptide (440 µg/mL), and using a 96-well microtiter plate peptide, serial dilution (200, 100, 50, 25, 12.5, and 6.2 µg/mL) was performed. 10 µL of inoculum (2 × 10 6 CFU/mL) was added to each well. Final volume in each well was 100 µL. Then they were incubated for 24 h at 37 • C, and the absorbance at 620 nm was measured using an Asys Expert Plus ELISA reader. For determining the minimum bactericidal concentration (MBC), a small sample was taken from each well using an inoculation loop, which was then spread on MHA plates and incubated overnight at 37 • C. MBC was considered to be the plate which exhibited no bacterial growth. Each of these tests was performed twice (n = 2).

MTT Assay
Cytotoxicity assays were performed as previously described [32]. Briefly, breast cancer cell lines (100 µL; 2.5 × 10 3 cells/well) were seeded in 96-well flat bottom tissue culture treated plates and were incubated at 37 • C in a 10% CO 2 humidified atmosphere for 24 h, allowing for cell adhesion. Then the media was removed, then 100 µL of FBS (5%) and 100 µL of peptide were added. The peptide final concentration was ranging from 200 to 6.25 µg/mL and the final FBS concentration was 2.5%. Plates were incubated 2 h at 37 • C in a 10% CO 2 humidified atmosphere (physiologic pH). Negative controls included medium and water. All controls were prepared in triplicate. Cell viability was determined using the MTT assay after 2 h [33]. For this, 10 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 2 h at 37 • C. Formazan crystals were clarified by centrifugation, the supernatant was discarded, and the crystals were dissolved in DMSO (100 µL). Absorbance (570 nm) was registered on a Bio-Rad 680 microplate reader. (n = 3).