A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

Abstract

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

Figure 1

Cytotoxic effect of LfB and LfcinB25 in the OSCC tumorigenic cell lines CAL27 (a) and SCC15 (b) and the immortalized nontumorigenic keratinocytes cell line Het-1A (c). The cells were incubated for 24 h with the LfB protein and the LfcinB25 peptide. After treatment, cell viability was determined by MTT assay and calculated as the percentage of average absorbance of each treatment relative to the average absorbance of the negative control. The maximum concentration of the LfB protein was 1.25 μM (100 μg/mL) and of LfcinB25 32 μM (100 μg/mL). Each treatment was done in triplicate.

Figure 2

Cytotoxic effect of LfcinB-derived peptides in the OSCC tumor cell lines CAL27 (a) and SCC15 (b) and the immortalized nontumorigenic keratinocytes cell line Het-1A (c). The cells were incubated for 24 h with the peptides and cell viability was determined by the MTT assay and calculated as the percentage of average absorbance of each treatment relative to the average absorbance of the negative control. The maximum concentration of the peptides used was LfcinB25, 32 μM; LfcinB(20–25), 101.5 μM; LfcinB-Pal, 67.3 μM; LfcinB(20–25)₄, 22.25 μM (all equivalent to 100 μg/mL). The data are expressed as the mean ± s.e.m. (n = 3). LfcinB(20–25)₄ cf LfcinB(20–25) had statistical significant differences at high concentration (100 μg/mL) (ANOVA, posttest Tukey, p < 0.05).

Figure 3

Cytotoxic activity of LfcinB(20–25)₄ in long-term treatments. (a) SCC15 and CAL27 cells were incubated with the peptide for 24, 48, 72, and 96 h. (b) SCC15 and CAL27 cells were treated for 24 h with the peptide and washed, and cells were incubated for 0, 24, 48, and 72 h in fresh culture medium. After the treatments, cell viability was determined by the MTT assay and calculated as the percentage of average absorbance of each treatment relative to the average absorbance of the negative control. Cell viability was evaluated 24, 48, and 72 h after treatments and was calculated as the percentage of average absorbance of treatments in relation to average absorbance of negative control. The concentrations of LfcinB(20–25)₄ and STA used were 22.25 μM (100 μg/mL) and 0.86 μM (0.4 μg/mL), respectively. Treatments were done in triplicate.

Figure 4

Cytotoxicity evaluation at different time points of treatment with LfcinB-derived peptides in the OSCC tumor cell lines CAL27 (a), SCC15 (b), and the immortalized nontumorigenic keratinocytes cell line Het-1A (c). The cells were incubated with LfcinB(20–25) or LfcinB(20–25)₄ at the indicated times. After treatment, cell viability was determined by the MTT assay and calculated as the percentage of average absorbance of each treatment relative to the average absorbance of the negative control. The concentration of the LfcinB(20–25)₄ used was 22.25 μM (100 μg/mL) and of LfcinB(20–25) was 101.5 μM (100 μg/mL). The STA concentration used was 0.86 μM (0.4 μg/mL) for CAL27 and 1.29 μM (0.6 μg/mL) for SCC15 and Het-1A. Each treatment was done in triplicate.

Figure 5

PI permeability in CAL27 and SCC15 cell lines evaluated by FACS. Cells were incubated with the different peptides for 1 h, after which they were harvested and incubated with PI in the dark for 10 min. Black plot: controls without treatment; gray plot: 1 hour of treatment with peptides, or 2.14 μM (1 μg/mL) STA control or 0.2% T-X100 control. The maximum concentration of the peptides used was 100 μg/mL equivalent to LfcinB25, 32 μM; LfcinB(20–25), 101.5 μM; LfcinB-Pal, 67.3 μM; and LfcinB(20–25)₄, 22.25 μM.

Figure 6

Cell morphology of CAL27 (a) and SCC15 (b) after treatment with LfcinB(20–25)₄ for 1, 12, or 24 h. The STA concentration used was 0.86 μM (0.4 μg/mL) for CAL27 and 1.29 μM (0.6 μg/mL) for SCC15. Photomicrographs were taken with a phase-contrast microscope.

Figure 7

Assessment of necrosis/apoptosis in nontumorigenic cell line Het-1A (a) and the tumorigenic cell line CAL27 (b). Cells were detached and incubated with different peptides indicated for 1 h, after which they were labeled with both Annexin V-FITC and PI, and analyzed by flow cytometry. 10 μM (4.66 μg/mL) STA and 0.2% T-X100 were used as controls. The maximum concentration of the peptides used was 100 μg/mL equivalent to LfcinB25, 32 μM; LfcinB(20–25), 101.5 μM; LfcinB-Pal, 67.3 μM; and LfcinB(20–25)₄, 22.25 μM. Photomicrographs were taken with a phase-contrast microscope. Barr = 100 μm.