The tetrameric peptide LfcinB (20-25)4 derived from bovine lactoferricin induces apoptosis in the MCF-7 breast cancer cell line

Abstract

The cytotoxic effect of the tetrameric peptide LfcinB (20-25)₄ against breast cancer cell line ATCC® HTB-22™ (MCF-7) was evaluated. The tetrameric peptide exhibited a concentration-dependent cytotoxic effect against MCF-7 cancer cells. The peptide at 22 µM had the maximum cytotoxic effect against MCF-7 cancer cells, reducing their cell viability to ∼20%. The cytotoxic effect of the tetrameric peptide against MCF-7 cells was sustained for 24 hours. Furthermore, the tetrameric peptide did not exhibit a significant cytotoxic effect against the non-tumorogenic trophoblastic cell line, which confirms their selectivity for breast cancer cell lines. The MCF-7 cells treated at 12.2 µM for 1 h exhibited morphological changes characteristic of apoptosis, such as rounded forms and cellular shrinkage. Furthermore, this peptide induces severe cellular damage to MCF-7 cells, mitochondrial membrane depolarization, and increase of cytoplasmic calcium concentration. Our results suggest that it has a significant selective cytotoxic effect against MCF-7 cells, which may be mainly associated with the apoptotic pathway. This peptide, which contains the RRWQWR motif, could be considered to be a promising candidate for developing therapeutic agents for the treatment of breast cancer.

Fig. 1. Cytotoxic effect of LfcinB (21–25)₄ peptide against MCF-7 and CRL-3271 cell lines after 2 h treatment at 37 °C. Cell viability percentage of breast cancer cell line MCF-7 (red) and human trophoblast cell line CRL-3271 (blue) can be observed. The data are expressed as the mean ± SD (n = 3). Statistically significant differences were found between the cytotoxic effect exhibited by the tetrameric peptide in both cell lines at 12.5 µg mL⁻¹* and 25, 50, 100 and 200 µg mL⁻¹**** (ANOVA, Sidak's multiple comparisons test, *p < 0.05; ****p < 0.0001).

Fig. 2. Cytotoxic effect of peptide LfcinB (21–25)₄ against MCF-7 and CRL-3271 cells after 2 and 24 h treatment. (A) MCF-7 cells were treated with peptide at concentrations: 15 µg mL⁻¹/3.3 µM (blue); 30 µg mL⁻¹/6.6 µM (orange) and 60 µg mL⁻¹/13.3 µM (red). (B) CRL-3271 cells were treated with peptide concentrations: at 5.5 µM (blue), 11 µM (orange) and 22 µM (red). Treatment time ± SD (n = 3) ANOVA post hoc Tukey, p < 0.05 *statistic significant differences with regard to maximum cytotoxic effect.

Fig. 3. Effect of peptide LfcinB (20–25)₄ on breast cancer cells MCF-7 (phase-contrast microscopy). Untreated cells (A); MCF-7 cells treated with tetrameric peptide (30 µg mL⁻¹; 12.2 µM) for 1 h (B) and 6 h (C).

Fig. 4. Cytoplasmic membrane integrity assays. MCF-7 cells were treated with tetrameric peptide LfcinB (20–25)₄ for 6 h in the presence of SYTO9/IP. Plain bars represent cells labelled with SYTO9 fluorochrome (cells with unaffected cytoplasmic membrane). Textured bars represent cells labeled with both SYTO9/PI fluorochromes (cells with affected cytoplasmic membrane). Positive control: cells treated with actinomycin for 24 h (blue); negative control: cells without treatment (orange); cells treated with peptide (green) at 15 µg mL⁻¹ (6.6 µM); 30 µg mL⁻¹ (12.2 µM) and 60 µg mL⁻¹ (24.4 µM). ± SD (n = 6).

Fig. 5. Apoptosis/necrosis assays via flow cytometry with Annexin-V and 7AAD. Live cells (blue), apoptotic cells (orange), and necrotic cells (red). Cells in absence of peptide (A), cells treated with actinomycin (B), cells treated with EDTA (C), cells treated with 30 µg mL⁻¹ of LfcinB (20–25)₄ for 1 h (D).

Fig. 6. Mitochondrial membrane depolarization assays. (A) Representative images of the flow cytometry of mitochondrial depolarization in MCF-7 cell line. (B) Depolarization percentage of C⁻: cells without treatment (blue), C⁺: MCF-7 cells treated with 10 µg mL⁻¹ actinomycin treatment for 24 hours (orange), cells treated with LfcinB (20–25)₄ peptide (30 µg mL; 12.2 µM) for (green) 30 min, 60 min and 120 min.

Fig. 7. Cytoplasmatic calcium measurement in MCF-7 cells treated with LfcinB (20–25)₄. (A) Negative control untreated cells and (B) cells treated with LfcinB (20–25)₄; the peptide (30 µg mL⁻¹) was injected at 5 min.