STRUCTURALLY MODIFIED IKAV PEPTIDES AS BIOLOGICALLY ACTIVE LIGANDS IN TRIPLE-NEGATIVE BREAST CANCER

Abstract

Introduction/Justification

Breast cancer remains a public health challenge, and triple-negative breast cancer (TNBC) is its most aggressive subtype, characterized by high recurrence, early metastasis, and lack of hormone receptors and HER2 expression. TNBC cells overexpress molecular targets, such as integrins, which play a key role in adhesion, migration, and signaling. Laminin-111, enriched in the breast tumor microenvironment, generates bioactive fragments, such as IKVAV, that selectively interact with integrins a3ß1 and a6ß1 associated with aggressive tumor behavior. These fragments participate in tumor cell growth regulation, supporting IKVAV as a biologically active ligand for integrin-targeting strategies. Structural modification by histidine (H) insertion enables radiolabeling with Tc, reinforcing its potential application in molecular imaging.

Objectives

To synthesize structurally modified IKVAV peptides incorporating a H residue and to evaluate their in vitro interaction as biologically active ligands in TNBC (MDA-MB-231) cells.

Materials and Methods

IKVAV-NH2, HIKVAV-NH2, and N-acetyl-HIKVAV-NH2 were synthesized by solid-phase peptide synthesis using the Fmoc/tBu strategy and MBHAR resin. Purification and characterization were performed by RP-HPLC and mass spectrometry. For growth curve analysis, MDA-MB-231 cells were seeded in 6-well plates (5 × 104 cells/well) and treated with the respective peptides (IKVAV-NH2: 151.6 µM; HIKVAV-NH2: 90.3 µM; N-acetyl-HIKVAV-NH2: 113.2 µM). Viable cells were counted on days 1, 3, 5, and 7, comparing with non-treated control (CT). Data are presented as mean ± SD and the statistical analyses were performed applying Student’s t-test (significance set at p < 0.05).

Results

IKVAV-NH2 presented a single, well-defined chromatographic peak and a compatible molar mass (528 g/mol), requiring no further purification. In contrast, HIKVAV-NH2 and N-acetyl-HIKVAV-NH2 initially showed two chromatographic peaks, with the minor peak corresponding to IKVAV-NH2, indicating incomplete H coupling. Co-injection and mass spectrometry confirmed this finding, necessitating additional purification. After purification, both peptides were obtained in pure form, with compatible molar masses (665 g/mol for HIKVAV-NH2 and 707 g/mol for N-acetyl-HIKVAV-NH2). Biological evaluation demonstrated that IKVAV-NH2 and HIKVAV-NH2 did not significantly affect MDA-MB-231 cell proliferation compared to CT: IKVAV-NH2 = (2.2 ± 0.7) × 105 versus CT = (2.0 ± 0.9) × 105 [day 7; p = 0.6322; n = 6]; HIKVAV-NH2 = (3.5 ± 0.4) × 105 versus CT = (3.1 ± 0.2) × 105 [day 7; p = 0.1444; n = 6]. In contrast, N-acetyl-HIKVAV-NH2 induced a significant increase in cell proliferation: N-acetyl-HIKVAV-NH2 = (7.6 ± 1.3) × 105 versus CT = (5.3 ± 0.9) × 105 [day 7; p = 0.0020; n = 5], indicating a distinct biological response following N-terminal acetylation. This effect may be associated with increased peptide stability in the biological environment, favoring prolonged cellular interaction.

Conclusion

Under the evaluated conditions, only N-acetyl-HIKVAV-NH2 significantly modulated MDA-MB-231 cell growth, suggesting that N-terminal acetylation influences peptide stability and biological activity. These findings support the relevance of structural modifications in peptide-based tumor-targeting strategies and highlight the need for further functional and preclinical investigations, including ???Tc radiolabeling for molecular imaging applications.