Evaluation of the CIB1R peptide derived from the cytoplasmic domain of neprilysin on cell migration in an in vitro model of lung cancer

Purpose – Lung cancer is characterized by high metastatic potential and poor clinical outcomes, underscoring the need for novel approaches to investigate mechanisms regulating tumor cell migration and invasion. The CIB1R peptide, derived from the cytoplasmic domain of neprilysin (NEP), has been proposed as a potential modulator of these processes. This study aimed to evaluate the effects of CIB1R and related peptides on cellular behaviors associated with metastasis in an in vitro model of non-small cell lung cancer. Materials and methods – Peptides were prepared and administered under serum-free conditions. Cell viability was assessed using the Alamar Blue assay. Migration and invasion were evaluated using Transwell (Boyden chamber) assays, while cell adhesion was quantified on Matrigel-coated surfaces. Peptide-associated fluorescence was analyzed using flow cytometry and confocal microscopy with FITC-labeled peptides. Membrane integrity was assessed by propidium iodide exclusion. Statistical analyses were performed using one-way ANOVA, followed by the Tukey post hoc test. A p-value < 0.05 was considered statistically significant. Results – CIB1R reduced cell migration and invasion in A549 cells in a dose-dependent manner without significantly affecting cell viability over 24 hours. However, similar inhibitory effects were observed with structurally related peptides, including a scrambled control sequence, indicating that the observed activity is not strictly sequence-specific. All peptides demonstrated strong cell-associated fluorescence signals, although the experimental design did not allow definitive discrimination between membrane-associated and intracellular localization. No significant increase in membrane permeability was detected under the conditions tested. Conclusions – These findings provide initial functional evidence that short cationic peptides derived from, or related to, the cytoplasmic domain of NEP can modulate cellular behaviors associated with tumor progression in vitro. The observed effects appear to be influenced by shared physicochemical properties rather than strictly sequence-specific mechanisms. Further studies are required to clarify the molecular basis of these effects and their relevance under physiological conditions.