Nuclear-Targeted Engineering of a Lunasin-Derived Peptide Enhances Antiproliferative Activity and AP-1-Associated Gene Modulation in A549 Lung Cancer Cells
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Abstract
Activator protein-1 (AP-1) is a transcriptional regulator involved in cancer-associated processes, including proliferation, invasion, and angiogenesis; however, its broad protein–DNA interaction interface limits direct targeting by conventional small molecules. This study aimed to evaluate whether nuclear-targeted engineering of a previously designed lunasin-derived peptide could enhance its antiproliferative and gene-regulatory activity in A549 lung adenocarcinoma cells. We hypothesized that the modified nuclear localization signal (NLS)-containing peptide would show stronger biological activity than the native/core peptide sequence. Two peptide forms were compared: Compound A, a 27-amino acid modified peptide containing a rigid EAAAK linker and an NLS motif, and Compound B, the native/core peptide. STRING and Cytoscape-based network analysis supported the selection of AP-1-associated downstream genes, including MMP9, CCND1, and VEGFA, for experimental validation. Importin-α docking suggested that Compound A could be accommodated within the NLS-recognition region and displayed broader receptor engagement than Compound B, with direct involvement of NLS-region residues. In A549 cells, both compounds reduced cell viability in a time- and concentration-dependent manner; however, Compound A showed consistently stronger antiproliferative activity, with IC₅₀ values of 52.69, 37.35, and 18.60 µM after 24, 48, and 72 h, respectively, compared with 238.26, 81.42, and 43.34 µM for Compound B. Microscopic examination further showed reduced cell density and treatment-associated morphological alterations. Exploratory RT-qPCR analysis showed that Compound A reduced MMP9 and VEGFA expression and produced a marked decreasing trend in CCND1 expression. In contrast, Compound B showed a non-significant reduction in MMP9 but non-significant increases in CCND1 and VEGFA, indicating a variable and non-suppressive transcriptional pattern. These findings suggest that nuclear-targeted modification enhanced the biological activity of the lunasin-derived peptide in A549 cells. Although direct mechanistic confirmation of AP-1 inhibition remains required, this study provides preliminary in vitro evidence supporting Compound A as a promising peptide candidate for further investigation.
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