Elmer Adilson Espino Zelaya¹, Felipe Pantoja Mesquita¹, Emerson Lucena da Silva¹, Thiago Loreto Mathos¹, Francisco Laio de Oliveira¹, Raquel Carvalho Montenegro¹, Pedro Filho Noronha de Souza¹

Colorectal cancer (CRC) is the third most common malignancy, with more than 1.9 million reported cases, and the second leading cause of cancer death, with more than 900,000 deaths annually. Despite advances in oncology, CRC has shown resistance to various conventional chemotherapy regimens. Nowadays, synthetic peptides (SPs) have emerged as new alternative molecules due to their anticancer properties. SPs possess high selectivity and lower toxicity compared to other small molecules and natural peptides due to the capability of select key amino acids and add to their final structure. Despite these promising advances, there are still no approved SPs for the treatment of CRC. In a previous study, the synthetic peptide PepKAA designed from a chitinase from Arabidopsis thaliana, exhibited antimicrobial and anticancer activity without toxicity to healthy cells or zebrafish model. Experimental data demonstrated the anticancer potential of PepKAA against gastric adenocarcinoma, CRC, breast cancer, and malignant melanoma. The inhibitory concentration for death of 50% (IC50) was obtained in the CRC cell line HCT116, with a value of 40.51 μM. Our previous work demonstrated that PepKAA selectively targets CRC cells by affecting several mechanisms of cancer development. Our study shows that PepKAA disrupts pathways involved in cancer survival and potentiates the action of 5-Fluorouracil (5-FU) in CRC. For this purpose, protein profiling in in HCT116 cells after treatment with PepKAA was analyzed using UHPLC MS/MS and PatternLab V. Fluorescence assays were performed to evaluate membrane integrity, ROS, cell viability, cell cycle, and invasion in the presence of 5-FU. The results showed that PepKAA induced early ROS accumulation and altered the proteomic profile of HCT116 cells, resulting in reduced levels of proteins involved in metabolism, cancer progression, oxidative stress response, DNA damage repair, and cell cycle regulation. Differential protein analysis revealed a higher abundance of proteins related to RNA splicing and cytoskeletal organization, and a lower abundance of proteins associated with translation regulation, mRNA metabolism, and deoxyribonuclease activity. These findings underscore the potential of PepKAA to modulate multiple cancer-related pathways, support biomarker discovery, and contribute to the identification of novel therapeutic targets.

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