Anamaria Falcão Pereira1, Jonas Costa de França1, Lais Lacerda Brasil de Oliveira1, Elmer Adilson Espino Zelaya1, Daiane Maria da Silva Brito1, Renata Rocha do Nascimento1, Marcus Vinícius Saldanha Ribeiro1, Bianca de Souza Bezerra1, Daniel Castro Freire1, Carlos Roberto Koscky Paier1, Pedro Filho Noronha de Souza1, Hermógenes David de Oliveira1, Mariana Lima Vale1
Colorectal cancer (CRC) is the second most common cancer in Brazil and the third most common cause of global mortality among men and women. Despite therapeutic advances, its treatment is still limited by dose-dependent toxic effects and tumor resistance. In this context, McLTP1 stands out, a lipid transfer protein isolated from Morinda citrifolia seeds, with analgesic, anti-inflammatory, antioxidant, neuroprotective properties; however, the cytotoxic potential of the McLTP1 has not yet been investigated in murine CRC cell line (CT26.WT). Recently, it was demonstrated that McLTP1 has antitumor effect in CT26.WT (murine colorectal adenocarcinoma cell line) tumor bearing mice model (unpublished data). Therefore, it is of interest to investigate the mechanisms involved in the antitumor effect of McLTP1, as well as its cytotoxic effects on this cell line. Thus, this study aims to explore, through the shotgun proteomic approach, the molecular mechanisms modulated by McLTP1, contributing to the understanding of its effects on CRC and guiding the deepening of future investigations into this potential adjuvant in CRC therapy. For this, the cytotoxicity of McLTP1 (0.024 to 200 micromolar) was evaluated in vitro in the CT26.WT cell line by the MTT assay for 48, 72 and 96 h. Then, the proteins from the cells treated with the McLTP1 IC50 and the control group were extracted, digested and the peptides purified for protein profile analysis by UHPLC-MS/MS (NPDM/UFC), with processing in PatternLab V. Differentially expressed proteins were selected based on p<0.05 and fold change greater than or equal to 1.5 or less than or equal to 0.5. Functional analysis included evaluation of protein-protein interactions via STRING (confidence greater than or equal to 0.7), gene ontology enrichment (RStudio) and mapping in metabolic pathways in the KEGG and Reactome platforms. McLTP1 demonstrated an in vitro cytotoxic effect on the CT26.WT cell line, with IC50 (104.7, 44.12 and 23.6 micromolar) after 48, 72 and 96 hours of incubation, respectively. Proteomic analysis identified 1,517 proteins, of which 69 were exclusive to the control group, 229 exclusive to the treated group and 1,219 common to both. Among these, 312 proteins were differentially expressed. Forty-five proteins are directly associated with relevant biological processes of the total proteins. Gene ontology (GO) analyses revealed that proteins unique to the control group were enriched for molecular functions related to phosphatase regulatory activity and enzyme inhibition, while proteins unique to the treated group showed greater association with cadherin binding. In the pathway mapping by KEGG, proteins involved in PI3K-Akt (mmu:75705) and mTOR (mmu:75705) signaling, in cellular metabolism (mmu:11429, mmu:229363, mmu:66902), in cytoskeleton organization (mmu:668303, mmu:20742, mmu:69654) and in transcriptional regulation (mmu:276770, mmu:75705) were highlighted. All 45 proteins showed association with cancer, including colorectal câncer. Among the total proteins identified, 45 are potentially involved in the cytotoxic effect of McLTP1 and are also related to cancer, including colorectal cancer. Therefore, the proteomic approach demonstrated the possible cytotoxic mechanisms of McLTP1 in the CT26.WT cell line based on the differential protein profile and further studies are needed to reinforce and deepen these findings.
Agradecimentos: The authors would like to thank the Multi-User Facility of Drug Research and Development Center of Federal University of Ceará for their technical support. This study was supported by the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP/Universal 2023) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Research Productivity Grant).