Jonas Costa de França¹, Anamaria Falcão Pereira¹, Renata Rocha do Nascimento¹, Bianca de Souza Bezerra¹, Marcus Vinícius Saldanha Ribeiro¹, Daniel Castro Freire¹, Daiane Maria da Silva Brito², Elmer Adilson Espino Zelaya², Lais Lacerda Brasil de Oliveira³, Carlos Roberto Koscky Paier³, Pedro Filho Noronha de Souza², Mariana Lima Vale¹

Colorectal cancer (CRC) ranks second among the most prevalent types of cancer in men and women in Brazil. The treatment of CRC still presents challenges, reinforcing the need for new therapeutic strategies. In this context, cannabinoids have demonstrated antitumor potential in preclinical studies, with emphasis on WIN55,212-2, a non-selective cannabinoid agonist, as it also presents neuroprotective potential in oxaliplatin-induced neuropathy models, a first-line antineoplastic agent in the treatment of metastatic CRC. Considering the paucity of studies on the effects and mechanisms of WIN55,212-2 in colorectal carcinoma cell line, the aim of this study was to identify key proteins involved in the antineoplastic effect of WIN55,212-2 in murine colorectal adenocarcinoma cell line (CT26.WT) through a shotgun proteomic approach. Initially, the cytotoxicity of WIN 55,212-2 was evaluated after 72 and 96 hours of treatment in the CT26.WT cell line. Initially, the cytotoxicity of WIN55,212-2 was evaluated after 72 and 96 hours of treatment in the CT26.WT cell line. After determining the IC50, protein extraction and digestion of cells treated and untreated with WIN55,212-2 were performed for subsequent analysis by shotgun proteomic. Membrane integrity, ROS production, the effect of the combination of WIN55,212-2 with oxaliplatin and the effect on the AKT/mTOR pathway by immunofluorescence were also evaluated. WIN55,212-2 obtained an IC50 of 6.69 μM and 5.4 μM at 72 and 96 hours of treatment. The total number of identified proteins was obtained and the genetic ontology and the network of interactions between them were analyzed. A total of 1148 proteins were obtained, 332 of which were exclusive to the control group and 116 to the treated group, while 700 were in both groups. Twenty-six proteins of this total are involved in biological processes such as protein processing and degradation, cytoskeleton reorganization, nucleotide metabolism and chemoresistance pathways. Analysis of interactions between proteins indicated alterations in relevant oncogenic signaling pathways, such as the PI3K/AKT/mTOR and NF-kB pathways. Moreover, the inhibitory effect of WIN 55,212-2 on the AKT/mTOR pathway, impairment of cell membrane integrity and induction of oxidative stress were observed. Finally, the combination of WIN 55,212-2 with oxaliplatin showed greater potency when compared to oxaliplatin alone. Thus, WIN 55,212-2 has potential as an antitumor agent in CRC cell lines, demonstrating its inhibitory effect on the AKT/mTOR pathway, its ability to modulate cellular processes critical to tumor progression and to potentiate the cytotoxic effects of oxaliplatin. These results not only highlight the relevance of nonselective cannabinoid agonists as a therapeutic alternative, but also highlight the importance of future investigations to explore the clinical applications of these compounds in the management of CRC.

Agradecimentos: The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), for their financial support, which was essential for carrying out this work, and the Central Analítica-UFC/CT-INFRA/MCTI-SISANO/Pró-Equipamentos CAPES for their technical assistance.