Enzymatic Laccase Nanoreactors Induce Apoptosis in MOLT-4-ALL Cells and Activate Prodrugs in a Synergetic Effect

Carlos A Medrano-Villagómez,1 Elizabeth Loredo-García,1,2 Jahaziel Gasperin-Bulbarela,2 Rafael Vazquez-Duhalt1 1Center for Nanosciences and Nanotechnology, National Autonomous University of Mexico, Ensenada, Baja California, Mexico; 2Ensenada Center for Scientific Research and Higher Education (CICESE), Ensenada, Baja California, MexicoCorrespondence: Rafael Vazquez-Duhalt, Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km 107 Carretera Tijuana‑Ensenada, Ensenada, Baja California, 22860, Mexico, Tel +52 777 133 6906, Email [email protected]: The search for novel cancer treatment strategies is of great interest. Recently, it has been reported that laccases from various sources exhibit anti-tumor effects. In addition, the use of nanometric platforms for delivering therapeutic agents at the cellular level is a promising approach for efficient cancer treatment. In this work, the cytotoxicity of Coriolopsis gallica laccase on human leukemia MOLT-4 cells was evaluated.Methods: Laccase was nanoconfined in a virus-like nanoparticle (VLPs) of the brome mosaic virus (BMV), and both free and nanoconfined preparations were evaluated the activation of prodrugs. The cytotoxicity was evaluated by neutral red to obtain the dose-response curve. Afterward, the death cell and mechanisms were characterized using flow cytometry of combinations of laccase (free and VLPs) with prodrugs (Doxorubicin, Irinotecan, and Procarbazine).Results: Laccase alone showed an apoptotic effect at a concentration of 0.35 μM (IC20), with a 49% of apoptotic cells at 24 hours. This effect was enhanced by the presence of doxorubicin (63.79%), irinotecan (43.44%), and procarbazine (53.27%) in the presence of both the free version (Lac) and the nano-encapsidated version (VLP-saLac). A similar effect was observed for the necroptosis population. Finally, the CI (Combination Index) was estimated by two different models, and the synergistic effect on cell death was confirmed.Discussion: The laccase pro-apoptotic effect in MOLT-4 cells has been demonstrated, increasing cytotoxicity by activating prodrugs in both free and nanoconfined forms. Laccase was confined in virus-like nanoparticles (VLPS) of the brome mosaic virus, and the cytotoxicity of both free and nanoconfined preparations was evaluated in the presence of prodrugs. Laccase alone showed a proapoptotic effect, and this effect was enhanced by the presence of prodrugs (doxorobucin, irinotecan, and procarbazine) for both free version (Lac) and the nanoencapsulated version (VLP-saLac). A synergistic effect on cell death was observed, with laccase exerting a pro-apoptotic effect in MOLT-4 cells and increasing cytotoxicity by activating prodrugs.Infographic on catalytic elements, treatment and in vitro evaluation for leukemia.Keywords: cancer, enzymatic nanoreactor, laccase, virus-based nanoreactor, virus-like particle