Tipos de mortes celulares induzidas pela terapia fotodinâmica com azul de metileno em células de câncer de mama murino in vitro e in vivo

Abstract

Photodynamic therapy (PDT) stands out as a minimally invasive therapeutic modality based on the activation of a photosensitizer by light at a specific wavelength in the presence of molecular oxygen, promoting the localized generation of reactive oxygen species capable of inducing selective cell death. Among the available photosensitizers, methylene blue (MB) has high availability, low cost, and favorable photophysical properties, making it a promising alternative for the treatment of neoplasms. However, gaps remain regarding the molecular mechanisms triggered by methylene bluemediated photodynamic therapy (MB-PDT) and the influence of dosimetric parameters on the activation of different forms of cell death. The present study aimed to evaluate the cell death mechanisms induced by MB-mediated PDT (MB-PDT) in in vitro and in vivo experimental models of 4T1 murine mammary carcinoma, using two photosensitizer concentrations (25 and 50 µM) and investigating the pathways of apoptosis, autophagy, necroptosis, and ferroptosis. Initially, the experimental parameters were established through cell viability assays, determining the methylene blue concentrations and defining the photodynamic protocol. Irradiation was performed using an InGaAlP diode laser at a wavelength of 660 nm, with a power output of 100 mW, spot area of 0.028 cm², energy of 2.8 J per point, energy density of 100 J/cm², irradiance of 3.57 W/cm², and exposure time of 28 s per point. Subsequently, cell migration assays, gene expression analysis by RT-qPCR, functional assessment of cell death pathways using specific pharmacological inhibitors, and histopathological and immunohistochemical characterization were performed in BALB/c mice bearing 4T1 cell-induced mammary carcinoma. The results demonstrated that MB-PDT significantly reduced tumor cell viability and migratory capacity. Gene expression analysis revealed modulation of markers associated with regulated cell death pathways, with a greater contribution of mechanisms related to autophagy and ferroptosis. Pharmacological inhibitor assays confirmed the functional contribution of ferroptosis to the cytotoxicity induced by the therapy. In the in vivo model, the treatment was shown to be safe, with no evidence of systemic toxicity. Although no significant differences in macroscopic tumor volume regression were observed during the experimental period, molecular and immunohistochemical analyses revealed alterations consistent with the activation of different immunogenic cell death pathways in the tumor tissue, including necroptosis. MB-PDT represents a promising antitumor strategy for the treatment of experimental mammary carcinoma, capable of inducing multiple regulated cell death pathways, particularly autophagy, necroptosis, and ferroptosis. These findings expand the understanding of the molecular mechanisms involved in photodynamic therapy and provide a basis for optimizing therapeutic parameters and developing translational protocols for breast cancer treatment.