Cardiovascular diseases and cancer are leading global causes of morbidity and mortality, necessitating advances in diagnosis and treatment. Doxorubicin (Doxo), a potent chemotherapy drug, causes long-term heart damage due to cardiotoxicity. Small extracellular vesicles (sEVs) carry bioactive molecules—such as proteins, lipids, and nucleic acids—that can modulate gene expression and signaling pathways in recipient cells, including cardiomyocytes. Through the delivery of cytokines, microRNAs, and growth factors, sEVs can influence cell survival, which plays a critical role in the development of cardiotoxicity. This study investigates the role of sEVs derived from breast cancer cells treated or not with Doxo and their potential to induce cardiomyocyte damage, thereby contributing to cardiotoxicity. We isolated sEVs from MCF-7 cells treated or not to Doxo using ultracentrifugation and characterized them through Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), and Western Blotting (WB) for the markers CD63, CD81, and TSG101. We analyzed cytokine profiles using a Multiplex Assay and Cytokine Membrane Array. We exposed Guinea pig cardiomyocytes to different concentrations of sEVs. We assessed their viability (MTT assay), shortening, reactive oxygen species (ROS–DHE dye) production, mitochondrial membrane potential (JC-1 dye), and calcium dynamics (FLUO-4 dye). We performed statistical analyses, including t-tests, ANOVA, Cohen’s d, and η² to validate the robustness of the results. Treatment of MCF-7 cells with 0.01 μM Doxorubicin resulted in increased sEVs production, particularly after 48 h of exposure (~1.79 × 10⁸ ± 2.77 × 10⁷ vs. ~5.1 × 10⁷ ± 1.28 × 10⁷ particles/mL, n = 3, p = 0.0019). These sEVs exhibited protein profiles in the 130–25 kDa range and 93–123 nm sizes. They carried cytokines including TNF-α, IL-1β, IL-4, IFN-γ, and IL-10. Exposure of cardiomyocytes to sEVs (0.025 μg/mL to 2.5 μg/mL) from both Doxo-treated and untreated cells significantly reduced cardiomyocyte viability, shortened cell length by up to 20%, increased ROS production, and disrupted calcium homeostasis and mitochondrial membrane potential, indicating severe cellular stress and cardiotoxicity. These findings suggest that Doxo enhances sEVs production from breast cancer cells, which plays a key role in cardiotoxicity through their cytokine cargo. The study highlights the potential of these sEVs as biomarkers for early cardiotoxicity detection and as therapeutic targets to mitigate cardiovascular risks in chemotherapy patients. Future research should focus on understanding the mechanisms by which Doxorubicin-induced sEVs contribute to cardiotoxicity and exploring their diagnostic and therapeutic potential to improve patient safety and outcomes in cancer therapy.

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.

Introducción. La entrada del rotavirus a la célula implica un mecanismo de múltiples pasos; las proteínas virales externas interaccionan con cuatro diferentes integrinas y Hsc70. Recientemente reportamos que la infección por rotavirus disminuye cuando se bloquea la proteína disulfuro-isomerasa de la superficie celular, lo que sugiere su interacción con el rotavirus en el proceso de entrada.Objetivo. Establecer la interacción del rotavirus con la proteína disulfuro-isomerasa en un sistema in vitro utilizando la proteína aislada de hígado bovino y, en un sistema celular, utilizando vellosidades intestinales de ratón y células MA104.Materiales y métodos. Se aisló la proteína disulfuro-isomerasa a partir de un homogenizado de hígado bovino utilizando anticuerpos anti-proteína disulfuro-isomerasa acoplados a agarosa mediante enlace hidrazona. La proteína disulfuro-isomerasa purificada se examinó por SDS-PAGE y Western blot y se utilizó para estudiar su interacción in vitro con rotavirus. Esta interacción se comparó con aquella observada en células MA104 y en las vellosidades intestinales de ratón.Resultados. La proteína disulfuro-isomerasa purificada mostró homogeneidad electroforética y fue capaz de unirse a rotavirus en un sistema in vitro. La interacción proteína-rotavirus fue detectada por ELISA de captura usando la proteína disulfuro-isomerasa bovina purificada y rotavirus de las cepas RRV y silvestre ECwt. La interacción de partículas de rotavirus purificadas con la proteína disulfuro-isomerasa celular se evidenció con ELISA, usando lisado celular después de la inoculación viral.Conclusión. La interacción rotavirus-proteína disulfuro-isomerasa fue demostrada in vitro, en células MA104 y en vellosidades intestinales de ratón lactante.

The therapeutic use of exogenous insulin of animal origin for the treatment of diabetes mellitus has been associated with the development of insulin antibodies, which raises concerns regarding the safety and efficacy of this formulation. With the advent of recombinant DNA technology and the generation of insulin with human-like sequences, the production of insulin antibodies has been reduced but not eliminated. As part of the analysis of immunogenicity induced by therapeutic proteins and to evaluate the immunogenic profile of three commercial formulations of human exogenous insulin (regular, neutral protamine Hagedorn (NPH), and glargine) we evaluated the presence of insulin antibodies in 29 serum samples from volunteers with type 2 diabetes, and we characterized their cross-reactivity, IgG subclass profile, and ability to form immune complexes. To this end, volunteers were classified into three groups according to their current insulin therapy: a) Regular and NPH insulin (n=10); b) NPH (n=9), and c) glargine (n=10). For the detection, characterization, and evaluation of the IgG subclasses we used an indirect ELISA test, and for the detection of immune complexes formed by insulin antibodies and exogenous insulin, capture ELISA. Our results evidenced that: i) Each insulin formulation was recognized differentially by the insulin antibodies suggesting a greater immunogenic potential for NPH insulin and a lower one for glargine insulin; ii) the IgG subclasses profile of insulin antibodies in humans was different for each formulation; the IgG3 subclass for NPH insulin was predominant in volunteers treated with regular and NPH insulin; iii) the presence of insulin antibodies was not associated with alterations in the metabolic parameters analyzed, and iv) immune complexes composed of exogenous insulin antibodies were not detected in the sera of diabetic volunteers. The results obtained in this study allowed us to conclude that there are differences between the immunogenic profile of the insulin formulations tested, which has significant value in the analysis of their immunogenicity with regard to their safety and efficacy. © 2018. Acad. Colomb. Cienc. Ex. Fis. Nat.