Análisis del mecanismo de muerte celular inducido por nanopartículas de oro recubiertas con quitosano en células leucémicas K562 y CEM: implicación de las especies reactivas de oxígeno.

La leucemia es un serio problema de salud a nivel mundial. Actualmente, las terapias convencionales afectan a células normales causando resistencia al tratamiento; por lo tanto, es necesario el desarrollo de nuevas terapias que puedan ser específicas para las células cancerosas y evitar la resistencia a la muerte celular por la inducción simultánea de diversas vías de muerte. Estudios recientes demuestran que tanto las nanopartículas de oro (AuNPs) como el quitosano tienen actividades biológicas interesantes que incluyen posibles efectos antitumorales. En este estudio, se analizó el mecanismo de muerte celular regulada (MCR), que inducen las nanopartículas de oro recubiertas con quitosano (AuNPs-Qts) en células leucémicas (K562 y CEM). Para ello, se sintetizaron AuNPs-Qts por el método de Turkevich. La muerte celular se evaluó por citometría de flujo, midiendo la exposición de fosfatidilserina y la permeabilidad de la membrana plasmática en células leucémicas y células mononucleares de sangre periférica (PBMC), como control sano no tumoral. La generación de ROS se midió utilizando DCFDA y en presencia del antioxidante N-acetilcisteína (NAC) como un inhibidor de ROS. Para analizar el daño mitocondrial se midió la pérdida del potencial de membrana mitocondrial mediante TMRE y el daño nuclear mediante el estudio de ϒ-H2AX, p53 y el análisis del ciclo celular. Para evaluar el efecto de las ROS en diferentes mecanismos de MCR, primero se analizó la formación de autofagosomas y se utilizó la spautina-1 para analizar la dependencia de autofagia; la activación de la caspasa efectora 3 y el Q-VD-OPH para analizar la dependencia de la apoptosis; y a la necrostatina-1 para analizar la dependencia de necroptosis. Los resultados obtenidos indican que las AuNPs-Qts inducen MCR dependiente de concentración en células leucémicas, mientras que muestran baja toxicidad en PBMC. Además, inducen la producción de ROS que generan daño mitocondrial y nuclear; induciendo MCR dependiente de ROS. Finalmente, los resultados demuestran que la MCR inducida por AuNPs-Qts en líneas celulares leucémicas es dependiente de la línea celular; induciendo apoptosis en células CEM y necroptosis en células K562. Estos resultados abren las puertas a próximos estudios que permitan probar su efectividad in vivo, además de la posibilidad de acoplar a las AuNPs-Qts con agentes que permitan abarcar más tipos de MCR, evitando así la resistencia de las células cancerosas al tratamiento. ABSTRACT Leukemia represent a serious health problem around the world. Currently, the available treatments present the disadvantages of affecting normal cells, and promoting treatment resistance. Therefore, the development of new therapies that can be specific to cancer cells and capable to avoid cell death resistance is needed. Recent studies show that both gold nanoparticles (AuNPs) and chitosan have interesting biological activities including potential antitumor effects. For this thesis, I synthetized chitosan-capped gold nanoparticles (AuNPs-Qts) by a chemical method (Turkevich), and analyzed their capacity to induce cell death in leukemic (K562 and CEM) and non-cancerous cells (PBMC). Cell death was measurement by the analysis of phosphatidylserine exposure, and plasma membrane permeabilization. ROS generation was measured using DCFDA and the antioxidant N-Acetyl Cystein (NAC) as a ROS inhibitor. Mitochondrial and nuclear damage were measured using TMRE and ϒ-H2AX, p53 and the cell cycle, respectively. Finally, to evaluate the effect of ROS in different mechanisms of regulated cell death, the role of autophagy, apoptosis and necroptosis was measured using spautin-1, Q-VD-OPH and necrostatin-1,respectively. Results show that AuNPs- Qts are cytotoxic in a dose-dependent manner in leukemia cells lines, while they showed low toxicity on PBMC. Additionally, they induced ROS production, which generated mitochondrial and nuclear damage and ROS dependent cell death. Finally, the results also show that cell death induced by AuNPs-Qts is dependent in the type of leukemic cell line, as they induce apoptosis in CEM and necroptosis in K562. These results open the doors to future studies to test efficiency in vivo, in addition to the possibility of coupling the AuNPs-Qts with other agents aiming at the simultaneous activation of different cell death pathways to overcome cell death resistance

Immunotherapies inducing immunogenic cell death in cancer: insight of the innate immune system

Cancer immunotherapies include monoclonal antibodies, cytokines, oncolytic viruses, cellular therapies, and other biological and synthetic immunomodulators. These are traditionally studied for their effect on the immune system’s role in eliminating cancer cells. However, some of these therapies have the unique ability to directly induce cytotoxicity in cancer cells by inducing immunogenic cell death (ICD). Unlike general immune stimulation, ICD triggers specific therapy-induced cell death pathways, based on the release of damage-associated molecular patterns (DAMPs) from dying tumour cells. These activate innate pattern recognition receptors (PRRs) and subsequent adaptive immune responses, offering the promise of sustained anticancer drug efficacy and durable antitumour immune memory. Exploring how onco-immunotherapies can trigger ICD, enhances our understanding of their mechanisms and potential for combination strategies. This review explores the complexities of these immunotherapeutic approaches that induce ICD, highlighting their implications for the innate immune system, addressing challenges in cancer treatment, and emphasising the pivotal role of ICD in contemporary cancer research

Chitosan gold nanoparticles induce cell death in HeLa and MCF-7 cells through reactive oxygen species production

Background: Nanotechnology has gained important interest, especially in the development of new therapies; the application of gold nanoparticles (AuNPs) in the treatment and detection of diseases is a growing trend in this field. As cancer represents a serious health problem around the world, AuNPs are studied as potential drugs or drug carriers for anticancer agents. Recent studies show that AuNPs stabilized with chitosan (CH) possess interesting biological activities, including potential antitumor effects that could be selective to cancer cells. Materials and methods: In this study, we synthesized sodium citrate-AuNPs and CHcapped AuNPs of 3–10 nm, and analyzed their cytotoxicity in cervical (HeLa) and breast (MCF-7) cancer cells, and in peripheral blood mononuclear cells (PBMCs). Then, we evaluated the clonogenic potential, cell cycle, nuclear alterations, caspase dependence, and reactive oxygen species (ROS) production in HeLa and MCF-7 cells after chitosan gold nanoparticles (CH-AuNPs) exposure. Results: Our data showed that CH-AuNPs are cytotoxic in a dose-dependent manner in the cancer cell lines tested, while they induce low cytotoxicity in PBMCs. Sodium citrate gold nanoparticles did not show cytotoxic effects. In both HeLa and MCF-7 cell lines, CH-AuNPs inhibit clonogenic potential without inducing cell cycle arrest or nuclear alterations. The cell death mechanism is specific for the type of cancer cell line tested, as it depends on caspase activation in HeLa cells, whereas it is caspase independent in MCF-7 cells. In all cases, ROS production is mandatory for cell death induction by CH-AuNPs, as ROS inhibition with N-acetyl cysteine inhibits cell death. Conclusion: Our results show that CH-AuNPs are selective for HeLa and MCF-7 cancer cells, rather than normal PBMCs, and that ROS production seems to be a conserved feature of the cell death mechanism induced by CH-AuNPs. These results improve the knowledge of CH-AuNPs and open the way to the design of new pharmacological strategies using these agents against cancer. Keywords: AuNPs, cancer, PBMC, nuclear alterations, cell cycle, RO

Bovine Dialyzable Leukocyte Extract IMMUNEPOTENT-CRP Induces Selective ROS-Dependent Apoptosis in T-Acute Lymphoblastic Leukemia Cell Lines

International audienceImmunotherapies strengthen the immune system to fight multiple diseases such as infections, immunodeficiencies, and autoimmune diseases, and recently, they are being used as an adjuvant in cancer treatment. IMMUNEPOTENT-CRP (I-CRP) is an immunotherapy made of bovine dialyzable leukocyte extract (bDLE) that has chemoprotective and immunomodulatory effects in different cellular populations of the immune system and antitumor activity in different cancer cell lines. Our recent results suggest that the antineoplastic effect of I-CRP is due to the characteristics of cancer cells. To confirm, we evaluated whether the selectivity is due to cell lineage or characteristics of cancer cells, testing cytotoxicity in T-acute lymphoblastic leukemia cells and their cell death mechanism. Here, we assessed the effect of I-CRP on cell viability and cell death. To determine the mechanism of cell death, we tested cell cycle, mitochondrial and nuclear alterations, and caspases and reactive oxygen species (ROS) and their role in cell death mechanism. Our results show that I-CRP does not affect cell viability in noncancer cells and induces selective cytotoxicity in a dose-dependent manner in leukemic cell lines. I-CRP also induces mitochondrial damage through proapoptotic and antiapoptotic protein modulation (Bax and Bcl-2) and ROS production, nuclear alterations including DNA damage (γ-H2Ax), overexpression of p53, cell cycle arrest, and DNA degradation. I-CRP induced ROS-dependent apoptosis in leukemic cells. Overall, here, we show that I-CRP cytotoxicity is selective to leukemic cells, inducing ROS-dependent apoptosis. This research opens the door to further exploration of their role in the immune system and the cell death mechanism that could potentially work in conjunction with other therapies including hematological malignances