Ferroptosis in acute leukemia / 中华医学杂志(英文版)

Ferroptosis is an iron-dependent cell death pathway that is different from apoptosis, pyroptosis, and necrosis. The main characteristics of ferroptosis are the Fenton reaction mediated by intracellular free divalent iron ions, lipid peroxidation of cell membrane lipids, and inhibition of the anti-lipid peroxidation activity of intracellular glutathione peroxidase 4 (GPX4). Recent studies have shown that ferroptosis can be involved in the pathological processes of many disorders, such as ischemia-reperfusion injury, nervous system diseases, and blood diseases. However, the specific mechanisms by which ferroptosis participates in the occurrence and development of acute leukemia still need to be more fully and deeply studied. This article reviews the characteristics of ferroptosis and the regulatory mechanisms promoting or inhibiting ferroptosis. More importantly, it further discusses the role of ferroptosis in acute leukemia and predicts a change in treatment strategy brought about by increased knowledge of the role of ferroptosis in acute leukemia.

Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection / 南方医科大学学报

OBJECTIVE@#To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection.@*METHODS@#The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs.@*RESULTS@#A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01).@*CONCLUSION@#DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.

Mechanism of programmed cell death in follicular atresia / 生理学报

Apoptosis and autophagy of follicular granulosa cells play an important regulatory role in the process of ovarian follicular atresia in animals. Recent studies have shown that ferroptosis and pyroptosis are also involved in the process of ovarian follicular atresia. Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation and reactive oxygen species (ROS) accumulation. Studies have confirmed that autophagy- and apoptosis-mediated follicular atresia also have typical characteristics of ferroptosis. Pyroptosis is a pro-inflammatory cell death dependent on Gasdermin protein, which can regulate ovarian reproductive performance by regulating follicular granulosa cells. This article reviews the roles and mechanisms of several types of programmed cell death independently or interactively regulating follicular atresia, in order to expand the theoretical research on follicular atresia mechanism and provide the theoretical reference for the mechanism of programmed cell death-induced follicular atresia.

Protección del ácido clorogénico frente a lesiones inducidas por venzo(a)pireno en Saccharomyces cerevisiae / Protection of chlorogenic acid against benzo(a)pyrene-induced lesions in Saccharomyces cerevisiae / Proteção do ácido clorogênico contra lesões induzidas por benzo(a)pireno em Saccharomyces cerevisiae

El objetivo del presente estudio fue analizar el efecto del ácido clorogénico, uno de los compuestos polifenólicos con mayor concentración en la infusión de Ilex paraguariensis, sobre el daño celular y molecular inducido por el benzo(a)pireno. La infusión de Ilex paraguariensis ("mate") es bebida por la mayoría de los habitantes de Argentina, Paraguay, sur de Brasil y Uruguay. La levadura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A y SX46Arad14() se utilizó como modelo eucariota. Las células en crecimiento exponencial se expusieron a concentraciones crecientes de benzo(a)pireno y a tratamientos combinados con una concentración de 250 ng/mL de benzo(a)pireno y ácido clorogénico a una concentración igual a la encontrada en la infusión de yerba mate. Luego de los tratamientos se determinaron fracciones de sobrevida, frecuencia mutagénica y roturas de doble cadena de ADN así como la modulación en la expresión de la proteína Rad14 a través de un análisis de Western Blot. Se observó un aumento significativo en las fracciones de sobrevida así como una disminución en la frecuencia mutagénica después de la exposición combinada con benzo(a)pireno y ácido clorogénico en comparación con los tratamientos con benzo(a)pireno como único agente. En la cepa mutante deficiente en la proteína Rad14 se observó un aumento significativo en la sensibilidad a benzo(a)pireno en comparación con la cepa SC7K lys2-3. En los tratamientos combinados de benzo(a)pireno y ácido clorogénico se observó una importante disminución de la letalidad. Con respecto a la determinación de roturas de doble cadena de ADN no se observó fraccionamiento cromosómico a la concentración de benzo(a)pireno utilizada en los experimentos. Los análisis de Western Blot mostraron un aumento en la expresión de la proteína Rad14 en las muestras tratadas con benzo(a)pireno como único agente en comparación con la muestra control. Adicionalmente se observó una disminución en la expresión de la proteína cuando en los tratamientos se utilizaron benzo(a)pireno y ácido clorogénico combinados. Los resultados indican que el ácido clorogénico disminuye significativamente la actividad mutagénica producida por el benzo(a)pireno, la cual no se encuentra relacionada con un incremento en la remoción de las lesiones inducidas por el sistema de reparación por escisión de nucleótidos.

The aim of this study was to analyze the effect of chlorogenic acid, a polyphenolic compound found at high concentrations in Ilex paraguariensis infusions, on cellular and molecular damage induced by benzo(a)pyrene. Ilex paraguariensis infusions ("mate") are consumed by most of the population in Argentina, Paraguay, southern Brazil and Uruguay. Saccharomyces cerevisiae yeast (SC7K lys2-3; SX46A and SX46Arad14( strains) were used as eukaryotic model organisms. Cells in an exponential growth phase were exposed to increasing concentrations of benzo(a)pyrene, as well as combined treatments of benzo(a)pyrene at a concentration of 250 ng/mL and chlorogenic acid at a concentration matching that which is commonly found in mate. Determinations of surviving fraction, mutagenic frequency and double strand DNA breaks induction were performed, along with the assessment of the modulation of the expression of protein Rad14 by Western Blot. A significant increase of surviving fractions and a decrease in mutagenic frequency were observed after exposure to benzo(a)pyrene plus chlorogenic acid, contrary to benzo(a)pyrene alone. A substantial increase in sensitivity to benzo(a)pyrene was observed for the Rad14 protein-deficient mutating strain when compared to the SC7K lys2-3 strain. An important decrease in lethality was observed when combined benzo(a)pyrene and chlorogenic acid treatments were applied. As for the determination of DSBs, no chromosomic fractionation was observed at the benzo(a)pyrene concentration tested in the experiments. Western Blot analysis showed an increase in the expression of protein Rad14 for samples treated with benzo(a)pyrene as a single agent when compared against the control sample. Additionally, the expression of this protein was observed to diminish when combined treatments with benzo(a)pyrene and chlorogenic acid were used. Results obtained indicate that chlorogenic acid significantly decreases the mutagenic activity of benzo(a)pyrene, which is not related to an increase in the removal of lesions induced by nucleotide excision repair system.

O objetivo deste estudo foi analisar o efeito do ácido clorogênico, um dos compostos polifenólicos com maior concentração na infusão de Ilex paraguariensis, sobre o dano celular e molecular induzido pelo benzopireno. A infusão de Ilex paraguariensis ("mate") é consumida pela maioria dos habitantes da Argentina, Paraguai, sul do Brasil e Uruguai. A levedura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A e SX46Arad14() foi utilizada como modelo eucariótico. Células em crescimento exponencial foram expostas a concentrações crescentes de benzopireno e tratamentos combinados foram realizados com uma concentração de 250 ng/mL de benzo(a)pireno e ácido clorogênico, igual à encontrada na infusão de erva-mate. Após os tratamentos, foram determinadas as frações de sobrevivência, frequência mutagênica e quebras de fita dupla do DNA, bem como a modulação na expressão da proteína Rad14 por meio de análise de Western Blot. Um aumento significativo nas frações de sobrevivência, bem como uma diminuição na frequência mutagênica foram observados após a exposição combinada de benzo(a)pireno e ácido clorogênico em comparação com tratamentos de agente único de benzo(a)pireno. Um aumento significativo na sensibilidade ao benzo(a)pireno foi observado na cepa mutante deficiente em proteína Rad14 em comparação com a cepa SC7K lys2-3. Nos tratamentos combinados de benzo(a)pireno e ácido clorogênico, observou-se uma diminuição significativa na letalidade. Com relação à determinação das quebras de fita dupla de DNA, não foi observado fracionamento cromossômico na concentração de benzo(a)pireno utilizada nos experimentos. A partir da análise de Western Blot, observou-se um aumento na expressão da proteína Rad14 nas amostras tratadas com benzo(a)pireno como agente único em relação à amostra controle. Além disso, uma diminuição na expressão da proteína foi observada quando combinados de benzo(a)pireno e ácido clorogênico foram usados ​​nos tratamentos. Os resultados obtidos neste trabalho indicam que o ácido clorogênico diminui significativamente a atividade mutagênica produzida pelo benzo(a)pireno, a qual não está relacionada a um aumento na remoção de lesões induzidas pelo sistema de reparo por excisão de nucleotídeos.

Copper induces damage, oxidative stress and cell death in endothelium of chronic intoxicated Wistar rats / El cobre induce daño, estrés oxidativo y muerte celular en el endotelio de ratas Wistar intoxicadas

SUMMARY: Reactive Oxygen Species (ROS) are part of the functional balance of various systems, they can generate cellular damage by oxidative stress associated with disease processes such as atherosclerosis, cardiovascular disease, diabetes, and aging. Some studies report that copper induces damage to the endothelium, which could be associated with cardiovascular pathologies. This study was an experimental comparative, prospective, longitudinal, and controlled clinical trial in a murine animal model. Twenty-four male Wistar rats were included, the distribution of the groups was time-depending chronic exposition to copper, and a control group. Results show gradual alterations in the groups treated with copper: areas with loss of the endothelium, signs of disorganization of smooth muscle fibers in the tunica media, as well as areas with the fragmentation of the elastic sheets. A significant statistical difference was observed in the active- Caspase-3 analysis expression in the aortic endothelium and endothelium of the capillaries and arterioles of the lung between the control group vs 300 ppm of copper. Expression of eNOS was detected in the endothelium of the aorta and vessels of the lung. Our study shows histological changes in the walls of the great vessels of intoxicated rats with copper, and the increment of inflammatory cells in the alveoli of the study model, mainly at a high dose of copper exposition. These results will be useful to understand more about the mediators involved in the effect of copper over endothelium and cardiovascular diseases in chronic intoxication in humans.

RESUMEN: Las Especies Reactivas de Oxígeno (ROS) son parte del equilibrio funcional de varios sistemas, pueden generar daño celular por estrés oxidativo asociado a procesos patológicos como aterosclerosis, enfermedades cardiovasculares, diabetes y envejecimiento. Algunos estudios informan que el cobre induce daños en el endotelio, lo que podría estar asociado a patologías cardiovasculares. Este estudio fue un ensayo clínico experimental comparativo, prospectivo, longitudinal y controlado en un modelo animal murino. Se incluyeron veinticuatro ratas Wistar macho, la distribución de los grupos fue la exposición crónica al cobre en función del tiempo y un grupo de control. Los resultados muestran alteraciones graduales en los grupos tratados con cobre: áreas con pérdida del endotelio, signos de desorganización de las fibras musculares lisas en la túnica media, así como áreas con la fragmentación de las láminas elásticas. Se observó una diferencia estadística significativa en la expresión del análisis de caspasa-3 activa en el endotelio aórtico y el endotelio de los capilares y arteriolas del pulmón entre el grupo de control frente a 300 ppm de cobre. Se detectó expresión de eNOS en el endotelio de la aorta y los vasos del pulmón. Nuestro estudio muestra cambios histológicos en las paredes de los grandes vasos de ratas intoxicadas con cobre, y el incremento de células inflamatorias en los alvéolos del modelo de estudio, principalmente a una alta dosis de exposición de cobre. Estos resultados serán útiles para comprender más sobre los mediadores involucrados en el efecto del cobre sobre el endotelio y las enfermedades cardiovasculares en la intoxicación crónica en humanos.

Análise proteômica de células BEAS-2B expostas a benzo[α]pireno e nicotinamida ribosídeo / Proteomic Analysis of BEAS-2B cells exposed to benzo[α]pyrene and nicotinamide riboside

Responsável por milhões de óbitos anuais e um grande custo para a saúde pública, o câncer é a segunda maior causa de mortes no mundo. Dentre seus diversos tipos, o câncer de pulmão, além da alta incidência, é um dos mais letais. A exposição a substâncias tóxicas provenientes da combustão de matéria orgânica, assim como o consumo de cigarro, são os principais responsáveis pela alta incidência de câncer de pulmão. Dentre estas substâncias, está o benzo[α]pireno (B[α]P), um carcinógeno completo, ou seja, capaz de iniciar e promover o processo de carcinogênese. Resultados anteriores obtidos pelo grupo demonstraram que células BEAS-2B expostas a 1 µM de B[α]P apresentaram alterações das concentrações de metabólitos intracelulares, indução de estresse redox e hipermetilação do DNA. A exposição a 1 µM de nicotinamida ribosídeo (NR), um dos precursores de NAD+, foi capaz de proteger as células BEAS-2B contra a transformação induzida por B[α]P, além de impedir totalmente que células não expostas a B[α]P formassem colônias em soft-agar. A utilização da proteômica neste trabalho permitiu verificar a abundância das proteínas nos quatro diferentes grupos de exposição: Controle, B[α]P, B[α]P + NR e NR. Após 120 h de exposição as células foram coletadas, as proteínas extraídas e preparadas para análise. Foram descobertas 3024 proteínas posteriormente analisadas com o objetivo de elucidar vias possivelmente envolvidas na proteção contra o processo de transfomação maligna. Os grupos NR e Controle demonstram ser mais parecidos em relação ao seu conteúdo, enquanto os grupos B[α]P e B[α]P + NR foram mais semelhantes entre si. A análise de proteínas exclusivas revelou menos processos relacionados ao reparo de DNA no grupo tratado apenas com B[α]P quando comparado com B[α]P + NR. A análise estatística do total de proteínas utilizando o teste ANOVA (p < 0,05, N = 5) revelou 564 proteínas diferencialmente expressas entre os grupos. A clusterização nos permitiu observar a diferença na abundância de proteínas entre os quatro tratamentos. As proteínas estão envolvidas em funções como a regulação do metabolismo, resposta a estresse, transdução de sinal, regulação de expressão gênica e morte celular. Um dos clusters (cluster 1), contendo 59 proteínas, revelou poucos processos na análise de enriquecimento, mas as proteínas contidas nele apresentam funções como controle da divisão celular, apoptose e proteção ao estresse redox. Nele podemos observar que, no geral, o tratamento com B[α]P aumentou a abundância de algumas proteínas, o que foi revertido no grupo B[α]P + NR. O tratamento apenas com NR diminuiu a abundância das proteínas contidas nesse cluster. Outro cluster (cluster 4) apresentou 51 proteínas de abundância diminuída durante a exposição ao B[α]P, o que se reverteu no grupo B[α]P + NR. As proteínas desse cluster estão envolvidas em etapas importantes da via glicolítica, de crescimento, adesão, migração e invasão celular. Apesar de ser descrito que a exposição a NR pode aumentar a eficiência do reparo de DNA, os resultados apresentados nesse trabalho indicam que o efeito protetor pode estar relacionado com a modulação do ciclo celular ou alterações na adesão celular

Responsible for millions of annual deaths and a great health expense, cancer is the second leading cause of death in the world. Among its many types, lung cancer, besides its high incidence, is also one of the most lethal. Exposure to toxic substances resulting from the combustion of organic matter, as well as cigarette consumption, are the mainly responsible for the high incidence of lung cancer. One of these substances is benzo[α]pyrene (B[α]P), a complete carcinogen, able to initiate and promote the carcinogenesis process. Results obtained previously demonstrated that BEAS-2B cells exposed to 1 µM BaP presented alterations in the levels of intracellular metabolites, induction of oxidative stress, and hypermethylation of DNA. The exposure to 1 µM nicotinamide riboside (NR), one of the precursors of NAD+, was able to protect BEAS-2B cells against the transformation induced by B[α]P, moreover, it also totally prevented the colonies formation on soft agar in cells not exposed to B[α]P. The use of proteomics allowed us to verify the abundance of proteins in the four different exposure groups: Control, B[α]P, B[α]P + NR e NR. After 120h of exposure, the cells were collected followed by the extraction of the proteins. A total of 3024 proteins were identified and analyzed aiming to elucidate possible pathways involved in the protective effect against the malignant transformation induced by B[α]P. The NR and Control groups showed to be more similar, while B[α]P and B[α]P + NR were more similar. The analysis of exclusive proteins revealed fewer processes related to DNA repair in B[α]P when compared with B[α]P + NR. The statistical analysis of the total proteins using the ANOVA test (p <0.5, N = 5) revealed 564 proteins differentially expressed between the groups. The heatmap showed the difference in protein abundance between the four treatments. Proteins are involved in functionssuch asthe regulation of metabolism, stress response, signal transduction, regulation of gene expression, and cell death. One of the clusters (cluster 1), containing 59 proteins, revealed a few processes in the enrichment analysis, but the proteins contained in it have functions such as control of cell division, apoptosis, and protection from redox stress. It is possible to observe, in general, treatment with B[α]P increased the abundance of some proteins, which was partially reversed in group B[α]P + NR. On the other hand, the NR treatment decreased the abundance of proteins contained in this cluster. Another cluster (cluster 4) showed 51 proteins of decreased abundance during exposure to B [α] P, which was partially reversed in group B[α]P + NR. The proteins in this cluster are involved in important stages of the glycolytic pathway, also in growth, adhesion, migration, and cell invasion. Although it has been described that exposure to NR can increase the efficiency of DNA repair, the results presented in this work indicate that the protective effect may be related to the modulation of the cell cycle or cell adehsion modifications

A new combination of naringin and trimetazidine protect kidney Mitochondria dysfunction induced by renal Ischemia / Reperfusion injury in rat

Abstract Ischemia/reperfusion (IR) injury leads to overproduction of Reactive Oxygen Species (ROS), and disrupts membrane potential that contributes to cell death. The aim of this study was to determine if naringin (NAR), trimetazidine (TMZ) or their combination, protect the kidney mitochondrial from IR injury. Forty rats were randomly allocated into five groups, harboring eight rats each: Sham, IR, NAR (100 mg/kg), TMZ (5 mg/kg) and NAR plus TMZ. Ischemia was induced by obstructing both renal pedicles for 45 min, followed by reperfusion for 4 hours. The mitochondria were isolated to examine the ROS, Malondialdehyde (MDA), Glutathione (GSH), mitochondrial membrane potential (MMP) and mitochondrial viability (MTT). Our findings indicated that IR injury resulted in excessive ROS production, increased MDA levels and decreased GSH, MMP and MMT levels. However, NAR, TMZ or their combination reversed these changes. Interestingly, a higher protection was noted with the combination of both, compared to each drug alone. We speculate that this combination demonstrates a promising process for controlling renal failure, especially with the poor clinical outcome, acquired with NAR alone. This study revealed that pretreatment their combination serves as a promising compound against oxidative stress, leading to suppression of mitochondrial stress pathway and elevation of GSH level.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells / 南方医科大学学报

OBJECTIVE@#To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism.@*METHODS@#MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells.@*RESULTS@#MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05).@*CONCLUSION@#IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.

Programmed Necrosis Inducers for Cancer Treatment / 中国医学科学院学报

Programmed necrosis,a mode of cell death independent of Caspase,is mainly mediated by receptor-interacting protein kinase-1 (RIPK1),receptor-interacting protein kinase-3 (RIPK3),and mixed lineage kinase domain-like protein (MLKL).Studies have demonstrated that programmed necrosis has the dual role of promoting and inhibiting tumor growth and thus we can control the development of tumor by regulating programmed necrosis.The drugs capable of inducing programmed necrosis show potential anti-tumor activity.In addition,inducing programmed necrosis is an effective way to overcome tumor resistance to apoptosis.This paper summarized the mechanisms of programmed necrosis and its relationship with tumors.We focused on the antitumor activity of programmed necrosis inducers including natural products,chemotherapeutic drugs,death receptor ligands,kinase inhibitors,inorganic salts,metal complexes,and metal nanoparticles.These agents will provide new therapeutic candidates for the treatment of tumors,especially the tumors acquiring resistance to apoptosis.

Acute kidney injury and regulated cell death / 生理学报

Acute kidney injury (AKI) refers to a clinical syndrome in which renal function declines rapidly in a short period of time caused by various pathological factors. During the development of AKI, renal tubules with the functions of reabsorption and excretion are prone to cell death due to external pathological stimuli, which is an important cause of impaired renal function. In recent years, a variety of new cell death pathways have been gradually recognized. Researchers have now found that regulated cell death (RCD), such as necroptosis, pyroptosis and ferroptosis, are important regulatory mechanisms of AKI. This article will summarize the research advances of various types of RCD involved in the process of AKI, aiming to deepen the understanding of AKI and provide innovative thoughts for the clinical treatment of AKI.

A Inibição do Metabolismo da Glicose por miR-34a e miR-125b Protege contra a Morte Celular de Cardiomiócitos Causada por Hiperglicemia / Inhibiting Glucose Metabolism By miR-34a and miR-125b Protects Against Hyperglycemia-Induced Cardiomyocyte Cell Death

Resumo Fundamento: É sabido que a resistência à insulina e a hiperglicemia são causas patológicas importantes no desenvolvimento de cardiomiopatia diabética (CMD). Entretanto, seus mecanismos moleculares precisos na patogênese da CMD ainda não estão claros. Objetivos: Estudos recentes revelam que os microRNAs (miRNAs) desempenham papéis essenciais na patogênese da CMD. Este projeto tem o objetivo de determinar os papéis de miR-34a e miR-125b na morte celular de cardiomiócitos causada por hiperglicemia. Métodos: Cardiomiócitos primários de ratos foram isolados e expostos a concentrações de glicose normais e altas. A viabilidade das células foi medida utilizando-se o ensaio MTT. As expressões de miR-34a e miR-125b foram detectadas por qRT-PCR. Alvos potenciais de miR-34a e miR-125b foram previstos pelo www.Targetscan.org, e validados a partir de tecidos cardíacos humanos. Um p<0,05 foi considerado significância estatística. Resultados: Demonstra-se neste estudo que o miR-34a e o miR-125b têm resposta celular reduzida no coração humano diabético. Além disso, os dados in vitro de cardiomiócitos primários de ratos demonstraram que o tratamento com glicose alta em curto prazo estimula a expressão de miR-34a e miR-125b. Demonstrou-se que, em condições de glicose alta, os cardiomiócitos de ratos apresentaram metabolismo de glicose intracelular, e a captação de glicose e a produção de lactato aumentaram significativamente. Foi identificado que as principais enzimas metabólicas da glicose, hexoquinase 2 (HK2) e lactato desidrogenase-A (LDHA) eram alvos diretos de miR-125b e miR-34a, respectivamente. A superexpressão de miR-125b e miR-34a poderia evitar a morte de celular de cardiomiócitos causada por hiperglicemia. Por fim, a recuperação de HK2 e LDHA em cardiomiócitos com superexpressão de miR-125b e miR-34a restaurou a sensibilidade de cardiomiócitos à hiperglicemia. Conclusões: Nossos resultados propõem um mecanismo molecular para proteção cardiovascular diabética mediada por microRNA e contribuirão para o desenvolvimento de estratégias de tratamento de disfunção cardiovascular associada a diabetes.

Abstract Background: It is well-known that insulin resistance and hyperglycemia are important pathological causes for the development of diabetic cardiomyopathy (DCM). However, its precise molecular mechanisms in the pathogenesis of DCM remain unclear. Objectives: Recent studies reveal that microRNAs (miRNA) play essential roles in the pathogenesis of DCM. This project aimed to determine the roles of miR-34a and miR-125b in hyperglycemia-induced cardiomyocyte cell death. Methods: Rat primary cardiomyocytes were isolated and exposed to normal and high concentrations of glucose. Cell viability was measured using MTT assay. Expressions of miR-34a and miR-125b were detected by qRT-PCR. Potential targets of miR-34a and miR-125b were predicted from www.Targetscan.org and validated from human heart tissues. A statistical significance of p<0.05 was considered. Results: The present study shows that miR-34a and miR-125b are downregulated in a human diabetic heart. Moreover, in vitro data from rat primary cardiomyocytes showed that short-term high glucose treatment stimulates miR-34a and miR-125b expressions. Under high glucose, it was found that rat cardiomyocytes displayed increased intracellular glucose metabolism, and glucose uptake and lactate production were significantly increased. It was also found that the key glucose metabolic enzymes, Hexokinase 2 (HK2) and Lactate dehydrogenase-A (LDHA), were direct targets of miR-125b and miR-34a, respectively. Overexpression of miR-125b and miR-34a could prevent hyperglycemia-induced cardiomyocyte cell death. Finally, the restoration of HK2 and LDHA in miR-125b and miR-34a overexpressed cardiomyocytes recovered the cardiomyocytes' sensitivity to hyperglycemia. Conclusion: Our results proposed a molecular mechanism for the microRNA-mediated diabetic cardiovascular protection and will contribute to developing treatment strategies for diabetes-associated cardiovascular dysfunction.

Orlistat induces ferroptosis-like cell death of lung cancer cells / 医学前沿

Aberrant de novo lipid synthesis is involved in the progression and treatment resistance of many types of cancers, including lung cancer; however, targeting the lipogenetic pathways for cancer therapy remains an unmet clinical need. In this study, we tested the anticancer activity of orlistat, an FDA-approved anti-obesity drug, in human and mouse cancer cells in vitro and in vivo, and we found that orlistat, as a single agent, inhibited the proliferation and viabilities of lung cancer cells and induced ferroptosis-like cell death in vitro. Mechanistically, we found that orlistat reduced the expression of GPX4, a central ferroptosis regulator, and induced lipid peroxidation. In addition, we systemically analyzed the genome-wide gene expression changes affected by orlistat treatment using RNA-seq and identified FAF2, a molecule regulating the lipid droplet homeostasis, as a novel target of orlistat. Moreover, in a mouse xenograft model, orlistat significantly inhibited tumor growth and reduced the tumor volumes compared with vehicle control (P < 0.05). Our study showed a novel mechanism of the anticancer activity of orlistat and provided the rationale for repurposing this drug for the treatment of lung cancer and other types of cancer.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

BACKGROUND: Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS: Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator defer-oxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION: These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Proteome profiling of different rat brain regions reveals the modulatory effect of prolonged maternal separation on proteins involved in cell death-related processes

BACKGROUND: Early-life stress in the form of maternal separation can be associated with alterations in offspring neurodevelopment and brain functioning. Here, we aimed to investigate the potential impact of prolonged maternal separation on proteomic profiling of prefrontal cortex, hippocampus and cerebellum of juvenile and young adult rats. A special attention was devoted to proteins involved in the process of cell death and redox state maintenance. METHODS: Long-Evans pups were separated from their mothers for 3 h daily over the first 3 weeks of life (during days 2-21 of age). Brain tissue samples collected from juvenile (22-day-old) and young adult (90-day-old) rats were used for label-free quantitative (LFQ) proteomic analysis. In parallel, selected oxidative stress markers and apoptosis-related proteins were assessed biochemically and by Western blot, respectively. RESULTS: In total, 5526 proteins were detected in our proteomic analysis of rat brain tissue. Approximately one tenth of them (586 proteins) represented those involved in cell death processes or regulation of oxidative stress balance. Prolonged maternal separation caused changes in less than half of these proteins (271). The observed alterations in protein expression levels were age-, sex- and brain region-dependent. Interestingly, the proteins detected by mass spectrometry that are known to be involved in the maintenance of redox state were not markedly altered. Accordingly, we did not observe any significant differences between selected oxidative stress markers, such as the levels of hydrogen peroxide, reduced glutathione, protein carbonylation and lipid peroxidation in brain samples from rats that underwent maternal separation and from the corresponding controls. On the other hand, a number of changes were found in cell death-associated proteins, mainly in those involved in the apoptotic and autophagic pathways. However, there were no detectable alterations in the levels of cleaved products of caspases or Bcl-2 family members. Taken together, these data indicate that the apoptotic and autophagic cell death pathways were not activated by maternal separation either in adolescent or young adult rats. CONCLUSIONS: Prolonged maternal separation can distinctly modulate expression profiles of proteins associated with cell death pathways in prefrontal cortex, hippocampus and cerebellum of juvenile rats and the consequences of early-life stress may last into adulthood and likely participate in variations in stress reactivity.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects

Apoptosis de neutrófilos en la infección asintomática por Virus de la Inmunodeficiencia Humana / Neutrophil apoptosis in asymptomatic Human Immunodeficiency Virus infection

En los últimos años se ha determinado que los neutrófilos son células altamente versátiles y sofisticadas, cuyas funciones van mucho más allá de la eliminación de los microorganismos. En la infección con el Virus de Inmunodeficiencia Humana (HIV), si bien el papel de los neutrófilos no está totalmente caracterizado, actualmente está claro que la relación entre los neutrófilos y el virus es mucho más compleja de lo que se pensaba. Los objetivos de este trabajo fueron evaluar en pacientes con infección asintomática, y sin tratamiento antirretroviral, el efecto de la infección por el HIV sobre la muerte celular de los neutrófilos y la expresión de receptores de superficie. En pacientes seropositivos sin tratamiento hubo un aumento de la apoptosis temprana de los neutrófilos en relación a los grupos controles. Esta apoptosis aumentada no depende de la activación de la vía extrínseca o intrínseca. En estos pacientes hubo un aumento de la expresión de TLR2 que, unido al aumento de la apoptosis temprana, podría ser indicativo de un fenotipo activado de los neutrófilos. En conclusión, este trabajo aporta información sobre aspectos relacionados con la apoptosis de los neutrófilos en estadios tempranos de la infección por HIV, contribuyendo así a una mayor comprensión acerca del efecto de este virus sobre componentes de la respuesta inmune innata.

In recent years it has been determined that neutrophils are highly versatile and sophisticated cells whose functions go far beyond the elimination of microorganisms. In Human Immunodeficiency Virus (HIV) infection, the role of neutrophils is not fully characterized but it is now clear that the relationship between neutrophils and HIV is much more complex than previously thought. The aims of this study were to evaluate the effect of HIV infection on neutrophil cell death and the expression of surface molecules on neutrophils in patients with asymptomatic infection and without antiretroviral treatment (ART). In HIV seropositive patients without antiretroviral therapy there was an increase in the early apoptosis of neutrophils in relation to the control groups. This increased apoptosis does not depend on the activation of the extrinsic or intrinsic pathway. In these patients there was an increase in the expression of TLR2 which, together with the increase of early apoptosis, could be indicative of an activated phenotype of neutrophils. In conclusion, this study provides information on aspects related to the apoptosis of neutrophils in early stages of HIV infection and therefore contributes to a better understanding of the effect of this virus on components of the innate immune response.

Nos últimos anos, determinou-se que os neutrófilos são células altamente versáteis e sofisticadas, cujas funções vão muito além da eliminação dos microrganismos. Na infecção pelo HIV, embora o papel dos neutrófilos não esteja totalmente caracterizado, atualmente fica bem claro que a relação entre os neutrófilos e o vírus é muito mais complexa do que se pensava anteriormente. Os objetivos deste trabalho foram avaliar em pacientes com infecção assintomática, e sem tratamento antirretroviral, o efeito da infecção pelo HIV na morte celular dos neutrófilos e a expressão de receptores de superfície. Nos pacientes soropositivos sem tratamento, houve um aumento da apoptose precoce dos neutrófilos em relação aos grupos controle.Esta apoptose aumentada não depende da ativação da via extrínseca ou intrínseca. Nestes pacientes, houve um aumento da expressão de TLR2 que, juntamente com o aumento da apoptose precoce, poderia ser indicativo de um fenótipo ativado dos neutrófilos. Em conclusão, este trabalho fornece informações sobre aspectos relacionados com a apoptose dos neutrófilos em estágios precoces da infecção pelo HIV, contribuindo desse modo para uma maior compreensão sobre o efeito deste vírus nos componentes da resposta imune inata.

Autophagy inhibitor 3-methyladenine could not modulate rotenone neurotoxicity in primary mesencephalic cell culture / El inhibidor de la autofagia 3-metiladenina no logra modular la neurotoxicidad de la rotenona en el cultivo primario de células mesencefálicas

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.

Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.

Síntese, avaliação biológica e modelagem molecular de potenciais antitumorais análogos da sanguinarina planejados por simplificação molecular / Synthesis, biological evaluation and molecular modeling of potential antitumors analogues from sanguinarine designed by molecular simplification

A sanguinarina é um alcaloide capaz de inibir Bcl-xL, uma proteína antiapoptótica que se encontra superexpressa em linhagens tumorais e que está frequentemente relacionada à resistência destas frente a quimioterápicos antineoplásicos. No intuito de identificar potenciais agentes antitumorais, o objetivo deste trabalho foi sintetizar três séries de análogos da sanguinarina planejados por simplificação molecular e avaliar sua atividade biológica. Dez N-benzil-naftil-aminas (3a-e; 4a-e) e dez arilisoquinolinas (6a-e; 7a-e) foram sintetizadas em duas a três etapas reacionais, utilizando-se métodos de aminação redutiva e acoplamento de Suzuki. Insucesso na etapa de reação de Heck impossibilitou a síntese da terceira série, benzofenantridínica, apesar de testadas diversas condições reacionais. Avaliação da citotoxicidade em linhagens de glioblastoma U87MG revelou que a série N-benzilnaftil-amina apresenta melhor atividade quando comparada às aril-isoquinolinas, sendo para ambas, observada atividade superior à temozolamida, principal fármaco para o tratamento de glioblastoma. Estudos em linhagem não tumorigênica MRC-5 demonstraram que os análogos foram significativamente superiores à sanguinarina em relação à seletividade. Os compostos mais mais promissores, 4a e 6e, induziram morte celular por apoptose e causaram despolarização da membrana mitocondrial, indicando morte apoptótica pela via extrínseca. Ademais, 4a interrompeu o ciclo interrompeu o ciclo celular na fase G2/M, indicando que o mesmo seria um agente ciclo celular específico. Simulações de dinâmica molecular sugerem que os compostos interagem com a proteína Bcl-xL principalmente por interações hidrofóbicas, e que o composto 4a apresentaria afinidade com o alvo semelhante à sanguinarina, embora esta tenha apresentado atividade superior em células U87. Perspectivas incluem estudos das vias de indução de morte celular, além da expansão do painel de células. Conclui-se, portanto, que os análogos da sanguinarina representam um arcabouço a ser explorado pelos químicos medicinais no desenvolvimento de potenciais antineoplásico

Sanguinarine is an alkaloid able to inhibit Bcl-xL, an antiapoptotic protein which is overexpressed in tumor cells and related to their resistance against antineoplastic chemotherapy. Regarding to develop potential antitumor agents, the aim of this work was the synthesis of three series of sanguinarine analogues designed by molecular simplification and their biological evaluation. Ten N-benzyl-naphtyl-amines (3a-e; 4ae) and ten aryl-isoquinolines (6a-e; 7a-e) were synthesized in two or three reaction steps through reductive amination and Suzuki coupling. Failure about Heck-type reaction had impaired the synthesis of the thirth series, benzophenanthridine, although several conditions were tested. Cytotoxicity evaluation against U87MG glioblastoma cell line showed that N-benzyl-naphtyl-amines are more active than aryl-isoquinolines and both series were superior to temozolamide, the main drug for glioblastoma treatment. Tests against non-tumorigenic cell MRC-5 indicated that the analogues were significantly superior to sanguinarine regarding selectivity. The most promising compounds, 4a e 6e, induced cell death by apoptosis and mitochondrial membrane depolarization, indicating apoptotic death by extrinsic pathway. 4a provide cell cycle arrest at G2/M phase, suggesting that it is a specific cell cycle agent. Molecular dynamics suggested that compounds interact with Bcl-xL mainly by hydrophobic interactions and 4a has affinity to the protein like sanguinarine, although the last showed superior activity against U87 cells. Perspectives include mechanistics studies about cell death pathway and expanding cell panel. In conclusion, sanguinarine anlogues represent a scaffold to be explored by medicinal chemists to the development of potential antitumor agent

Ferroptosis in respiratory diseases / 生理学报

Ferroptosis is a novel form of regulated cell death which is dependent on iron and reactive oxygen species (ROS) and associated with the accumulation of lipid peroxides. It is obviously different from other cell death types in terms of morphology, biochemistry, genetics, etc. Also, it is related to the production of iron catalyzed lipid peroxides which is triggered by non-enzymatic or enzymatic reactions. Ferroptosis has been proved to be involved in hematological diseases, cardio-cerebrovascular diseases, liver and kidney diseases. This paper will review the definition, mechanism, inducers of ferroptosis, as well as the function of ferroptosis in respiratory system. We expect to present a new concept for respiratory research and suggest potential targets for clinical prevention and treatment of respiratory diseases.

Cell death of intestinal epithelial cells in intestinal diseases / 生理学报

Gut injury continues to be the devastating and unpredictable critical illness associated with increased cell death of intestinal epithelial cells (IECs). The IECs, immune system and microbiome are the interrelated entities to maintain normal intestinal homeostasis and barrier integrity. In response to microbial invasion, IEC cell death occurs to maintain intestinal epithelium function and retain the continuous renewal and tissue homeostasis. But the imbalance of IEC cell death results in increased intestinal permeability and barrier dysfunction that leads to several acute and chronic intestinal diseases, such as intestinal ischemia/reperfusion (I/R), sepsis, inflammatory bowel diseases (IBD), necrotizing enterocolitis (NEC), etc. During the pathophysiological state, the excessive IEC apoptotic cell death leads to a chronic inflammatory condition, later switches to necroptotic cell death mechanism that induces more pathological features than apoptosis and may also induce other lytic cell death mechanisms like pyroptosis and ferroptosis to increase the pathogenesis of the intestinal diseases. But still, there remains gaps in the fundamental knowledge about the IEC cell death mechanisms in chronic intestinal diseases. Together, a deep understanding of the specific cell death mechanisms underlying chronic intestinal diseases, including sepsis, IBD, NEC, and intestinal I/R, is desperately needed to develop emerging novel promising therapeutic strategies. This review aims to show how the acute and critical illness in the gut are driven by IEC cell death mechanism, such as apoptosis, necrosis, necroptosis, pyroptosis, and ferroptosis.