Idarubicin-induced oxidative stress and apoptosis in cardiomyocytes: An in vitro molecular approach

Idarubicin (IDA) is an anthracycline antibiotic, frequently used for the treatment of various human cancers. In vivo rodent model studies have identified a variety of possible adverse outcomes from IDA including heart effects like increased heart weights, myocardial histopathological injury, electrocardiogram abnormalities, and cardiac dysfunction. Despite significant investigations, the molecular mechanisms responsible for the cardiotoxicity of IDA have not been fully clarified. The aim of the current study was to investigate the effects of IDA on the HL-1 cardiac muscle cell. Different concentrations of IDA (10−6, 10−5, 10−4, and 10−3 M) were used at different time (6, 12, 24, and 48 h) periods, and the Cell Counting Kit-8 (CCK-8); 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe method; and enzyme-linked immunosorbent assay (ELISA) were used to detect the oxidative stress level. In addition, we used network analysis to predict IDA-induced cardiotoxicity. The TUNEL assay, qRT-PCR, ELISA assay, and Western blotting detection of related apoptotic factors including caspase family, Bax, and Bcl-2. Overall, we found that IDA was generally more toxic at high concentrations or extended durations of exposure. At the same time, IDA can increase the content of reactive oxygen species (ROS), malondialdehyde (MDA), and decrease the level of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in cells, and increase the content of lactate dehydrogenase (LDH) and nitric oxide synthase (NOS) in the medium. Network analysis showed that the apoptosis signaling pathway was activated; specifically, the caspase family was involved in the signal pathway. The results of the TUNEL assay, qRT-PCR, ELISA, and Western blot found that IDA can activate apoptotic factors. The mechanism may be related to the activation of apoptosis signaling pathway. These results indicate that the cardiotoxic effects of IDA are most likely associated with oxidative stress and ROS formation, which finally ends in apoptotic factors’ activation and induction of cell apoptosis.

Elevated Central Carbon Metabolism - a Hallmark for Senescent Cells in Aging Human Hematopoietic Stem Cell Compartment

Comprehensive proteomic studies of HSC derived from bone marrow of healthy human subjects (n = 59) in different age groups (range: 20 - 72 years) showed that aging HSCs are characterized not only by myeloid lineage skewing, senescence associated secretory phenotype (SASP), accumulation of reactive oxygen species (ROS), anti-apoptosis, but prominently by elevated glycolysis, glucose uptake, and accumulation of glycogen. This is caused by a subset of HSC that has become more glycolytic than others and not on a per cell basis. Subsequent comparative transcriptome studies of HSCs from human subjects >60 years versus those from <30 years have confirmed this association of elevated glycolysis with aging transcriptome signature. Provided with this background and based on glucose metabolism levels, we have developed a method to isolate human HSCs (CD34+ cells) from bone marrow into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GU high, GU inter, GU low). For human subjects >60 years old (n=9), the proportions of these subsets are: GU high= 5.4+3.5 %, GU inter= 66.4+22.5 %, GU low= 28.2+21.7 %. For subjects <30 years (n=5), the proportions are GU high= 1.7+1.5 %, GU inter= 66.5+36.9 %, GU low= 31.8+36.7. Single-cell RNA-sequencing (scRNA-seq) studies and gene ontology analysis of biological processes revealed that, compared to the GU inter and GU low subsets, the GU high cells showed a significantly higher expression of genes involved in myeloid development, inflammation response (AIF1, CASP2, ANXA1, ZFP36), anti-apoptosis (GSTP1, NME1, BCL2, DMNT1, BAX), cell cycle checkpoint (MCL1, CDK1, CDK4, EIF5A), histone regulation (BCL6, EGR1, KDM1A, MLLT3), b-galactosidase, and significantly lower expressions of genes involved in lymphoid development, and of MDM4, MDM2, FOXP1, SOX4, RB1. Functional studies indicated that the glycolytic enzymes were elevated in elderly HSCs, and the GU low subset corresponded to primitive and more pluripotent HSCs than the GU interand GU high subsets. Pathway analyses have then demonstrated that the GU high subset is associated with up-regulated p53 as well as JAK/STAT signaling pathways, characteristic of senescent HSCs observed in murine models. Applying Gene Set Enrichment Analysis (GSEA) algorithms, we have compared the scRNA-seq data of CD34+ cells derived from young (<30 years) versus older (>60 years) subjects, as well as the scRNA-seq data from GU high subset versus GU inter and GU lowsubsets from each individual subject (n = 6). The results are shown in Figure 1. In analogy to the comparison between old (>60 years) versus young (<30 years) HSCs (CD34+ cells), GSEA of the GU high versus GU inter and GU low subsets shows the same pattern of changes - significant upregulation of gene-set expressions for (a) inflammatory response (b) G2M checkpoint, (c) MTORC1, (d) ROS, (Fig. 1B), (e) allograft rejection; and down-regulation of gene-set expressions for (f) pluripotency, (g) androgen response, (h) UV response (Fig. 1C) as well as (i) interferon-a induction during SARS-CoV2-infection (data not shown in Fig. 1). Thus, our novel findings of elevated glycolysis coupled with significant activation of MTORC1 in the senescent cells of the HSC compartment have provided evidence for the important role of calorie restriction (CR) for healthy aging of HSCs. In numerous animal models, aging has been shown to be driven by the nutrient-sensing MTORC1 network. In animal models of aging, CR has been reported to deactivate the MTOR pathway, thus slowing aging and delaying diseases of aging. Conclusion: In a series of multi-omics studies, we have demonstrated that the GU high subset is identical to the senescent cells (SCs) in human HSC compartment. Studies in animal models have shown that SCs in murine bone marrow are responsible for driving the aging process, and elimination of this subset by inhibitors of anti-apoptotic factors is able to rejuvenate hematopoiesis in mice. Our present results have provided cellular and molecular evidence that SCs in human HSC compartment are also dependent on anti-apoptotic factors, elevated MTORC1 as well as increased glycolysis for survival. Inhibition of MTORC1 or glycolysis, either by specific inhibitors or by CR, may eliminate senescent HSCs and promote rejuvenation of human hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Terminalia chebula reduces melanoma cell proliferation and increases apoptosis by inhibiting tyrosinase

Recently, research on using plant-derived drugs as anticancer agents has been greatly developed. Terminalia chebula (T. chebula) Retz., the mature fruit of T. chebula, contains active ingredients that have antioxidant, antimicrobial, and antitumor effects. Because traditional Chinese medicine components have unstable physical and chemical properties, such as poor solubility, fast decomposition, and high irritation, drug nanocarriers that effectively increase their solubility and reduce irritation are desirable. Additionally, T. chebula fruit can inhibit tyrosinase activity. This study aimed to determine whether T. chebula nanostructured lipid carriers can regulate the proliferation and apoptosis of melanoma cells by inhibiting tyrosinase activity. Tyrosinase activity in drug-treated melanoma cells was detected by MTT and DOPA oxidation assays, and cell proliferation rate was observed. Western blot (MTT) was used to detect the activity of apoptotic factors, and flow cytometry was used to determine the apoptotic rate. The results showed that tyrosinase activity and cell proliferation rate decreased with increasing drug concentration in cultured cells. Furthermore, the inhibitory effect was concentration-dependent. Additionally, cells cultured with a high concentration of T. chebula nanostructured lipid carriers could activate apoptotic factors and increase cell apoptosis. Thus, this study showed that T. chebula fruit nanostructured lipid carriers could inhibit the proliferation of melanoma cells and increase apoptosis by inhibiting tyrosinase activity, demonstrating a new plant-derived drug for melanoma treatment.

Study on the Critical Roles of Apoptosis in Asthma Disease

Inflammation, and remodeling in airways are the two crucial characteristics of asthma, a common respiratory disease. In asthma pathophysiology, the recruitment of granulocytes finally results in inflammation, leading to lung damage. In this regard, failure to clear inflammatory cells by programmed cell death, apoptosis will cause the prolongation of inflammation. On the other hand, in airway epithelial cells, apoptosis may occur, resulting in airway remodeling. Hence, dysregulation of apoptosis has been suggested to contribute to the development of asthma. Importantly, knowledge of the factors related to apoptotic cascade seems vital to explore various pharmacological interventions for the treatment of asthma. In this review, we highlight several important apoptotic and anti-apoptotic factors contributing either to inflammatory cells or airway epithelial cells involved in asthma pathogenesis.

Exosomes Derived from Bone Marrow Mesenchymal Stem Cells (BMSC) Inhibit Apoptosis Factors Caspase-3 and Caspase-9 to Promote the Repair of Cardiomyocytes

This study assesses the effect of exosomes derived from bone marrow mesenchymal stem cells (MSCs) on cardiomyocytes by inhibiting the apoptotic factors Caspase-3 and Caspase-9. Cell purity was evaluated under a microscope and exosomes were obtained by ultracentrifugation from the culture supernatant of BMSCs. Tunable resistive pulse sensing (TRPS) method analyzed the concentration distribution of exosomes particle size, and specific surface antigens were examined by flow cytometry. Exosomes were used to process cardiomyocytes to detect cardiomyocyte repair. After plasmid interference technology, the effect of exosomes on caspase-3 and caspase-9 expression was detected by western blot. The activity of cardiomyocytes was analyzed by CCK-8. Exosomes can promote the viability of cardiomyocytes. The mRNA and protein levels of GLUT3 in cardiomyocytes were significantly increased. Exosomes can inhibit cardiomyocyte apoptosis by down-regulating the expression of apoptosis-related proteins. Exosomes can improve the function and promote the repair of myocardium by inhibiting the expression of apoptotic factors Caspase-3 and Caspase-9.

STUDY OF THE PHARMACOLOGICAL ACTIVITY OF NOVEL EPOR/CD131 HETERORECEPTOR AGONISTS IN MICE WITH ENDOTHELIAL-SPECIFIC EXPRESSION OF MUTANT POLG GENE

The aim of the research was to study antiatherosclerotic and endothelial kinds of a protective activity of peptides mimicking an erythropoietin a-helix B tertiary structure with laboratory codes EP-11-1 (UEHLERALNSS), EP-11-2. (UEQLERALNCS), EP-11-3 (UEQLERALNTS).Materials and methods. The study was conducted on 96 C57Bl/6J male double transgenic Polgmut/mut/Cdh5-CRE mice. Atherosclerosis was induced by a balloon injury accompanied by Western diet. Then, for 27 days, the drugs under study were administered once per 3 days at the dose of 20 μg/kg. On the 28th day, the animals were euthanized and the area of atherosclerotic plaques was collected for an assessment. The expression of genes associated with the processes of inflammation, apoptosis, and angiogenesis was determined in the tissues of the aorta. In addition, the endothelial protective effect of peptides in isolated segments of the thoracic aorta of wild and transgenic ransgenic Polgmut/mut mice was studied.Results. The assessment of the plaque size in the animals with the Polgmut/mut/Cdh5-CRE genotype against the background of the peptides under study did not reveal statistically significant differences in comparison to control. However, a quantitative PCR showed a statistically significant decreased expression of pro-apoptotic factors p-53 and Bax, and also increase the expression of anti-apoptotic factor Bcl-2 against the background of the peptides EP-11-1 and EP-11-2 administration. The administration of EP-11-1 and the original peptide pHBSP resulted in a statistically significant decrease in the Bax/Bcl-2 ratio. Compounds EP-11-1, EP-11-2, and EP-11-3 were more effective than the original peptide pHBSP, in reducing the increased expression of genes for inflammatory markers iNos, intercellular adhesion molecules Icam-1, Vcam-1 and E-selectin. The use of EP-11-1 led to a more efficient, in comparison with pHBSP, restoration of endothelial-dependent vasodilation of the aortic segments in mice with endothelial-specific overexpression of the mutant Polg gene.Conclusion. The study carried out on a murine model of the endothelial-specific expression of mutant gamma polymerase has shown that derivatives of the pHBSP peptide with laboratory codes EP-11-1, EP-11-2, EP-11-3, obtained by BLAST-searching for groups of pHBSP related peptides, have atheroprotective and endothelial protective kinds of a protective activity, which is more pronounced in comparison with the original peptide pHBSP.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

Effects of Acute Diquat Poisoning on Liver Mitochondrial Apoptosis and Autophagy in Ducks

Diquat (DQ) is an effective herbicide and is widely used in agriculture. Due to persistent and frequent applications, it can enter into aquatic ecosystem and induce toxic effects to exposed aquatic animals. The residues of DQ via food chain accumulate in different tissues of exposed animals including humans and cause adverse toxic effects. Therefore, it is crucial and important to understand the mechanisms of toxic effects of DQ in exposed animals. We used ducks as test specimens to know the effects of acute DQ poisoning on mechanisms of apoptosis and autophagy in liver tissues. Results on comparison of various indexes of visceral organs including histopathological changes, apoptosis, autophagy-related genes, and protein expression indicated the adverse effects of DQ on the liver. The results of our experimental trial showed that DQ induces non-significant toxic effects on pro-apoptotic factors like BAX, BAK1, TNF-α, caspase series, and p53. The results revealed that anti-apoptotic gene Parkin was significantly upregulated, while an upward trend was also observed for Bcl2, suggesting that involvement of the anti-apoptotic factors in ducklings plays an important role in DQ poisoning. Results showed that DQ significantly increased the protein expression level of the autophagy factor Beclin 1 in the liver. Results on key autophagy factors like LC3A, LC3B, and p62 showed an upward trend at gene level, while the protein expression level of both LC3B and p62 reduced that might be associated with process of translation affected by the pro-apoptotic components such as apoptotic protease that inhibits the occurrence of autophagy while initiating cell apoptosis. The above results indicate that DQ can induce cell autophagy and apoptosis and the exposed organism may resist the toxic effects of DQ by increasing anti-apoptotic factors.

A Study on Mesoporous Silica Loaded With Novel Photosensitizers HCE6 and Oxaliplatin for the Treatment of Cholangiocarcinoma

PurposeCholangiocarcinoma (CCA) is a malignant tumor with a high incidence. The therapeutic effect of conventional chemotherapy and radiotherapy is not obvious. Photodynamic therapy (PDT) is an ideal modality to fight cancer, and the nature of photosensitizer limits its application in clinical therapy. The aim of this study was to explore a novel mode of drug delivery for the intervention of bile duct cancer.MethodsOxaliplatin and photosensitizer HCE6 were loaded with mesoporous silica nanoparticles (MSNs) to synthesize Oxaliplatin/HCE6-MSNs (OH-MSNs); the structure of OH-MSNs was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), the drug release rate was detected by high performance liquid chromatography; the cellular activity, apoptosis level, and the expression levels of intracellular apoptosis and autophagy-related factors of OH-MSNs on cholangiocarcinoma cells were observed by CCK-8, flow cytometry, colony formation assay, and Western blot; the effects of OH-MSNs on cholangioma growth were observed by mouse tumor formation, immunohistochemistry, and tissue Tunel staining.ResultsThe release of OH-MSNs to Oxaliplatin was enhanced under acidic conditions; compared with Oxaliplatin or O-MSNs, OH-MSNs showed more potent killing effects against cholangiocarcinoma cells (P<0.05), and exerted notably inhibitory effects on the activity of cholangiocarcinoma cells (P<0.05), promoted their apoptosis (P<0.05), and greatly facilitated the expression of pro-apoptotic factors and autophagic factors in cholangiocarcinoma cells (P<0.05), and markedly inhibited the expression of anti-apoptotic factors and autophagic inhibitory factors (P<0.05); moreover, OH-MSNs could significantly suppress the growth of mouse cholangiocarcinoma (P<0.05) and induce apoptosis of tumor cells compared with Oxaliplatin or O-MSNs (P<0.05).ConclusionMSNs loading greatly increases the killing effect of Oxaliplatin on cholangiocarcinoma cells and upgrades the autophagic level of cholangiocarcinoma cells, while OH-MSNs synthesized by further loading HCE6 have a more apparent killing effect on cholangiocarcinoma cells.