Comparing Two Methods for the Isolation of Exosomes.

Exosomes are membrane-bound nanovesicles released by cells into their extracellular environment. They carry different types of RNA including mRNA which may be useful in the diagnosis of various diseases. Exosome isolation has been a challenge because of their small size; therefore, two exosome isolation methods were compared in this study. The Exoquick-TC PLUS™ exosome isolation kit (kit) was compared with the classic ultracentrifugation (UC) method for exosome isolation. In samples obtained using both methods, cryo-electron microscopy showed round or slightly elongated vesicles with diameters ranging from 50 to 150 nm and delimited by a bilayered membrane. Dynamic light scattering resulted in multiple peaks for kit exosomes, whereas a single peak was observed for UC exosomes. Significantly, more total RNA was present in UC exosomes in contrast to kit exosomes (P < 0.0001). This was reflected in subsequent mRNA analysis using qPCR, where UC exosomes had lower Ct values compared to kit exosomes. In conclusion, exosome characterization revealed the presence of exosomes in both UC and the kit samples. The kit samples presented additional peaks from DLS which might be due to impurities. Overall, due to a higher total RNA and mRNA content, UC is a better option for subsequent mRNA analysis; nevertheless, the kit can still be used if an ultracentrifuge is not available as four out of the five genes selected were detected and quantified using the kit.

Pharmacogenes that demonstrate high association evidence according to CPIC, DPWG, and PharmGKB.

Background: Different levels of evidence related to the variable responses of individuals to drug treatment have been reported in various pharmacogenomic (PGx) databases. Identification of gene-drug pairs with strong association evidence can be helpful in prioritizing the implementation of PGx guidelines and focusing on a gene panel. This study aimed to determine the pharmacogenes with the highest evidence-based association and to indicate their involvement in drug-gene interactions. Methodology: The publicly available datasets CPIC, DPWG, and PharmGKB were selected to determine the pharmacogenes with the highest drug outcome associations. The upper two levels of evidence rated by the three scoring methods were specified (levels A-B in CPIC, 3-4 in DPWG, or 1-2 levels in PharmGKB). The identified pharmacogenes were further ranked in this study based on the number of medications they interacted with. Results: Fifty pharmacogenes, with high to moderately high evidence of associations with drug response alterations, with potential influence on the therapeutic and/or toxicity outcomes of 152 drugs were identified. CYP2D6, CYP2C9, CYP2C19, G6PD, HLA-B, SLCO1B1, CACNA1S, RYR1, MT-RNR1, and IFNL4 are the top 10 pharmacogenes, where each is predicted to impact patients' responses to ≥5 drugs. Conclusion: This study identified the most important pharmacogenes based on the highest-ranked association evidence and their frequency of involvement in affecting multiple drugs. The obtained data is useful for customizing a gene panel for PGx testing. Identifying the strength of scientific evidence supporting drug-gene interactions aids drug prescribers in making the best clinical decision.

Modulation of ATP8B1 Gene Expression in Colorectal Cancer Cells Suggest its Role as a Tumor Suppressor.

AIM: The study aims to understand the role of tumor suppressor genes in colorectal cancer initiation and progression. BACKGROUND: Sporadic colorectal cancer (CRC) develops through distinct molecular events. Loss of the 18q chromosome is a conspicuous event in the progression of adenoma to carcinoma. There is limited information regarding the molecular effectors of this event. Earlier, we had reported ATP8B1 as a novel gene associated with CRC. ATP8B1 belongs to the family of P-type ATPases (P4 ATPase) that primarily function to facilitate the translocation of phospholipids. OBJECTIVE: In this study, we attempt to implicate the ATP8B1 gene located on chromosome 18q as a tumor suppressor gene. METHODS: Cells culture, Patient data analysis, Generation of stable ATP8B1 overexpressing SW480 cell line, Preparation of viral particles, Cell Transduction, Generation of stable ATP8B1 knockdown HT29 cell line with CRISPR/Cas9, Generation of stable ATP8B1 knockdown HT29 cell line with shRNA, Quantification of ATP8B1 gene expression, Real-time cell proliferation and migration assays, Cell proliferation assay, Cell migration assay, Protein isolation and western blotting, Endpoint cell viability assay, Uptake and efflux of sphingolipid, Statistical and computational analyses. RESULTS: We studied indigenous patient data and confirmed the reduced expression of ATP8B1 in tumor samples. CRC cell lines were engineered with reduced and enhanced levels of ATP8B1, which provided a tool to study its role in cancer progression. Forced reduction of ATP8B1 expression either by CRISPR/Cas9 or shRNA was associated with increased growth and proliferation of CRC cell line - HT29. In contrast, overexpression of ATP8B1 resulted in reduced growth and proliferation of SW480 cell lines. We generated a network of genes that are downstream of ATP8B1. Further, we provide the predicted effect of modulation of ATP8B1 levels on this network and the possible effect on fatty acid metabolism-related genes. CONCLUSION: Tumor suppressor gene (ATP8B1) located on chromosome 18q could be responsible in the progression of colorectal cancer. Knocking down of this gene causes an increased rate of cell proliferation and reduced cell death, suggesting its role as a tumor suppressor. Increasing the expression of this gene in colorectal cancer cells slowed down their growth and increased cell death. These evidences suggest the role of ATP8B1 as a tumor suppressor gene.

Emerging Pharmacologic Therapies for Heart Failure With Reduced Ejection Fraction.

The global burden of heart failure has reached epidemic proportions with tremendous health and economic consequences. Sodium glucose cotransporter 2 inhibitors, vericiguat, and omecamtiv mecarbil are novel agents that promise to blunt the high residual risk of heart failure with reduced ejection fraction. We review the vast knowledge base that has rapidly materialized for these agents and is poised to shape the current and future trends and recommendations in heart failure pharmacotherapy.

Le fardeau planétaire que représente l'insuffisance cardiaque (IC) a atteint des proportions épidémiques en plus d'avoir d'énormes répercussions sur la santé et l'économie. Le vericiguat et l'omécamtiv mécarbil sont des inhibiteurs du cotransporteur sodium-glucose de type 2 (SGLT2) novateurs et prometteurs qui donnent de l'espoir dans la réduction du risque résiduel élevé d'insuffisance cardiaque avec fraction d'éjection réduite. Nous examinons à l'heure actuelle la vaste base de connaissances qui s'est rapidement constituée pour ces agents et qui s'apprête à façonner les tendances et recommandations actuelles et futures relatives à la pharmacothérapie de l'IC.

Transcriptomics-Based Characterization of the Toxicity of ZnO Nanoparticles Against Chronic Myeloid Leukemia Cells.

INTRODUCTION: Zinc oxide nanoparticles (ZnO NPs) have recently attracted attention as potential anti-cancer agents. To the best of our knowledge, the toxicity of ZnO NPs against human chronic myeloid leukemia cells (K562 cell line) has not been studied using transcriptomics approach. OBJECTIVE: The goals of this study were to evaluate the capability of ZnO NPs to induce apoptosis in human chronic myeloid leukemia cells (K562 cells) and to investigate the putative mechanisms of action. METHODS: We used viability assay and flowcytometry coupled with Annexin V-FITC and propidium iodide to investigate the toxicity of ZnO NPs on K562 cells and normal peripheral blood mononuclear cells. Next we utilized a DNA microarray-based transcriptomics approach to characterize the ZnO NPs-induced changes in the transcriptome of K562 cells. RESULTS: ZnO NPs exerted a selective toxicity (mainly by apoptosis) on the leukemic cells (p≤0.005) and altered their transcriptome; 429 differentially expressed genes (DEGs) with fold change (FC)≥4 and p≤0.008 with corrected p≤0.05 were identified in K562 cells post treatment with ZnO NPs. The over-expressed genes were implicated in "response to zinc", "response to toxic substance" and "negative regulation of growth" (corrected p≤0.05). In contrast, the repressed genes positively regulated "cell proliferation", "cell migration", "cell adhesion", "receptor signaling pathway via JAK-STAT" and "phosphatidylinositol 3-kinase signaling" (corrected p≤0.05). Lowering the FC to ≥1.5 with p≤0.05 and corrected p≤0.1 showed that ZnO NPs over-expressed the anti-oxidant defense system, drove K562 cells to undergo mitochondrial-dependent apoptosis, and targeted NF-κB pathway. CONCLUSION: Taken together, our findings support the earlier studies that reported anti-cancer activity of ZnO NPs and revealed possible molecular mechanisms employed by ZnO NPs to induce apoptosis in K562 cells.

Cytotoxic activity of the novel heterocyclic compound G-11 is primarily mediated through intrinsic apoptotic pathway.

Natural and chemically synthesized heterocyclic compounds have been explored for their potential use as anticancer agents. We had synthesized non-natural heterocyclic analogs and evaluated their anti-tumor activity by measuring effect on cell proliferation and induction of apoptosis in different cell lines. Previously, we identified a pyrazole-containing compound (G-11) showing cytotoxic effect towards leukemia and lymphoma cell lines. In this study, we further investigated the mechanistic aspects of anticancer properties of G-11 in HL-60 cell line. We demonstrated that cytotoxic effect of G-11 is mediated by caspase-dependent apoptosis. However, the involvement of mitochondrial dysfunction induced by G-11 was independent of caspases. G-11 triggered generation of ROS, caused disruption of mitochondrial transmembrane potential, increased release of cytochrome c to the cytosol, and altered the expression of Bcl-2 and Bax proteins. These results suggest significant involvement of intrinsic apoptotic pathway. This study comprehensively details the possible mechanisms of action of a novel heterocyclic compound which could find its potential use as an anticancer agent.

Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-ß-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

The pyridone-annelated isoindigo (5'-Cl) induces apoptosis, dysregulation of mitochondria and formation of ROS in leukemic HL-60 cells.

BACKGROUND/AIMS: In our quest to develop an isoindigo with improved efficacy and bioavailability, we recently synthesized a series of novel substituted pyridone-annelated isoindigo and evaluated their antiproliferative effects. We identified the compound [(E)-1-(5'-Chloro-2'-oxoindolin-3'-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f] quinoline-8-carboxylic acid], abbreviated as 5'-Cl, which shows selective antiproliferative activities against various cancer cell lines mediated through apoptosis. Here we have investigated the molecular mechanisms underlying the apoptotic activity of 5'-Cl in the human promyelocytic leukemia HL-60 cells. METHODS: We employed different methods to determine the apoptotic pathways triggered by 5'-Cl in HL-60 cells, using flow cytometry, nuclear staining, caspases activation, mitochondria functioning, generation of reactive oxygen species (ROS) and Western blotting techniques. RESULTS: Low concentrations (1-8 µM) of 5'-Cl inhibited the growth of HL-60 cells in a dose and time-dependent manner. Cytotoxicity of this compound is found to be mediated by a caspase-dependent apoptosis. Also, there were indications of caspase independent apoptosis as z-VAD-FMK failed to fully rescue the cells from 5'-Cl-induced apoptosis. In addition, the compound triggered generation of Reactive Oxygen Species (ROS), caused depolarization of the mitochondrial inner membrane, decreased the level of cellular ATP, modulated the expression and phosphorylation of Bcl-2 leading to loss of its association with Bax and increased the release of cytochrome c to the cytosol of treated cells. The effects of 5'-Cl on mitochondria and apoptosis were substantially blocked in the presence of a combination between z-VAD-FMK and either of the ROS scavenger N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). CONCLUSION: We demonstrated that the growth inhibitory effects of 5'-Cl in HL-60 cells involve multiple pathways of apoptosis and dysregulation of mitochondrial functions.

Identification of STAT3-independent regulatory effects for protein inhibitor of activated STAT3 by binding to novel transcription factors.

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/ß-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

Probabilistic modeling of DNA mismatch repair effects on cell cycle dynamics and iododeoxyuridine-DNA incorporation.

Previous studies in our laboratory have described increased and preferential radiosensitization of mismatch repair-deficient (MMR(-)) HCT116 colon cancer cells with 5-iododeoxyuridine (IUdR). Indeed, our studies showed that MMR is involved in the repair (removal) of IUdR-DNA, principally the G:IU mispair. Consequently, we have shown that MMR(-) cells incorporate 25% to 42% more IUdR than MMR(+) cells, and that IUdR and ionizing radiation (IR) interact to produce up to 3-fold greater cytotoxicity in MMR(-) cells. The present study uses the integration of probabilistic mathematical models and experimental data on MMR(-) versus MMR(+) cells to describe the effects of IUdR incorporation upon the cell cycle for the purpose of increasing IUdR-mediated radiosensitivity in MMR(-) cells. Two computational models have been developed. The first is a stochastic model of the progression of cell cycle states, which is applied to experimental data for two synchronized isogenic MMR(+) and MMR(-) colon cancer cell lines treated with and without IUdR. The second model defines the relation between the percentage of cells in the different cell cycle states and the corresponding IUdR-DNA incorporation at a particular time point. These models can be combined to predict IUdR-DNA incorporation at any time in the cell cycle. These mathematical models will be modified and used to maximize therapeutic gain in MMR(-) tumors versus MMR(+) normal tissues by predicting the optimal dose of IUdR and optimal timing for IR treatment to increase the synergistic action using xenograft models and, later, in clinical trials.