An update on recent studies of extracellular vesicles and their role in hypercoagulability in thalassemia (Review).

Thromboembolic events are a significant clinical concern in thalassemia and hemoglobinopathies, highlighting the need for new strategies to treat and detect these specific hematologic complications. In recent years, extracellular vesicles (EVs) have garnered interest due to their role in cell-to-cell communication, including angiogenesis, immune responses and coagulation activation. Their multifaceted role depends on the cellular origin and cargo, making them potential diagnostic biomarkers and therapeutic agents. The present review highlights recent advances in understanding the involvement of EVs in hypercoagulability in thalassemia, the characterization of circulating EVs and the potential for using EVs as predictive biomarkers. ß-Thalassemia intermedia exhibits a high incidence of thromboembolic events, contributing to significant morbidity and mortality. Advanced technologies have enabled the profiling and characterization of circulating EVs in patients with ß-thalassemia through various techniques, including flow cytometry, proteomic studies, reverse transcription-quantitative PCR, transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Microparticles from splenectomized ß-thalassemia/hemoglobin E patients induce platelet activation and aggregation, potentially contributing to thrombus formation. The abundance of these microparticles, primarily released from platelets and damaged red cells, may have a role in thromboembolic events and other clinical complications in thalassemia. This suggests a promising future for EVs as diagnostic and predictive biomarkers in thalassemia management.

MicroRNA expression profiles associated with the metastatic ability of MDA­MB­231 breast cancer cells.

Breast cancer is an important worldwide public health concern. The incidence rate of breast cancer increases every year. The primary cause of death is metastasis, a process by which cancer cells spread from a primary site to secondary organs. MicroRNAs (miRs/miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level. Dysregulation of certain miRNAs is involved in carcinogenesis, cancer cell proliferation and metastasis. Therefore, the present study assessed miRNAs associated with breast cancer metastasis using two breast cancer cell lines, the low-metastatic MCF-7 and the highly metastatic MDA-MB-231. miRNA array analysis of both cell lines indicated that 46 miRNAs were differentially expressed when compared between the two cell lines. A total of 16 miRNAs were upregulated in MDA-MB-231 compared with MCF-7 cells, which suggested that their expression levels may be associated with the highly invasive phenotype of MDA-MB-231 cells. Among these miRNAs, miR-222-3p was selected for further study and its expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Under both non-adherent and adherent culture conditions, the expression levels of miR-222-3p in the MDA-MB-231 cell line were higher than those noted in the MCF-7 cell line under the same conditions. Suppression of endogenous miR-222-3p expression in MDA-MB-231 cells using a miR-222-3p inhibitor resulted in a 20-40% reduction in proliferation, and a ~30% reduction in migration, which suggested that the aggressive phenotype of MDA-MB-231 cells was partly regulated by miR-222-3p. Bioinformatic analysis of miR-222-3p using TargetScan 8.0, miRDB and PicTar identified 25 common mRNA targets, such as cyclin-dependent kinase inhibitor 1B, ADP-ribosylation factor 4, iroquois homeobox 5 and Bcl2 modifying factor. The results of the present study indicated that miR-222-3p was potentially associated with the proliferation and migratory ability of the MDA-MB-231 cell line.

Up-regulation of microRNA 101-3p during erythropoiesis in ß-thalassemia/HbE.

Ineffective erythropoiesis is the main cause of anemia in ß-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in ß-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in ß-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of ß-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in ß-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of ß-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe ß-thalassemia/HbE compared to normal controls and mild ß-thalassemia/HbE. SUB1 gene expression was significantly lower in severe ß-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and ß-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in ß-thalassemia/HbE via other target genes.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter.

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells.

BACKGROUND: Previous studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown. METHODS: We generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics. RESULTS: PC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP. CONCLUSIONS: Suppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells. GENERAL SIGNIFICANCE: Our results highlight the possibility of the use of PC as an anti-cancer drug target.

Protein expression profiles that underpin the preventive and therapeutic potential of Moringa oleifera Lam against azoxymethane and dextran sodium sulfate-induced mouse colon carcinogenesis.

Previous studies in a mouse model have indicated the anticancer potential of boiled Moringa oleifera pod (bMO)-supplemented diets; however, its molecular mechanisms are still unclear. Therefore, the present study aimed to explore the protein expression profiles responsible for the suppressive effect of bMO supplementation on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced mouse colon carcinogenesis. Analysis by gel electrophoresis and liquid chromatography-tandem mass spectrophotometry demonstrated that there were 125 proteins that were differentially expressed in mouse colon tissues between 14 experimental groups of mice. The differentially expressed proteins are involved in various biological processes, such as signal transduction, metabolism, transcription and translation. Venn diagram analysis of the differentially expressed proteins was performed in six selected mouse groups, including negative control, positive control mice induced by AOM/DSS, the AOM/DSS groups receiving preventive or therapeutic bMO diets and their bMO-supplemented control groups. This analysis identified 7 proteins; 60S acidic ribosomal protein P1 (Rplp1), fragile X mental retardation, cystatin 9, round spermatids protein, zinc finger protein 638, protein phosphatase 2C (Ppm1g) and unnamed protein product as being potentially associated with the preventive and therapeutic effects of bMO in AOM/DSS-induced mouse colon cancer. Analysis based on the search tool for interactions of chemicals (STITCH) database predicted that Rplp1 interacted with the apoptotic and inflammatory pathways, whereas Ppm1g was associated only with inflammatory networks. This proteomic analysis revealed candidate proteins that are responsible for the effects of bMO supplementation, potentially by regulating apoptotic and inflammatory signaling networks in colorectal cancer prevention and therapy.

c-Myc directly targets an over-expression of pyruvate carboxylase in highly invasive breast cancer.

Here we showed that the c-Myc oncogene is responsible for overexpression of pyruvate carboxylase (PC) in highly invasive MDA-MB-231 cells. Pharmacological inhibition of c-Myc activity with 10074-G5 compound, resulted in a marked reduction of PC mRNA and protein, concomitant with reduced cell growth, migration and invasion. This growth inhibition but not migration and invasion can be partly restored by overexpression of PC, indicating that PC is a c-Myc-regulated pro-proliferating enzyme. Analysis of chromatin immunoprecipitation sequencing of c-Myc bound promoters revealed that c-Myc binds to two canonical c-Myc binding sites, locating at nucleotides -417 to -407 and -301 to -291 in the P2 promoter of human PC gene. Mutation of either c-Myc binding site in the P2 promoter-luciferase construct resulted in 50-60% decrease in luciferase activity while double mutation of c-Myc binding sites further decreased the luciferase activity in MDA-MB-231 cells. Overexpression of c-Myc in HEK293T cells that have no endogenous c-Myc resulted in 250-fold increase in luciferase activity. Mutation of either E-boxes lowered luciferase activity by 50% and 25%, respectively while double mutation of both sites abolished the c-Myc transactivation response. An electrophoretic mobility shift assay using nuclear proteins from MDA-MB-231 confirmed binding of c-Myc to both c-Myc binding sites in the P2 promoter. Bioinformatic analysis of publicly available transcriptomes from the cancer genome atlas (TCGA) dataset revealed an association between expression of c-Myc and PC in primary breast, as well as in lung and colon cancer tissues, suggesting that overexpression of PC is deregulated by c-Myc in these cancers.

miRNA Regulation of Glucose and Lipid Metabolism in Relation to Diabetes and Non-alcoholic Fatty Liver Disease.

Glucose and lipids are important nutrients because they provide most of the energy for the cells. A pre-translational regulation by microRNAs (miRNAs) plays a pivotal role in cellular metabolism by targeting the key rate-limiting enzymes of relevant pathways to fine-tune control of metabolic homeostasis. Aberrant expression of these miRNAs can result in an over or under expression of those key enzymes, contributing to the etiology of diabetes and non-alcoholic fatty liver disease (NAFLD). Here we discuss recent studies of various miRNAs that control insulin sensitivity, hepatic glucose production and de novo lipogenesis and how aberrant expression of these miRNAs contributes to the pathophysiology of diabetes and NAFLD in animal models. We also review the current application of circulating miRNAs as biomarkers for diagnosis or disease monitoring in diabetes and NAFLD.

Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells.

We recently showed that the anaplerotic enzyme pyruvate carboxylase (PC) is up-regulated in human breast cancer tissue and its expression is correlated with the late stages of breast cancer and tumor size [Phannasil et al., PloS One 10, e0129848, 2015]. In the current study we showed that PC enzyme activity is much higher in the highly invasive breast cancer cell line MDA-MB-231 than in less invasive breast cancer cell lines. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with 13C6-glucose and 13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction in viability and proliferation rates suggesting the perturbation of pathways that are involved in cancer invasiveness. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.