Microvesicles of osteoblasts modulate bone marrow mesenchymal stem cell-induced apoptosis to curcumin in myeloid leukemia cells.

Microvesicles (MVs) derived from bone marrow niche components have an important role in genetic reprogramming and subsequent drugs induce apoptosis in leukemic cells. Here, we have found that undertreatment of curcumin or daunorubicin, the cross-talk through MVs of KG-1-bone marrow mesenchymal stem cells (BMSCs), significantly downregulates the expression of the survival gene osteopontin (OPN), CXCL-12, IL-6 (interleukin-6), STAT-3, and VCAM-1 (vascular cell adhesion molecule 1) in treated-KG-1 cells as well as exclusively upregulates CXCL-12 in BMSCs. Drug treated-cell populations' MVs of both single cultured osteoblasts (OBs) and cocultured KG-1 + BMSCs + OBs similarly upregulate survival mediators' OPN, CXCL-12, IL-6, STAT-3, and VCAM-1 in treated-KG-1 cells. Likewise, isolated MVs from KG-1 cells or communication between KG-1, BMSCs, and OBs treated by drugs increase the expression of genes OPN, CXCL-12, IL-6, STAT3, and VCAM-1 by OBs. MVs derived from KG-1 + BMSCs + OBs reduce drug-induced apoptosis in KG-1 cells. This suggests MVs-mediated information transfer is a procedure whereby OBs could overcome BMSCs-induced apoptosis in drug-treated-KG-1 cells.

Inhibitory Effects of Arsenic Trioxide and Thalidomide on Angiogenesis and Vascular Endothelial Growth Factor Expression in Leukemia Cells

Acute myeloid leukemia (AML) is a blood disorder characterized by uncontrolled proliferation of myeloid progenitors and decrease in the apoptosis rate. The vascular endothelial growth factor (VEGF) promotes blood vessel regeneration which might play important roles in development and progression of neoplasia. Our previous studies focused on cytotoxicity and anticancer effects of arsenic trioxide (ATO) and thalidomide (THAL) as an anti-VEGF compound in the AML cell model. ATO also affects regulatory genes involved in cell proliferation and apoptosis. The aim of present study was to examine the effects of ATO and THAL alone and in combination on U937 and KG-1 cells , with attention to mRNA expression for VEGF isoforms. Growth inhibitory effects was assessed by MTT assay and apoptosis induction was determined by Annexin/PI staining. mRNA expression levels were evaluated by real-time PCR. Our data indicated that ATO (1.618µM and 1µM in KG-1 and U937 cell lines respectively), THAL (80µM and 60µM) and their combination inhibited proliferation and induced apoptosis in our cell lines. mRNA expression of VEGF (A, B) decreased while C and D isoforms did not show any significant changes. Taken together, according to the obtained results, the VEGF autocrine loop could be a target as a therapeutic strategy for cases of AML.

Osteopontin plays a unique role in resistance of CD34+/CD123+ human leukemia cell lines KG1a to parthenolide.

OBJECTIVES: To determine if parthenolide (PTL) is cytotoxic for leukemia-like KG1a cells and if it involves in certain molecular-mediated resistance, especially osteopontin (OPN). METHODS: PTL/daunorubicin (DNR)-treated KG1a cells were examined for viability using MTT and colony-formation assay, and stained for apoptosis using AV/PI. The gene and protein expression were evaluated by qReal-time PCR and Western blotting analysis, respectively. OPN gene was inhibited by OPN siRNA. The cells were stained for various fractions using PE anti-CD34, FITC anti-CD38 and PerCP anti-CD123. RESULTS: Cell viability and proliferation assay exhibited KG1a cells are relatively refractory to used concentrations of PTL. OPN mRNA and protein levels increased in response to PTL. Suppression of OPN with siRNA increased the cytotoxic effects of PTL on KG1a cells. PTL treatment and OPN siRNA suppression in KG1a cells resulted in a decrease of mRNA expression of AKT, mTOR, ß-catenin, and Phosphatase and tensin homolog (PTEN). The sub-population cells of CD34+ and CD123+ from KG1a cells are enriched by PTL treatment. CONCLUSION: Parthenolide in spite of the reduction in gene expression of AKT, mTOR or beta-catenin, stimulates the OPN expression in KG1a cells. The OPN expression pattern in KG1a cells could be compatible with CD34+/CD123+ subtype enrichment by PTL which in turn implies OPN's unique role in resistance of cell populations characterized by CD34+/CD123+ phenotype.

Parthenolide Induces Apoptosis in Committed Progenitor AML Cell line U937 via Reduction in Osteopontin.

BACKGROUND: Interfering with cell proliferation and survival is a critical role for antineoplastic drugs leading to cell death through induction of apoptosis. Alternative treatments with herbal extracts offer insights into acute myeloid leukemia (AML) therapy. Parthenolide (PTL), an extract from feverfew, induces apoptosis in primary human leukemia stem cells (LSCs) and bulk leukemic cell populations. Osteopontin (OPN) preserves cell viability in response to anticancer agents and its receptors could be utilized for therapeutic targeting of cancer cells. METHODS: U937 cells were cultured in RPMI 1640 with concentrations of 2, 4, 6, 8, and 10 µM PTL for 20-24 hours for MTT assays. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI as Annexin V+/PI- and Annexin V+/PI+ to measure early and late apoptosis, respectively. Quantitative real-time PCR was used to measure OPN gene expression using the 2(-ΔΔCt) method. The PTL-treated cells were stained with FITC-CD38 antibody for flow cytometry analyses. Data were compared using one-way analysis of variance (ANOVA) by SPSS 19. RESULTS: Parthenolide inhibited growth of U937 cells with IC25 and IC50 values of 4 and 5.8 µM, respectively. Death induction with PTL was apoptotic. Flow cytometry showed a significant decrease in the percentage of CD38+ U937 cells in response to PTL. Osteopontin gene expression decreased in response to PTL. CONCLUSION: PTL induced apoptosis and reduced OPN gene expression in U937 cells.

Erythrocytic phosphatidylserine exposure and hemostatic alterations in ß-thalassemia intermediate patients.

INTRODUCTION: Hypercoagulable state is one of the common findings in beta-thalassemia intermedia (ß-TI), particularly in splenectomized patients, with infrequent blood transfusion. Abnormality of the red blood cells (RBC) membrane due to oxidative damage is suggestive of possible etiologies. Membrane lipid peroxidation increases the exposure of phosphatidylserine (PS) that plays a role in the activation of coagulation factors V and X, subsequently initiating thrombosis. Our aim of this study was to find the probable correlation of the alteration of the PS on the RBC outer membrane with the hypercoagulable state in the ß-TI patients. MATERIALS AND METHODS: Our cross-sectional study was conducted on 39 splenectomized ß-TI patients and 38 age-matched healthy controls. The mean age was 37 years. Analysis of the PS exposure on the RBCs was performed by fluorescein isothiocyanate (FITC) conjugated AV protein. Measurement of the coagulation factors X, V and antithrombin III (AT-III) was performed. We also checked the D-dimer levels. Analysis was performed by SPSS16. RESULTS: Fluorescence of FITC-Annexin V labeling on patients RBCs were higher than healthy controls; (2.8 ± 2.2%) of the patients versus (0.4 ± 0.18%) in the control group and was statistically significant (P < 0.05). Mean levels of factor X and AT-III of the patients as compared with the control group decreased and showed significant difference (P < 0.05). CONCLUSIONS: Circulation of thalassemic RBCs, which abnormally possess PS on RBC membrane outer surface, suggests the possibility of the gradual consumption of the coagulation factors in the presence of a chronic coagulability state.