Menthol induces extracellular vesicle regulation of apoptosis via ATG3 and caspase-3 in acute leukemic cells.

Leukemia is one of the most deadly cancers in Thailand. Natural compounds have been developed for cancer treatment. Menthol, a peppermint compound, has shown pharmacological properties such as anti-cancer activity. However, the mechanism of menthol inducing extracellular vesicles in leukemic cells is not yet understood. In this study, we investigated the effects of menthol on leukemic extracellular vesicles and their role in apoptosis. NB4 and Molt-4 leukemic cells were cultured with menthol in various concentrations and times. Bioinformatic analysis was used to investigate target proteins of extracellular vesicle and apoptosis, followed by mRNA and protein expression by RT‒PCR and western blotting, respectively. Our findings indicate that menthol inhibits leukemic cell proliferation and increases extracellular vesicles. Furthermore, menthol treated leukemic extracellular vesicles induce apoptosis and upregulate the expression of ATG3 and caspase-3 in both mRNA and protein levels. These results suggest that menthol has an antileukemic effect through ATG3 and caspase-3 in apoptosis of leukemic extracellular vesicles.

Increased autophagy leads to decreased apoptosis during ß-thalassaemic mouse and patient erythropoiesis.

ß-Thalassaemia results from defects in ß-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both ßIVS2-654-thalassaemic mice and ß-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in ß-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both ß-thalassaemic mice and ß-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in ß-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in ß-thalassaemia.

Internalization of cell-derived microparticles triggers endothelial pro-inflammatory responses.

BACKGROUND: Increased numbers of circulating microparticles (MPs) have long been documented in thalassemia and are considered as a contributing factor in developing the thromboembolic events (TEEs), which are associated with endothelial dysfunction. Indeed, the cellular and molecular mechanisms by which MPs and endothelial cells interact and their consequences remain poorly investigated. OBJECTIVE: The present study aims to compare the biological effects of MPs obtained from healthy subjects and ß-thalassemia/HbE patients on endothelial pro-inflammatory responses. METHODS: MPs isolated from plasma by two-step centrifugation from 10 healthy donors, 19 splenectomized and 30 non-splenectomized ß-thalassemia/HbE patients were first characterized for their cellular origins, then counted and incubated with primary human umbilical vein endothelial cells (HUVECs). Internalization of MPs into HUVECs and their induction on endothelial cell activation and pro-inflammatory responses were determined. RESULTS: MPs either from healthy or ß-thalassemia/HbE patients could become internalized into endothelial cells, but unlike MPs from healthy donors and non-splenectomized patients, MPs from splenectomized patients were the most active and induced the 2-fold up-regulation of pro-inflammatory genes, IL1B, CXCL8, and CCL2 and 4-fold increase in interleukin-1ß. In addition, MPs from both healthy subjects and splenectomized patients at 106/ml failed to trigger the secretion of endothelial IL-6 and IL-8 while higher MP concentration at 5 × 106/ml significantly induced this secretion. CONCLUSIONS: Plasma MPs isolated from splenectomized ß-thalassemia/HbE patients are capable of triggering pro-inflammatory responses from endothelial cells reflected at both gene and protein levels.

Microparticles from ß-thalassaemia/HbE patients induce endothelial cell dysfunction.

Thromboembolic complication occurs frequently in ß-thalassaemia/HbE patients, particularly in splenectomised patients. Endothelial cells play an important role in thrombosis. There is strong evidence of endothelial cell activation and dysfunction in ß-thalassaemia. Microparticles (MPs) are associated with thrombosis and endothelial cell dysfunction in many diseases including ß-thalassaemia. However, the effect of thalassaemic-MPs on endothelial cells mediating thrombus formation has not been elucidated. In this study, the effects of circulating MPs from ß-thalassaemia/HbE patients on endothelial cell functions were investigated. The results showed that MPs directly induce tissue factor, interleukin (IL)-6, IL-8, intracellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin expression in human umbilical vein endothelial cells (HUVECs). Notably, the levels of these endothelial cell activation markers were significantly increased in HUVECs treated with MPs obtained from splenectomised ß-thalassaemia/HbE patients when compared to MPs from non-splenectomised patients or normal subjects. The increased endothelial cell activation ultimately lead to increased monocyte-endothelial cell adhesion. THP-1 and HUVECs adhesion induced by MPs from normal subjects, non-splenectomised and splenectomised patients increased to 2.0 ± 0.4, 2.3 ± 0.4 and 3.8 ± 0.4 fold, respectively when compared to untreated cells. This finding suggests that MPs play an important role on thrombosis and vascular dysfunction in ß-thalassaemia/HbE disease, especially in splenectomised cases.

Microparticles from splenectomized ß-thalassemia/HbE patients play roles on procoagulant activities with thrombotic potential.

Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in ß-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized ß-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin+) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in ß-thalassemia/HbE patients.

Effect of Tumor Necrosis Factor-Alpha on Erythropoietin and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in ß-Thalassemia/Hemoglobin E Patients.

OBJECTIVE: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in ß-thalassemia and might contribute to ineffective erythropoiesis. However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and erythropoietin receptor (EPOR) expression involved in proliferation of ß-thalassemia/hemoglobin (Hb) E erythroid progenitor cells compared with cells from healthy subjects. MATERIALS AND METHODS: CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry. RESULTS: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and ß-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for ß-thalassemia/Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells. CONCLUSION: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation. It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in ß-thalassemia/Hb E patients.

Interferon-gamma induced nitric oxide-mediated apoptosis of anemia of chronic disease in rheumatoid arthritis.

Proinflammatory cytokines play a role in the pathogenesis of anemia of chronic disease (ACD), which is a common cause of anemia in rheumatoid arthritis (RA). Anemia in RA is associated with increased apoptosis of erythroid cells. However, there is unclear information on the mechanism of ACD in the disease. The purpose of this study is to investigate the role of cytokines on nitric oxide-mediated apoptosis in erythroid progenitor cells of ACD in RA patients. Erythroid progenitor cells from healthy subjects and RA patients with ACD were treated with cytokines like interleukin-1ß, tumor necrosis factor-α, and interferon-γ at concentrations of 2, 20, and 40 ng/ml for 14 days. Cell viability and cell apoptosis were analyzed by trypan blue staining and flow cytometry, respectively. The results showed that the highest effect of cytokines on reduction cell viability and induction cell apoptosis was found in 20 ng/ml IFN-γ-treated cells of RA patients. In addition, IFN-γ showed significantly increased nitric oxide production and iNOS mRNA expression, which was measured by Griess assay and real-time PCR, respectively. The percentage of cell apoptosis and NO production was reduced after an inducible nitric oxide synthase inhibitor, SMT, treatment. In conclusion, IFN-γ could induce nitric oxide production-mediated apoptosis process, which might be involved in the pathogenesis of ACD in RA patients.

Cytokine-induced apoptosis of beta-thalassemia/hemoglobin E erythroid progenitor cells via nitric oxide-mediated process in vitro.

BACKGROUND/AIM: ß-Thalassemia/hemoglobin E (ß-thal/HbE) is a common hereditary anemia in Thailand. Ineffective erythropoiesis due to apoptosis and decreased lifespan of circulating thalassemic red blood cells are the major causes of anemia. Changes to bone marrow microenvironment could contribute to apoptotic events. This study examined the effects of cytokines interleukin-1ß, tumor necrosis factor-α and interferon-γ on apoptosis of ß-thal/HbE erythroid progenitor cells in vitro, including nitric oxide-mediated apoptotic processes. METHODS: Percent apoptosis of erythroid progenitor cells from 5 ß-thal/HbE patients and 5 normal control subjects was examined using flow cytometry. In addition, the inducible nitric oxide synthase (iNOS) mRNA level and nitrite production were measured using quantitative PCR and the Griess method, respectively. RESULTS: Upon cytokine treatment, a higher percent apoptosis was obtained with ß-thal/HbE erythroid progenitor cells compared with control, and the maximum effect was observed using 20 ng/ml interferon-γ on day 14 of culture. There was an increase in iNOS mRNA level and a concomitant elevation of nitrite concentration in culture medium. Apoptosis and nitrite level were abrogated when ß-thal/HbE and control cells were treated with S-methylisothiourea sulfate, an iNOS inhibitor. CONCLUSION: The marked sensitivity of erythroid progenitor cells from ß-thal/HbE patients to cytokine-induced apoptosis via an NO-mediated process reflects a proapoptotic status of such thalassemic red blood cells.