Pinostrobin, a fingerroot compound, regulates miR-181b-5p and induces acute leukemic cell apoptosis.

Pinostrobin (PN) is the most abundant flavonoid found in fingerroot. Although the anti-leukemic properties of PN have been reported, its mechanisms are still unclear. MicroRNAs (miRNAs) are small RNA molecules that function in posttranscriptional silencing and are increasingly being used in cancer therapy. The aims of this study were to investigate the effects of PN on proliferation inhibition and induction of apoptosis, as well as the involvement of miRNAs in PN-mediated apoptosis in acute leukemia. The results showed that PN reduced cell viability and induced apoptosis in acute leukemia cells via both intrinsic and extrinsic pathways. A bioinformatics approach and Protein-Protein Interaction (PPI) network analysis revealed that ataxia-telangiectasia mutated kinase (ATM), one of the p53 activators that responds to DNA damage-induced apoptosis, is a crucial target of PN. Four prediction tools were used to predict ATM-regulated miRNAs; miR-181b-5p was the most likely candidate. The reduction in miR-181b-5 after PN treatment was found to trigger ATM, resulting in cellular apoptosis. Therefore, PN could be developed as a drug for acute leukemia; in addition, miR-181b-5p and ATM may be promising therapeutic targets.

Pinostrobin induces acute leukemia cell apoptosis via the regulation of miR-410-5p and SFRP5.

AIMS: This study attempted to explore the mechanisms involved in pinostrobin (PN)-mediated acute leukemia cell apoptosis regulated by miR-410-5p. MATERIAL AND METHODS: NB4 and MOLT-4 cells were cultured and treated with PN at the IC50 concentration. Apoptosis was examined by Annexin V-FITC/PI staining. RT-qPCR was used to measure the expression of caspase-3, BAK, BCL-W, and MCL-1. The target protein of PN was identified using LC-MS/MS followed by bioinformatic analysis. TargetScan, DIANA, and miRDB were used for the prediction of miRNAs involved in the PN-induced apoptosis mechanism. miRNA mimic transfection, RT-qPCR, and western blot analysis were performed to evaluate the regulatory effect of miRNA on its target and the involvement of miRNA in apoptosis induction by PN. In addition, the synergistic effect of PN and daunorubicin (DNR) were investigated by using the MTT assay. KEY FINDINGS: The results showed that PN reduced cell viability and induced apoptosis in both leukemia cell lines. From the LC-MS/MS and bioinformatics analysis, SFRP5 and miR-410-5p were selected as a potential PN target protein and miRNA, respectively. After miRNA mimic transfection, miR-410-5p, which is an onco-miRNA, was decreased and led to increased apoptosis in both cell lines, indicating that this miRNA is involved in PN-mediated apoptosis mechanisms. Moreover, PN demonstrated a synergistic effect with DNR, suggesting that PN may be used in combination with conventional chemotherapy drugs. SIGNIFICANCE: PN regulates the expression of miR-410-5p and SFRP5 to promote apoptosis in acute leukemia cells. It could be developed as an alternative treatment for leukemia in the future.

Anti-leukemic effect of menthol, a peppermint compound, on induction of apoptosis and autophagy.

Background: Menthol, a natural compound in peppermint leaves, has several biological activities, including antioxidant, anti-inflammatory, antiviral, antibacterial and anticancer properties. This study revealed the anti-leukemic effects and its underlying mechanisms of the menthol related apoptosis signaling pathway and autophagy in both NB4 and Molt-4 leukemic cell lines. Methods: Both leukemic cells were treated with menthol in various concentration. Cell viability was assessed using MTT assay, whereas apoptosis and autophagy were analyzed by flow cytometry using Annexin V-FITC/PI and anti-LC3/FITC antibodies staining, respectively. Apoptotic and autophagic related gene and protein expression were detected using RT-qPCR and western blot analysis, respectively. Moreover, STITCH database was used to predicts the interaction between menthol and proposed proteins. Results: Menthol significantly decreased cell viability in NB4 and Molt-4 cell lines in dose dependent manner. In combination of menthol and daunorubicin, synergistic cytotoxic effects were observed in leukemic cells. However, there was a minimal effect found on normal, peripheral blood mononuclear cells (PBMCs). Moreover, menthol significantly induced apoptosis induction via upregulation of caspase-3, BAX, p53 and downregulation of MDM2 mRNA expression. Autophagy was also induced by menthol through upregulating ATG3 and downregulating mTOR mRNA expression. For protein expression, menthol significantly increased caspase-3 whereas decreased mTOR in both leukemic cells. Conclusions. These results suggest that menthol exhibits cytotoxic activities by inhibition of cell proliferation, induction of apoptosis and autophagy through activating the caspase cascade, altering BAX and p53/MDM2, and regulating autophagy via the ATG3/mTOR signaling pathway.

Sesamin Acts as Anti-leukemic Compound Interacting with Novel Phosphoprotein Targets and Inducing Apoptosis in Leukemic Cells.

Leukemia is one of the high-incidence cancers that is characterized by an abnormal production of immature white blood cells. Subject to many reports on the side effects of conventional chemotherapy, herbs and natural compounds have been studied as an alternative medicine. In this study, sesamin, a lignan in sesame seed with pharmaceutical functions including anti-cancer, was chosen and treated with MOLT-4 and NB4 leukemic cell lines in various concentrations for 24 and 48 hours. The effect of sesamin on cell inhibition and expression levels of apoptotic genes in leukemic cell lines were investigated by MTT assay and real-time PCR, respectively. Moreover, apoptotic proteins were studied by mass spectrometry and bioinformatics tools to investigate the relation between sesamin and targeted proteins. Results showed that sesamin increased cell inhibition in both cell lines in dose- and time-dependent manner. Levels of caspase-3, -7, -8, and -9 gene expressions significantly increased, while BCL-2 decreased drastically in sesamin-treated cells. From bioinformatics study, PARP4, IPPK and caspase family proteins were found to be involved in sesamin that induced apoptosis in leukemic cells. Besides, doxorubicin, a chemotherapeutic drug, also shared the same protein targets as sesamin in apoptosis pathway. Sesamin demonstrates its potential to enhance cell inhibition and promotes cell apoptosis in both MOLT-4 and NB4 leukemic cell lines. This study will benefit the development of sesamin as an effective anti-leukemia drug in the future.

Bone marrow-mesenchymal stem cell-derived extracellular vesicles affect proliferation and apoptosis of leukemia cells in vitro.

Mesenchymal stem cells (MSCs) have been proposed to have potential for tissue engineering and cell therapy due to their multilineage differentiation potential and ability to secrete numerous paracrine factors, including extracellular vesicles (EVs). Increasing evidence has demonstrated that MSC-derived EVs (MSC-EVs) are able to induce the repair of tissue damage and regulate the immune system. However, their role in cancer development is still unclear. Reports have suggested that whether MSC-EVs have an inhibitory or promoting effect on cancer is dependent on the type of cancer. In this study, the role of MSC-EVs in the regulation of leukemic cell growth in vitro was investigated. The EVs were collected from conditioned media of MSCs by ultrafiltration using a 10 kDa molecular weight cutoff (MWCO) filter. The isolated MSC-EVs were comprised of microvesicles and exosomes, as examined by the size of vesicles and exosomal proteins, CD81 and flotillin-1. Cell proliferation, cell cycle status, apoptosis, and gene expression were examined in the leukemic cell lines NB4 and K562 after treatment with MSC-EVs. Suppression of cell proliferation and induction of apoptosis was observed. Gene expression analysis revealed differential expression of apoptotic-related genes in NB4 and K562. MSC-EVs increased the expression of BID and BAX and decreased expression of BCL2, indicating the induction of intrinsic apoptosis in NB4. In contrast, MSC-EVs increased the expression of the death receptor gene TRAILR2 and cell cycle regulator genes P21 and CCNE2 in K562. In conclusion, MSC-EVs partially induce leukemic cell apoptosis, and thus may have potential for the development of supportive therapies for leukemia.

Punicalagin, a pomegranate compound, induces apoptosis and autophagy in acute leukemia.

BACKGROUND: Punicalagin is the major phenolic compound found in pomegranate peels. It has several reported medical benefits, including antioxidant, anti-inflammatory, and anticancer properties. The present study investigated the anti-leukemic effects and the molecular mechanism of punicalagin on NB4 and MOLT-4 leukemic cell lines. METHODS: Leukemic cells were treated with punicalagin and cell viability was determined using MTS assay. Apoptosis and autophagy were analyzed by flow cytometry using Annexin V-FITC/PI and anti-LC3/FITC antibodies staining, respectively. Apoptotic and autophagic mRNA expression were determined using reverse transcription-quantitative PCR. STITCH bioinformatics tools were used to predict the interaction between punicalagin and its proposed target proteins. RESULTS: Results indicated that punicalagin decreased NB4 and MOLT-4 cell viability in a dose-dependent manner. Punicalagin, in combination with daunorubicin, exhibited synergistic cytotoxic effects. Punicalagin induced apoptosis through the upregulation of caspase-3/-8/-9, Bax and the downregulation of Bcl-2 expression. Punicalagin also promoted autophagy via the downregulation of mTOR and the upregulation of ULK1 expression. Cyclooxygenase-2 and toll-like receptor 4 were found to be involved in punicalagin-induced cell death in punicalagin-targeted protein interactions. CONCLUSIONS: These results suggest that punicalagin exerts cytotoxic activities by suppressing proliferation and promoting apoptosis and autophagy by activating the caspase cascade, altering Bax and Bcl-2, and regulating autophagy via mTOR/ULK1 signaling.

Bioactive peptide isolated from sesame seeds inhibits cell proliferation and induces apoptosis and autophagy in leukemic cells.

Leukemia is the most common type of hematological malignancies. Several natural products including bioactive peptides have been explored and studied for their anti-leukemic activities. In the present study, anti-leukemic peptide, IGTLILM (IM-7), was isolated and identified from the protein hydrolysate of sesame seeds by reverse phase-solid phase extraction, off-gel fractionation and nano LC-MS/MS. The cytotoxic effects of IM-7 were studied in MOLT-4 and NB4 acute leukemic cell lines using an MTT assay. The induction of apoptosis and autophagy was investigated by flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies, respectively. The mRNA alterations of apoptotic and autophagic-related genes were determined by reverse transcription-quantitative PCR. The present study found that IM-7 inhibited the proliferation of MOLT-4 and NB4 cells in dose-dependent manner, but it showed a minimal effect on healthy mononuclear cells. IM-7 activated apoptosis and autophagy through the upregulation of CASP3, ULK1 and BECN1 and the downregulation of BCL2. In addition, IM-7 enhanced the cytotoxic effect of the anti-leukemic drug, daunorubicin. The findings suggested that IM-7 was potent to suppress the proliferation of MOLT-4 and NB4 leukemic cells and induce apoptosis and autophagy through the regulation of caspase 3-Bcl-2 and ULK1-Beclin1, respectively.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines.

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

A novel peptide isolated from garlic shows anticancer effect against leukemic cell lines via interaction with Bcl-2 family proteins.

Leukemia is a group of cancer caused by the abnormal proliferation and differentiation of hematopoietic stem cells. Efforts geared toward effective therapeutic strategies with minimal side effects are underway. Peptides derived from natural resources have recently gained special attention as alternative chemotherapeutic agents due to their minimal adverse effects. In the present study, the aim was to isolate peptides from garlic (Allium sativum) and investigate their anticancer activity against leukemic cell lines. The protein extract of A. sativum was pepsin-digested to obtain protein hydrolysate followed by sequential purification methods. A novel anticancer peptide, VKLRSLLCS (VS-9), was identified and characterized by mass spectrometric analysis. The peptide was demonstrated to significantly inhibit the cell proliferation of MOLT-4 and K562 leukemic cell lines while exhibiting minimal inhibition against normal PBMC. Particularly, VS-9 could induce apoptosis and upregulate mRNA levels of caspase 3, caspase 8, caspase 9, and Bax while downregulating Bcl-2, Bcl-xL, and Bcl-w. Molecular docking of VS-9 with the anti-apoptotic Bcl-2 protein family suggested that VS-9 could bind the binding groove of the BH3 domain on target proteins. Protein-peptide interaction analysis by affinity chromatography and LC-MS/MS further showed that VS-9 could bind Bcl-2 proteins. Results suggest VS-9 as a potential garlic-derived novel anticancer peptide possessing apoptosis-inducing properties against leukemic cell lines via anti-apoptotic Bcl-2 protein family.

Autophagy and apoptosis induction by sesamin in MOLT-4 and NB4 leukemia cells.

Sesamin, the major furofuran lignan found in the seeds of Sesamum indicum L., has been investigated for its various medicinal properties. In the present study, the anti-leukemic effects of sesamin and its underlying mechanisms were investigated in MOLT-4 and NB4 acute leukemic cells. Leukemic cells were treated with various concentrations of sesamin. Cell viability was determined using an MTT assay. Flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies was applied to detect the level of apoptosis and autophagy, respectively. Reverse transcription-quantitative PCR was performed to examine the alterations in the mRNA expression of apoptotic and autophagic genes. In addition, bioinformatics tools were used to predict the possible interactions between sesamin and its targets. The results revealed that sesamin inhibited MOLT-4 and NB4 cell proliferation in a dose-dependent manner. In addition, sesamin induced both apoptosis and autophagy. In sesamin-treated cells, the gene expression levels of caspase 3 and unc-51 like autophagy activating kinase 1 (ULK1) were upregulated, while those of mTOR were downregulated compared with in the control. Notably, the protein-chemical interaction network indicated that caspase 3, mTOR and ULK1 were the essential factors involved in the effects of sesamin treatment, as with anticancer agents, such as rapamycin, AZD8055, Torin1 and 2. Overall, the findings of the present study suggested that sesamin inhibited MOLT-4 and NB4 cell proliferation, and induced apoptosis and autophagy through the regulation of caspase 3 and mTOR/ULK1 signaling, respectively.

Bioinformatics and experimental studies of anti-leukemic activity from 6-gingerol demonstrate its role in p53 mediated apoptosis pathway.

6-gingerol is a traditional medicine that possesses anti-cancer activity against several types of cancer. However, the mechanism of action still remains unclear. Therefore, this study explored the effects of 6-gingerol on anti-leukemic mechanisms in NB4, MOLT4, and Raji leukemic cell. Results indicated that 6-gingerol inhibited cell proliferation and induced cell apoptosis in these 3 cell lines. Moreover, 6-gingerol was shown to increase the mRNA expression of the caspase family thereby suggesting that 6-gingerol induced apoptosis through the caspase-dependent pathway. To explore the signaling pathway regulating 6-gingerol induced apoptosis, we utilized and integrated the network pharmacology approach together with experimental investigations. Targets of 6-gingerol were identified from ChEMBL and STITCH databases, which were used for constructing the protein-protein interaction (PPI) network. Results from the PPI network indicated that p53 was a key regulator. Moreover, it was found that 6-gingerol could increase the levels of p53 mRNA in all leukemic cell lines. Thus, 6-gingerol has shown to have anti-cancer activity. In addition, p53, BAX and BCL2 could be involved in the apoptosis pathway of these leukemic cells. This study is anticipated to be useful for the development of 6-gingerol as an anti-leukemic drug in the future.

Combination of ferric ammonium citrate with cytokines involved in apoptosis and insulin secretion of human pancreatic beta cells related to diabetes in thalassemia.

BACKGROUND: Diabetes mellitus (DM) is a common complication found in ß-thalassemia patients. The mechanism of DM in ß-thalassemia patients is still unclear, but it could be from an iron overload and increase of some cytokines, such as interleukin1-ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The objective of this study was to study the effect of interaction between ferric ammonium citrate (FAC) and cytokines, IL-1ß and TNF-α, on 1.1B4 human pancreatic ß-cell line. METHODS: The effect of the combination of FAC and cytokines on cell viability was studied by MTT assay. Insulin secretion was assessed by the enzyme-linked immunosorbent assay (ELISA). The reactive oxygen species (ROS) and cell apoptosis in normal and high glucose condition were determined by flow cytometer. In addition, gene expression of apoptosis, antioxidant; glutathione peroxidase 1 (GPX1) and superoxide dismutase 2 (SOD2), and insulin secretory function were studied by real-time polymerase chain reaction (Real-time PCR). RESULTS: The findings revealed that FAC exposure resulted in the decrease of cell viability and insulin-release, and the induction of ROS and apoptosis in pancreatic cells. Interestingly, a combination of FAC and cytokines had an additive effect on SOD2 antioxidants' genes expression and endoplasmic reticulum (ER) stress. In addition, it reduced the insulin secretion genes expression; insulin (INS), glucose kinase (GCK), protein convertase 1 (PSCK1), and protein convertase 2 (PSCK2). Moreover, the highest ROS and the lowest insulin secretion were found in FAC combined with IL-1ß and TNF-α in the high-glucose condition of human pancreatic beta cell, which could be involved in the mechanism of DM development in ß-thalassemia patients.

Diallyl disulfide induces apoptosis and autophagy via mTOR pathway in myeloid leukemic cell line.

Leukemia is a hematological malignancy which is produced by uncontrolled proliferation of leukocyte precursors. Currently, alternative medicines, using herb extracts, have been developed for cancer treatment. In this study, the effect of diallyl disulfide (DADS) on the induction of apoptosis and autophagy was investigated in K562 and NB4 myeloid leukemia cells. Leukemia cells were treated with various concentrations of DADS for 24 and 48 h. The percentage of cell viability was measured using an MTT assay. The percentages of apoptosis and autophagy were analyzed by staining with annexin-FITC and anti-LC3 FITC-conjugated antibodies, respectively. Then, the stained cells were detected by flow cytometry. In addition, PP242, a mammalian target rapamycin (mTOR) inhibitor, was used to study the involvement of the mTOR pathway in DADS-induced apoptosis and autophagy. mTOR mRNA expression was measured by real-time PCR. The results showed that DADS decreased cell viability and increased the percentage of cell apoptosis in a dose- and time-dependent manner. mTOR expression was significantly decreased in DADS- and mTOR inhibitor-treated cells. The highest percentages of apoptosis and autophagy were shown in cells treated with 100 µg/ml DADS combined with 10 µM of the mTOR inhibitor. According to our results, DADS could induce apoptosis and autophagy via the mTOR pathway in both K562 and NB4 myeloid leukemia cell lines.

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.

p53 and nitric oxide are involved in cytokine-induced apoptosis in Kasumi-1 and Molt-4 Leukemics cells.

BACKGROUND: Immunotherapy has been developed to treat cancers. There are many signaling pathways involved in cytokine induced apoptosis of many cancers but their role remains unclear in some cancers such as leukemia. OBJECTIVE: To investigate the involvement of the nitric oxide (NO) and p53 tumor suppressor gene in apoptotic pathways induced by cytokines in leukemic cell lines. METHODS: Leukemic cell lines, Kasumi-1 (AML-M2) and Molt- 4 (ALL) were treated with cytokines, interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ). The effect of cytokines on the induction cell apoptosis was analysed by flow cytometry. In addition, nitric oxide production and p53 protein levels were measured by using the Griess method and Western blot, respectively. RESULTS: Upon cytokine treatment, there was a significant increase in the percentage of cell apoptosis in both leukemic cell lines. The highest apoptosis was shown in 40 U/ml IFN-γ treated cells. In addition, nitric oxide and p53 protein increased in IFN-γ treated cells. There was a reduction of apoptosis and p53 level after adding the inducible nitric oxide synthase inhibitor, SMT. CONCLUSION: p53 and nitric oxide are involved in the mediation of apoptosis induced by cytokines in Kasumi-1 and Molt-4 leukemic cell lines.

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.

Nitric oxide and caspase 3 mediated cytokine induced apoptosis in acute leukemia.

BACKGROUND: Leukemia is characterized by the uncontrolled accumulation of white blood cells. Recently, cytokines have been used in immunotherapy, which is a new strategy for leukemia treatment. OBJECTIVE: To investigate the effect of cytokines on induction of apoptosis in acute leukemic cell lines; HL-60, MV4-11, K-562 and Molt-4 and in addition, to study the involvement of nitric oxide (NO) in apoptotic pathways. METHODS: Leukemic cell lines were incubated with cytokines; interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma in various concentrations and for variable periods of time. The percent apoptosis and caspase 3 activation were examined by flow cytometry. Moreover, NO production and inducible nitric oxide synthase (iNOS) mRNA were measured by using Griess method and Real-time PCR, respectively. RESULTS: Cytokines caused a time and dose-dependent induction of apoptosis in leukemic cell lines. The highest cell apoptosis was found in K-562 treated with 40 U/ml interferon-gamma for 48 hours; this correlated with the result of cell growth inhibition and caspase 3 activation. NO and iNOS mRNA were increased in cytokines treated cells. Moreover, apoptosis was reduced by SMT, an iNOS inhibitor, which confirms the possible involvement of NO in the apoptotic pathway. CONCLUSION: Cytokines especially interferon-y induced apoptosis in acute leukemia via NO and caspase 3 pathway.