Apoptosis In Human Leukemia Cells Research Articles

Reprogramming of RNA m6A Modification Is Required for Acute Myeloid Leukemia Development.

Hematopoietic homeostasis is maintained by hematopoietic stem cells (HSCs), and it is tightly controlled at multiple levels to sustain the self-renewal capacity and differentiation potential of HSCs. Dysregulation of self-renewal and differentiation of HSCs leads to the development of hematologic diseases, including acute myeloid leukemia (AML). Thus, understanding the underlying mechanisms of HSC maintenance and the development of hematologic malignancies is one of the fundamental scientific endeavors in stem cell biology. N 6-methyladenosine (m6A) is a common modification in mammalian messenger RNAs (mRNAs) and plays important roles in various biological processes. In this study, we performed a comparative analysis of the dynamics of the RNA m6A methylome of hematopoietic stem and progenitor cells (HSPCs) and leukemia-initiating cells (LICs) in AML. We found that RNA m6A modification regulates the transformation of long-term HSCs into short-term HSCs and determines the lineage commitment of HSCs. Interestingly, m6A modification leads to reprogramming that promotes cellular transformation during AML development, and LIC-specific m6A targets are recognized by different m6A readers. Moreover, the very long chain fatty acid transporter ATP-binding cassette subfamily D member 2 (ABCD2) is a key factor that promotes AML development, and deletion of ABCD2 damages clonogenic ability, inhibits proliferation, and promotes apoptosis of human leukemia cells. This study provides a comprehensive understanding of the role of m6A in regulating cell state transition in normal hematopoiesis and leukemogenesis, and identifies ABCD2 as a key factor in AML development.

Discovery of novel selective phosphodiesterase‑1 inhibitors for the treatment of acute myelogenous leukemia

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. PDE1 (Phosphodiesterase 1) is a subfamily of the PDE super-enzyme families that can hydrolyze the second messengers cAMP and cGMP simultaneously. Previous research has shown that suppressing the gene expression of PDE1 can trigger apoptosis of human leukemia cells. However, no selective PDE1 inhibitors have been used to explore whether PDE1 is a potential target for treating AML. Based on our previously reported PDE9/PDE1 dual inhibitor 11a, a series of novel pyrazolopyrimidinone derivatives were designed in this study. The lead compound 6c showed an IC50 of 7.5 nM against PDE1, excellent selectivity over other PDEs and good metabolic stability. In AML cells, compound 6c significantly inhibited the proliferation and induced apoptosis. Further experiments indicated that the apoptosis induced by 6c was through a mitochondria-dependent pathway by decreasing the ratio of Bcl-2/Bax and increasing the cleavage of caspase-3, 7, 9, and PARP. All these results suggested that PDE1 might be a novel target for AML.

Ligustrum vulgare leaves and fruit extracts induce apoptosis of human leukemia cells

Background and purpose: Cytotoxic effects of Ligustrum vulgare leaves on HeLa cervical tumor cells suggested that Ligustrum vulgare extracts should be investigated as potential anticancer agents. Therefore, we examined a potential antileukemic activity of methanolic extracts of Ligustrum vulgare leaves and fruit extracts on two types of leukemia cells, MOLT-4 and JVM-13, lymphocytes isolated from the blood of 33 chronic lymphocytic leukemia (CLL) patients and on mononuclear leukocytes isolated from the blood of 18 healthy individuals.Material and methods: The cytotoxicity of examined extracts was measured by MTT assay and LDH activity test. The antiapoptotic potential of tested extracts was measured by Annexin V/7AAD flowcytometric assay.Results: The results showed that both extracts exhibited a moderate cytotoxic effect on all three types of leukemia cells. The Ligustrum vulgare leaf extract was the most effective on MOLT-4 cells, the fruit extract on JVM-13 cells and both extracts were equally effective on CLL cells. In addition, none of the tested extracts was toxic to healthy mononuclear cells. Both extracts acted by inducing apoptosis of leukemic cells.Conclusion: Ligustrum vulgare extracts exhibit significant antileukemic potential and should be further investigated.

Effect of fresh soy milk and its compounds on apoptosis in human leukemic cells and peripheral blood mononuclear cells


 
 
 
 Purpose: To determine the effect of fresh soy milk and its compounds (coumestrol, daidzein and genistein) on apoptosis in human leukemic cells and peripheral blood mononuclear cells (PBMC).
 Methods: The apoptotic effect of fresh soy milk and its compounds on human leukemic cells (K562, Jurkat, U937) and PBMC was determined by flow cytometry. The PBMC from healthy donors were isolated by conducting density gradient centrifugation principle. Lymphoprep and cytotoxicity of the compounds was evaluated by 3-(4,5- ‘;1dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) MTT) assay.
 Results: PBMC treatment of daidzein and genistein had a significantly higher half-maximal inhibitory concentration (IC50) value (p < 0.01) and (p < 0.0001), compared to the leukemia cells. In addition, soy milk had a significantly higher IC50 value (p < 0.05) in PBMC than the leukemia cells. Coumestrol induced a significantly increased apoptotic cells in K562, U937 cells (p < 0.01) and Jurkat cells (p < 0.05). However, daidzein only increased apoptosis (p < 0.05) in K562 and U937. Genistein reported a significantly increased apoptotic cell in K562, Jurkat (p < 0.01) and U937 cells (p < 0.001). Nonetheless, in PBMC, significant apoptosis by the compounds did not occur. However, only soy milk induced a significantly increased apoptosis in PBMC (p < 0.05).
 Conclusion: The findings indicate that coumestrol, daidzein, genistein and soy milk inhibits the viability of leukemia cells by apoptosis induction, but they are non-toxic toward normal cells. Therefore, the results suggest that the compound alone, and thus, soy milk might have a therapeutic effect in leukemia.
 
 
 

Relative effect of Malayer Shahani and Asgari grapes seed extract on inducing apoptosis in human leukemia cells.

Previous studies have suggested that consuming fruit and vegetable can lower the risk of several cancers, including breast, colorectal, and lung cancers. The present study aims to investigate the in vitro anticancer effects of Shahani and Asgari grape seed extract (GSE) grown in Malayer City of Iran on HL-60 cancer. However, to the best of the author's knowledge, it is the first time in this study that the antiproliferative effect of Shahani and Asgari GSE is compared. Shahani and Asgari GSE Was extraction white method of Liquid/liquid extraction with ethyl acetate. Then assessing cytotoxic activities of Shahani and Asgari GSE on the HL-60 cells was tested using MTT assay. The results show that compared with the control group, seed extract of both Shahani and Asgari at the various concentrations (25, 50, 100, and 200 μg/ml) had a significantly inhibitory effect on HL-60 cell proliferation that was dose dependent. However, Shahani GSE at different concentrations (50, 100, and 200 μg/ml) indicated a significantly higher inhibitory effect compared to Asgari GSE. In addition, GSE can induce cell cycle arrest at G0/G1 cells. Furthermore, GSE of Asgari and Shahani remarkably increased the induction of HL-60 cell apoptosis depending on its dose. However, at the concentration of 200 μg/ml, GSE induced cell necrosis rather than apoptosis. Seed extract of both Shahani and Asgari at the various concentrations had a significantly inhibitory effect on HL-60 cell proliferation that was dose dependent.

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells.

Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL-60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas-induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase-3-like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V-positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide-induced apoptotic U937 cells with either cystatin C or D resulted in a dose-dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1-9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL-60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

D-Pinitol treatment induced the apoptosis in human leukemia MOLT-4 cells by improved apoptotic signaling pathway

Cancer is still remain as a global burden with the 18.1 million and 9.6 million new cases and mortlities, respectively estimated globally. Leukemia may arise at all ages varied from the infants to elders. In this exploration, we planned to evaluate the antiproliferative effect of D-pinitol on human leukemia MOLT-4 cells. Anticancer potential of D-pinitol was examined using MTT assay. Reactive oxygen species (ROS) generation was studied by fluorescence microscopic method using DCFH-DA staining. Apoptotic morphological alterations were determined by dual staining (acridine orange and ethidium bromide). Western blot and ELISA methods were employed to study apoptotic protein expression. D-pinitol treatment significantly induced cytotoxicity in human leukemia MOLT-4 cells. We observed that D-pinitol induces the generation of ROS in MOLT-4 cells. Further, we noticed that D-pinitol significantly induced apoptosis in a dosage dependent manner. Moreover, western blot and ELISA based analysis revealed that D-pinitol elevated the Bax, Caspase-3, Caspase-9 and attenuated the Bcl-2 expression in leukemic cancer cell. Our findings suggest that D-pinitol treatment induces the apoptosis in human leukemic cells by generating intracellular ROS and modulating apoptotic protein expression.

Melittin-A Natural Peptide from Bee Venom Which Induces Apoptosis in Human Leukaemia Cells.

Bee venom is a very complex mixture produced and secreted by the honeybee (Apis mellifera). Melittin is a major component of bee venom that accounts for about 52% of its dry mass. A vast number of studies have been dedicated to the effects of melittin’s regulation of apoptosis and to the factors that induce apoptosis in various types of cancer such as breast, ovarian, prostate, lung. The latest evidence indicates its potential as a therapeutic agent in the treatment of leukaemia. The aim of our present study is to evaluate melittin’s ability to induce apoptosis in leukaemia cell lines of different origin acute lymphoblastic leukaemia (CCRF-CEM) and chronic myelogenous leukaemia (K-562). We demonstrated that melittin strongly reduced cell viability in both leukaemia cell lines but not in physiological peripheral blood mononuclear cells (PMBCs). Subsequent estimated parameters (mitochondrial membrane potential, Annexin V binding and Caspases 3/7 activity) clearly demonstrated that melittin induced apoptosis in leukaemia cells. This is a very important step for research into the development of new potential anti-leukaemia as well as anticancer therapies. Further analyses on the molecular level have been also planned (analysis of proapoptotic genes expression and DNA damages) for our next research project, which will also focus on melittin.

Dasmahapatra G, Rahmani M, Dent P, Grant S. The tyrphostin adaphostin interacts synergistically with proteasome inhibitors to induce apoptosis in human leukemia cells through a reactive oxygen species (ROS)–dependent mechanism. Blood. 2006;107(1):232-240.

Dasmahapatra G, Rahmani M, Dent P, Grant S. The tyrphostin adaphostin interacts synergistically with proteasome inhibitors to induce apoptosis in human leukemia cells through a reactive oxygen species (ROS)–dependent mechanism. Blood. 2006;107(1):232-240.

The Effect of Furanocoumarin Derivatives on Induction of Apoptosis and Multidrug Resistance in Human Leukemic Cells.

Background: The insensitivity of cancer cells to therapeutic agents is considered to be the main cause of failure of therapy and mortality of patients with cancer. A particularly important problem in these patients is the phenomenon of multidrug resistance, consisting of abnormal, elevated expression of transport proteins (ABC family). The aim of this research included determination of IC50 values of selected furanocoumarins in the presence and absence of mitoxantrone in leukemia cells and analysis of changes in apoptosis using anexinV/IP and Casp3/IP after 24 h exposure of cell lines to selected coumarins in the presence and absence of mitoxantrone in IC50 concentrations. Methods: Research was conducted on 3 cell lines derived from the human hematopoietic system: HL-60, HL-60/MX1 and HL-60/MX2. After exposure to coumarin compounds, cells were subjected to cytometric analysis to determine the induction of apoptosis by two methods: the Annexin V test with propidium iodide and the PhiPhiLux-G1D2 reagent containing caspase 3 antibodies. Results: All of the furanocoumarin derivatives studied were found to induce apoptosis in leukemia cell lines. Conclusions: Our results clearly show that the furanocoumarin derivatives are therapeutic substances with antitumor activity inducing apoptosis in human leukemia cells with phenotypes of resistance.

Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism

Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism

The Role of E4BP4 in Apoptosis of Human Leukemia Cells in Culture

Apoptosis is a physiological and genetically controlled process of cell death important for development and cellular homeostasis. Several diseases can be attributed to an imbalance between cellular proliferation and attrition, brought on by inadequate or excessive apoptosis. In cancer, a lack of apoptosis causes excessive accumulation of unwanted cells. Apoptosis is primarily regulated via altered expression of key genes and modulation of signal transduction pathways. Our laboratory focuses on understanding the early apoptosis events triggered by chemotherapeutic agents in cell culture models of leukemia. Previous studies have shown that the E4BP4 (adenovirus E4 binding protein 4) gene in upregulated in correlation with apoptosis. E4BP4 upregulation has been implicated in induction of the pro‐apoptotic BIM protein, a member of the Bcl‐2 family of proteins. In studies presented here, we have utilized CRISPR‐mediated genomic editing technology to knock down E4BP4 expression. Three clones with reduced E4BP4 expression were analyzed for their response to Dexamethasone (Dex), a synthetic glucocorticoid and anti‐leukemic agent. While E4BP4 knockdown did not alter the ability of cells to undergo apoptosis in response to Dex, two of the three clones showed a reduction in the extent of BIM upregulation. Residual E4BP4 expression in our knock down clones may be sufficient to evoke an apoptotic response. Further efforts are underway to understand downstream pathways regulated by E4BP4.Support or Funding InformationThis research is funded by a NIH SCORE SC3 award to RDM (GM081099) and a MARC‐U* STAR scholarship to Erick Mitchell‐Velasquez.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Critical role of H2O2 in mediating sanguinarine-induced apoptosis in prostate cancer cells via facilitating ceramide generation, ERK1/2 phosphorylation, and Par-4 cleavage

Natural products are a major source of potential anticancer agents, and in order to develop improved and more effective cancer treatments, there is an immense need in exploring and elucidating their mechanism of action. Sanguinarine (SNG), a quaternary benzophenanthridine alkaloid, has been shown to induce cytotoxicity in various human cancers and suppresses various pro-tumorigenic processes such as invasion, angiogenesis, and metastasis in different cancers. Lack of understanding the anticancer mechanism(s) of SNG has impeded the development of this molecule as a potential anticancer agent. Earlier, we have reported that SNG induces reactive oxygen species (ROS)-dependent ceramide (Cer) generation and Akt dephosphorylation, leading to the induction of apoptosis in human leukemic cells. In the present study, we demonstrate that SNG has potent anti-proliferative activity against prostate cancer cells. Our data suggest that SNG induces Cer generation via inhibiting acid ceramidase and glucosylceramide synthase, two important enzymes involved in Cer metabolism. Furthermore, we demonstrate that SNG induces ROS-depended extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation, and prostate apoptosis response-4 (Par-4) cleavage, leading to the induction of apoptosis in human prostate cancer cells. Overall, our findings provide molecular insight into the role of ROS signaling in the anticancer mechanism(s) of SNG. This may provide the basis for its use as a nontoxic and an effective therapeutic agent in the treatment of prostate cancer.

Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations.

Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5–10.8 μM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.

Holotoxin A₁ Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells.

Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus) japonicus. We previously showed that cladoloside C2, the 25(26)-dihydro derivative of holotoxin A1, induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1-induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1 induced apoptosis, acid sphingomyelinase (SMase) and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1–induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

Effects of endoplasmic reticulum stress on autophagy and apoptosis of human leukemia cells via inhibition of the PI3K/AKT/mTOR signaling pathway.

The present study aimed to explore the regulatory effects of endoplasmic reticulum stress (ERS) on the phosphoinositide 3‑kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, and its subsequent effects on autophagy and apoptosis of human leukemia cells. Human leukemia cells were cultured and treated with various concentrations of tunicamycin for 0, 24, 48, 72 and 90h. Subsequently, human leukemia cells were assigned into the ER activation group, which was treated with 100ng/ml tunicamycin, the ER activation + TO901317 (PI3K inhibitor) group, and the control group. An MTT assay was conducted to detect cell proliferation. In addition, a monodansylcadaverine (MDC) assay was used to detect the formation of autophagosomes and Annexin V‑fluorescein isothiocyanate/propidium iodide double staining was used to examine cell apoptosis. Western blotting was performed to detect the expression levels of 78‑kDa glucose‑regulated protein (GRP78), phosphorylated (p)‑protein kinase R‑like endoplasmic reticulum kinase (PERK), p‑α subunit of eukaryotic initiation factor 2 (eIF2α), microtubule‑associated protein 1A/1B‑light chain 3 (LC3), caspase‑3, CCAAT‑enhancer‑binding protein homologous protein (CHOP), PI3K, AKT and mTOR. Treatment with 100ng/ml tunicamycin for 72h was considered the optimal condition for further experiments. Compared with in cells prior to treatment, human leukemia cells treated with tunicamycin exhibited increased expression of p‑PERK, p‑eIF2α and GRP78 after 72h (P<0.05). In addition, the expression levels of mTOR, AKT and PI3K were decreased in the ER activation group compared with in the control and ER activation + TO901317 groups (P<0.05). Compared with in the control group, cell proliferation was inhibited and MDC fluorescence intensity was increased in the ER activation group (P<0.05). Furthermore, compared with in the control and ER activation + TO901317 groups, western blotting indicated that the expression levels of LC3‑II were increased in the ER activation group (P<0.05). The apoptotic rate was also higher in the ER activation group compared with in the control group (P<0.05), and caspase‑3 and CHOP expression was elevated in the ER activation group (P<0.05). These findings indicated that ERS may induce autophagy and apoptosis of human leukemia cells via inhibiting the PI3K/AKT/mTOR signaling pathway.

Inactivation of PBX3 and HOXA9 by down-regulating H3K79 methylation represses NPM1-mutated leukemic cell survival.

Acute myeloid leukemia (AML) with an NPM1 mutation (NPMc+) has a distinct gene expression signature and displays molecular abnormalities similar to mixed lineage leukemia (MLL), including aberrant expression of the PBX3 and HOXA gene cluster. However, it is unclear if the aberrant expression of PBX3 and HOXA is essential for the survival of NPM1-mutated leukemic cells.Methods: Using the gene expression profiling of TCGA and E-MTAB-3444 datasets, we screened for high co-expression of PBX3 and HOXA9 in NPMc+ leukemia patients. We performed NPMc+ depletion and overexpression experiments to examine aberrant H3K79 methylation through epigenetic regulation. Through RNA interference technology and small-molecule inhibitor treatment, we evaluated the effect of methyl-modified H3K79 on cell survival and explored the possible underlying mechanism.Results: We showed that NPMc+ increased the expression of PBX3 and HOXA9, which are both poor prognosis indicators in AML. High PBX3 and HOXA9 expression was accompanied by increased dimethylated and trimethylated H3K79 in transgenic murine Lin-Sca-1+c-Kit+ cells and human NPMc+ leukemia cells. Using chromatin immunoprecipitation sequencing (ChIP-seq) assays of NPMc+ cells, we determined that hypermethylated H3K79 was present at the expressed HOXA9 gene but not the PBX3 gene. PBX3 expression was positively regulated by HOXA9, and a reduction in either PBX3 or HOXA9 resulted in NPMc+ cell apoptosis. Importantly, an inhibitor of DOT1L, EPZ5676, effectively and selectively promoted NPMc+ human leukemic cell apoptosis by reducing HOXA9 and PBX3 expression.Conclusion: Our data indicate that NPMc+ leukemic cell survival requires upregulation of PBX3 and HOXA9, and this action can be largely attenuated by a DOT1L inhibitor.

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aimsImatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM. MethodshUC-MSC-Exo were isolated and identified. K562 cells were then treated or not with IM (1 µmol/L) in combination with hUC-MSC-Exo (50 µg/mL). Cell viability and apoptosis were determined by cell counting kit 8 (CCK-8) and annexin V/propidium iodide (PI) double staining, respectively. Apoptotic proteins, caspase and their cleaved forms were detected by Western blot. ResultsIt was shown that hUC-MSC-Exo alone had no effect on cell viability and apoptosis of K562 cells. However, hUC-MSC-Exo promoted IM-induced cell viability inhibition and apoptosis. Moreover, hUC-MSC-Exo enhanced the increased Bax expression and the decreased Bcl-2 expression that were induced by IM. Compared with IM alone, caspase-9 and caspase-3 were further activated by combination of hUC-MSC-Exo with IM. Finally, the effects of hUC-MSC-Exo on K562 cells could be reversed by pretreatment of K562 cells with caspase inhibitor Z-VAD-FMK (30 µmol/L) DiscussionThese results indicate that hUC-MSC-Exo enhanced the sensitivity of K562 cells to IM via activation of caspase signaling pathway. Therefore, combining IM with hUC-MSC-Exo could be a promising approach to improve the efficacy of CML treatment.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model.Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.