Triptolide cooperates with chemotherapy to induce apoptosis in acute myeloid leukemia cells

OBJECTIVE: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. MATERIALS AND METHODS: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. RESULTS: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. CONCLUSION: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.     

Decitabine activates specific caspases downstream of p73 in myeloid leukemia

The demethylating effect of 5-aza-2' deoxycytidine (decitabine, DAC) has been well characterized. The molecular events downstream of methylation inhibition are less well known. Here, DAC was shown to induce apoptosis in acute myeloid leukemia (AML) cells (p53 mutant and wild type) but not in epithelial or normal peripheral blood mononuclear cells. Apoptosis was characterized by activation of the mitochondrial but not the receptor death pathway, as demonstrated by the release of cytochrome c and loss of mitochondrial membrane potential. Western blotting and enzyme assays showed that caspase-3, but not caspase-6 or caspase-8, were activated. Decitabine induced expression of the cell cycle inhibitor p21, arresting AML cell lines in G1 of the cell cycle. Expression of p21 was induced irrespective of the methylation status of its promoter, mediated instead via reexpression of the tumor suppressor p73, an upstream regulator of p21. The promoter of p73 was hypermethylated in AML cell lines in vitro and in primary AML cells ex vivo but not in DAC-resistant epithelial cells. In conclusion, DAC acts on leukemic myeloid cells via caspase activation, which may be dependent on demethylation of the hypermethylated p73 promoter and consequent reexpression of p73.     

Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.     

端粒酶基因反义核酸促进顺铂诱导白血病细胞凋亡

目的探讨人类端粒酶逆转录酶(hTERT)基因反义寡核苷酸(ASODN)对顺铂诱导原代培养的急性髓细胞白血病(AML-M2)、慢性髓细胞白血病(CML)细胞凋亡的影响.方法采用锥虫蓝拒染法观察hTERTASODN与顺铂联合对AML、CML细胞生存的影响.琼脂糖凝胶电泳分析细胞凋亡的DNA断裂;通过流式细胞仪对细胞凋亡峰进行定量分析.结果hTERT基因ASODN作用于AML、CML细胞24h再加入顾铂对AML、CML细胞的生存均具有很明显的抑制作用,分别同正义寡核苷酸(SODN)联合顺铂组、单用顺铂组进行比较,差异有显著性(P＜0.01).hTERTASODN作用于AML、CML细胞24h再加入顺铂,经琼脂糖凝胶电泳即可见到DNA梯形条带,并且凋亡细胞百分率(42.68%,35.72%)分别与SODN联合顺铂组(29.02%,23.84%)、单用顺铂组(27.53%,21.02%)进行比较,差异有显著性(P＜0.01).结论hTERT基因反义寡核苷酸可促进顺铂诱导AML、CML细胞凋亡.     

Deregulation of apoptosis in acute myeloid leukemia

Apoptosis, or programmed cell death, is central to the development and homeostasis of the hematopoietic system. Dysregulation of apoptosis plays an important role in the development of a variety of human pathologies, including cancer, autoimmune diseases and neurodegenerative disorders. Particularly, studies carried out in the last years have shown that leukemia cells invariably have abnormalities in one or more apoptotic pathways, determining a survival advantage of these cells over their normal counterpart. Furthermore, abnormalities in the apoptotic response also play a role in the development of drug resistance by leukemic cells. The identification of the different components of the apoptotic pathways has enabled the detection of various biochemical defects present in leukemic cells compared to their normal counterpart. These defects contribute to the survival advantage of the leukemic clone over the normal hematopoietic cells and are also frequently associated with a low rate of response to standard chemotherapy treatment and with poor survival. Furthermore, these findings have also lead to the identification of many potential apoptotic targets for the development of new drugs targeting anti-apoptotic molecules abnormally expressed or regulated in leukemic cells. Many of these drugs restore the sensitivity of leukemic cells to apoptotic stimuli and some of them are under investigation at a clinical level.     

Butyrate-stable monosaccharide derivatives induce maturation and apoptosis in human acute myeloid leukaemia cells

The rapid degradation and subsequent lack of efficacy of n-butyric acid in vivo has been improved by the synthesis of monosaccharide stable pro-drugs of butyric acid. We studied the effects of D1 (O-n-butanoyl-2,3-O-isopropylidene-alpha- D -mannofuranoside), G1 (1-O-n-butanoyl- D , L -xylitol), and F1 (1-O-n-butanoyl 2,3-O-isopropylidene- D , L -xylitol) on the maturation and proliferation of AML cell lines HL 60 and FLG 29.1 and of purified blast cells from 10 cases of de novo acute myeloid leukaemia (AML). AML cell maturation was measured by surface antigen expression, morphology and cytochemistry. Toxicology in mice was also evaluated (DL50 1000 mg/kg). In HL 60 cells G1 and D1 increased the expression of CD15 and CD11a (presenting 62% of promyelo-metamyelocytes), and in 7/10 cases of primary AMLs that of CD11a, CD11b, CD15, and myeloperoxidase. D1, G1 and F1 induced a dose-dependent inhibition of tritiated thymidine uptake. Apoptosis (evaluated by flow cytometry and agarose gel electrophoresis) was induced in AML blasts by D1 and F1 (79% and 94% respectively for HL 60 cells) and, with less effect, by G1 (27%). The persistence of maturative and apoptotic activity in these new pro-drugs of butyric acid, hydrolysed only inside the tumour cell, suggests a possible use in differentiation therapy of myelodysplastic syndromes and AMLs.     

FLT3 inhibitors in acute myeloid leukemia

The fms-like tyrosine kinase 3 (FLT3) plays an important role in both normal and malignant hematopoiesis. Activating mutations in the FLT3 receptor can be detected in approximately 30% of acute myeloid leukemias (AMLs) and are associated with a distinctly poor clinical outcome for patients. There are now several classes of FLT3 inhibitors in development with varying degrees of potency and selectivity for the target, including several in late-phase clinical trials in combination with chemotherapy. Major clinical responses in AML patients receiving single-agent FLT3 inhibitors have been rare, although transient peripheral blood blast reduction is common. Given such biological suggestion and preclinical activity, FLT3 inhibitors hold promise in improving the outcome of patients with mutant FLT3 AML. This review summarizes the current attempts to target this molecule, with emphasis on the validity of the target, the results of the clinical trials evaluating the FLT3 inhibitors in AML, the optimal use of these compounds and the mechanisms of resistance.     

Immune checkpoint inhibitors in acute myeloid leukemia

Understanding the immune biology of AML and designing rational approaches to target or harness the immune environment to improve outcomes is an area of intense research in AML. There are two primary immune checkpoint harnessing modalities under clinical evaluation in AML: T-cell (such as PD1 inhibitors nivolumab and pembrolizumab) and macrophage (such as the anti-CD47 antibody magrolimab) These work synergistically with hypomethylating agents. Patients who do not achieve complete or partial responses based on IWG criteria often achieve durable stable disease or hematologic improvement, which may provide meaningful benefit for patients, even in the absence of traditional response unlike cytotoxic therapies. Patients should ideally be prospectively selected for CPI based therapies based on pre-treatment biomarkers, as there are definite populations that are more likely to respond. Immune toxicities are often mistaken for infection or other adverse event; however, if identified and treated early and aggressively with steroids, immune toxicity outcomes can be improved. Therefore, in the formative stage of development ideally only centers with experience in immune therapies should perform CPI studies in AML.     

Small-molecule XIAP antagonist restores caspase-9 mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells

Clinical outcome in patients with primary nodal diffuse large B-cell lymphomas (DLBCLs) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including X-linked inhibitor of apoptosis protein (XIAP). XIAP suppresses apoptosis through inhibiting active caspase-3, caspase-7, and caspase-9. In this study, we investigated to see if the small-molecule XIAP antagonist 1396-12 induces cell death in cultured lymphoma cells of patients with DLBCL. Treatment with this XIAP antagonist resulted in relief of caspase-3 inhibition and in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy-refractory and -responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal-center B cells from healthy donors. XIAP antagonist-sensitive samples were characterized by high expression levels of XIAP, relatively low expression levels of Bcl-2, and by constitutive caspase-9 activation. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in cultured DLBCL cells and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers, suggesting the possibility of predefining patients most likely to benefit from XIAP antagonist therapy.     

shRNA-Mediated BAALC knockdown affects proliferation and apoptosis in human acute myeloid leukemia cells

Brain and acute leukemia, cytoplasmic (BAALC) is a novel molecular marker indicating an inferior outcome in acute myeloid leukemia (AML) with normal cytogenetics. The biological function of BAALC is largely unknown. In this study, BAALC gene expression in an acute myeloid leukemia cell line KG1a was knocked down by a small hairpin RNA (shRNA). The expression of BAALC mRNA and protein in the knockdown cells was significantly inhibited. The proliferation and apoptosis status in the knockdown cells were investigated. The growth curves and FACS analysis demonstrated that BAALC gene knockdown resulted in decreased proliferation and enhanced apoptosis in KG1a cells. These results indicated that BAALC may act as an adverse prognostic factor through prompting proliferation and inhibiting apoptosis in leukemia cells.     

普萘洛尔对急性髓系细胞白血病U937细胞增殖和凋亡的影响

目的:探究普萘洛尔对急性髓系细胞白血病U937细胞的增殖和凋亡的影响及可能的机制。方法:采用CCK8法检测不同药物对U937细胞增殖率的影响,分别为普萘洛尔(0、200、400、800、1000、2 000μmol/L)作用24、48、72 h,阿糖胞苷单用(0、10、50、100、200、400、800 nmol/L)24 h,普萘洛尔(400μmol/L)联合阿糖胞苷(10、50、100、200、400、800 nmol/L)作用24 h。普萘洛尔(0、200、400、800μmol/L)作用U937细胞24 h后,用流式细胞术检测细胞的凋亡和周期、线粒体膜电位的变化,用碱性彗星实验检测DNA的损伤,Caspase-Glo试剂盒检测Caspase-3、8、9相对活性的变化,Western blot法检测Bcl-2、Bax蛋白表达量的变化。结果:普萘洛尔可以有效抑制U937细胞的增殖,其效应具有一定的时间(200、400μmol/L,P0.05)和剂量相关性(0、200、400、800μmol/L,P0.01),普萘洛尔还可协同阿糖胞苷的抗肿瘤效应。流式、彗星、Caspase、Western blot结果分别表明普萘洛尔可增加细胞凋亡率,增加G_0/G_1期和减少G_2/M期细胞比例,降低线粒体膜电位水平,诱导DNA损伤,增强Caspase3、8、9活性,上调Bax的表达、下调Bcl-2的表达,且在实验浓度范围内与剂量呈相关性(P0.05)。结论:普萘洛尔可抑制急性髓系细胞白血病U937的增殖并诱导其凋亡,这一机制可能与其诱导G_0/G_1期细胞阻滞、损伤DNA,激活内、外源性凋亡途径有关。     

Chronic myeloid leukemia lysate-loaded dendritic cells induce T-cell responses towards leukemia progenitor cells

Treatment of chronic myeloid leukemia with tyrosine kinase inhibitors, such as imatinib mesylate, dasatinib and nilotinib, results in high rates of cytogenetic and molecular responses. However, in many patients, minimal residual disease is detected by molecular techniques. Since chronic myeloid leukemia cells are particularly good targets for immune surveillance mechanisms, we explored active specific immunotherapy using leukemia lysate-loaded dendritic cells in vitro. Our data show the potency of dendritic cell-based vaccination strategies for the induction of T cell-mediated responses to eradicate minimal residual disease.     

LncRNA-H19对急性髓系白血病细胞增殖和凋亡的影响

目的探索长链非编码RNA-H19对急性髓系白血病细胞增殖和细胞凋亡的调控作用。方法采用RAN干扰技术对急性髓系白血病细胞系HL-60、THP-1和U937的LncRNA-H19进行表达沉默,用实时荧光定量PCR(qPCR)检测LncRNA-H19在细胞中的表达情况。用MTT检测细胞增殖情况,用流式细胞术检测细胞凋亡情况。结果三种白血病细胞系分别被3条si-RNA干扰后,LncRNA-H19表达均明显下调。MTT结果显示,RNA干扰后,LncRNA-H19干扰细胞组的细胞增殖明显低于阴性对照组,而流式细胞术显示,siH19干扰细胞组的细胞凋亡明显高于阴性对照组,差异有统计学意义(P0.05)。结论急性髓系白血病细胞中,LncRNA-H19具有调控急性髓系白血病细胞增殖和细胞凋亡的功能。     

Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes

Background

Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors.

Design and Methods

Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5’ aza-2’-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents.

Results

PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5’ aza-2’-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes.

Conclusions

On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.     

Sildenafil and vardenafil,types 5 and 6 phosphodiesterase inhibitors,induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells

Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3'5' cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-alpha (SDF-1alpha). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.