Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 μg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2α and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells. Jin and Song contributed equally to this article.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells

Chrysin is a natural, biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammation, anti-cancer, and anti-oxidation properties. The mechanism by which chrysin initiates apoptosis remains poorly understood. In the present report, we investigated the effect of chrysin on the apoptotic pathway in U937 human promonocytic cells. We show that chrysin induces apoptosis in association with the activation of caspase 3 and that Akt signal pathway plays a crucial role in chrysin-induced apoptosis in U937 cells. Furthermore, we have shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002 significantly, enhanced apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3, and PLC-gamma1 cleavage by chrysin. Together, these findings suggest that the Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to chrysin and raise the possibility that combined interruption of chrysin and PI3K/Akt-related pathways may represent a novel therapeutic strategy in hematological malignancies.     

GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.     

Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells

Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia‐inducible factors (HIFs) activity, which are key effectors of hypoxia‐mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down‐regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF‐independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.     

L-Ascorbic acid induces apoptosis in acute myeloid leukemia cells via hydrogen peroxide-mediated mechanisms

L-Ascorbic acid (LAA) is being investigated clinically for the treatment of patients with acute myeloid leukemia (AML) based on the observed effects of LAA on AML progenitor cells in vitro. However, the mechanism for LAA-induced cytoreduction remains to be elucidated. LAA at concentrations of 0.25-1.0 mM induced a dose- and time-dependent inhibition of proliferation in three AML cell lines and also in leukemic cells from peripheral blood specimens obtained from three patients with AML. In contrast, ovarian cancer cell lines were only minimally affected. Flow cytometric analysis showed that LAA at concentrations of 0.25-1.0 mM could significantly induce apoptosis in the AML cell lines. LAA induced oxidation of glutathione to oxidized form (GSSG) and subsequent H(2)O(2) accumulation in a concentration-dependent manner, in parallel to induction of apoptosis. The direct role of H(2)O(2) in the induction of apoptosis in AML cells was clearly demonstrated by the finding that catalase could completely abrogate LAA-induced apoptosis. Induction of apoptosis in LAA-treated AML cells involved a dose-dependent increase of Bax protein, release of cytochrome C from mitochondria to cytosol, activation of caspase 9 and caspase 3, and cleavage of poly[ADP-ribose]polymerase. In conclusion, LAA can induce apoptosis in AML cells, and this is clearly due to H(2)O(2) which accumulates intracellularly as a result of oxidation of reduced glutathione by LAA.     

Escherichia coli induces apoptosis in human monocytic U937 cells through the Fas/FasL signaling pathway

Apoptosis is a genetically regulated cellular suicide mechanism that plays an essential role in development and in defense of multicellular organism. Escherichia coli (E. coli) can induce monocyte apoptosis; however, the mechanism is not clear. This study determines if Fas/FasL regulates E. coli-induced human monocyte line U937 cell apoptosis. We found that infection of U937 cells with E. coli induced rapid cell death in a dose- and time-dependent manner displaying the characteristic features of apoptosis. Moreover, opsonized E. coli induced U937 apoptosis with a higher apoptotic rate (53.29 ± 5.83%) than non-opsonized E. coli (19.37 ± 2.56%). Studying the underlying mechanisms we found that the E. coli-induced apoptosis was associated with a more prominent induction expression of Fas/FasL in a time- and dose-dependent manner. Furthermore, E. coli treatment resulted in a significant increase in the levels of DR5, TRAIL, and FADD, but exerted no statistically significant effects on the levels of DR4. The activity of caspase-8 enzyme increased in infection groups, positively correlated with apoptosis rate. Taken together, these results clearly indicate that receptor-mediated phagocytosis of E. coli induces apoptosis. Moreover, our findings suggest a possible regulatory role of Fas/FasL in the pathway of E. coli infection.     

Enhancement of esculetin on arsenic trioxide-provoked apoptosis in human leukemia U937 cells

In order to overcome chemotherapy resistance, many laboratories are searching for agents that increase the sensitivity of cancer cells to anticancer drugs. Arsenic trioxide (As₂O₃) is widely used in treating human acute polymyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As₂O₃. Esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of HL-60 cells as well as enhanced taxol-induced apoptosis in HepG2 cells, thereby displaying anticancer potential. In this study, esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. Since inhibitors of MAPKs have modulated the GSH-redox state and enhanced the sensitivity of leukemia cells to As₂O₃-provoked apoptosis, we monitored the effect of combining esculetin and As₂O₃ (2.5 μM) on the GSH level. Our study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As₂O₃-induced GSH depletion. We found that the As₂O₃ (2.5 μM) treatment slightly induced apoptosis and the pretreatment of esculetin enhanced the As₂O₃-provoked apoptosis significantly. In addition, esculetin enhanced the effect of As₂O₃ on caspase activation in U937 cells. We compared the combined esculetin and As₂O₃ treatment to the As₂O₃ treated alone. The combined esculetin and As₂O₃ treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As₂O₃-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As₂O₃-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal-mitochondrial pathway of apoptosis.     

Recognition by TNF-alpha of the GPI-anchor glycan induces apoptosis of U937 cells

Tumor necrosis factor-alpha (TNF-alpha) binds to TNF-alpha receptors (TNFR) to produce a hexameric (TNF-alpha)(3)-(TNFR)(3) structure that stimulates apoptosis. We found by using ELISA that TNF-alpha binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm-Horsfall glycoprotein. These binding abilities were inhibited by 10(-6)M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) beta1 -->phosphate-->6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-alpha to hAP. Thus, TNF-alpha binds to the GlcNAcbeta1-->phosphate-->6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-alpha is related to its physiological functions, human lymphoma U937 cells were used. TNF-alpha stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-alpha-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-alpha stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-alpha)(3)-(TNFR)(3)-(GPI-anchored glycan)(3) complex. The GPI-anchored glycoprotein involved remains to be identified.     

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.Only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis.¹ This extremely poor prognosis is mainly caused by treatment failure due to chemotherapy resistance. This resistance is often a multifactorial phenomenon that can include enhanced expression or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R).^{2, 3} The IGF-1R stimulates proliferation, protects cells from apoptosis and has been implicated in the development and maintenance of various cancers.^{4, 5} Several oncogenes require an intact IGF-1R pathway for their transforming activity⁶ and moreover, disruption or inhibition of IGF-1R activity has been shown to inhibit the growth and motility of a wide range of cancer cells in vitro and in mouse models.^{4, 5} IGF-1Rs are membrane receptors and binding of their ligand, the insulin-like growth factor-1 (IGF-1), results in receptor phosphorylation and activation of MAPK and PI3K/Akt signaling.⁴ Importantly, IGF-1, normally produced by the liver and bone marrow stromal cells, can stimulate the proliferation of cancer cells in vitro and genetic manipulations that reduce IGF-1 signaling can lead to decreased tumor growth.^{7, 8}In hematological malignancies, a role for IGF-1 signaling has been demonstrated in multiple myeloma (MM) where it stimulates growth and potently mediates survival.⁹ Several anti-IGF-1R strategies have been shown to inhibit MM growth.^{10, 11} In AML, expression of the IGF-1R and IGF-1 was detected in AML cell lines and primary AML blasts and stimulation with IGF-1 can promote the growth of AML cells.^{12, 13, 14} In addition, neutralizing IGF-1R antibodies and the tyrosine kinase inhibitors (TKIs) NVP-AEW541 and NVP-ADW742, have been shown to inhibit proliferation and to induce apoptosis.^{15, 16}In addition to its mitogenic and anti-apoptotic roles, directly influencing tumor development, IGF-1R appears to be a critical determinant of response to numerous anti-cancer therapies, including TKIs and chemotherapy.^{2, 3, 17, 18, 19, 20, 21, 22} In AML, activated IGF-1R signaling has been linked to cytarabine resistance, a drug included in every AML treatment schedule.¹⁷ Notably, in several cancer cell lines, a small subpopulation of drug-tolerant cancer cells exists that maintains their viability, after treatment with a lethal drug dose, via engagement of the IGF-1R.¹⁸The activity of the IGF-1R is tightly controlled at multiple levels, including their processing, endocytosis, trafficking and availability of its ligands.⁴ Ligand bioavailability is partly controlled by the family of secreted insulin-like growth factor-binding protein (IGFBP1 to IGFBP6), which can bind to IGFs therewith regulating the interaction of these ligands to their receptors. However, as IGFBPs are able to induce IGF-dependent and IGF-independent effects, the results of several studies on their role in cancer cell survival appeared to be controversial and complex.^{23, 24} In addition to IGFBPs, various IGFBP-related proteins have been identified.^{23, 25} One of these is the IGFB-related protein 1, also known as insulin-like growth factor-binding protein-7 (IGFBP7). IGFBP7 has 30% homology to IGFBP1 to IGFBP6 in its N-terminal domain and functions predominantly as a tumor suppressor.^{23, 24, 25, 26} In contrast to IGFBP1 to IGFBP6, which bind to the IGFs,²³ IGFBP7 is a secreted protein that can directly bind to the IGF-1R and thereby inhibits its activity.²⁷ The abundance of IGFBP7 is inversely correlated with tumor progression in hepatocellular carcinoma.²⁸ Importantly, decreased expression of IGFBP7 has been associated with therapy resistance^{29, 30} and increasing IGFBP7 levels can inhibit melanoma and breast cancer growth.^{31, 32} IGFBP7 was originally identified as being involved in Raf-mediated apoptosis and senescence³³ and also has been shown to induce senescence in mesenchymal stromal cells.³⁴We established that IGFBP7 induces a cell cycle block and apoptosis in AML cells and cooperates with chemotherapy in the induction of leukemia cell death. AML patients with low IGFBP7 expression have a worse outcome than patients with high IGFBP7 expression, indicating that AML patients might benefit from a combination therapy consisting of chemotherapy and IGFBP7. Our results define IGFBP7 as a focus to enhance chemotherapy efficacy and improve AML patient survival.     

Common signaling networks characterize leukemia-initiating cells in acute myeloid leukemia

Identification and characterization of leukemia-initiating cells (LICs) is important to understand leukemogenesis and develop novel therapies for leukemia. In this issue of Cell Stem Cell, Gibbs et?al. (2012) demonstrate that common active signaling pathways in LICs may be targeted to treat acute myeloid leukemia.     

Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells

Arachidonic acid (AA) participates in a reacylation/deacylation cycle of membrane phospholipids, the so-called Lands cycle, that serves to keep the concentration of this free fatty acid in cells at a very low level. To manipulate the intracellular AA level in U937 phagocytes, we have used several pharmacological strategies to interfere with the Lands cycle. We used inhibitors of the AA reacylation pathway, namely thimerosal and triacsin C, which block the conversion of AA into arachidonoyl-CoA, and a CoA-independent transacylase inhibitor that blocks the movement of AA within phospholipids. In addition, we used cells overexpressing group VIA phospholipase A(2), an enzyme with key roles in controlling basal fatty acid deacylation reactions in phagocytic cells. All of these different strategies resulted in the expected increase of cellular free AA but also in the induction of cell death by apoptosis. Moreover, when used in combination with any of the aforementioned drugs, AA itself was able to induce apoptosis at doses as low as 10 muM. Blocking cyclooxygenase or lipoxygenases had no effect on the induction of apoptosis by AA. Collectively, these results indicate that free AA levels within the cells may provide an important cellular signal for the onset of apoptosis and that perturbations of the mechanisms controlling AA reacylation, and hence free AA availability, may decisively affect cell survival.     

Tryptophol induces death receptor (DR) 5-mediated apoptosis in U937 cells

Tryptophol is a natural component isolated from vinegar produced from the boiled extract of black soybean. We have reported that tryptophol induces apoptosis in U937 cells via activation of caspase-8 followed by caspase-3. Tryptophol, however, did not affect human peripheral blood lymphocytes (PBL). In this study, we found that tryptophol enhances formation of a death-inducing signaling complex including death receptor (DR) 5. Cell viability and induction of apoptosis by tryptophol was reduced by transfection with decoy receptor (DcR) 1. These results indicate that tryptophol induces apoptosis through DR5 and that the resistance of PBL to tryptophol-induced apoptosis might be due to competition from DcR1.     

Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells

Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.     

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.     

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G₂/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.     

Isolated ricin B-chain-mediated apoptosis in U937 cells

We have previously reported that ricin, a toxic lectin that inhibits protein synthesis induced apoptotic cell death. In this study, we have found that isolated ricin CM-B-chain, which has no effect on cellular protein synthesis, induced DNA fragmentation in U937 cells in a dose- and time-dependent manner, albeit it required a longer incubation time and higher concentration than those of holotoxin ricin. Z-Asp-CH2-DCB, a caspase family inhibitor and serine protease inhibitor, 3,4-dichloroisocoumarine (DCI) effectively inhibited the CM-B-chain-mediated DNA fragmentation as well as in ricin. Thus, like ricin, multiple proteases with different substrate specificity may also be involved in the CM-B-chain-mediated apoptotic pathway. Furthermore, BFA inhibited both ricin- and CM-B-chain-mediated DNA fragmentation, suggesting an intracellular vesicle transport system through the Golgi complex may be involved in the apoptotic induction by these proteins as a common feature. On the other hand, cycloheximide (CHA) strongly increased the CM-B-chain-mediated DNA fragmentation, but inhibited ricin-mediated DNA fragmentation. The opposite effects of CHA may reflect the difference in the apoptotic mechanism between ricin and CM-B-chain. In conclusion, our results suggest that ricin-B-chain can induce apoptosis through its lectin activity, but the underlying mechanism may be distinct from that of ricin in which the A-chain contributes profoundly to the apoptotic induction.     

Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD. cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibtor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD. cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.