U937 Apoptosis Research Articles

Inactivation of Bcl‐2 through IκB kinase (IKK)‐dependent phosphorylation mediates apoptosis upon exposure to 4‐hydroxynonenal (HNE)

Apoptosis of macrophage foam cells loaded with modified/oxidized lipids is implicated in destabilization of advanced atherosclerotic plaques in humans. Concentration of HNE, main aldehydic product of plasma LDL peroxidation, elevates in atherosclerotic lesions as well as in cultured cells under oxidative stress. Although this reactive aldehyde has been shown to promote apoptosis with the involvement of p38 MAPK and JNK in various mammalian cell lines, roles of B-cell lymphoma 2 (Bcl-2) family proteins remain to be deciphered. We demonstrated that HNE-induced apoptosis was accompanied by concurrent downregulations of antiapoptotic Bcl-x(L) and Mcl-1 as well as upregulation of proapoptotic Bak. Furthermore, phoshorylation of Bcl-2 at Thr56, Ser70, and probably more phosphorylation sites located on N-terminal loop domain associated with HNE-induced apoptosis in both U937 and HeLa cells while ectopic expression of a phospho-defective Bcl-2 mutant significantly attenuated apoptosis. In parallel to this, HNE treatment caused release of proapoptotic Bax from Bcl-2. Pharmacological inhbition of IKK inhibited HNE-induced Bcl-2 phosphorylation. Similarly, silencing IKKα and -β both ended up with abrogation of Bcl-2 phosphorylation along with attenuation of apoptosis. Moreover, both IKKα and -β coimmunoprecipitated with Bcl-2 and in vitro kinase assay proved the ability of IKK to phosphorylate Bcl-2. In view of these findings and considering HNE inhibits DNA-binding activity of nuclear factor-κB (NF-κB) through prevention of IκB phosphorylation/ubiquitination/proteolysis, IKK appears to directly interfere with Bcl-2 activity through phosphorylation in HNE-mediated apoptosis independent of NF-κB signaling.

Decitabine, a DNA methyltransferase inhibitor, induces apoptosis in human leukemia cells through intracellular reactive oxygen species generation

The DNA methyltransferase inhibitor decitabine, 5-aza-2'-deoxycytidine, has been found to exert anti-metabolic and anticancer activities when tested against various cultured cancer cells. Furthermore, decitabine has been found to play critical roles in cell cycle arrest and apoptosis in various cancer cell lines; however, these roles are not well understood. In this study, we investigated decitabine for its potential anti-proliferative and apoptotic effects in human leukemia cell lines U937 and HL60. Our results indicated that treatment with decitabine resulted in significantly inhibited cell growth in a concentration- and time-dependent manner by the induction of apoptosis. Decitabine-induced apoptosis in U937 and HL60 cells was correlated with the downregulation of anti-apoptotic Bcl-2, XIAP, cIAP-1 and cIAP-2 protein levels, the cleavage of Bid proteins, the activation of caspases and the collapse of mitochondrial membrane potential (MMP). However, apoptosis induced by decitabine was attenuated by caspase inhibitors, indicating an important role for caspases in decitabine responses. The data further demonstrated that decitabine increased intracellular reactive oxygen species (ROS) generation. Moreover, N-acetyl-L-cysteine, a widely used ROS scavenger, effectively blocked the decitabine-induced apoptotic effects via inhibition of ROS production and MMP collapse. These observations clearly indicate that decitabine-induced ROS in human leukemia cells are key mediators of MMP collapse, which leads to apoptosis induction followed by caspase activation.

3,5-Disubstituted-thiazolidine-2,4-dione analogs as anticancer agents: Design, synthesis and biological characterization

A series of 2,5-disubstituted-thiazolidine-2,4-dione analogs based on the newly identified lead 1, a potential anticancer agent via the inhibition of the Raf/MEK/extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades, were synthesized and biologically characterized. A new lead structure, 15, was identified to have improved anti-proliferative activities in U937 cells, to induce apoptosis in U937, M12 and DU145 cancer cells, and to arrest U937 cells at the S-phase. Furthermore, Western blot analysis demonstrated a correlation of the anti-proliferative activity and blockade of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Collectively, these results strongly encourage further optimization of 15 as a new lead with multi-target properties to develop more potent compounds as anticancer agents.

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

BackgroundThe natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo.Methodology and ResultsHF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells.SignificanceOur data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

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.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937

Background Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. Methods Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. Results Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. Conclusions These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. General significance This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.

Gallic Acid Inhibits Cell Viability and Induces Apoptosis in Human Monocytic Cell Line U937

In the present study, we investigated the effects of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a polyhydroxyphenolic compound, isolated from Rhus chinensis, on the human monocytic lymphoma cell line U937. In vitro experiments showed that treating U937 cells with various amounts of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA induces apoptosis, we examined the gene expression of p53, nuclear factor κB (NF-κB), and inhibitor of NF-κB (I-κB) after treating the cells with GA and found that expression levels of the genes for p53 and NF-κB increased and that for I-κB decreased. The results obtained from western blotting with U937 cells showed up-regulation of NF-κB protein and down-regulation of proliferating cell nuclear antigen and I-κB protein. These results demonstrate that GA efficiently induces apoptosis in U937 cells and that GA is a potential chemotherapeutic agent against lymphoma.

Preferential involvement of both ROS and ceramide in fenretinide-induced apoptosis of HL60 rather than NB4 and U937 cells

Leukemic cells responding to apoptosis-inducing drugs can be varied in terms of the mechanisms of action. Fenretinide, a synthetic retinoid, is worth of study as a promising candidate for apoptosis-based therapy of leukemia. Yet, it remains unclear whether this drug exerts the similar mechanisms on different leukemic cells. Here, we report a comparative analysis of fenretinide-induced apoptosis in three acute myeloid leukemic (AML) cell lines including HL60, NB4 and U937. Through a series of antagonist assays, we revealed similarities and differences of mechanisms involved in these three cell lines. Antioxidant vitamin C completely abrogated fenretinide-induced apoptosis in all cell lines, demonstrating that ROS is an essential and common mediator. However, the apoptotic effects of fenretinide could be blocked by ceramide synthase inhibitor fumonisin B 1 only in HL60 rather than the other two. Moreover, fumonisin B 1 was unable to inhibit the generation of ROS in fenretinide-treated HL60 cells, indicating that ROS may function as upstream stimulus of ceramide-mediated apoptosis. These comparative results strongly suggest that the apoptotic response induced by fenretinide in HL60 involves both ROS and ceramide, whereas drug-induced apoptosis in NB4 and U937 requires ROS but is independent of ceramide. Differentiated modes of action exerting on AML may guide the use of this apoptosis-inducing drug, and hence advance our knowledge about the nature of cancer-specific responses to this drug.

The Synergistic Effect of DNA Methylation Inhibitor 5-Azacytidine and Bcl-2 Signaling Blockade ABT-737 Combination Treatment Induce Apoptosis In AML

AbstractAbstract 3311Bcl-2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). ABT-737 is a small molecule BH3 mimetic that binds tightly to a hydrophobic cleft on Bcl-2 and Bcl-XL and exerts its proapoptotic function by preventing antiapoptotic Bcl-2 family members from sequestering activating BH3 proteins (Oltersdorf et al., Nature 2005). We have reported that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induces the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway (Konopleva et al., Cancer Cell 2006). The ABT-737 related clinical compound, ABT-263, is undergoing Phase I/II studies in chronic lymphocytic leukemia, with initial signs of clinical activity. However, the main side effect is thrombocytopenia resulting from inhibition of Bcl-XL. Hence, combinations of ABT-737 with non-cytotoxic agents are desirable to take full advantage of ABT236's unique spectrum of biophysical and preclinical activities.In this project, we studied pharmacologic interactions between ABT-737 and the DNA methyltransferase inhibitor 5-azacytidine (5-azaC). 5-azaC is a cytidine analog with clinical activity in myelodysplastic syndromes (MDS) and in AML. Since recent studies indicate that 5-azaC induce DNA damage in p53-dependent fashion, we tested the hypothesis that the pro-apoptotic effects of 5-azaC/ABT-737 combination are related to non-redundant activation of BH3-only proteins and mitochondrial apoptosis in AML cells with wt p53.In vitro, 5-azaC and ABT-737 in combination for 72 hrs induced growth inhibition and apoptosis in AML cell lines OCI-AML3, MOLM-13 and U937 in a highly synergistic, dose-dependent fashion, with combination indices (CIs) ranging from 0.1 to 0.22. These effects were observed even at low concentrations (5-azaC 100nM and ABT-737 10nM, at 10:1 ratio). In contrast, no synergistic apoptosis was seen in p53-null HL-60 cells. Likewise, ABT-737/5-azaC induced apoptosis in a synergistic fashion in OCI-AML3 cells infected with vector control shRNA (CI=0.04) but failed to induce cell death in OCI-AML3 p53 shRNA cells, indicating critical p53-dependent mechanisms of cell death. In primary AML samples sensitive to ABT-737 alone (n=3), synergistic and additive effects were seen. The combined effects of 5-azaC and ABT-737 were further investigated in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice injected with cells from a patient with primary refractory AML. Seven days after leukemia transplantation, mice were treated with vehicle, ABT-737 (intraperitoneally (IP), 75mg/kg/day for 10 days), 5-azacytidine (IP, 4mg/kg/day for 5 days) or with the combination. Engraftment of patient leukemia cells was analyzed by the immunohistochemical detection of CD45-positive cells in bone marrow and spleen seven weeks after transplantation. Both, ABT-737 and 5azaC each exerted anti-leukemic effects, as evidenced by significant reduction in leukemia cells in bone marrow and spleen (10% and 3% CD45+ cells detectable in 1/3 mice in ABT-737 and 5-azaC groups, respectively), no CD45+ AML cells were detected in organs of 3/3 mice treated with the combination. No overt hemorrhage was detected in the animals.In summary, the combination of 5-azaC and ABT-737 induces synergistic cells death in AML cell lines and in a subset of primary AML samples in a p53-dependent fashion. The mechanisms of this pharmacologic interactions including the p53-dependent upregulation of BH3-only proteins, described by us for ABT-737/MDM2 inhibitor combinations, are currently under investigation. Results suggest that this therapeutic strategy can be successfully utilized in AML patients with low mutation rate and unimpaired signaling of p53. Disclosures:No relevant conflicts of interest to declare.

The high level of RANTES in the ectopic milieu recruits macrophages and induces their tolerance in progression of endometriosis

RANTES (C-C chemokine, regulated on activation, normal T cell expressed and secreted) is involved in progression of endometriosis, but the precise mechanism is understood inadequately. This study is to elucidate the roles of RANTES in macrophage recruitment and tolerance in the endometriotic milieu. The expression of RANTES was analyzed by immunohistochemistry. The cell co-cultures were applied to simulate the endometriotic milieu to investigate the regulation of RANTES secretion and its receptor CCR1 expression. Transwell migration assay was used for chemotaxis of U937 cells (macrophage line) to endometrial stromal cells (ESCs) and/or human pelvic mesothelial cells. The expression of CCR1 was analyzed by RT-PCR and qPCR in transcription and by western blot in translation respectively. Concentrations of RANTES, IL10, and IL12p70 were determined by ELISA. The phenotype of U937 cells and apoptosis of ESCs were analyzed by flow cytometry. We have found that the expression of RANTES is significantly higher in the endometriotic tissue and eutopic endometrium than that of the normal endometrium without endometriosis. The combination of 17β-estradiol and dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin increases significantly RANTES secretion in the endometriosis-associated cell co-culture which can recruit more macrophages, upregulate CCR1 expression, and induce tolerant phenotype, which inhibits the apoptosis of ESC in the milieu. In conclusion, the higher levels of RANTES in the ectopic milieu facilitate the onset and progression of endometriosis by macrophage recruitment and tolerance that in turn inhibits apoptosis and enhances growth of ESC.

Rimonabant-induced apoptosis in leukemia cell lines: Activation of caspase-dependent and -independent pathways

Rimonabant (SR141716), a cannabinoid CB1 receptor antagonist known for anti-obesity activity, has more recently been shown to inhibit tumor cell growth. Here we demonstrated the antitumor potential of SR141716 in leukemia-derived cell lines and its low toxicity in normal cells (PBMC). SR141716 (1–20μM range of doses) reduced Jurkat and U937 cell number by activating death signals as well as affecting cell cycle progression. The most prominent response in U937 to SR141716 was a G0/G1 block, while in Jurkat cells there was activation of cell death processes. SR141716-treated cells exhibited the morphological and biochemical features of apoptosis and to some extent necrosis. Apoptotic mode of cell death was confirmed in both cell lines by analysis of cell morphology, phosphatidylserine exposure and DNA fragmentation. Moreover, the drug was found to induce an early and robust mitochondrial membrane depolarization. In Jurkat cells the apoptotic process was typically caspase-dependent, while in U937 caspase-independent pathways were also activated. The contribution of PARP activation to SR141716-induced apoptosis in U937 was suggested by protein PARylation, AIF release and apoptosis reversal by PARP inhibitors. Moreover, SR141716 negatively modulated, especially in U937, the PI3K/AKT pathways. In conclusion, our data indicate that SR141716 elicits alternative response and/or cell death pathways depending on the cell type affected.

Abstract 667: Mechanisms underlying synergistic interactions between the CDK inhibitor flavopiridol (Alvocidib) and the BH3 mimetic GX15-070 (Obatoclax) in human multiple myeloma cells

Abstract To establish a mechanistic basis for a strategy combining CDK inhibitors and Bcl-2 antagonists, in vitro and in vivo interactions between flavopiridol (FP) and GX15-070 (GX) were investigated in human multiple myeloma (MM) cells. The activity of this regimen was also examined in primary CD138+ MM cells and in a murine MM xenograft model. Co-administration of sub-toxic concentrations of FP interacted synergistically with GX to induce apoptosis in U266, RPMI8226, H929, OPM-2, and MM.1S, as well as IL-6-dependent ANBL6 and KAS 6/1 MM cells. Combination treatment also effectively killed MM cells in the presence of stromal cells or growth factors. The regimen was active against multiple drug-resistant MM cell lines, including bortezomib/Velcade- (U266/VR and OPM-2/VR), Revlimid- (8226/R10R), dexamethasone- (MM.1R), melphalan- (8226/LR5), and doxorubicin-resistant (8226/Dox40) MM cells. FP also sharply promoted GX lethality in primary CD138+ MM cells, while sparing normal CD138- bone marrow cells. GX induced clear declines in Mcl-1 and Bcl-xL levels which were potentiated by FP, whereas Bcl-2 expression did not change. Significantly, FP/GX overcame cytoprotective effects mediated by ectopic Mcl-1, Bcl-xL, or Bcl-2 expression. Notably, BH3-only protein expression profiling revealed multiple changes: a) FP, in the presence or absence of GX, triggered a clear increase in Bim expression (both BimEL and BimL isoforms); b) FP/GX co-treatment sharply induced Bik (∼35kDa species) and Noxa (∼27 kDa species),; c) co-exposure (24hr) to FP and GX induced a clear reduction in Puma expression; and c) no changes in the expression of other BH3-only proteins occurred. These findings were confirmed in various MM cell lines, including multiple drug-resistant cells, and were associated with Bak/Bax activation, Bax translocation, cytochrome C release, and caspase activation. Importantly, Bim, Bik, or Noxa knock-down by stable transfection with shRNA dramatically diminished FP/GX lethality. Lastly, co-administration of FP and GX significantly suppressed tumor growth, reflected by diminished tumor size and bioluminescence intensity, in athymic nude mice inoculated with 8226 cells stably expressing luciferase. These events were associated with pronounced apoptosis, manifested by caspase 3 activation and PARP degradation. Western blot analysis also revealed down-regulation of Mcl-1, as well as up-regulation of Bik and Noxa in tumor tissues following FP/GX co-administration. Collectively, these findings provide a mechanistic framework for a strategy combining CDK inhibitors with Bcl-2 antagonists in which down-regulation of anti-apoptotic proteins and induction of BH3-only proteins cooperate to trigger synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 667.

Abstract 3974: TIAF1 blocks p53 suppression of SMAD promoter activation

Abstract p53 participates in the TGF-β signaling. How p53 regulates SMAD gene transcription is largely unknown. TIAF1 is a TGF-β1-induced factor and a downstream effector of the TGF-β signaling. TIAF1 is known to control nuclear localization of Ser15-phosphorylated p53. Ectopic TIAF1 induces growth inhibition and apoptosis of monocytic U937 and many non-fibroblasts, via upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. Wild type p53 suppresses the transcriptional activation of SMAD gene, whereas isoform p53γ induces the gene activation. TGF-β1 increases the physical association of TIAF1 with Smad4, and when overexpressed, TIAF1 inhibits Smad4-dependent promoter activation. In combination, TIAF1 and p53 support the promoter activation regulated by endogenous or ectopic Smad4, indicating that TIAF1 blocks p53 suppression of the SMAD promoter activation, and vice versa. Without Ser46 phosphorylation in p53 (p53ΔS46), p53ΔS46 alone, or in the co-presence of TIAF1, abolishes the Smad4-regulated promoter activation. These observations suggest a critical role of Ser46 in p53 in controlling SMAD transcriptional activation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3974.

Induction of apoptosis in human leukaemic cell lines K562, HL60 and U937 by diethylhexylphthalate isolated from Aloe vera Linne.

We investigated the effect of diethylhexylphthalate (DEHP) from Aloe vera Linne on the apoptosis of human leukaemic cell lines K562, HL60 and U937 to examine its pharmacological activity. At a level of 10 microg mL(-1) DEHP a significant anti-leukaemic effect was observed for all three cell lines, as measured by clonogenic assay. After treatment with 10 microg mL(-1) DEHP for 4 h, agarose gel electrophoresis and flow cytometric analysis confirmed the occurrence of apoptosis. These results indicate that DEHP isolated from Aloe vera Linne has a potent antileukaemic effect, and thus represents a new type of pharmacological activity with respect to human leukaemic cells.

Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

To explore the mechanisms involved in Staphylococcus aureus (S. aureus) invading human monocytic U937 cells. S. aureus were added to U937 cells at multiplicity of infections (MOI) of 20:1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry analysis. Akt and nuclear factor-kappaB (NF-kappaB) activities were detected by Western blotting. Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated (phosphorylated) Akt and NF-kappaB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-kappaB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. S. aureus can stimulate the apoptosis of U937 cells. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-kappaB.

Effect of YC-1, a HIF Inhibitor,On Apoptosis of Leukemia Cell.

Abstract 4432 ObjectThis study was purposed to investigate the expression of HIF-1α and VEGF in leukemia cell lines and the effect of YC-1 on the regulation of HIF-1α and VEGF and the induction of apoptosis in U937 cell, exploring the mechanism of apoptosis after treatment of YC-1. MethodsRT-PCR was used to determine the levels of HIF-1α mRNA and VEGF mRNA in K562,U937 and Jurkat cells. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour, 8 hours,16 hours and 24 hours respectively, the changes of morphologic features were observed by DAPI staining under fluorescent microscope and the apoptotic rates were assayed by flow cytometry with Annexin V-FITC/PI staining; the levels of HIF-1α mRNA and VEGF mRNA were measured with RT-PCR ; the protein expression of HIF-1α, VEGF, Bax,Bcl-2 and Caspase-3 were measured by Western blot. ResultsHIF-1α mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour,8 hours,16hours and 24hours respectively, the typical apoptotic morphologic features of U937 cells were observed under fluorescent microscope and the apoptotic rates were significantly increased in a time-dependent manner, they were (4.87±0.70)%, (27.27±2.00)%, (51.53±2.81) and (60.5±3.20)% respectively, the level of VEGF mRNA reduced, while the level of HIF-1α mRNA had no obviously changes. Furthermore, the expression of HIF-1α, VEGF and Bcl-2 decreased, while the expression of Bax and Caspase-3 increased in a time-dependent manner. ConclusionHIF-1α mRNA and VEGF mRNA are expressed in leukemia, YC-1 has significant effect of down-regulation the protein expression of HIF-1α and VEGF and induction of apoptosis in U937, its mechanisms may involve in up-regulating Bax/Bcl-2 ratio and expression of Caspase-3. Disclosures:No relevant conflicts of interest to declare.

Targeting CDK9 Dramatically Potentiates ABT-737-Induced Apoptosis in Human Multiple Myeloma Cells through a Bim-Dependent Mechanism.

Abstract 297 Background:High expression of the anti-apoptotic Bcl-2 family protein Mcl-1 is a critical survival mechanism in human multiple myeloma (MM) cells, and a key barrier to apoptosis induced by the Bcl-2/-xL antagonist ABT-737. De novo expression of the short-lived protein Mcl-1 is controlled transcriptionally by CDK9, a catalytic subunit of the PTEF-b transcription elongation complex (CDK9/cyclin T) that phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) to promote elongation of nascent transcripts. Thus, approaches targeting CDK9 may enhance ABT-737 efficacy in MM cells. Methods:To test this hypothesis, we employed the pan-CDK inhibitor roscovitine and a highly-selective CDK9 inhibitor (CDK9i) in drug-naïve and drug-resistant MM cell lines, as well primary plasma cells. Results:Co-administration of non- or sub-toxic concentrations of roscovitine or CDK9i interacted in a highly synergistic manner with ABT-737 (e.g., 150-300 nM) to induce apoptosis in U266, RPMI 8226, H929, OPM-2, and MM.1S MM cells, as well as IL-6-depedent ANBL-6 and KAS 6/1 cells. In each case, Combination Index values were significantly less than 1.0. These regimens were fully active in various drug-resistant MM cells, including U266/VR and OPM-2/VR bortezomib-resistant cells, RPMI 8226/RR lenalidomide-resistant cells, MM.1R dexamethasone-resistant cells, RPMI 8226/LR5 melphalan-resistant cells, and RPMI 8226/Dox40 doxorubicin-resistant cells. They also effectively killed MM cells in the presence of stromal cells or growth factors (i.e., IL-6, IGF-1, BAFF, and APRIL). Co-administration of roscovitine significantly enhanced ABT-737 lethality in primary CD138+ MM cells, while largely sparing CD138− bone marrow cells. Notably, the majority of MM cell lines exhibited high expression of cyclin T1 rather than cyclin T2a/b, in association with constitutively-activated CDK9, manifested by high levels of phosphorylation of CDK9 and RNA pol II CTD at Ser2 and 5. Exposure of MM cells to either roscovitine or CDK9i strikingly reduced RNA pol II CTD phosphorylation at both sites (particularly Ser5), but did not affect CDK9 and cyclin T1 levels. Whereas treatment with ABT-737 alone led to a modest but clear increase in Mcl-1 expression, roscovitine or CDK9i completely blocked this event and substantially down-regulated basal Mcl-1 expression. Notably, similar phenomena occurred in U266/VR cells, which displayed higher basal levels of Mcl-1, an important candidate resistance mechanism, compared to parental cells. Moreover, knock down of Mcl-1 by stable transfection with shRNA dramatically increased U266 sensitivity to ABT-737. Co-treatment of MM cells with ABT-737 and roscovitine or CDK9i did not affect expression of other anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL, but induced a Bcl-2 cleavage fragment. Expression profiling of BH3-only proteins in MM cells co-exposed to roscovitine and ABT-737 revealed a modest or a marked increase in expression of Bim (both EL and L isoforms) and Noxa (particularly a ∼27 kDa species), respectively, accompanied by a clear decrease in Puma levels. No changes in expression of other BH-3-only proteins (e.g., Bid, Bad, Bik, BMF, Hrk) were observed. Interestingly, knock down of Bim by stable transfection with shRNA dramatically diminished roscovitine/ABT-737 lethality, whereas Noxa knock-down by shRNA only partially protected cells from this regimen. In contrast, both shRNA approaches substantially protected U266 cells from bortezomib lethality. Conclusions:Collectively, these findings provide a mechanistic framework for targeting transcription by CDK9 inhibitors to diminish ABT-737-mediated Mcl-1 upregulation and to downregulate basal Mcl-1 levels, resulting in highly synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents. They also suggest that the BH3-only protein Bim may be required for apoptosis triggered by this strategy, while up-regulation of Noxa may amplify MM cell killing in this setting. Disclosures:No relevant conflicts of interest to declare.

A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases, Cdc25C and PP2Acalpha, which are coregulators of the G(2)-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS.

Retracted: Heat‐shock pretreatment inhibits sorbitol‐induced apoptosis in K562, U937 and HeLa cells

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 +/- 0.3 degrees C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase-3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase-3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members.

Epac inhibits apoptosis of human leukocytes

cAMP is known to participate in the regulation of apoptosis in leukocytes. Depending on the cell type, pro- and antiapoptotic effects of cAMP have been described. Thus far, most of the cAMP-dependent effects have been attributed to the activation of PKA. However, Epac proteins (direct cAMP targets and guanine nucleotide exchange factors for Ras-like GTPases) have been shown recently to contribute to cAMP-dependent regulation of apoptosis. Therefore, we investigated the effects of the selective Epac activators 8-pCPT and Sp on apoptosis in human leukocytic cells (U937, HL-60, primary human mononuclear cells). We report here that Epac activation inhibits leukocyte apoptosis significantly.