The BH3-only protein Noxa is stimulated during apoptosis of chronic lymphocytic leukemia cells triggered by M2YN, a new plant-derived extract

Deficiency of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL) cells. M2Yn is a natural extract from plants of central Asia, identified for its antiangiogenic properties and its ability to block the migration of malignant cells. Here, we report that in vitro treatment of cells derived from CLL patients with M2Yn results in internucleosomal DNA fragmentation, phosphatidylserine externalization, mitochondrial membrane depolarization, caspase-3 activation and cleavage of the caspase substrate PARP-1. The extents of these effects depend on the patients and are mostly comparable to those of flavopiridol or hyperforin, two known plant-derived apoptosis inducers of CLL cells. M2Yn does not modulate Mcl-1 expression, while downregulation of this antiapoptotic protein is involved in the action of flavopiridol. By contrast, M2Yn, like hyperforin, upregulates the Noxa protein, possibly by inhibiting proteasomal activity. This BH3-only protein is known to trigger the activation of the pro-apoptotic protein Bak through displacement of the Mcl-1/Bak complex at the mitochondrial membrane, as actually observed here in M2Yn-treated cells. Our data, therefore, show that M2Yn can induce the caspase-dependent mitochondrial pathway of apoptosis in CLL cells via a mechanism resembling that of hyperforin. Our data also confirm that the BH3-only protein Noxa is a relevant target for CLL therapy.     

The BH3-only member Noxa causes apoptosis in melanoma cells by multiple pathways

The molecular causes for resistance of melanoma to apoptosis are currently only partly understood. In the present study, we examined gene transfer and expression of the proapoptotic BH3-only protein Noxa as an alternative approach to chemotherapy and investigated the molecular mechanisms regulating Noxa-induced apoptosis. Noxa gene transfer caused dysregulation of both mitochondria and, as shown for the first time, also the endoplasmic reticulum, resulting in the accumulation of reactive oxygen species. Interestingly, expression of Noxa not only triggered the classical mitochondrial caspase cascade, but also resulted in the activation of apoptosis signal-regulating kinase1 and its downstream effectors c-Jun N-terminal kinase and p38. The activation of these kinases was abolished by antioxidants. Moreover, inhibition of the kinases by RNA interference or pharmacological inhibitors significantly attenuated Noxa-induced apoptosis. Thus, our data provide evidence for the involvement of multiple pathways in Noxa-induced apoptosis that are triggered at mitochondria and the endoplasmic reticulum, and suggest Noxa gene transfer as a complementary approach to chemotherapy.     

Regulation of the proapoptotic BH3-only protein BIM by glucocorticoids, survival signals and proteasome in chronic lymphocytic leukemia cells.

Glucocorticoids induce apoptosis in chronic lymphocytic leukemia (CLL) cells through a caspase-dependent mechanism. However, their mechanism of action remains unknown. We have studied the regulation of the proapoptotic BH3-only Bcl-2 interacting mediator of cell death (BIM) in CLL cells. We demonstrate that glucocorticoids upregulate BIM at protein and mRNA levels. We have investigated the ability of different survival signals, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), stromal cell-derived factor-1alpha (SDF-1alpha), interleukin 4 (IL-4) and B-cell receptor (BCR) activation, to influence the levels of BIM and its induction by glucocorticoids. TPA downregulates BIM(EL) by extracellular signal-regulated kinase (ERK)-mediated BIM phosphorylation and further proteasome-mediated degradation. However, SDF-1alpha and BCR activation induce transient BIM phosphorylation, without protein degradation. Proteasome inhibitors do not modify the levels of BIM with respect to untreated cells. However, they induce apoptosis and inhibit TPA-induced BIM(EL) degradation, leading to its accumulation. In conclusion, the results implicate BIM in glucocorticoid-induced apoptosis in CLL cells. BIM(EL) phosphorylation through the ERK pathway targets the protein for proteasomal degradation.     

Garlic arrests MDA-MB-435 cancer cells in mitosis, phosphorylates the proapoptotic BH3-only protein BimEL and induces apoptosis

Components of garlic (Allium sativum) can cause disruption of microtubules, cell cycle arrest, and apoptosis in cancer cells. We show here that a water-soluble extract of garlic arrested MDA-MB-435 cancer cells in mitosis and caused apoptosis. The proapoptotic BH3-only, bcl-2 family protein BimEL, which in healthy cells can be tightly sequestered to the microtubule-associated dynein motor complex, was modified after garlic treatment. The main effect of garlic on BimEL was a considerable increase in a phosphorylated form of the protein. This phosphorylation(s), probably partly dependent on c-jun N-terminal kinase activity, promoted mitochondrial localisation of BimEL. Furthermore, inhibition of extracellular signal-regulated kinases 1/2 increased the amount of another form of BimEL present in the mitochondrial cellular fraction. Treatment of cells with the garlic compound diallyl disulphide had similar effects on BimEL. The results indicate that the apoptotic effect of garlic and a combination of garlic and the inhibitor of extracellular signal-regulated kinases 1/2 in MDA-MB-435 cells partly is due to modifications that are necessary for translocation of the proapoptotic protein BimEL to mitochondria where it executes its proapoptotic function.     

Cyclin-dependent kinase inhibitor Roscovitine induces apoptosis in chronic lymphocytic leukemia cells.

A new class of cell cycle inhibitors is currently entering clinical trials. These drugs exert their activity by inhibition of cyclin-dependent kinases (cdk) and induce cell cycle arrest and apoptosis in cancer cells. Roscovitine, a cdk2-inhibitor that is in preclinical evaluation, induced apoptosis in B-CLL cells at doses that were not cytotoxic for normal human B cells. At 20 microM, Roscovitine induced apoptosis in 21 of 28 B-CLL samples and was equally effective in zap-70-positive or -negative samples. Caspase-3 was cleaved in B-CLL cells exposed to Roscovitine and the pancaspase inhibitor z.VAD.fmk-blocked Roscovitine-induced apoptosis. Expression of the proapoptotic protein Bak was increased and Bax cleavage and conformational change was observed in Roscovitine-treated B-CLL cells. Antiapoptotic proteins Mcl-1 and XIAP were downregulated, but the expression of Bcl-2 remained unchanged. In contrast to previous reports in cancer cell lines, Roscovitine treatment was not accompanied by nuclear accumulation of p53. Cyc202 (R-Roscovitine) is in early clinical trials in cancer patients. Given its powerful effects on zap-70-positive and -negative B-CLL cells, but not on normal lymphocytes, Roscovitine might be an attractive drug to be tested in this incurable disease.     

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) cells develop resistance to nucleoside analogs over time. This chemoresistance may be caused by selection for B-CLL cells with defects in the particular apoptosis pathway triggered by these drugs. Therefore, anticancer agents that induce apoptosis through alternative pathways might be useful in treating chemoresistant B-CLL. Farnesyltransferase inhibitors (FTIs) are a class of synthetic drugs with definite molecular targets, which have demonstrated cytotoxicity against leukemic cell lines. We have studied the ex vivo effect of the FTI BMS-214662 on cells from 18 patients with B-CLL. Low concentrations (<1 microM) of BMS-214662 prevented farnesylation of the chaperone marker HDJ-2 and had no effect on Akt activation. BMS-214662 induced apoptosis in B-CLL cells from all patients studied, including those showing resistance to cladribine and fludarabine ex vivo and in vivo. Treatment with BMS-214662 induced loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, proapoptotic conformational changes of Bax and Bak, reduction in Mcl-1 levels and activation of caspases 9 and 3. The general caspase inhibitor Z-VAD-fmk did not prevent BMS-214662-induced cell death. These results indicate that BMS-214662 may be a useful drug for treating B-CLL and, in particular, an alternative for the therapy of purine analog-resistant or relapsed B-CLL.     

BH3-only protein BIK induces caspase-independent cell death with autophagic features in Bcl-2 null cells

The BH3-only protein BIK normally induces apoptotic cell death. Here, we have investigated the role of BCL-2 in BIK-induced cell death using Bcl-2+/+ and Bcl-2-/- mouse embryo fibroblasts. Ectopic expression of BIK in Bcl-2-/- cells resulted in enhanced cell death compared to Bcl-2+/+ cells. In these cells, while caspase-8 was activated, there was no significant activation of caspase-9 and 3. There was no detectable mitochondrial to cytosolic release of cytochrome-c. However, there was significant redistribution of AIF from mitochondria to the nucleus. The extent of BIK-induced cell death was augmented by treatment with the pancaspase inhibitor, zVAD-fmk. The Bcl-2 null cells expressing BIK exhibited autophagic features such as cytosolic vacuoles, punctate distribution of LC3 and enhanced expression of Beclin-1. The survival of BIK-expressing Bcl-2-/- cells was enhanced in the presence of PI3 kinase inhibitors 3-methyladenine and Wortmannin and also by depletion of Atg5 and Beclin-1. Death of BIK-expressing Bcl-2-/- cells treated with zVAD-fmk was increased under caspase-8 depletion. Our results suggest enhanced expression of BIK in the Bcl-2 deficient cells leads to cell death with autophagic features and the extent of such cell death could be increased by inhibition of caspases.     

Prodigiosin induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia.

We have previously reported that prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) induces apoptosis in human hematopoietic cancer cell lines with no marked toxicity in nonmalignant cell lines. In this study, we demonstrate that prodigiosin induces apoptosis of B-cell chronic lymphocytic leukemia (B-CLL) cells (n=32 patients). The dose-response for the cytotoxic effect of prodigiosin was analyzed in cells from 12 patients showing an IC(50) of 116+/-25 nM. Prodigiosin induced apoptosis of B-CLL cells through caspase activation. We also analyzed the cytotoxic effect of prodigiosin in T cells from B-CLL samples and no differences were observed with respect to leukemia cells. This is the first report showing that prodigiosin induces apoptosis in human primary cancer cells.     

The BH3-only protein BimL overrides Bcl-2-mediated apoptosis resistance in melanoma cells

Melanoma cells are characterized by apoptosis deficiency coinciding with reduced expression of the proapoptotic Bcl-2 protein Bim. An adenoviral vector was constructed with the Bim_L cDNA controlled by an inducible promoter. Highly efficient apoptosis induction and abrogated cell proliferation was seen in melanoma cells upon Bim_L overexpression. Loss of mitochondrial membrane potential, release of mitochondrial apoptogenic factors and caspase-9 processing indicated the activation of mitochondrial apoptosis pathways. Bim_L activated both Bax and Bak, as shown by siRNA knockdown and activation-specific antibodies. Of note, Bim_L overrode the apoptosis blockade by Bcl-2 overexpression or by Bax/Bak single knockdown. The high efficacy correlated to Bim_L interaction with all antiapoptotic Bcl-2 family members in melanoma cells, shown by co-immunoprecipitation analyses for Bcl-2, Bcl-x_L, Mcl-1 and Bcl-w. Thus, Bim_L reveals an outstanding proapoptotic potential in melanoma cells, and strategies for its re-expression appear of interest. These have been reported for B-Raf inhibitors, and their efficacy may be partly attributed to Bim_L.     

BH3-only proteins: integrated control point of apoptosis

Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins, Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53.     

ZGDHu-1 promotes apoptosis of chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. We aimed to examine the proapoptotic effects of a novel proteasome inhibitor, N,N'-di-(m-methylphenyi)-3,6- dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide?(ZGDHu-1), on CLL cells. B lymphocytes were isolated from CLL patients and normal healthy controls, and treated with various concentrations of ZGDHu-1 for different days. CLL cell viability was detected by MTT assay. The apoptosis, mitochondrial membrane potential (?ψm) and reactive oxidative species (ROS) were examined by flow cytometry. The expression of caspase-3 and Bcl-2/Bax ratio was detected by western blotting. ZGDHu-1 significantly reduced the viability of CLL cells and induced apoptosis in comparison to the control cells (both P<0.05). Normal peripheral B cells were resistant to the apoptosis-inducing effects of ZGDHu-1. Apoptosis induced by ZGDHu-1 was accompanied with generation of ROS, loss of ?ψm, downregulation of Bcl-2 and increase of caspase-3 cleavage. Results of this study indicate that ZGDHu-1 is a promising specific treatment for CLL in the clinic.     

Loss of Hus1 sensitizes cells to etoposide-induced apoptosis by regulating BH3-only proteins

The Rad9-Rad1-Hus1 (9-1-1) cell cycle checkpoint complex plays a key role in the DNA damage response. Cells with a defective 9-1-1 complex have been shown to be sensitive to apoptosis induced by certain types of genotoxic stress. However, the mechanism linking the loss of a functional 9-1-1 complex to the cell death machinery has yet to be determined. Here, we report that etoposide treatment dramatically upregulates the BH3-only proteins, Bim and Puma, in Hus1-deficient cells. Inhibition of either Bim or Puma expression in Hus1-knockout cells confers significant resistance to etoposide-induced apoptosis, whereas knockdown of both proteins results in further resistance, suggesting that Bim and Puma cooperate in sensitizing Hus1-deficient cells to etoposide treatment. Moreover, we found that Rad9 collaborates with Bim and Puma to sensitize Hus1-deficient cells to etoposide-induced apoptosis. In response to DNA damage, Rad9 localizes to chromatin in Hus1-wild-type cells, whereas in Hus1-deficient cells, it is predominantly located in the cytoplasm where it binds to Bcl-2. Taken together, these results suggest that loss of Hus1 sensitizes cells to etoposide-induced apoptosis not only by inducing Bim and Puma expressions but also by releasing Rad9 into the cytosol to augment mitochondrial apoptosis.     

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.     

Sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis by hyperthermia

We recently reported that, in cultured leukemic T lymphocytes and promyelocytic cells, a mild heat shock treatment (1 h at 42 degrees C) induced a long lasting stimulation of the apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). On the opposite, no effects were recorded toward normal human T lymphocytes. The apoptogenic efficiency of TRAIL in leukemic lymphocytes is linked to the long lasting increased ability of TRAIL to recognize and bind DR4 and DR5 receptors during hyperthermia. Here, we have analyzed whether this new apoptotic co-treatment could be relevant toward primary cells from patients suffering of chronic lymphocytic leukemia. Analysis of samples from 24 patients with different ages, sex and disease stages revealed that half of them had lymphocytes that, once isolated and analyzed in vitro, positively responded (increase of cell death) to the heat shock plus TRAIL co-treatment. Analysis of the level of expression of various anti-apoptotic proteins in the cell samples revealed a great heterogeneity between patients and no clear relationships could be drawn. Nevertheless, most cell samples that were sensitive to TRAIL plus heat shock induced apoptosis displayed a higher level of cell surface DR4 and DR5 receptors than the non-sensitive counterparts. Hence, analysis of the level of TRAIL surface receptors is a prerequisite for future clinical applications based on this protocol.     

MZ3 induces apoptosis in human leukemia cells

Purpose 4-(4-Bromophenyl)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmi-dazole-1-carboxamide (MZ3) is one of the synthesized combretastatin-A-4 analogues and has been reported that it displayed a promising specific activity against leukemia cell lines. Our purpose was to investigate the mechanism of MZ3’s cytotoxicity. Methods Cytotoxicity was measured by MTT method, apoptosis was measured by flow cytometry. DNA fragmentation was tested by agarose gel electrophoresis. Mitochondrial membrane potential (ΔΨm) was detected by JC1 staining and flow cytometry, while intracellular reactive oxygen species (ROS) was detected by 5-(and-6)-carboxy-2′-7′-dichlorofluorescin diacetate staining and flow cytometry. Protein expression was analyzed by western blotting. In vivo activity of MZ3 was assayed through severe combined immunodeficiency (SCID) mice model of human leukemia engrafts. Results MZ3 exhibited high anti-cancer activity in six leukemia cell lines, including two drug-resistant cell lines. MZ3 induced DNA fragmentation, and caused an elevation of ROS and a loss of ΔΨm in HL60 cells. MZ3 also induced the activation of caspase-3, influenced the expression of Bcl-2 family members, MAPKs and other proteins relative to mitochondria-induced apoptosis. In addition, N-acetylcysteine cannot inhibit HL60 cell apoptosis caused by MZ3. Furthermore, a prolonged survival time was observed after treatment with MZ3 in SCID mice model of human leukemia engrafts. Conclusions MZ3 is a potent compound against leukemia cell lines both in vitro and in vivo, and the mitochondrial pathway mediated by Bcl-2 protein family and MAPKs might be involved in signaling MZ3-induced apoptosis.     

Drug-induced apoptosis in chronic lymphocytic leukemia.

Glucocorticoids and fludarabine are able to induce typical features of apoptosis in CLL lymphocytes.Cysteinyl aspartate specific proteases (caspases) play a key biochemical role in the apoptotic pathway. Caspase activation following cytotoxic stimuli leads to highly specific proteolytic cleavage of functionally important cellular enzymes. One of them is poly ADP-ribose) polymerase (PARP). To some extent caspase activation seems to be under the control of the Bcl-2 family of interacting proteins. We determined the role of Bcl-2-family proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), activation of caspase-3 (CPP32/Yama) and activation of PARP in CLL apoptosis. All 21 analyzed CLL samples expressed Bcl-2 and Bax. Four of 13 (31%) samples with a low Bcl-2/Bax ratio exhibited in vitro prednisolone resistance, whereas eight of nine (88%) samples with a high Bcl-2/Bax ratio were in vitro resistant (相似文献   

The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.