STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells

Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.     

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.     

Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents

Some, but not all, of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) induce apoptosis as shown by cell shrinkage, chromatin condensation, and DNA fragmentation in three human cell lines, HL-60 promyelocytic, Jurkat T lymphoma, and Hut-78 s.c. lymphoma cells. This chemical selectivity, together with the lack of apoptotic activity against rat Leydig cells, argues against a general cell poisoning effect. PBOX-6, a potent member of the series, caused activation of a member of the caspase-3 family of proteases. In addition, the caspase-3-like inhibitor z-DEVD-fmk, but not the caspase-1-like inhibitor z-YVAD-fmk prevented PBOX-6-induced apoptosis, suggesting that caspase 3-like proteases are involved in the mechanism by which PBOX compounds induce apoptosis. The release of cytochrome c into the cytosol in HL-60 cells in response to PBOX-6 suggests that this cellular response may be important in the mechanism by which PBOX-6 induces apoptosis. However, reactive oxygen intermediates do not play a key role in PBOX-6-induced apoptosis because neither the free radical scavenger TEMPO nor the antioxidant N-acetylcysteine had any effect on PBOX-6-induced apoptosis. The apoptotic induction seems independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR) that binds these pyrrolobenzoxazepines with high affinity, due to the lack of correlation between their affinities for the receptor and their apoptotic potencies, their high apoptotic activity in PBR-deficient cells such as Jurkats, and their lack of apoptotic induction in PBR-rich rat Leydig cells. These PBOXs also can overcome nuclear factor-kappaB-mediated resistance to apoptosis. This suggests an important potential use of these compounds in drug-resistant cancers.     

(-)-Gossypol acts directly on the mitochondria to overcome Bcl-2- and Bcl-X(L)-mediated apoptosis resistance

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family, including Bcl-2 and Bcl-X(L), contributes to malignant transformation and subsequent resistance to traditional chemotherapeutics. Thus, these proteins represent attractive targets for novel anticancer agents. The small molecule, gossypol, was initially investigated as a contraceptive agent, but subsequently has been shown to possess anticancer properties in vitro and in vivo. Recently gossypol has been found to bind to Bcl-X(L) and, with less affinity, to Bcl-2. Here we investigate the ability of the (-) enantiomer of gossypol, (-)-gossypol, to overcome the apoptosis resistance conferred by Bcl-2 or Bcl-X(L) overexpression in Jurkat T leukemia cells. (-)-Gossypol potently induced cell death in Jurkat cells overexpressing Bcl-2 (IC50, 18.1+/-2.6 micromol/L) or Bcl-X(L) (IC50, 22.9+/-3.7 micromol/L). Vector-transfected control cells were also potently killed by (-)-gossypol (IC50, 7.0+/-2.7 micromol/L). By contrast, the chemotherapy drug etoposide only induced efficient killing of vector-transfected cells (IC50, 9.6+/-2.3 micromol/L). Additionally, (-)-gossypol was more efficient than etoposide at inducing caspase-3 activation and phosphatidylserine externalization in the setting of Bcl-2 or Bcl-X(L) overexpression. (-)-Gossypol-induced apoptosis was associated with Bak activation and release of cytochrome c from mitochondria, suggesting a mitochondrial-mediated apoptotic mechanism. Moreover, (-)-gossypol treatment of isolated mitochondria purified from Bcl-2-overexpressing cells also resulted in cytochrome c release, indicating a possible direct action on Bcl-2 present in the mitochondrial outer membrane. Taken together, these results suggest that (-)-gossypol is a potent and novel therapeutic able to overcome apoptosis resistance by specifically targeting the activity of antiapoptotic Bcl-2 family members. (-)-Gossypol may be a promising new agent to treat malignancies that are resistant to conventional therapies.     

黄芪浸膏粉诱导Jurkat clone E6-1细胞凋亡

目的利用黄芪浸膏粉(黄芪)诱导白血病细胞JurkatcloneE6-1(Jurkat)凋亡并探讨其作用机理。方法不同浓度(0~12.5mg/ml)黄芪与Jurkat孵育120小时后流式细胞仪上检测细胞凋亡;黄芪10mg/ml与Jurkat孵育120小时后,流式细胞仪上检测细胞中caspase3、9活性变化及Bcl-2含量改变。结果①黄芪诱导Jurkat凋亡,随剂量的增加,活细胞逐渐减少。在12.5mg/ml时凋亡达高峰,为(80.55±1.82)%,并出现坏死细胞,为(12.49±1.88)%。②黄芪诱导Jurkat凋亡伴有caspase3、9的激活,caspase3由(0.57±0.16)%升至(84.53±4.85)%;caspase9由(0.25±0.16)%升至(20.72±4.87)%,caspase抑制剂zVAD-fmk在抑制caspase3、9激活的同时抑制细胞凋亡发生。③黄芪对Jurkat中Bcl-2含量无影响,黄芪作用前后Bcl-2含量分别为(21.20±0.12)%、(24.81±0.14)%。结论黄芪通过不依赖Bcl-2的途径诱导Jurkat凋亡,在此过程中,caspase39、的激活十分重要。     

Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways

This study was undertaken to characterize preclinical cytotoxic interactions for human malignancies between the multikinase inhibitor sorafenib (BAY 43-9006) and proteasome inhibitors bortezomib or MG132. Multiple tumor cell lines of varying histiotypes, including A549 (lung adenocarcinoma), 786-O (renal cell carcinoma), HeLa (cervical carcinoma), MDA-MB-231 (breast), K562 (chronic myelogenous leukemia), Jurkat (acute T-cell leukemia), MEC-2 (B-chronic lymphocytic leukemia), and U251 and D37 (glioma), as well as cells derived from primary human glioma tumors that are likely a more clinically relevant model were treated with sorafenib or bortezomib alone or in combination. Sorafenib and bortezomib synergistically induced a marked increase in mitochondrial injury and apoptosis, reflected by cytochrome c release, caspase-3 cleavage, and poly(ADP-ribose) polymerase degradation in a broad range of solid tumor and leukemia cell lines. These findings were accompanied by several biochemical changes, including decreased phosphorylation of vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta, and Akt and increased phosphorylation of stress-related c-Jun NH2-terminal kinase (JNK). Inhibition of Akt was required for synergism, as a constitutively active Akt protected cells against apoptosis induced by the combination. Alternatively, the JNK inhibitor SP600125 could also protect cells from apoptosis induced by the combination, indicating that both inhibition of Akt and activation of JNK were required for the synergism. These findings show that sorafenib interacts synergistically with bortezomib to induce apoptosis in a broad spectrum of neoplastic cell lines and show an important role for the Akt and JNK pathways in mediating synergism. Further clinical development of this combination seems warranted.     

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo

This study first investigates the anticancer effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) in human nonsmall cell lung cancer cells, A549. Plumbagin has exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21 and reduced amounts of cyclinB1, Cdc2, and Cdc25C. Plumbagin treatment also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Blockade of p53 activity by dominant-negative p53 transfection partially decreased plumbagin-induced apoptosis and G2/M arrest, suggesting it might be operated by p53-dependent and independent pathway. Plumbagin treatment triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that c-Jun NH2-terminal kinase (JNK) is a critical mediator in plumbagin-induced cell growth inhibition. Activation of JNK by plumbagin phosphorylated p53 at serine 15, resulting in increased stability of p53 by decreasing p53 and MDM2 interaction. SP600125 (anthra [1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone), a specific inhibitor of JNK, significantly decreased apoptosis by inhibiting the phosphorylation of p53 (serine 15) and subsequently increased the interaction of p53 and MDM2. SP6000125 also inhibited the phosphorylation of Bcl-2 (Ser70) induced by plumbagin. Further investigation revealed that plumbagin's inhibition of cell growth effect was also evident in a nude mice model. Taken together, these results suggest a critical role for JNK and p53 in plumbagin-induced G2/M arrest and apoptosis of human nonsmall cell lung cancer cells.     

肿瘤坏死因子凋亡诱导配体诱导白血病细胞凋亡中抵抗机制的初步探讨

目的初步探讨肿瘤坏死因子凋亡诱导配体(TRAIL)诱导白血病细胞发生凋亡过程中可能存在的抵抗机制。方法用流式细胞仪检测经 TRAIL 处理后,白血病细胞系 K562、CEM 凋亡情况及线粒体跨膜电位的改变;采用 Western blot 检测 TRAIL 处理后细胞凋亡相关蛋白 Bcl-xL、Bax 及内源性半胱天冬酶(caspase)-8表达情况;并用 ELISA 法检测核转录因子(NF)-kB 活性变化。结果经TRAIL 处理后,K562、CEM 细胞出现不同程度的凋亡,其凋亡指数分别为29.98%、14.1%,后者显著低于前者(P<0.001);线粒体跨膜电位下降,分别为73.25%、25.4%(P<0.01);CEM 细胞中内源性caspase-8的表达水平低于 K562细胞,并且两者均表现出 Bcl-xL 表达上升、Bax 表达下降,在 CEM 细胞中 Bcl-xL/Bax 比例为18.8,显著高于 K562细胞 Bcl-xL/Bax 的比值(5.1);并且在 TRAIL 处理 CEM细胞后早期(2 h)即表现出 NF-kB 活性增加(0.48 μmol·L~(-1)·mg~(-1)蛋白),高于 K562细胞(0.326μmol·L~(-1)·mg~(-1)蛋白(P<0.001)。结论 TRAIL 诱导白血病细胞凋亡过程中,CEM 细胞对药物抵抗原因可能与 CEM 细胞内源性 caspase-8表达水平较低、线粒体内膜敏感程度降低、NF-kB 活性早期增加以及 Bcl-2家族蛋白的表达变化有关。     

Rapamycin and UCN-01 synergistically induce apoptosis in human leukemia cells through a process that is regulated by the Raf-1/MEK/ERK, Akt, and JNK signal transduction pathways

Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-xL and diminished the expression of cyclin D1 and p34(cdc2). Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin-mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.     

Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells

Interactions between histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo2 ligand, were examined in human leukemia cells (e.g., U937, Jurkat, and HL-60). Simultaneous exposure of cells to 100-ng/ml TRAIL with either 1-mM sodium butyrate or 2- micro M suberoylanilide hydroxamic acid resulted in a striking increase in leukemic cell mitochondrial damage, caspase activation, and apoptosis. Lethal effects were significantly diminished in U937 cells ectopically expressing dominant-negative caspase-8, dominant-negative Fas-associated death domain, CrmA (receptor pathway), or Bcl-2 or Bcl-X(L) (mitochondrial pathway). Analysis of mitochondrial events in U937 cells exposed to TRAIL/HDAC inhibitors revealed enhanced Bid activation and Bax translocation, loss of mitochondrial membrane potential, and cytoplasmic release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor. No changes were observed in expression of FLICE-like inhibitory protein, TRAIL receptors, or reactive oxygen species generation. TRAIL/HDAC inhibitor-induced apoptosis triggered caspase-dependent cleavage of p21(WAF1/CIP1); moreover, enforced expression of a nuclear localization signal deletant form of p21(WAF1/CIP1) significantly diminished lethality. Lastly, p27(KIP1), pRb, X-linked inhibitor of apoptosis, and Bcl-2 displayed extensive proteolysis. These findings indicate that coadministration of TRAIL with HDAC inhibitors synergistically induces apoptosis in human myeloid leukemia cells and provide further evidence that simultaneous activation of the extrinsic and intrinsic pathways in such cells leads to a dramatic increase in mitochondrial injury and activation of the caspase cascade.     

AS601245 (1,3-benzothiazol-2-yl (2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile): a c-Jun NH2-terminal protein kinase inhibitor with neuroprotective properties

Recent evidence suggests that activation of the c-Jun NH2-terminal protein kinase (JNK) signal transduction pathway may play a role in ischemia-induced cell death. Thus, preventing the activation of JNK, or c-Jun phosphorylation could be neuroprotective. In the current study, we report that a small molecule, AS601245 (1,3-benzothiazol-2-yl (2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile), which has been shown to inhibit the JNK signaling pathway, promotes cell survival after cerebral ischemia. In vivo, AS601245 (40, 60, and 80 mg/kg) administered i.p. provided significant protection against the delayed loss of hippocampal CA1 neurons in a gerbil model of transient global ischemia. This effect is mediated by JNK inhibition and therefore by c-Jun expression and phosphorylation. A significant neuroprotective effect of AS601245 administered either by i.p. injection (6, 18, and 60 mg/kg) or as i.v. bolus (1 mg/kg) followed by an i.v. infusion (0.6 mg/kg/h) was also observed in rats after focal cerebral ischemia. These data suggest that the use of JNK inhibitors such as AS601245 may be a relevant strategy in the therapy of ischemic insults.     

Sulindac independently modulates extracellular signal-regulated kinase 1/2 and cyclic GMP-dependent protein kinase signaling pathways

Colorectal cancer is the second leading cause of cancer mortality in the United States. Substantial human and animal data support the ability of nonsteroidal anti-inflammatory drugs to cause regression of existing colon tumors and prevent new tumor formation. The mechanism by which the nonsteroidal anti-inflammatory drug sulindac prevents tumor growth is poorly understood and seems complex as sulindac can modulate several growth-related signaling pathways. Sulindac metabolites simultaneously (a) increase cellular cyclic GMP and subsequently activate cyclic GMP-dependent protein kinase (PKG); (b) activate c-jun NH2-terminal kinase (JNK); (c) inhibit extracellular signal-regulated kinase 1/2 (ERK1/2); and (d) decrease beta-catenin protein expression at times and doses consistent with apoptosis. The purpose of this study was to determine if PKG, ERK1/2, JNK, and beta-catenin are independent targets for sulindac in vitro. Pharmacologic activation of PKG with YC-1 increases JNK phosphorylation and induces apoptosis in colon cancer cells without modulating ERK1/2 phosphorylation or beta-catenin protein expression. Inhibition of ERK1/2 with U0126 induces apoptosis but fails to activate JNK phosphorylation or down-regulate beta-catenin protein expression. Cotreatment with U0126 and YC-1 synergistically increases apoptosis in colorectal cancer cells and recapitulates the effects of sulindac treatment on ERK1/2, JNK, and beta-catenin. These results indicate that sulindac metabolites modulate ERK1/2 and PKG pathways independently in colon cancer cells and suggest that the full apoptotic effect of sulindac is mediated by more than one pathway. Using similar combinatorial approaches in vivo may provide more effective, less toxic chemopreventive and chemotherapeutic strategies. Such therapies could dramatically reduce the incidence and death rate from colorectal cancer.     

二硫化二砷诱导T淋巴细胞白血病细胞株凋亡的机制研究

目的探讨二硫化二砷诱导T淋巴细胞白血病细胞株凋亡的分子机制。方法将不同浓度的二硫化二砷与T淋巴细胞白血病细胞株共培养。采用CCK-8方法测定细胞活性;AnnexinⅤ-PI方法测定细胞凋亡率,采用实时定量PCR在基因水平测定凋亡相关因子Bcl-2和Bax的表达。结果随着二硫化二砷药物浓度的增高,细胞活性减低,随着药物作用时间的延长,细胞活性逐渐减弱。其差异均有统计学意义。细胞凋亡结果显示：二硫化二砷药物浓度越高,细胞凋亡率越高,随着药物作用时间的延长,细胞凋亡率也越高。其差异均有统计学意义。实时荧光定量结果显示：与对照组比较,10μmol/L二硫化二砷与CEM细胞共孵育48 h,Bax/Bcl-2的比例升高（P〈0.05）。结论二硫化二砷以时间和剂量依赖的方式抑制CEM细胞生长和诱导凋亡,诱导凋亡的机制与Bax/Bcl-2的比率增加有关。     

[6]-Shogaol,a dietary phenolic compound,induces oxidative stress mediated mitochondrial dependant apoptosis through activation of proapoptotic factors in Hep-2 cells

Ginger (Zingiber officinale) is a well-known herb used in ethnomedicine. [6]-shogaol, a phenolic nature is a major constituent of ginger. In this study, we investigated the anticancer activity of [6]-shogaol in Laryngeal cancer (Hep-2) cells. We demonstrated the effects of [6]-shogaol on the cell growth and apoptosis in Hep-2 cells were analyzed by the generation of reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔYm), DNA damage and apoptotic morphological changes were analyzed by AO/EtBr, AO and Hoechst staining. Further, apoptotic protein expressions were analyzed by western blot analysis. Our results indicated that [6]-shogaol induces apoptosis as evidenced by loss of cell viability, enhanced ROS, lipid peroxidation results in altered mitochondrial membrane potential, increased DNA damage in Hep-2 cells. Further, the prooxidant role of [6]-shogaol inhibit Bcl-2 expression with the simultaneous up-regulation of Bax, Cytochrome c, Caspase-9 and -3 protein expressions were observed in Hep-2 cells. Thus, [6]-shogaol induces apoptosis in Hep-2 cells through inducing oxidative damage and modulate apoptotic marker expressions. Therefore, [6]-shogaol might be used as a therapeutic agent for the treatment of laryngeal cancer.     

c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation

Apoptotic and mitogenic stimuli activate c-Jun NH2-terminal kinases (JNKs) in T cells. Although T cells express both JNK1 and JNK2 isozymes, the absence of JNK2 alone can result in resistance to anti-CD3-induced thymocyte apoptosis and defective mature T cell proliferation. Similar defects in thymocyte apoptosis and mature T cell proliferation, the latter due to reduced interleukin 2 production, are also caused by JNK1 deficiency. Importantly, T cell function was compromised in Jnk1(+/-)Jnk2(+/-) double heterozygous mice, indicating that JNK1 and JNK2 play similar roles in regulating T cell function. The reduced JNK dose results in defective c-Jun NH2-terminal phosphorylation in thymocytes but not in peripheral T cells, in which nuclear factors of activated T cells (NK-ATs)-DNA binding activity is affected. Thus, JNK1 and JNK2 control similar functions during T cell maturation through differential targeting of distinct substrates.     

Dephosphorylation targets Bcl-2 for ubiquitin-dependent degradation: a link between the apoptosome and the proteasome pathway.

Injury of the endothelial cells by the induction of apoptotic cell death may play an important role in the pathophysiology of atherosclerosis and the progression of inflammatory diseases. Here, we demonstrate an essential role for the ubiquitin-dependent proteasome complex in stimulus-induced degradation of the antiapoptotic protein Bcl-2. Bcl-2 is specifically degraded after stimulation of human endothelial cells with tumor necrosis factor (TNF)-alpha in a process that is inhibited by specific proteasome inhibitors. In addition, the mutation of the potential ubiquitin-acceptor amino acids of Bcl-2 provides protection against TNF-alpha- and staurosporine-induced degradation in vitro and in vivo. Moreover, mimicking phosphorylation of the putative mitogen-activated protein (MAP) kinase sites of the Bcl-2 protein (Thr 56, Thr 74, and Ser 87) abolishes its degradation, suggesting a link between the MAP kinase pathway to the proteasome pathway. Finally, inhibition of Bcl-2 degradation either by suppressing ubiquitin-dependent proteasomal degradation or by mimicking continuous phosphorylation of the putative MAP kinase sites in the Bcl-2 protein confers resistance against induction of apoptosis. Thus, the degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome and may thereby amplify the activation of the caspase cascade.     

SODD和Survivin在化疗药物诱导白血病细胞凋亡中的作用

为了研究死亡结构域沉默子（SODD）、survivin、caspase3、caspase8及caspase9在化疗药物诱导白血病细胞凋亡中的变化,以进一步明确凋亡调控基因的调控机制和寻找药物作用新靶点,采用Annexin V/PI双标记流式细胞术检测化疗药物作用后Jurkat细胞凋亡发生率;采用免疫印迹法分析SODD、caspase3、caspase8及caspase9蛋白表达的变化;采用RT-PCR检测survivin mRNA表达的变化;采用免疫组织化学SABC法检测survivin蛋白表达的变化。结果表明：SODD及survivn高表达均抑制肿瘤细胞凋亡,VCR具有时间依赖性特异下调SODD蛋白的功能并能有效诱导Jurkat细胞凋亡,在该凋亡过程中caspase3、caspase8酶原呈时间依赖性逐渐被水解剪切,而caspase9及survivin均无明显变化趋势。结论：SODD参与了VCR诱导白血病细胞凋亡过程,VCR通过下调SODD表达并启动受体配体介导途径诱导Jurkat细胞凋亡,在该过程中线粒体凋亡途径并未被启动。     

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo

In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.     

Potentiation of the lethality of the histone deacetylase inhibitor LAQ824 by the cyclin-dependent kinase inhibitor roscovitine in human leukemia cells

Interactions between the novel histone deacetylase inhibitor LAQ824 and the cyclin-dependent kinase inhibitor roscovitine were examined in human leukemia cells. Pretreatment (24 hours) with a subtoxic concentration of LAQ824 (30 nmol/L) followed by a minimally toxic concentration of roscovitine (10 micromol/L; 24 hours) resulted in greater than additive effects on apoptosis in U937, Jurkat, and HL-60 human leukemia cells and blasts from three patients with acute myelogenous leukemia. These events were associated with enhanced conformational changes in Bax; mitochondrial release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor; and a marked increase in caspase activation. LAQ824/roscovitine-treated cells displayed caspase-dependent down-regulation of p21(CIP1) and Mcl-1 and a pronounced caspase-independent reduction in X-linked inhibitor of apoptosis (XIAP) expression. The lethality of this regimen was significantly attenuated by ectopic expression of XIAP, a nuclear localization signal-defective p21(CIP1) mutant, Mcl-1, and Bcl-2. Combined exposure to LAQ824 and roscovitine resulted in a significant reduction in XIAP mRNA levels and diminished phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Notably, roscovitine blocked LAQ824-mediated differentiation. Finally, LAQ824 and roscovitine individually and in combination triggered an increase in generation of reactive oxygen species; moreover, coadministration of the free radical scavenger N-acetylcysteine prevented LAQ824/roscovitine-mediated mitochondrial injury and apoptosis. Collectively, these findings suggest that combined treatment of human leukemia cells with LAQ824 and roscovitine disrupts maturation and synergistically induces apoptosis, lending further support for an antileukemic strategy combining novel histone deacetylase and cyclin-dependent kinase inhibitors.