The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells

Mitogen-activated protein kinase (MAPK) is activated in the majority of melanomas, and its activity is essential for cell survival. In this report, we examined the effects of a novel raf inhibitor BAY 43-9006 on melanoma cell viability and intracellular signaling and found that it induces apoptosis through a caspase-independent mechanism. At concentrations that suppress extracellular signal-regulated kinase (ERK) phosphorylation, BAY 43-9006 dephosphorylates Bad on Ser(75) and Ser(99), activates Bak and Bax, and reduces the mitochondrial transmembrane potential. BAY 43-9006 (sorafenib) down-modulates the levels of Bcl-2 and Bcl-X(L) in a MAPK-independent manner in A2058 and SKMEL5 melanoma cells but not in the more resistant A375 cells. Of the three lines tested, only A375 cells were rescued from BAY 43-9006-induced apoptosis by knocking down Bad. BAY 43-9006 induced poly(ADP-ribose) polymerase cleavage and the mitochondrial release of cytochrome c and SMAC. However, the pan-caspase inhibitor Z-VAD-fmk had only a modest protective effect against the drug, suggesting that BAY 43-9006-induced apoptosis is largely caspase independent. BAY 43-9006 but not the MAP/ERK kinase inhibitors PD98059 or U0126 induced the nuclear translocation of apoptosis-inducing factor (AIF) in A2058 and SKMEL5 cells, and the introduction of a small interfering RNA (siRNA) for AIF partially protected these cells from BAY 43-9006-induced apoptosis. The AIF siRNA had little effect in A375 cells, in which drug-induced AIF release was negligible. These data indicate that in sensitive cell lines, BAY 43-9006-induced apoptosis is independent of Bad dephosphorylation and caspase activation and largely mediated through the nuclear translocation of AIF.     

Melphalan-induced apoptosis in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim complex

Multiple myeloma (MM) is a rapidly fatal plasma-cell malignancy that evolves mainly in the bone marrow. Melphalan is widely used to treat patients with MM but as yet its mechanisms of action are poorly documented. In the current study, we demonstrate that melphalan induces a drastic downregulation of Mcl-1L, Bcl-x(L) and BimEL in human melphalan-sensitive myeloma cells while the most potent proapoptotic isoforms, BimL and S, are affected to a lesser extent. Moreover, Mcl-1L and BimEL disappearance is associated with the generation of proapoptotic cleaved forms generated by a caspase cleavage. In myeloma cells, we have previously shown that Mcl-1 neutralizes the proapoptotic function of Bim and therefore, prevents the activation of death effectors. In this study, we demonstrate that melphalan disrupts the Mcl-1/Bim complex whereas the Bcl-2/Bim complex is not modified. The disappearance of full length Mcl-1 allows the release of Bim isoforms, particularly L and S, which can exert their proapoptotic function and leads to Bax activation and cytochrome c release. Thus, we can hypothesize that the cleaved 26 kDa proapoptotic Mcl-1 and the 19 and 12 kDa of Bim, generated during melphalan treatment could contribute to the amplification loop of apoptosis.     

The DNA damage-induced decrease of Bcl-2 is secondary to the activation of apoptotic effector caspases

Apoptosis induced by DNA-damaging agents or radiation mainly proceeds through death receptor-independent caspase activation. The release of mitochondrial apoptogenic proteins, such as cytochrome c, into the cytoplasm leading to Apaf1-dependent activation of caspase-9 is a key event in this pathway. The permeability of the mitochondrial outer membrane is regulated by the various pro- and antiapoptotic Bcl-2 family proteins, and it is thought that DNA damage triggers apoptosis through the downregulation of antiapoptotic Bcl-2. Using murine embryonic fibroblasts (MEF) deficient and proficient in Apaf1, we show that DNA-damaging agents and radiation lead to a decline in Bcl-2 protein only in wt MEF, but not in apaf1(-/-) MEF, which are defective in the activation of effector caspases and apoptosis. In contrast, the induction of proapoptotic Noxa, the activation of Bax, the cytoplasmic release of cytochrome c, as well as a drop of the mitochondrial transmembrane potential Deltapsim are equally observed in wt and apaf1(-/-) MEF following DNA damage. Moreover, the loss of Bcl-2 protein occurring in wt MEF can be prevented by caspase inhibition. Hence, the activation of proapoptotic Bcl-2 family proteins rather than the downregulation of antiapoptotic Bcl-2 mediates the primary signal in the DNA damage-induced release of mitochondrial apoptogenic proteins in MEF.     

Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells

The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.     

2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process

The effects of 2-Methoxyestradiol (2ME)-induced apoptosis was examined in human leukemia cells (U937 and Jurkat) in relation to mitochondrial injury, oxidative damage, and perturbations in signaling pathways. 2ME induced apoptosis in these cells in a dose-dependent manner associated with release of mitochondrial proteins (cytochrome c, AIF), generation of reactive oxygen species (ROS), downregulation of Mcl-1 and XIAP, and inactivation (dephosphorylation) of Akt accompanied by activation of JNK. In these cells, enforced activation of Akt by a constitutively active myristolated Akt construct prevented 2ME-mediated mitochondrial injury, XIAP and Mcl-1 downregulation, JNK activation, and apoptosis, but not ROS generation. Conversely, 2ME lethality was potentiated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Furthermore, in U937 cells, the hydrogen peroxide scavenger catalase and a superoxide dismutase (SOD) mimetic, TBAP, blocked these events, as well as Akt inactivation. Interruption of the JNK pathway by pharmacologic or genetic (e.g. siRNA) means attenuated 2ME-induced mitochondrial injury, XIAP and Mcl-1 downregulation, and apoptosis. Collectively, these findings suggest a hierarchical model of 2ME-related apoptosis induction in human leukemia cells in which 2ME-induced oxidative injury represents a primary event resulting in Akt inactivation, leading, in turn, to JNK activation, and culminating in XIAP and Mcl-1 downregulation, mitochondrial injury, and apoptosis. They also suggest that in human leukemia cells, the Akt pathway plays a critical role in mediating the response to oxidative stress induced by 2ME.     

Failure to activate caspase 3 in phorbol ester-resistant leukemia cells is associated with resistance to apoptotic cell death

The protein kinase C (PKC)-specific inhibitor, Ro-31-8220, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with the PKC-specific inhibitor RO-31-8220. For this, we used the U937 human leukemia cell line and a phorbolmyristate acetate (PMA)-resistant derivative cell line, R-U937. Ro-31-8220 treatment of U937 cells leads to apoptosis, which is accompanied by activation of caspase 3 (as measured by decreased levels of the 32kDa inactive form and increased proteolytic cleavage of phospholipase C (PLC)-gamma1). The broad-range caspase inhibitor z-VAD-fmk inhibits this induction of apoptosis, supporting a direct link between caspase activation and Ro-31-8220 induction of apoptosis. This activation of apoptosis is also accompanied by release of cytochrome c, but not by altered expression of Bcl-2 family protein or IAP family proteins. In R-U937 cells, Ro-31-8220 fails to cause release of cytochrome c, activation of caspase 3, or apoptosis. Activation of Akt occurs to a greater extent in the R-U937 cells than the U937 cells and thus might be related to protection from Ro-31-8220-induced apoptosis.     

Human THP-1 monocytic leukemic cells induced to undergo monocytic differentiation by bryostatin 1 are refractory to proteasome inhibitor-induced apoptosis

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. We demonstrated that treatment of THP-1 human monocytic leukemia cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death through an apoptotic pathway. Apoptosis in THP-1 cells induced by Z-LLL-CHO involved a cytochrome c-dependent pathway, which included the release of mitochondrial cytochrome c, activation of caspase-9 and -3, and cleavage of Bcl-2 into a shortened 22-kDa fragment. Induction of apoptosis by protease inhibitor also was detected in U937 and TF-1 leukemia cell lines and cells obtained from acute myelogenous leukemia patients but not in normal human blood monocytes. Treatment of human blood monocytes with Z-LLL-CHO did not induce apoptosis or Bcl-2 cleavage in these cells that rarely proliferate. Interestingly, when THP-1 cells were induced to undergo monocytic differentiation by bryostatin 1, a naturally occurring protein kinase C activator, they were no longer susceptible to apoptosis induced by Z-LLL-CHO. Bryostatin 1-induced differentiation of THP-1 cells was associated with growth arrest, acquisition of adherent capacity, and expression of membrane markers characteristic of blood monocytes. Likewise, differentiated THP-1 cells were refractory to Z-LLL-CHO-induced cytochrome c release, caspase activation, and Bcl-2 cleavage. Resistance to Z-LLL-CHO-induced apoptosis in differentiated THP-1 cells was not due to cell cycle arrest. These findings show that the action of proteasome inhibitors is mediated primarily through a cytochrome c-dependent pathway and induces apoptosis in leukemic cells that are not differentiated.     

DADS通过MAPK和PI3K/Akt 信号通路下调Mcl-1诱导白血病细胞凋亡*

目的:二烯丙基二硫（DADS）为天然植物大蒜中的提取物,能抑制多种肿瘤细胞生长,本文探讨丝裂原激活的蛋白激酶（MAPKs）、3- 磷酸肌醇激酶（PI 3K/Akt）信号通路和Bcl- 2 家族成员在DADS诱导的人白血病HL- 60细胞凋亡中的作用。方法:利用流式细胞术检测DADS 诱导的白血病细胞凋亡,Western blot研究MAPKs和PI 3K/Akt信号通路在DADS 诱导的人白血病HL- 60细胞凋亡中的变化及对Bcl- 2 家族凋亡相关蛋白表达的影响。结果:DADS呈浓度和时间依赖性地诱导人白血病HL- 60细胞凋亡,在此过程中ERK/MAPK 和PI 3K/Akt信号通路被抑制,而p38MAPK 信号通路被激活,ERK/MAPK 和PI 3K/Akt信号通路通过降低Mcl-1（myeloid cell leukemia-1）和升高Bax 的表达诱导人白血病细胞凋亡,而p38MAPK 则不是通过调控Mcl-1 和Bax 的表达诱导人白血病细胞凋亡,进一步利用RNA干扰技术沉默Mcl-1 基因可增加DADS对HL- 60细胞增殖抑制和诱导凋亡作用。结论:MAPK 和PI 3K/Akt信号通路通过下调Mcl-1 的表达参与了DADS诱导的HL- 60细胞凋亡作用。      

Bisindolylmaleimide IX is a potent inducer of apoptosis in chronic lymphocytic leukaemic cells and activates cleavage of Mcl-1.

New agents are required for the treatment of chronic lymphocytic leukaemia (CLL). We show here that a protein kinase C inhibitor, bisindolylmaleimide IX, is a potent inducer of apoptosis in CLL cells, and investigate the mechanisms by which this is induced. Bisindolylmaleimide IX induced a conformational change and subcellular redistribution of Bax from the cytosol to the mitochondria, resulting in the release of the proapoptotic mediators cytochrome c, Smac and Omi/HtrA2 from the mitochondrial inner membrane space. This was followed by the activation of caspase-9 as the apical caspase and subsequent activation of effector caspases. CLL cells undergoing apoptosis showed a rapid caspase-mediated cleavage of Mcl-1, an antiapoptotic member of the Bcl-2 family implicated in CLL survival and poor prognosis. This cleavage was mediated primarily by caspase-3. Cleavage of Mcl-1 may provide a feed-forward amplification loop, resulting in the rapid induction of apoptosis. Bisindolylmaleimide IX or a related derivative may be of clinical use in the treatment of CLL.     

Acacetin induces apoptosis in human T cell leukemia Jurkat cells via activation of a caspase cascade

Flavonoids are naturally occurring antioxidants, with several flavonoids shown to have chemopreventive effects on cancer. We investigated the effects of the flavonoid acacetin on human T cell leukemia Jurkat cells. Acacetin inhibited the proliferation of Jurkat cells by inducing apoptosis in a concentration- and time-dependent manner. Acacetin-induced cell death was characterized by changes in nuclear and cell morphology. Treatment of Jurkat cells with acacetin also induced caspase-3, -8 and -9 activities in a time-dependent manner. Acacetin-induced apoptosis was blocked by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor and a caspase-8 inhibitor, but not by a caspase-9 inhibitor. In addition, acacetin promoted the expression of FAF1, phosphor-FADD, Apaf-1 and cytochrome c. Acacetin-induced apoptosis was also accompanied by upregulation of Bax, and downregulation of Bcl-2. Taken together, these results suggest that acacetin may induce apoptosis in T cell leukemia cells, possibly by activating the Fas-mediated pathway. These findings may help in designing cancer therapeutic and chemopreventive agents.     

Involvement of proapoptotic Bcl-2 family members in parthenolide-induced mitochondrial dysfunction and apoptosis

Parthenolide is a sesquiterpene lactone responsible for the bioactivities of Feverfew. Besides its potent anti-inflammatory effect, this compound has recently been reported to induce apoptosis in cancer cells, possibly through mitochondrial dysfunction. In the present study, we attempted to examine parthenolide-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members. Using a human colorectal cancer cell line COLO205, we first demonstrated that parthenolide acted through the cell death receptor pathway to activate caspase 8. Following caspase 8 activation, Bid, a proapoptotic Bcl-2 member, was cleaved and this cleavage then triggered Bax conformational changes and Bax translocation from cytosol to mitochondrial membrane. Meanwhile, another proapoptotic protein, Bak, was up-regulated and oligomerized on the mitochondrial membrane. All these alterations were found to be prerequisite for the subsequent release of proapopototic mitochondrial proteins, including cytochrome c and Samc, in parthenolide-treated cells. Moreover, selective inhibition of caspase 8 activity by a synthetic caspase inhibitor (IETD-FMK) or overexpression of a viral protein (CrmA) suppressed the cleavage of Bid, conformational changes of Bax, cytochrome c release, and apoptosis. Therefore, the proapoptotic Bcl-2 family members are important mediators relaying the cell death signaling elicited by parthenolide from caspase 8 to downstream effector caspases such as caspase 3, and eventually to cell death.     

Resveratrol-mediated sensitisation to TRAIL-induced apoptosis depends on death receptor and mitochondrial signalling

Natural food products such as resveratrol have gained considerable attention as cancer chemopreventive agents. In the present study, we investigated the potential of resveratrol to overcome the resistance of tumour cells against TRAIL. While resveratrol enhanced TRAIL-induced apoptosis through G1 cell cycle arrest and survivin depletion, resveratrol failed to sensitise cells with high expression levels of Bcl-2 or FADD-DN. Interestingly, overexpression of Bcl-2 or FADD-DN did not interfere with resveratrol-mediated cell cycle arrest or survivin depletion, but blocked release of cytochrome c and Smac from mitochondria into the cytosol, enhanced caspase activation and apoptosis upon combined treatment with resveratrol and TRAIL indicating that overexpression of Bcl-2 or FADD-DN decoupled the effect of resveratrol on the cell cycle and apoptosis. Similarly, cell cycle arrest at G1 using the cell cycle specific inhibitor mimosine or downregulation of survivin expression by antisense oligonucleotides failed to enhance TRAIL-induced apoptosis in Bcl-2- or FADD-DN-transfected cells. Likewise, inhibition of caspase activity using the broad range caspase inhibitor zVAD.fmk did not interfere with resveratrol-mediated cell cycle arrest and survivin depletion, while blocking apoptosis upon combined treatment with resveratrol and TRAIL. Thus, resveratrol is a potent sensitiser for TRAIL in certain tumours. However, it may be ineffective in others, e.g. in tumours with enhanced Bcl-2 expression or defective death receptor signalling.     

The Bcl-2 transgene protects T cells from renal cell carcinoma-mediated apoptosis.

PURPOSE: Tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor immunity. Using coculture experiments, it has been shown that tumor lines stimulate T-cell apoptosis by a pathway involving a mitochondrial permeability transition and cytochrome c release. Activated T cells express abundant levels of Bcl-2, an antiapoptotic molecule that would be expected to confer resistance to such tumor-mediated killing. We examined the mechanism by which Bcl-2 is dysregulated in T cells exposed to the renal tumor line SK-RC-45, and we determined whether overexpressing Bcl-2 protects T cells from tumor-mediated apoptosis. EXPERIMENTAL DESIGN: Activated T lymphocytes and Jurkat cells transfected or not transfected with Bcl-2 were exposed to SK-RC-45 for 48-72 h. After coculture, lymphocytes were analyzed for Bcl-2 expression using Western analysis and for tumor-induced apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling. The role of SK-RC-45-stimulated caspase activation in degrading T-cell Bcl-2 was assessed using a pan-caspase inhibitor, as well as a specific inhibitor of caspase-9. RESULTS: The renal cell carcinoma cell line SK-RC-45 sensitizes peripheral blood activated T lymphocytes and Jurkat cells to apoptosis by a mechanism that involves degradation of the antiapoptotic protein Bcl-2. The SK-RC-45-induced modulation of lymphocyte Bcl-2 levels was largely caspase independent because pretreatment of T cells with pan-caspase inhibitor III or an inhibitor of caspase-9 had minimal or no effect on stabilizing the protein, although it did provide protection against apoptosis. Overexpression of Bcl-2 protected Jurkat cells from tumor-mediated killing. CONCLUSIONS: Bcl-2 inhibition is a mechanism by which tumors may render lymphocytes sensitive to other tumor-derived, proapoptotic stimuli.     

Inactive caspase 3 activates Akt in human leukemia cells susceptible or resistant to apoptosis induced by phorbol ester

Phorbol 12-myristate 13-acetate (PMA) is a protein kinase C (PKC) activator and tumor promoter that induces terminal differentiation in human myeloid leukemia cells. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest and apoptosis. In the present studies, we determined the effect of high intracellular levels of the anti-apoptosis Bcl-2 protein on caspase 3 activation and cyctochrome c release during phorbol ester 12-myristate 13-acetate (PMA)-induced apoptosis. For this, we used the U937 cells, Bcl-2 overexpressed U937 cells (U937/Bcl-2) and the PMA-resistant derivative cell line R-U937. The G1 arrest of U937 cells and U937/Bcl-2 cells induced by treatment with 20 nM PMA is associated with cyclin A down-regulation and accumulation of p21, cdks inhibitor. However, PMA had no effect on the levels of cyclin A expression and p21 expression under the same conditions of time and concentration of PMA in the R-U937 cells. Treatment with 20 nM PMA for 24 h produced morphological features of apoptosis and DNA fragmentation in U937 and U937/Bcl-2 cells, but not in R-U937 cells. This was associated with the caspase 3 activation and cyctochrome c release. R-U937 cells exhibited less cytochrome c release and sustained phosphorylation level of Akt during PMA-induced apoptosis. These findings indicate that R-U937 cells are resistant to PMA-induced apoptosis by a mechanism of the signaling defect in the activation of the caspase 3 that is involved in the execution of apoptosis.     

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax)

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.     

Sensitization to CD95 ligand-induced apoptosis in human glioma cells by hyperthermia involves enhanced cytochrome c release

CD95L-induced apoptosis involves caspase activation and is facilitated when RNA and protein synthesis are inhibited. Here, we report that hyperthermia sensitizes malignant glioma cells to CD95L- and APO2L-induced apoptosis in the absence, but not in the presence, of inhibitors of RNA and protein synthesis. Hyperthermia does not alter CD95 expression at the cell surface and does not modulate the morphology of CD95-mediated cell death on electron microscopy. Bcl-2 gene transfer inhibits apoptosis and abrogates the sensitization mediated by hyperthermia. Hyperthermia does not overcome resistance to apoptosis conferred by the viral caspase inhibitor, crm-A, indicating the absolute requirement for the activation of crm-A-sensitive caspases, probably caspase 8, for apoptosis. CD95L-evoked DEVD-amc-cleaving caspase activity is enhanced by hyperthermia, suggesting that hyperthermia operates upstream of caspase processing to promote apoptosis. There is no uniformly enhanced processing of three caspase 3 substrates, poly-ADP ribose polymerase (PARP), protein kinase C (PKC) delta and DNA fragmentation factor (DFF) 45. Yet, hyperthermia promotes CD95L-evoked DNA fragmentation. Interestingly, hyperthermia enhances the CD95L-evoked release of cytochrome c in the absence, but not in the presence, of CHX. In contrast, the reduction of the mitochondrial membrane potential is enhanced by hyperthermia both in the absence and presence of CHX, and enhanced cytochrome c release is not associated with significantly enhanced caspase 9 processing. The potentiation of cytochrome c release at hyperthermic conditions in the absence of CHX is abrogated by Bcl-2. Thus, either hyperthermia or inhibition of protein synthesis by CHX potentiate cytotoxic cytokine-induced apoptosis. These pathways show no synergy, but rather redundance, indicating that CHX may function to promote apoptosis in response to cytotoxic cytokines by inhibiting the synthesis of specific proteins whose synthesis, function or degradation is temperature-sensitive.     

Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA).

Interactions between the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) and the cyclin-dependent kinase (CDK) inhibitor flavopiridol (FP) were examined in human leukemia cells. Simultaneous exposure (24 h) of myelomonocytic leukemia cells (U937) to SAHA (1 microM) and FP (100 nM), which were minimally toxic alone (1.5 +/- 0.5% and 16.3 +/- 0.5% apoptosis respectively), produced a dramatic increase in cell death (ie 63.2 +/- 1.9% apoptotic), reflected by morphology, procaspase-3 and -8 cleavage, Bid activation, diminished DeltaPsi(m), and enhanced cytochrome c release. FP blocked SAHA-mediated up-regulation of p21(CIP1) and CD11b expression, while inducing caspase-dependent Bcl-2 and pRb cleavage. Similar interactions were observed in HL-60 and Jurkat leukemic cells. Enhanced apoptosis in SAHA/FP-treated cells was accompanied by a marked reduction in clonogenic surivival. Ectopic expression of either dominant-negative caspase-8 (C8-DN) or CrmA partially attenuated SAHA/FP-mediated apoptosis (eg 45 +/- 1.5% and 38.2 +/- 2.0% apoptotic vs 78 +/- 1.5% in controls) and Bid cleavage. SAHA/FP induced-apoptosis was unaffected by the free radical scavenger L-N-acetyl cysteine or the PKC inhibitor GFX. Finally, ectopic Bcl-2 expression marginally attenuated SAHA/FP-related apoptosis/cytochrome c release, and failed to restore clonogenicity in cells exposed to these agents. Together, these findings indicate that SAHA and FP interact synergistically to induce mitochondrial damage and apoptosis in human leukemia cells, and suggest that this process may also involve engagement of the caspase-8-dependent apoptotic cascade.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.