Mechanism of fenretinide (4-HPR)-induced cell death

4-HPR (fenretinide) is a synthetic analog of retinoic acid (RA) whose potential as a chemopreventative agent has gained support from in vitro and animal experiments and in limited clinical trials. Comparative analyses of cellular, biochemical, and molecular properties of fenretinide with RA using various tissue culture cells reveal that a key distinction between these two retinoids lies in the ability of fenretinide to induce programmed cell death, also known as apoptosis. Here we review the composite evidence for induction of apoptosis in fenretinide-treated cells. Assays used to validate apoptosis in various cell types are also summarized. Apoptosis in response to fenretinide primarily occurs by a receptor-independent mechanism, which is accompanied by increases in signaling molecules, e.g., ceramide, and cysteine-dependent aspartate-directed proteases, termed caspases, including execution caspase-3. Both caspase-3 inhibitor DEVD-CHO and ceramide synthase inhibitor fumonisin B₁ (FB₁) block fenretinide-induced apoptosis. Increase in caspase-3 appears to result from fenretinide-elicited stabilization of procaspase-3 zymogen. We also review apoptotic regulatory proteins such as inhibitor of apoptosis (IAPs) and second mitochondria-derived activator of caspase (SMACs) that participate in the coordinate control of caspase activities. The existence of a large number of proteins capable of modulating apoptosis via activation or inhibition of caspases, coupled with the fact that both the initiation and execution phases of apoptosis utilize pre-existing zymogens, which, once set in motion, culminates in an irreversible apoptotic cascade, raise the possibility that the on/off switch of apoptosis is linked to an intricate intracellular regulatory network, capable of responding to external stimuli such as fenretinide. This network functions to provide checks/balances of the need for apoptosis as well as to minimize and prevent untimely errors in apoptosis. We suggest that dynamic and coordinated regulation of apoptosis by such a hypothetical network in vivo may involve co-localization of pro- and anti-apoptotic proteins and their respective activators/inhibitors in a macromolecular modular unit which we propose to be named caspasomes. Fenretinide also induces apoptosis by elevating reactive oxygen species (ROS), unrelated to changes in ceramide-caspases. Thus multiple, distinct pathways contribute to the induction of apoptosis by fenretinide.     

The inherent cellular level of reactive oxygen species: One of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As₂O₃)-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As₂O₃. Low concentration of As₂O₃ was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As₂O₃, and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As₂O₃. Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As₂O₃-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As₂O₃.     

Mechanisms of fenretinide-induced apoptosis

Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization.     

Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis.

The nature of the signaling pathway(s) which initiate drug-triggered apoptosis remains largely unknown and is of fundamental importance in understanding cell death induced by chemotherapeutic agents. Here we show that in the leukemic cell lines U937 and HL-60, daunorubicin, at concentrations which trigger apoptosis, stimulated two distinct cycles of sphingomyelin hydrolysis (approximately 20% decrease at 1 microM) within 4-10 min and 60-75 min with concomitant ceramide generation. We demonstrate that the increase in ceramide levels, which precedes apoptosis, is mediated by a neutral sphingomyelinase and not by ceramide synthase. Indeed, potent ceramide synthase inhibitors such as fumonisin B1 did not affect daunorubicin-triggered sphingomyelin hydrolysis, ceramide generation or apoptosis. In conclusion, we provide evidence that daunorubicin-triggered apoptosis is mediated by a signaling pathway which is initiated by an early sphingomyelin-derived ceramide production.     

Mechanisms of free-radical induction in relation to fenretinide-induced apoptosis of neuroblastoma

The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro l-argenine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A(2) or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.     

Effector mechanisms of fenretinide-induced apoptosis in neuroblastoma

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.     

Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells

Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI), is approved for the second-line treatment of chronic myeloid leukemia (CML) in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML) are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA). We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b⁺ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b⁺ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨ_m) in CD11b⁺ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH₂, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH₂ cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH₂ cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies. Accepted: 26 November 1999     

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

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

Background

The 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 Results

HF 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.

Significance

Our 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.     

A link between apoptosis and degree of phosphorylation of high mobility group A1a protein in leukemic cells

Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated.     

Accumulation of major stress protein 70kDa protects myeloid and lymphoid cells from death by apoptosis

The major heat shock protein, hsp70, is known to contribute to the mechanisms of cell protection against a variety of stress and cytotoxic factors, providing an increase of cell survival. Whether hsp70 could be implicated in the rescue of cells from stress-induced death proceeding on apoptotic pathway is not well established. Here we report that susceptibility of myeloid and lymphoid cell lines to apoptosis induced by heat shock or ethanol coincides with hsp70 content and can be modulated by changes in expression of this protein. Cells of lymphoid and myeloid lines differing in basal and inducible level of the protein were tested. The cells containing higher amounts of hsp70 (U937, Jurkat, Molt4) were more resistant to the apoptosis-inducing stimuli then cells which accumu-late lower amounts of the protein (HL60) and especially those lacking the protein (NSO). Inhibition of hsp70 accumulation by quercetin made cells more susceptible to the same apoptotic inducer. Enhancement of hsp70 expression by previous heating or by liposomal delivery of the exogenic protein to the cells lacking hsp70 made them more resistant to apoptosis. The possible mechanisms of the hsp70 protective effect in apoptosis are discussed.     

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl₂) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (ΔΨ m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl₂-induced mitochondrial Δ Ψ m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl₂-induced HIF-1α protein accumulation. Moreover, inducible expression of HIF-1α gene dose not alter DFO and CoCl₂-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl₂-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1α-independent mechanisms, DFO and CoCl₂-induced apoptosis involves different initiating signal pathways that remain to be investigated.     

Ceramide signaling in fenretinide-induced endothelial cell apoptosis

Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.     

SMT-A07, a 3-(Indol-2-yl) indazole derivative, induces apoptosis of leukemia cells in vitro

N-(2-(1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide (SMT-A07) is a novel 3-(Indol-2-yl) indazole derivative. The anticancer activities in vitro and the cell apoptosis-induction abilities of SMT-A07 on human leukemia HL60 and NB4 cell lines were investigated in this study. The results of MTT assay showed SMT-A07 was a potential and highly efficient antitumor compound with IC₅₀ values ranging from 0.09 to 1.19 μM in five leukemia cell lines. SMT-A07 treatment for 24 h caused the increment of apoptosis rate from 6.88 to 49.72% in HL60 cells and from 8.72 to 56.28% in NB4 cells by flow cytometry analysis. Agarose gel electrophoresis showed DNA fragmentation that appeared after cells were exposed to SMT-A07. After SMT-A07 incubation, DAPI staining revealed the presence of DNA fragmentation, and perinuclear apoptotic body. SMT-A07 also resulted in a loss of ΔΨm in both HL60 and NB4 cells by JC-1 staining. Moreover, apoptosis-related proteins were examined by western blotting to explore the mechanism of its cytotoxicity. SMT-A07 exposure caused down-regulation and cleavage of procaspase-8, procaspase-3, Bid, PARP and up-regulation of cleaved caspase-8, cleaved caspase-3, PARP (Cleaved Fragment). In addition, the presence of pan-caspase inhibitor BOC-D-FMK prevented cells from caspase-3 activation, PARP cleavage, and subsequent apoptosis. Our study demonstrates that SMT-A07 displays an apparent antitumor activity with extensive anti-leukemia spectrum, and SMT-A07 can induce the apoptosis of HL60 and NB4 cells activation of the caspase cascade, which deserves further development.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c mice

Fumonisin B₁ is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B₁ toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B₁ on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B₁ subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B₁ treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 281–289, 1998