Caspase-dependent and -independent mechanisms in apoptosis induced by hydroquinone and catechol metabolites of remoxipride in HL-60 cells

The hydroquinone and catechol like metabolites, NCQ344 and NCQ436 respectively, of the antipsychotic remoxipride have recently been demonstrated to induce apoptosis in myeloperoxidase (MPO)-rich human bone marrow progenitor and HL-60 cells [S.M. McGuinness, R. Johansson, J. Lundstrom, D. Ross, Induction of apoptosis by remoxipride metabolites in HL-60 and CD34+/CD19- human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia, Chem. Biol. Interact. 121 (1999) 253-265]. In the present study, we determined the molecular mechanisms of apoptosis induced by these remoxipride metabolites in HL-60 cells. Our results show that apoptosis was accompanied by phosphatidylserine (PS) exposure, activation of caspases-9, -3, -7 and DNA cleavage. In HL-60 cells treated with the hydroquinone NCQ344 and catechol NCQ436, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp. fluoromethyl ketone (Z-VAD.FMK) blocked DNA cleavage and activation of caspases-9, -3/-7. In addition, PS exposure was significantly but not completely inhibited by Z-VAD.FMK. These results demonstrate that although Z-VAD.FMK inhibitable caspases are necessary for maximal apoptosis induced by NCQ344 and NCQ436, additional caspase-independent processes may orchestrate changes leading to PS exposure during apoptosis induced by the remoxipride polyphenolic metabolites.     

Benzene and the genotoxicity of its metabolites. I. Transplacental activity in mouse fetuses and in their dams

Benzene and some of its metabolites (hydroquinone, phenol, catechol, 1,2,4-benzenetriol, p-benzoquinone, o,o'-biphenol, p,p'-biphenol) have been tested for their capability to induce micronuclei in bone marrow cells of pregnant mice and, transplacentally, in fetal liver cells. Dams are scarcely sensitive to the genotoxic activity of benzene and its metabolites while the latter are able to produce only evident toxic effects. Benzene and hydroquinone transplacentally induce micronuclei in fetal liver cells while all other metabolites show weak or negative genotoxicity, although they produce severe cellular toxicity.     

Characterization of the phenol monooxygenase gene from Chromobacterium violaceum: Potential use for phenol biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol.     

Benzene and the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depression in mouse bone marrow cells

Benzene (880 mg/kg) and 4 of its metabolites, i.e., phenol (265 mg/kg), hydroquinone (80 mg/kg), catechol (40 mg/kg), and p-benzoquinone (5-20 mg/kg) have been tested for their capability to induce micronuclei in bone marrow cells of male mice after oral administration or intraperitoneal injection. Oral administration of benzene shows more activity than intraperitoneal injection, whereas the metabolites show more activity if administered by the latter method. The respective genotoxic strengths of the benzene metabolites are the following: hydroquinone much greater than phenol greater than catechol = p-benzoquinone. This last is active when administered orally.     

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.     

Interactive inhibition of erythroid 59Fe utilization by benzene metabolites in female mice

Using radioiron uptake into erythrocytes as a measure of hematopoiesis, it was demonstrated that benzene inhibited bone marrow function in female mice. Hydroquinone was marginally effective, but the inhibition occurred only at the highest dose tested (100 mg/kg). The combination of phenol and hydroquinone was more effective in reducing erythrocyte production than either chemical given alone. Catechol given alone was not inhibitory but when phenol was added to catechol, erythropoiesis was suppressed, as observed for the phenol and hydroquinone combination. It appears that benzene toxicity may be the result of cooperative inhibitory effects produced by its metabolites.     

Methanogenic degradation of hydroquinone and catechol via reductive dehydroxylation to phenol

Abstract Fermentative degradation of hydroquinone, catechol, and phenol was demonstrated with nearly-homogeneous mixed methanogenic cultures obtained from freshwater sediments and sewage sludge by enrichment with the respective phenolic substrates. Gram-negative short rods predominated in these cultures, together with hydrogen- and acetate-utilizing methanogens. Acetate and methane were the only degradation products. Bacteria enriched with hydroquinone or catechol also degraded phenol and p -hydroxy-benzoate, but not resorcinol or resorcylic acids. Phenol was formed as an intermediate during catechol and hydroquinone degradation, indicating that reductive dehydroxylation was the primary event in degradation of these substrates. Inhibition experiments with bromoethanesulfonate and acetylene indicated that catechol, hydroquinone, and phenol degradation depended on a syntrophic co-operation of fermenting bacteria and hydrogen-oxidizing methanogens.     

Two benzene metabolites, catechol and hydroquinone, produce a synergistic induction of micronuclei and toxicity in cultured human lymphocytes

A mixture of two benzene metabolites, hydroquinone and catechol, produces a striking synergistic genotoxic response in cultured human lymphocytes. This was demonstrated using an anti-kinetochore antibody modification of the micronucleus assay. Treatment with hydroquinone alone or in combination with phenol produced a 3-fold increase in micronucleated cells over background. Treatment with catechol or phenol alone and in combination produced only minor increases in the number of micronucleated cells. In contrast, simultaneous treatment with equimolar (75 microM) concentrations of hydroquinone and catechol resulted in a greater than 16-fold induction of micronucleated cells. Given an additivity model, 20 additional micronucleated cells would be expected (after correcting for background frequencies), yet 140 were observed. Further analysis revealed that over 90% of the micronucleated cells stained positively for kinetochores, indicating a high probability that these micronuclei contain entire chromosomes. This synergistic response appears to occur only at equimolar levels of hydroquinone and catechol. These results suggest that these metabolites are acting together to disrupt the mitotic spindle and interfere with chromosome segregation. These data provide further support for the hypothesis that multiple metabolites acting in concert are involved in the benzene-induced genotoxicity and leukemia in humans.     

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.     

Induction of sister-chromatid exchanges in human lymphocytes by microsomal activation of benzene metabolites

Metabolic activation of the benzene metabolites, catechol, hydroquinone, and phenol, by rat-liver microsomes and an NADPH-generating system (S9 mix) caused an increased induction of sister-chromatid exchanges (SCEs) in cultured human lymphocytes. There were different optimal concentrations of S9 mix for converting each benzene metabolite into further reactive forms that could induce SCE-forming lesions. The data indicate that catechol and hydroquinone can be optimally metabolized to produce reactive species, presumably benzo(semi)quinones, under conditions of lower metabolic activity than those necessary for phenol and benzene.     

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.     

Metabolomics using 1H-NMR of apoptosis and Necrosis in HL60 leukemia cells: differences between the two types of cell death and independence from the stimulus of apoptosis used

High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.     

Antitumour and antimalarial activity of artemisinin–acridine hybrids

Artemisinin–acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2–4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.     

Induction of phospholipid hydroperoxide glutathione peroxidase in human polymorphonuclear neutrophils and HL60 cells stimulated with TNF-alpha

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is characterized as an important enzyme for protecting cells from oxidative stress-induced apoptosis and regulating the production of leukotrienes and prostanoids in cells overexpressing PHGPx. We studied whether the expression level of PHGPx fluctuates in polymorphonuclear leukocytes (PMNs) which were exposed to reactive oxygen species (ROS) and inflammatory cytokines at an inflammation site. Human peripheral PMNs up-regulated the expression level of PHGPx following culture with TNF-alpha, but not with IL-1beta, IL-8, and GRO. The up-regulated PHGPx expression was also observed in neutrophil-like cells that differentiated from the human leukemia cell line HL60 only after stimulation with TNF-alpha. However, macrophage-like differentiated HL60 cells and other cell lines, A498, ECV304, HeLa, U937, and HEK293, showed no increase in the PHGPx expression. This up-regulation of PHGPx was inhibited by treatment with the anti-oxidants, pyrrolidine dithiocarbamate, and N-acetyl-L-cysteine, and by inhibitors of NFkappaB and Src kinases. The stimulation of neutrophil-like differentiated HL60 cells with TNF-alpha induced activation of NFkappaB and c-Src kinase, and the activation was attenuated by treatment with the anti-oxidants. Up-regulation in neutrophil-like HL60 cells was also observed following exposure to H(2)O(2). These results indicate that activation of NFkappaB and/or Src kinases through ROS signaling may be involved in the up-regulation of the PHGPx in human PMNs stimulated by TNF-alpha.     

A novel method for the separation and quantitation of benzene metabolites using high-pressure liquid chromatography

A method for the separation of benzene metabolites using reverse-phase high-pressure liquid chromatography is described. The antoxidant, ascorbic acid is added to an aqueous mixture of 1,2,4-benzenetriol, hydroquinone, catechol, and phenol, to prevent autooxidation. The eluting solvents are equilibrated with nitrogen, degassed, and maintained under a nitrogen atmosphere during the analysis. A highly resolved and reproducible profile of the metabolites is achieved under these conditions. This method should prove useful in a number of pharmacokinetic studies where the biotransformation of the parent compound to autooxidizable species such as polyphenols and quinones precludes analysis under aerobic conditions.     

Pterostilbene and 3'-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.     

Phenol degradation by yeasts isolated from industrial effluents

Yeast strains of the genera Aureobasidium, Rhodotorula and Trichosporon were isolated from stainless steel effluents and tested for their ability to utilize phenol as the sole carbon source. Fourteen strains grew in the presence of up to 10 mm phenol. Only the strain Trichosporon sp. LE3 was able to grow in the presence of up to 20 mm phenol. An inhibitory effect was observed at concentrations higher than 11 mm, resulting in reduction of specific growth rates. Phenol degradation was a function of strain, time of incubation and initial phenol concentration. All strains exhibited activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission. Addition of glucose and benzoate reduced the phenol consumption rate, and both substrates were used simultaneously. Glucose concentrations higher than 0.25% inhibited the induction of phenol oxidation by non-proliferating cells and inhibited phenol oxidation by pre-induced cells.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.     

Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (elL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70-fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR I thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant elL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCRI did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of elL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.