Antifungal activity of miltefosine against both azole-susceptible and -resistant Aspergillus strains

Miltefosine, an alkylphosphocholine, has been approved recently for the treatment of visceral leishmaniasis. Miltefosine has shown promise as a treatment for paracoccidioidomycosis, and has mixed activity against other fungi and yeast. There are limited data on the in-vitro activity of miltefosine against azole-resistant and -susceptible Aspergillus spp. As such, the aim of this study was to determine the in-vitro activity of miltefosine against Aspergillus strains. Miltefosine was tested against 108 azole-susceptible and -resistant Aspergillus strains isolated from Iran and other countries using the broth microdilution method. Miltefosine was found to be effective against azole-resistant Aspergillus isolates, with minimum inhibitory concentrations (MICs) ranging from 1.562 to 6.25 µg/mL. MIC₅₀ and MIC₉₀ were 1.562 and 3.125 µg/mL, respectively. Miltefosine had a higher geometric mean MIC (2.459 µg/mL) for wild-type Aspergillus isolates than itraconazole (0.220 µg/mL) and voriconazole (0.298 µg/mL). No significant difference was found between miltefosine MICs for azole-resistant Aspergillus isolates and azole-susceptible Aspergillus isolates (P>0.05). Miltefosine appears to have good in-vitro activity against azole-resistant Aspergillus strains, according to these findings. Furthermore, the findings suggest that miltefosine could be used to treat infections caused by azole-resistant Aspergillus spp.     

Effects of miltefosine on membrane permeability and accumulation of [Tc]-hexakis-2-methoxyisobutyl isonitrile, 2-[F]fluoro-2-deoxy-d-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3-4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.     

Effect of miltefosine on erythrocytes

BackgroundMiltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-level ([Ca²⁺]_i). The present study explored, whether miltefosine elicits eryptosis.MethodsCell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence.ResultsA 48 h exposure to miltefosine (?4.9 μM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca²⁺]_i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca²⁺.ConclusionMiltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca²⁺ entry.     

Miltefosine enhances phagocytosis but decreases nitric oxide production by peritoneal macrophages of C57BL/6 mice

Miltefosine is an anticancer drug currently used to treat visceral and cutaneous leishmaniasis, also presents a broad-spectrum of fungicidal and antiamoebae activities. It acts on the metabolism of phospholipids and glycoproteins of the membrane of parasites. Our study aimed to evaluate the effects of miltefosine (0.4 to 50.0 μg/mL) on the phagocytosis and nitric oxide production by macrophages of C57BL/6 mice to clarify the immunomodulatory effects of the drug on macrophages of C57BL/6, strain mice that is biased to Th1 response. Peritoneal macrophages were in vitro treated with miltefosine and phagocytosis of sensitized or nonsensitized Saccharomyces cerevisiae was assessed. NO production was evaluated by Griess reaction. In the concentration of 1.6 μg/mL and 50.0 μg/mL, miltefosine increased phagocytosis of non-opsonized S. cerevisiae in 59.7% and 214.3%, respectively. For phagocytosis through opsonin receptors, miltefosine (50.0 μg/mL) increased the phagocytic index in 208.6% (p=0.04, paired t test). Miltefosine (50.0 μg/mL) decreased in 39.3% NO production by macrophages. However, treatment with miltefosine (50.0 μg/mL) after infection of macrophages with Leishmania amazonensis increased NO production in 73.4% (p=0.01, Wilcoxon test). Our data showed that, besides the antimicrobial effect of miltefosine, the drug showed immunomodulatory effects on macrophages of C57BL/6 mice, improving phagocytosis and decreasing NO production, but was able to increase NO production when macrophages were previously infected with L. amazonensis. These results suggest that miltefosine may favor the better evolution of infectious diseases by improving the innate immune response of macrophages.     

Treatment of leishmaniasis with miltefosine: 2008 status

Background: Miltefosine is the first recognized oral treatment for leishmaniasis. It was first registered, in 2002, for Indian visceral leishmaniasis, and was reviewed by the present author in 2005. Question: Miltefosine is now being used for the full range of clinical leishmaniasis. The present review addresses non-clinical and clinical advances since 2005. Methods: PubMed was accessed for all articles on miltefosine from 2005 to 2008. Results/conclusions: Miltefosine is effective and can be recommended for visceral disease in India and in Ethiopia, and for cutaneous disease in Colombia and Bolivia. For unusual forms of disease that require long periods of treatment such as diffuse cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis, oral miltefosine is probably the treatment of choice.     

Phase II study of miltefosine 6% solution as topical treatment of skin metastases in breast cancer patients

Topical treatment of skin metastases with a cytotoxic agent is attractive for its easy self-administration and absence of major systemic interference. Miltefosine exerts its cytotoxicity by acting on cell membrane phospholipids and can be administered topically. Twenty breast cancer patients with progression of skin metastases were treated with a 6% solution of miltefosine, which was topically administered once daily during the first week and twice daily thereafter. Sixteen out of 20 patients also had metastatic disease at other sites. Concomitant systemic treatment when ongoing for at least 2 months prior to study entry was permitted, and consisted of chemotherapy and hormonal therapy in seven and nine patients, respectively. Prior palliative cytotoxic and hormonal therapy had been administered to 11 and 19 patients, respectively. No grade 3 and 4 toxicity occurred. Miltefosine therapy was discontinued in two patients due to nausea and in one patient due to skin toxicity. Grade 1 and 2 adverse skin reactions, and nausea and vomiting were seen in 11 and two patients, respectively. In 18 patients evaluable for response, four partial responses were noted (response rate 22%), while seven patients had stable disease. Three partial responses were observed in patients in whom the skin lesions were smaller than 1.5 cm2. Median duration of response was 2.5 months and median time to progression for all patients was 1.9 months. In this study topically applied miltefosine for metastatic skin lesions of breast cancer showed modest activity in a relatively heavily pretreated patient population, without serious systemic toxicity.     

Methamphetamine induces apoptosis in an immortalized rat striatal cell line by activating the mitochondrial cell death pathway

Methamphetamine is a neurotoxic drug of abuse known to cause cell death both in vitro and in vivo. Nevertheless, the molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that methamphetamine-induced apoptosis is associated with early (2 h) overexpression of bax, decreases of mitochondrial membrane potential and oxygen consumption as well as release of cytochrome c from mitochondria. In addition, activated caspase-9 was detected at 4 h post-METH exposure. Cell death was detectable by annexin V and propidium iodide staining after 8 h of methamphetamine exposure. At that time, the majority of the cells were stained by annexin V alone, with some cells being stained for both annexin V and propidium iodide. Moreover, cleavage of caspase-3, poly (ADP-ribose) polymerase and DNA fragmentation-related factor 45 was detected at 8 h post drug treatment. These results indicate that methamphetamine-induced apoptotic cell death results from early overexpression of bax, reduction of mitochondrial respiration and membrane potential and release of mitochondrial cytochrome c with subsequent activation of the caspase cascade.     

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death,DNA damage and cell cycle arrest in human cervical cancer HeLa cells

ContextBufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity.ObjectiveThe cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro.Materials and methodsThe cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control.ResultsThe compound had strong effect on the cells (IC₅₀ = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells.ConclusionsBersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.     

Cytotoxic effect and apoptosis induction by Bothrops leucurus venom lectin on tumor cell lines

Neoplastic transformation is the abnormal proliferation of cells. These transformations are often related to changes in cell surface glycoconjugates which can be detected by lectins. We evaluated the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus venom as well as its cytotoxicity and hemolysis activity. The phosphatidylserine externalization and mitochondrial membrane potential were also determined. BlL exhibited cytotoxic activity against all tumor cell lines tested by induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis.     

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50μM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.     

The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: A comparison with the effect of doxorubicin

In the present study, we investigated the anticancer activity of WP 631, a new anthracycline analog, in weakly doxorubicin-resistant SKOV-3 ovarian cancer cells. We studied the time-course of apoptotic and necrotic events: the production of reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in human ovarian cancer cells exposed to WP 631 in the presence and absence of an antioxidant, N-acetylcysteine (NAC). The effect of WP 631 was compared with the activity of doxorubicin (DOX), the best known first-generation anthracycline.Cytotoxic activity was determined by the MTT assay. The morphological changes characteristic of apoptosis and necrosis in drug-treated cells were analyzed by double staining with Hoechst 33258 and propidium iodide (PI) using fluorescence microscopy. The production of reactive oxygen species and changes in mitochondrial membrane potential were studied using specific fluorescence probes: DCFH₂-DA and JC-1, respectively.The experiments showed that WP 631 was three times more cytotoxic than DOX in the tested cell line. It was found that the new anthracycline analog induced mainly apoptosis and, marginally, necrosis. Apoptotic cell death was associated with morphological changes and a decrease in mitochondrial membrane potential. In comparison to DOX, the novel bisanthracycline induced a significantly higher level of ROS and a greater drop in the membrane potential.The results provide direct evidence that the novel anthracycline WP 631 is considerably more cytotoxic to human SKOV-3 ovarian cancer cells than doxorubicin. The drug can produce ROS, which are immediately involved in the induction of apoptotic cell death.     

Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.     

Hesperetin induces an apoptosis-triggered extrinsic pathway and a p53- independent pathway in human lung cancer H522 cells

We studied the chemoprevention property of hesperetin on H522 cells using MTT, an apoptosis assay, an analysis of cell cycle progression, and the mitochondrial membrane potential, and apoptotic marker gene expression was determined using quantitative PCR. Hesperetin enhanced apoptotic cell death and mitochondrial membrane potential loss in H522 cells. Hesperetin up-regulated the levels of Fas, FADD, and caspase-8 expression and downregulted the levels of caspase-3 and caspase-9, p53, and Bax expression in H522 cells. This study shows that hesperetin induces apoptosis in H522 cells via a pathway independentof p53 and Bax but triggers the death-receptor Fas-initiated FADD/ caspase-8-dependent apoptotic pathway.     

A gold(I) phosphine complex selectively induces apoptosis in breast cancer cells: implications for anticancer therapeutics targeted to mitochondria

Bis-chelated gold(I) phosphine complexes have shown great potential as anticancer agents, however, their efficacy has been limited by their high toxicity and lack of selectivity for cancer cells. Here, we have investigated the anticancer activity of a new bis-chelated Au(I) bidentate phosphine complex of the novel water soluble ligand 1,3-bis(di-2-pyridylphosphino)propane (d2pypp). We show that this gold complex [Au(d2pypp)(2)]Cl, at submicromolar concentrations, selectively induces apoptosis in breast cancer cells but not in normal breast cells. Apoptosis was induced via the mitochondrial pathway, which involved mitochondrial membrane potential depolarisation, depletion of the glutathione pool and caspase-3 and caspase-9 activation. The gold lipophilic complex was accumulated in mitochondria of cells, driven by the high mitochondrial membrane potential. To address the molecular basis of the observed selectivity between the two cell lines we investigated the effect of the gold complex on the thioredoxin/thioredoxin reductase system in normal and cancer breast cells. We show that [Au(d2pypp)(2)]Cl inhibits the activities of both thioredoxin and thioredoxin reductase and that this effect is more pronounced in the breast cancer cells. This difference may account for the selective cell death seen in the breast cancer cells but not in the normal cells. Our investigation has led to new insights into the mechanism of action of bis-chelated gold(I) diphosphine complexes and their future development as mitochondria targeted chemotherapeutics.     

9-Oxo-(10E,12E)-octadecadienoic acid,a cytotoxic fatty acid ketodiene isolated from eggplant calyx,induces apoptosis in human ovarian cancer (HRA) cells

9-Oxo-(10E,12E)-octadecadienoic acid (9-EE-KODE), which is isolated from the calyx of eggplants, exhibits cytotoxic activity against human ovarian cancer (HRA) cells. The aim of the present study is to clarify the action mechanism of 9-EE-KODE leading to cell death. After the treatment of 9-EE-KODE in HRA cells, we found intracellular DNA fragmentation, surface-exposure of phosphatidylserine in the outer cell membrane, and increased caspase-3/7 activities in the HRA cells. The dissipation of mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, down-regulation of Bcl-2, and up-regulation of Bax levels were also found in 9-EE-KODE-treated cells in a dose-dependent manner. These results demonstrated that 9-EE-KODE induced apoptosis in HRA cells via the mitochondrial regulation pathway.

     

The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes

Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and F_oF₁-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca²⁺ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca²⁺ homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death.     

Protective effects of peroxisome proliferator-activated receptor agonists on human podocytes: proposed mechanisms of action

BACKGROUND AND PURPOSE Peroxisome proliferator-activated receptor (PPAR) agonists exert anti-albuminuric effects. However, the nephroprotective effects of these drugs remain to be fully understood. We have investigated whether gemfibrozil, GW0742 and pioglitazone protect human podocytes against nutrient deprivation (ND)-induced cell death and the role of mitochondrial biogenesis as a cytoprotective process. EXPERIMENTAL APPROACH Immortalized human podocytes were pre-treated with the PPAR agonists and exposed to ND (5?h) under normoxia, hypoxia or in the presence of pyruvate. Cell death was measured at the end of the ND and of the recovery phase (24?h). Mitochondrial mass, cytochrome c oxidase (COX) subunits 1 and 4 were measured as markers of mitochondrial cell content, while membrane potential as an index of mitochondrial function. PGC-1α, NRF1 and Tfam expression was studied, as crucial regulators of mitochondrial biogenesis. KEY RESULTS Cell pre-treatment with gemfibrozil, GW0742, or pioglitazone significantly decreased the ND-induced cell loss, necrosis and apoptosis. These effects were attenuated by hypoxia and potentiated by pyruvate. Pre-treatment with these drugs significantly increased mitochondrial cell content, while it did not affect mitochondrial function. In all these experiments pioglitazone exerted significantly larger effects than gemfibrozil or GW0742. CONCLUSIONS AND IMPLICATIONS Gemfibrozil, GW0742 and pioglitazone may exert direct protective effects on human podocytes. Mitochondrial biogenesis is a cell response to the PPAR agonists related to their cytoprotective activity. These results provide a mechanistic support to the clinical evidence indicating PPAR agonists as disease-modifying agents for glomerular diseases.     

A key role for caspase-2 and caspase-3 in the apoptosis induced by 2-chloro-2'-deoxy-adenosine (cladribine) and 2-chloro-adenosine in human astrocytoma cells

Both the anticancer agent 2-chloro-2'-deoxy-adenosine (Cladribine) and its derivative 2-chloro-adenosine induce apoptosis of human astrocytoma cells (J Neurosci Res 60:388-400, 2000). In this study, we have analyzed the involvement of caspases in these effects. Both compounds produced a gradual and time-dependent activation of "effector" caspase-3, which preceded the appearance of the nuclear signs of apoptosis, suggesting a temporal correlation between these two events. Moreover, the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (fmk) suppressed both caspase-3 activation and apoptosis induction. "Initiator" caspase-9 and caspase-8 were only marginally activated at later times in the apoptotic process. Accordingly, at concentrations that selectively inhibit these caspases, neither N-benzyloxycarbonyl-Leu-Glu-His-Asp-fmk nor N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fmk could prevent adenosine analog-induced cell death. To definitively rule out a role for the caspase-9/cytochrome c-dependent mitochondrial pathway of cell death, neither adenosine analog had any effect on mitochondrial membrane potential, which was instead markedly reduced by other apoptotic stimuli (e.g., deoxyribose, NaCN, and betulinic acid). Consistently, although the latter triggered translocation of mitochondrial cytochrome c to the cytoplasm, no cytosolic accumulation of cytochrome c was detected with adenosine analogs. Conversely, 1 to 7 h after addition of either adenosine analog (i.e., before the appearance of caspase-3 activation), caspase-2 activity was surprisingly and markedly increased. The selective caspase-2 inhibitor N-benzyloxy carbonyl-Val-Asp-Val-Ala-Asp-fmk significantly reduced both adenosine analogs-induced caspase-2 activation and the associated cell death. We conclude that adenosine analogs induce the apoptosis of human astrocytoma cells by activating an atypical apoptotic cascade involving caspase-2 as an initiator caspase, and effector caspase-3. Therefore, these compounds could be effectively used in the pharmacological manipulation of tumors characterized by resistance to cell death via either the mitochondrial or caspase-8/death receptor pathways.     

Necrotic pathway in human osteosarcoma Saos-2 cell death induced by chloroacetaldehyde

Chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, may be responsible for serious adverse effects like encephalopathy in ifosfamide chemotherapy. In this study, we demonstrate that chloroacetaldehyde, but not ifosfamide, induces cell death in human osteosarcoma Saos-2 cells and we investigated the mechanism by which this occurs. Chloroacetaldehyde above 30 micromol/l induced significant cell death in a time-dependent manner. Thiol compounds such as N-acetyl cysteine, glutathione and dithiothreitol protected the cells against chloroacetaldehyde-induced cell death, although other nonthiol compounds and the antioxidative enzymes superoxide dismutase and catalase did not, suggesting that reactive oxygen species might not mediate cell death. In cells exposed to chloroacetaldehyde, levels of both total thiols and glutathione were significantly reduced. Chloroacetaldehyde also collapsed the mitochondrial membrane potential of these cells, induced the release of cytochrome c from mitochondria to the cytosol and significantly reduced cellular ATP levels during the course of death. The mitochondrial potential collapse was also prevented by thiol compounds. Flow cytometric analyses by means of annexin-V and propidium iodide double staining and immunofluorescence staining of active caspase-3 revealed that cells subjected to a lethal dose of chloroacetaldehyde displayed features characteristic of necrosis and that caspase-3 was not activated in response to chloroacetaldehyde. Taken together, these findings suggest that Saos-2 cells exposed to chloroacetaldehyde die by necrosis resulting from a decrease in intracellular thiols, disruption of the mitochondrial membrane potential and the depletion of cellular ATP.     

Mitochondrial-derived ROS in edelfosine-induced apoptosis in yeasts and tumor cells

Aim： To investigate whether a similar process mediates cytotoxicity of 1-O- octadecyl-2-O-methyl-rac-glycero-3-phosphocholine （ET- 18-OCH3, edelfosine） in both yeasts and human tumor cells. Methods： A modified version of a previously described assay for the intracellular conversion of nitro blue tetrazolium to formazan by superoxide anion was used to measure the generation of reactive oxygen species （ROS）. Apoptotic yeast cells were detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. DNA fragmentation and the generation of ROS were measured by cytofluorimetric analysis in Jurkat cells. Results： Edelfosine induced apoptosis in Saccharomyces cerevisiae, as assessed by TUNEL assay. Meanwhile, edelfosine induced a time- and concentration-dependent generation of ROS in yeasts. Rotenone, an inhibitor of the mitochondrial electron transport chain, prevented ROS generation and apoptosis in response to edelfosine in S cerevisiae. α-Tocopherol abrogated the edelfosineinduced generation of intracellular ROS and apoptosis. Edelfosine also induced an increase of ROS in human leukemic ceils that preceded apoptosis. The overexpression of Bcl-2 by gene transfer abrogated both ROS generation and apoptosis induced by edelfosine in leukemic cells. Changes in the relative mito- chondrial membrane potential were detected in both yeasts and Jurkat cells. Conclusion： These results indicate that edelfosine induces apoptosis in yeasts in addition to human tumor cells, and this apoptotic process involves mitochondria, likely through mitochondrial-derived ROS. These data also suggest that yeasts can be used as a suitable cell model in elucidating the antitumor mechanism of action of edelfosine.