Hepatoprotective effects of sea cucumber ether-phospholipids against alcohol-induced lipid metabolic dysregulation and oxidative stress in mice.

Sea cucumber is widely consumed as food and folk medicine in Asia, and its phospholipids are rich sources of dietary eicosapentaenoic acid enriched ether-phospholipids (ether-PLs). Emerging evidence suggests that ether-PLs are associated with neurodegenerative disease and steatohepatitis. However, the function and mechanism of ether-PLs in alcoholic liver disease (ALD) are not well understood. To this end, the present study sought to investigate the hepatoprotective effects of sea cucumber ether-PLs, including plasmenyl phosphatidylethanolamine (PlsEtn) and plasmanyl phosphatidylcholine (PlsCho), and their underlying mechanisms. Our results showed that compared with EtOH-induced mice, ether-PL treated mice showed improved liver histology, decreased serum ALT and AST levels, and reduced alcohol metabolic enzyme (ALDH2 and ADH1) expressions. Mechanistic studies showed that ether-PLs attenuated "first-hit" hepatic steatosis and lipid accumulation evoked by alcohol administration. Moreover, PlsEtn more effectively restored endogenous plasmalogen levels than PlsCho, thereby enhancing hepatic antioxidation against "second-hit" reactive oxygen species (ROS) due to the damaged mitochondria and abnormal ethanol metabolism. Taken together, sea cucumber ether-PLs show great potential to become a natural functional food against chronic alcohol-induced hepatic steatosis and lipid metabolic dysregulation.

Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

Evaluation of in vitro and in vivo anti-inflammatory activity of biologically active phospholipids with anti-neoplastic potential in porcine model.

BACKGROUND: This study aims to investigate the anti-inflammatory effect of biologically active phospholipids (BAP) used in preparations for clinical practice in humans. Until date, except anti-neoplastic ability, little is known about anti-inflammatory property of the phospholipids. METHODS: While the course of bacterially induced acute pneumonia and markers of inflammation were studied in in vivo system in pigs orally supplemented with BAP, the pro- and anti-inflammatory response of lipopolysaccharide-stimulated porcine monocyte-derived macrophages to 24 h- and 48 h-treatment by BAP was investigated in in vitro system. In vivo, the animal health status was monitored and pro-inflammatory IL-1ß and IL-8 in sera were detected by ELISA during the experiment, while bronchoalveolar lavage fluids (BALF) and the lungs were examined post-mortem. Total and differential counts of white blood cell (WBC) were determined in blood and BALF. In vitro, mRNA expression of pro-inflammatory (TNF-α, IL-1ß, CXCL10) and anti-inflammatory (IL-10 and Arg1) cytokines, and level of activated caspase 1 and phosphorylated protein kinase C epsilon (pPKCϵ), were studied using qRT-PCR and Western blot, respectively. For the purposes of both systems, 6 animals were used in each of the BAP-supplemented and the control groups. RESULTS: In vivo, BAP had a positive influence on the course of the disease. The immunomodulatory effects of BAP were confirmed by lower levels of IL-1ß, IL-8, and a lower WBC count in the supplemented group in comparison with the control group. A lower percentage of lung parenchyma was affected in the supplemented group comparing to the control group (on average, 4% and 34% of tissue, respectively). In vitro, BAP suppressed mRNA expression of mRNA for IL-10 and all pro-inflammatory cytokines tested. This down-regulation was dose- and time-dependent. Arg1 mRNA expression remained unaffected. Further dose- and time-dependent suppression of the activated caspase 1 and pPKCϵ was detected in macrophages when treated with BAP. CONCLUSIONS: Our results demonstrate that BAP has anti-inflammatory and immunomodulatory properties, thus emphasizing the potential of this compound as a natural healing agent.

Vitamin E prevents lipid raft modifications induced by an anti-cancer lysophospholipid and abolishes a Yap1-mediated stress response in yeast.

We have previously established that the anti-cancer lysophospholipid edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, Et-18-OCH(3)) induces cell death in yeast by selective modification of lipid raft composition at the plasma membrane. In this study we determined that alpha-tocopherol protects cells from the edelfosine cytotoxic effect, preventing the internalization of sterols and the plasma membrane proton pump ATPase, Pma1p. Two non-mutually exclusive hypotheses were considered to explain the protective effect of alpha-tocopherol: (i) its classical antioxidant activity is necessary to break progression of lipid peroxidation, despite the fact Saccharomyces cerevisiae does not possess polyunsaturated fatty acids and (ii) due to its complementary cone shape, insertion of alpha-tocopherol could correct membrane curvature stress imposed by edelfosine (inverted cone shape). We then developed tools to distinguish between these two hypotheses and dissect the structural requirements that confer alpha-tocopherol its protective effect. Our results indicated its lipophilic nature and the H donating hydroxyl group from the chromanol ring are both required to counteract the cytotoxic effect of edelfosine, suggesting edelfosine induces oxidation of membrane components. To further support this finding and learn more about the early cellular response to edelfosine we investigated the role that known oxidative stress signaling pathways play in modulating sensitivity to the lipid drug. Our results indicate the transcription factors Yap1 and Skn7 as well as the major peroxiredoxin, Tsa1, mediate a response to edelfosine. Interestingly, the pathway differed from the one triggered by hydrogen peroxide and its activation (measured as Yap1 translocation to the nucleus) was abolished by co-treatment of the cells with alpha-tocopherol.

Bisxanthones from Hypericum japonicum: inhibitors of PAF-induced hypotension.

The CHCl3-soluble part (HJ) of the MeOH extract from Hypericum japonicum afforded new bisxanthones, jacarelhyperols A (1) and B (2). The structures were elucidated by spectral methods. These compounds and HJ showed significant inhibitory effects against PAF-induced hypotension by an in vivo evaluation method, which can reveal the pharmacological action of the test material.

Evaluation of agonists and antagonists acting at Group I metabotropic glutamate receptors in the thalamus in vivo.

Recordings were made from single neurones in the ventrobasal thalamus of anaesthetised rats in order to evaluate the properties of several agonists and antagonists of Group I mGlu receptors. The selective mGlu1 receptor antagonist LY367385 was found to reduce excitatory responses to iontophoretically applied ACPD and DHPG whereas the mGlu5 agonist CHPG was resistant to antagonism. The antagonists LY367366 and LY393053 reduced responses to all three agonists, but without reducing responses to NMDA or AMPA. Although AIDA was also found to reduce mGlu agonist-evoked responses, this antagonist also produced significant reductions in responses to NMDA and AMPA. These data suggest that there are functional mGlu1 and mGlu5 receptors in the thalamus. Furthermore, LY367385 is a useful tool for investigating mGlu1 functions whereas LY367366 and LY393053 have a broader spectrum of action. The usefulness of AIDA as an antagonist in physiological experiments would appear to be limited by its effects against NMDA and AMPA.

Sensitivity of K562 and HL-60 cells to edelfosine, an ether lipid drug, correlates with production of reactive oxygen species.

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane-targeting anticancer ether lipid drug has been shown previously in vitro to be capable of initiating oxidative processes in cells. Here we study two human leukemia cell lines (HL-60 and K562) that have different sensitivities to edelfosine; HL-60 cells are more sensitive than K562 cells. To determine whether edelfosine alters the sensitivity of these lines to an oxidative stress, cells were subjected to the oxidative stress of iron(II) plus ascorbate and then monitored for free radical formation, membrane integrity, and cytotoxicity. The HL-60 cell was sensitive to the ether lipid drug in clonogenic and dye exclusion assays; a lipid-derived free radical was generated by this sensitive cell in the presence of small amounts of Fe2+ and ascorbate as detected by electron paramagnetic resonance and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. There was also simultaneous generation of an ascorbate-free radical, which has been shown to estimate cellular oxidative flux. In contrast, the K562 cell was resistant to edelfosine cytotoxicity in all assays and did not generate either lipid-derived or ascorbate-free radicals. Subcellular homogenates of the HL-60 cell generated both radicals when exposed to the drug, but homogenates of K562 did not generate either, suggesting that differential drug uptake or intracellular drug localization is not the cause of the difference in oxidation. Trypan blue uptake by the HL-60, but not the K562 cells, measured under the same conditions as the oxidation experiments, demonstrated a loss of membrane impermeability with similar time and concentration dependence, suggesting a causal relationship of membrane damage and radical generation. Complementary studies of HL-60 cell membrane integrity with propidium iodide impermeability and light scatter using the flow cytometer showed a concentration dependence that was similar to radical generation. Biochemical studies of the fatty acids of the HL-60 cell revealed more highly polyunsaturated lipids in the cells. Cellular antioxidant enzymes and vitamin E contents of the two cell lines were similar. We conclude that there is a time- and concentration-dependent generation of important oxidations by the sensitive HL-60 cells exposed to the membrane-targeted ether lipid, but the resistant K562 cells are oxidatively silent. This may be due in part to the differences in fatty acid polyunsaturation of the cellular membranes. The difference in oxidative susceptibility could be the basis for drug resistance to this membrane-specific anticancer agent.

Apoptosis triggered by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is prevented by increased expression of CTP:phosphocholine cytidylyltransferase.

A HeLa cell line was constructed for the regulation of CTP:phosphocholine cytidylyltransferase (CCT) expression via a tetracycline-responsive promoter to test the role of CCT in apoptosis triggered by exposure of cells to the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Basal CCT expression in the engineered HeLa cell line was the same as in control HeLa cells lines, and CCT activity and protein were elevated 25-fold following 48 h of induction with doxycycline. Increased CCT expression prevented ET-18-OCH3-induced apoptosis. Acylation of exogenous lysophosphatidylcholine circumvented the requirement for CCT activity by providing an alternate route to phosphatidylcholine, and heightened CCT expression and lysophosphatidylcholine supplementation were equally effective in reversing the cytotoxic effect of ET-18-OCH3. Neither CCT overexpression nor lysophosphatidylcholine supplementation allowed the HeLa cells to proliferate in the presence of ET-18-OCH3, indicating that the cytostatic property of ET-18-OCH3 was independent of its effect on membrane phospholipid synthesis. These data provide compelling genetic evidence to support the conclusion that the interruption of phosphatidylcholine synthesis at the CCT step by ET-18-OCH3 is the primary physiological imbalance that accounts for the cytotoxic action of the drug.

The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine.

The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G2/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH3 which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.

Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent.

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH3 could be stably associated with liposomes, less free ET-18-OCH3 would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH3. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH3 liposome compositions were minimally hemolytic. For example, whereas the HI5 (drug concentration required to cause 5% human red cell lysis) was 5-6 microM for free ET-18-OCH3, it was approximately 250 microM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH3, (4:3:1:2) and 640 microM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH3 (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH3 in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH3.

Effects of iron supplementation and ET-18-OCH3 on MDA-MB 231 breast carcinomas in nude mice consuming a fish oil diet.

Lipid peroxidation products can be cytotoxic. Our objectives were (1) to use two pro-oxidants (iron and a pro-oxidative drug) to selectively increase lipid peroxidation in the implanted human breast tumours of mice consuming fish oil and (2) to kill the cancer cells without harming normal host tissues. The theoretical basis for selective cytotoxicity is that normal cells are better able to handle oxidative stress than cancer cells. Male athymic nude mice, consuming an AIN-76 diet, were injected s.c. with MDA-MB 231 human breast carcinoma cells. Three weeks later, all mice had palpable tumours, 3-10 mm in diameter, and diets were changed to modified AIN-76 diets containing 19% menhaden fish oil and 1% corn oil with or without supplemental 0.3% ferric citrate. After 2 weeks, half of the mice on each diet (19% fish oil with or without supplemental ferric citrate) were injected (three times per week for 2 weeks) with the ether-lipid drug edelfosine (ET-18-OCH3). The concentration of lipid peroxidation products in tumours (as measured by thiobarbituric acid-reactive substances, TBARS) was significantly increased by both ferric citrate and ET-18-OCH3. The TBARS in livers were not increased, nor was there evidence of other harmful side-effects to the host mice. The addition of iron enhanced tumour cell death whereas ET-18-OCH3 suppressed tumour cell mitosis. The use of iron supplementation combined with ET-18-OCH3 resulted in the slowest growth rate, lowest mitotic index, highest level of lipid peroxidation products and increased the cytotoxic index in tumours without detectable harm to the host. That iron supplementation increased tumour suppression beyond that expected from the increase in the concentration of TBARS in the tumour merits further investigation.

The effect of edelfosine on CTP:cholinephosphate cytidylyltransferase activity in leukemic cell lines.

Analogs of ether phospholipids have been shown to have selective anti-neoplastic activity. The compounds are known to inhibit phospholipid biosynthesis. This paper examines the effect of the alkyl-lysophospholipid, edelfosine, on the rate-limiting enzyme, CTP:cholinephosphate cytidylyltransferase, in de novo phosphatidylcholine synthesis in sensitive and resistant leukemic cell lines. Enzyme activity was measured by the incorporation of 14C-phosphocholine into CDP-choline by lysates of HL60 and K562; cells demonstrated inhibition of incorporation of 14C-phosphocholine in HL60 cell lysates but no inhibition in K562 lysates. Partial purification of cytidylyltransferase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting demonstrated similarity between the enzyme isolated from each cell line. Cloning and sequencing of cytidylyltransferase cDNA of HL60 cells was accomplished using a probe encoding the entire protein sequence of the K562 cytidylyltransferase gene. A substitution at nucleotide 751 from A in the HL60 cell cDNA clone to G in the K562 cDNA clone resulted in a change in amino acid number 251 from lysine (positively charged) in the HL60 enzyme to glutamic acid (negatively charged) in the K562 enzyme. This negative charge in the lipid-binding domain of the K562 enzyme may result in a weaker binding of edelfosine and the observed decrease in activity, as evidenced by resistance to edelfosine by K562 cells.

Response of human multiple myeloma-derived cell lines to alkyl-lysophospholipid.

Clinical studies using high-dose chemotherapy and autologous bone marrow transplantation in multiple myeloma report improvement in tumor regression. The potential success of this approach may be compromised because of the myeloma cell burden. A promising approach is chemopurging of bone marrow with alkyl-lysophospholipids (ALP), a new class of antitumor agents. Using a limiting dilution assay, we tested ALP against several drug-resistant human myeloma cell lines: RPMI 8226/S, 8226/DOX40, 8226/LR-5, 8226/MR-20, 8226/DOX1V, and 8226/MDR10V. IC50 values were approximately 6 microg/mL, for all cell lines evaluated. ALP was effective against all cells, with mean log kills ranging from 2.48 to 4.95. Cytotoxicity was temperature-dependent. Survival curves of cell lines exposed to ALP showed a steep slope during the first 4 hours of exposure, with little additional cell kill after 24 hours. Dose-response curves after drug exposure for 4 and 24 hours reached plateaus at 75 and 25 microg/mL, respectively. Increasing concentrations of ALP caused a sustained increase in resting [Ca2+]i of sufficient magnitude to induce cell death. These results suggest that ALP is an effective cytotoxic agent even in cells known to be resistant to alkylating agents and other classes of cytotoxic drugs.

Platelet activating factor and lyso-phosphatidylcholines from strawberry.

Lipids from strawberry fruits, leaves, achenes and pollen were separated into classes by TLC, purified by HPLC and tested for biological activity. A lipid fraction from fruits with the same chromatographic behaviour as authentic platelet activating factor (PAF) showed identical biological activity, namely, dose-dependent aggregation of washed rabbit platelets, inhibition of aggregation by CV 3988, platelet desensitization to PAF and vice versa, and loss of activity by alkaline hydrolysis and recovery of activity by reacetylation. The presence of PAF was confirmed by FAB mass spectrometry. Lyso-phosphatidylcholines, including lyso-PAF, were also found in all the plant parts tested.

Synthesis and biological evaluation of phosphate triester alkyl lysophospholipids (ALPs) as novel potential anti-neoplastic agents.

Phosphate triester derivatives of the anti-neoplastic alkyl lysophospholipids (ALP) have been prepared as novel potential therapeutic agents. In particular, simple, symmetrical phosphate triesters have been prepared, using phosphorochloridate chemistry. The compounds have been fully characterized by a range of techniques, and assayed for their inhibition of DNA synthesis by mammalian cells in culture. The compounds are generally inhibitory towards DNA synthesis in the micromolar range. However, the magnitude of the effect varies greatly with the phosphate structure. In particular, there is a clear trend towards decreasing activity with increasing alkyl chain length. Thus, short-chain dialkyl phosphate esters appear more effective than the reference compound hexadecyl phosphatidyl choline.

SRI 62-834, a cyclic ether analogue of the phospholipid ET-18-OCH3, displays long-lasting beneficial effect in chronic relapsing experimental allergic encephalomyelitis in the Lewis rat. Comparison with cyclosporin and (Val2)-dihydrocyclosporin effects in clinical, functional and histological studies.

The therapeutic effect of the ether phospholipid SRI 62-834, which lacks the characteristics of an immunosuppressive agent, was compared with those of two immunosuppressive drugs, cyclosporin and valine2-dihydrocyclosporin, in a rat model of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). Drug treatment was initiated at the beginning of the first spontaneous remission on day 15 and was discontinued on day 31. Whereas the untreated rats experienced two paralytic relapses around days 21 and 31, the progression of CR-EAE was prevented during the period of drug administration. Protection with both cyclosporin and its derivative was complete, but SRI 62-834 only attenuated the clinical disease. The absence of paralytic symptoms was reflected by a distinct reduction in mononuclear cell infiltration in the central nervous system at days 21 and 31 in treated animals. The main difference between the two drug classes became apparent after withdrawal of therapy. Discontinuation of SRI 62-834 resulted in a long-lasting beneficial effect, with the rats remaining clinically normal and showing no histopathological changes. However, cyclosporin only delayed the clinical symptoms which reappeared after cessation of treatment. The exacerbated paralytic relapse, which followed about 1 week later and was associated with severe perivascular cell infiltrates and tissue destruction, subsequently became chronic in several animals. By contrast, withdrawal of valine2-dihydrocyclosporin partially prevented disease relapse and markedly reduced severity of symptoms without progression of a chronic disease. These results demonstrate the clear differences in the mode of action of these compounds in CR-EAE and suggest that SRI 62-834 could be an interesting candidate for the treatment of multiple sclerosis.

Ether lipids in cancer chemotherapy.

There is considerable evidence that certain ether lipids such as 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine represent a new class of antineoplastic agents. These ether lipids have been shown to be cytotoxic for a wide variety of tumors. The site of action of ether lipids appears to be the cell membrane, where they inhibit the biosynthesis of phosphatidylcholine as well as the activity of protein kinase C. However, it remains uncertain that these effects represent the only mechanism for the cytotoxic action of these ether lipids. Further experiments to elucidate the molecular mechanisms of the cytotoxic action of these compounds are necessary. Clinical phase I pilot trials have been completed showing encouraging tumor response in a small number of patients treated. Some of these ether lipids are selectively cytotoxic to leukemic cells sparing of normal cells within a certain dose range. Thus, they are uniquely suitable for purging residual leukemic cells from remission marrow used for autologous bone marrow transplantation. A phase I/II study of autologous bone marrow transplantation in acute leukemia using bone marrow cells treated with ether lipids is in progress.

Purging of acute myeloid leukemic cells by ether lipids and hyperthermia.

Ether lipids (EL) and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of EL (ET-18-OCH3, ET-16-NHCOCH3, or BM 41.440) and hyperthermia on the growth of hematopoietic progenitors, myeloid leukemic cell lines, and leukemic cells obtained from patients with acute myeloid leukemia (AML) were examined to determine if this combination resulted in a greater selective killing of leukemic cells than that achieved by either EL or heat alone. When the cells were treated simultaneously with EL (50 micrograms/mL) and hyperthermia (42 degrees C) for one hour, the killing of leukemic cell line cells was enhanced considerably. Among the three EL, however, the combination of ET-18-OCH3 and heat seemed to be the most cytotoxic to leukemic cell line cells with no effect on the growth of hematopoietic progenitors. An increase in the duration of treatment with ET-18-OCH3 to four hours with heat added during the last hour resulted in a further reduction of leukemic cell line cells while sparing 50% of hematopoietic progenitors after cryopreservation. The combined treatment with ET-18-OCH3 and heat also inhibited the growth of leukemic progenitors obtained from AML patients by 97% to 100%. These data indicate that the combined treatment with EL and hyperthermia might offer an efficient means to eliminate myeloid leukemic cells in vitro.