Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.

A change in all ceramide species during chemically induced apoptosis of HL-60 cells was determined using electrospray tandem mass spectrometry. Ceramides of C16:0, C24:1 and C26:1 increased significantly 4 h after the addition of actinomycin D, when the activation of caspase-3 was maximal. Addition of catalase, which inhibited apoptosis, the activation of caspase-3-like protease, and the release of cytochrome c from mitochondria to cytosol caused by actinomycin D or daunorubicin, significantly inhibited the increase of these ceramides at all time points. Ceramides of C16:0, C24:1, C18:0, C22:1 and C26:1 increased significantly 4 h after the addition of daunorubicin to HL-60 cells. Catalase also significantly inhibited the increase of these ceramides induced by daunorubicin. Based on time courses of events and inhibition studies, it is concluded that the increase of ceramides is downstream from both generation of hydrogen peroxide and cytochrome c release from mitochondria and takes place almost simultaneously with the activation of caspase-3.

Docosahexaenoic acid and vitamin E can reduce human monocytic U937 cell apoptosis induced by tumor necrosis factor.

The effects of polyunsaturated fatty acids and vitamin E on tumor necrosis factor (TNF)-induced apoptosis of human monocytic U937 cells was explored to assess to what extent these nutrients could attenuate apoptosis. Preincubation of U937 cells with arachidonic acid for 24 h did not affect TNF-induced apoptosis. Eicosapentaenoic acid slightly but significantly reduced the proportion of apoptotic cells only when apoptosis was induced by TNF without cycloheximide (CHI). In contrast, preincubation with docosahexaenoic acid (DHA) greatly (40 approximately 70%) attenuated apoptosis induced by stimulation with either TNF or TNF + CHI for 3 h. The inhibition of apoptosis was accompanied by enrichment of DHA in membrane phospholipids, indicating that DHA probably exerted its inhibitory activity after being incorporated into the phospholipids. Vitamin E also played a role as a partial inhibitor of apoptosis 3 h after TNF addition. This vitamin could further reduce the apoptosis of DHA-treated cells, and such an additive effect was obvious when apoptosis was induced at a low frequency. Longer-range stimulation of U937 cells with TNF showed that inhibition of apoptosis by preincubating cells with either DHA or vitamin E was not significant 9 h after TNF addition, but that preincubation with both DHA and vitamin E could reduce the proportion of apoptotic cells even at this time point. Our findings suggested that ingestion of nutrients such as DHA and vitamin E might exert beneficial effects on organ dysfunction associated with various TNF-related diseases.

Involvement of hydrogen peroxide and hydroxyl radical in chemically induced apoptosis of HL-60 cells.

Effects of three kinds of antagonists against reactive oxygen species were evaluated at the same time in chemically induced apoptosis of human leukemic HL-60 cells. Apoptosis of HL-60 cells induced by actinomycin D, H7, 1-beta-D-arabinofuranosylcytosine, and daunorubicin was inhibited significantly by radical scavengers (vitamin E, N-acetyl-L-cysteine, and mercaptoethanol), catalase, and a spin trap, N-t-butyl-alpha-phenylnitrone. These results suggest that hydrogen peroxide and hydroxyl radical are common mediators of apoptosis caused by these chemicals with apparently different functional mechanisms. The consumption of vitamin E to inhibit apoptosis induced by actinomycin D was undetectable, suggesting that the generation of reactive oxygen species during apoptosis was not very extensive. Radicals were suggested to be a mediator of apoptosis of HL-60 cells induced by cisplatin based on the observations that the above inhibitors, except catalase, effectively inhibited apoptosis by the drug.

Quantitative analysis of ceramide molecular species by high performance liquid chromatography.

A method was developed for quantitative analysis of molecular species of ceramide (N-acyl-sphingosine) and dihydroceramide (N-acyl sphinganine) by high performance liquid chromatography (HPLC). Various N-acyl chain-containing ceramides or dihydroceramides were semi-synthesized as standard materials and allowed to react with anthroyl cyanide, a fluorescent reagent. Anthroyl derivatives of ceramide and dihydroceramide containing C16, C18, C20, C22, and C24 saturated N-acyl chain could be completely separated to each molecular species by reversed-phase HPLC equipped with fluorescence detector, although some ceramide molecular species containing monoenoic acyl chain were eluted together with saturated dihydroceramide species. Ceramide molecular species could be quantified using N-heptadecanoyl or N-tricosanoyl sphingosine as an internal standard, and the lower detection limit was below 1 pmol. This method was applied to the analysis of sphingomyelin and free ceramide in U937 cells. The analysis of the ceramide obtained by hydrolysis of sphingomyelin of U937 cells revealed that the ceramide moiety was mainly composed of N-palmitoyl sphingosine, N-nervonoyl sphingosine, and N-lignoceroyl sphingosine, representing 50.0, 27.4, and 6.7% of sphingomyelin, respectively. The total free ceramide and dihydroceramide of U937 cells was determined to be 254 +/- 5 pmol/10(6) cells. Major molecular species of the free ceramide fraction were N-lignoceroyl, N-palmitoyl, and N-nerovonoyl sphingosine, representing 27.6%, 26.6%, and 13.6% of this fraction, respectively. Different distribution of free ceramide molecular species from sphingomyelin species may suggest that selective metabolism of molecular species occurs in the synthesis or degradation of sphingomyelin. These results indicate that the picomole level of molecular species of ceramide and dihydroceramide is successfully determined by fluorescence HPLC and that this newly developed method may be useful to reveal the metabolism and function of ceramide and related compounds in cultured cells.

Distinctive inhibitory activity of docosahexaenoic acid against sphingosine-induced apoptosis.

The effect of supplementation of docosahexaenoic acid (DHA) on the apoptosis of HL60 cells was examined using N-acetyl sphingosine (C2-ceramide) and sphingosine as apoptosis-inducing agents. Although C2-ceramide-induced apoptosis was not affected by DHA supplementation, sphingosine-induced apoptosis was reduced almost to the background level by preincubation with 10 microM DHA for 24 h. Among the fatty acids, only DHA appeared to be endowed with the ability to reduce sphingosine-induced apoptosis, whereas, other unsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA), did not show this activity. Incubation of HL60 with DHA within 6 h did not affect the apoptosis, suggesting that DHA probably expressed the inhibitory activity after modulation of the membrane fatty acid composition. DHA also attenuated the apoptosis induced by dimethylsphingosine and H-7, but not by calphostin C, indicating that enrichment of DHA in membranous phospholipid does not necessarily prevent all of the apoptosis associated with the inhibition of protein kinase C. The mechanism of the inhibition against sphingosine-induced apoptosis by DHA remains to be further explored. However, the inhibition of cytosolic phospholipase A2 (cPLA2) may be involved in the mechanism, because distinctive inhibitory activity of DHA against cPLA2 has been demonstrated [M. Shikano, Y. Masuzawa, K. Yazawa, K. Takayama, I. Kudo, K. Inoue, Biochim. Biophys. Acta, 1212, 1994, 211-216], and arachidonyl trifluoromethylketone, a specific inhibitor of cPLA2, attenuated the apoptosis induced by sphingosine.

Evaluation of a trans configuration for the apoptosis-inducing activity of ceramide.

The requirement of a trans double bond for the biological action of ceramide was assessed by comparing the apoptosis-inducing activity of various ceramide analogs. The cis isomer and an acetylene type derivative of sphingosine were chemically synthesized, and the 2-amino moiety was acylated with hexanoic acid. These cell-permeable ceramide derivatives were compared with N-hexanoyl sphingosine (C6-Cer) or N-hexanoyl dihydrosphingosine (C6-DH-Cer) in their activity to induce apoptosis of HL60. Either the cis isomer of C6-Cer (C6-cis-Cer) or a triple bond derivative (C6-TRP-Cer) induced apoptosis when assessed by fluorescence microscopy of the morphological changes and electrophoretic analysis of DNA C6-TRP-Cer yielded the highest percentage of apoptotic cells corresponding to three times that was induced by C6-Cer. C6-cis-Cer also showed stronger activity than C6-Cer. The minimum amounts of C6-TRP-Cer and C6-cis derivative required to induce apoptosis were 0.1 and 0.5 microM, respectively, while 1 microM C6-Cer was required to exhibit the activity. C6-DH-Cer showed very low but significant activity above 10 microM. N-acetyl-sphingosine (C2-Cer) induced more apoptotic cells than C6-Cer, and C2-TRP-Cer was much more potent than C2-Cer. These observations suggest that the trans configuration of ceramide is not necessarily essential for the activity to induce apoptosis. In addition, distinctive activity of C6- or C2-TRP-Cer suggests that this ceramide analog might be useful for developing a new type of antitumor drug.

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants.

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.

Alteration of beta-D-glucan from edible mushroom after injection into mouse peritoneal cavity.

Tritium-labeled antitumor beta-D-glucan derivative (T-labeled glucan) was prepared from the branched beta-1,3-D-glucan of an edible mushroom, Volvariella volvacea, by its periodate oxidation followed by reduction with NaBT4. Twenty-three hours after T-labeled glucan had been injected into the mouse peritoneal cavity, about 3% of the total radioactivity injected was found in the mouse serum. In spite of the fact that T-labeled glucan had no affinity to the anion-exchange column before injection, about 50% of the labeled beta-D-glucan in the serum thus obtained was adsorbed onto the column. The labeled beta-D-glucan fraction eluted from the column by salt gradient showed antitumor activity in vivo.

Relationship among malondialdehyde, TBA-reactive substances, and tocopherols in the oxidation of rapeseed oil.

Relationship among malondialdehyde (MDA), 2-thiobarbituric acid (TBA)-reactive substances (TBA-RS), and tocopherols in the oxidation of rapeseed oil was investigated. MDA was determined by a new HPLC method with chemical derivatization. When the oil was heated at 170 degrees C, TBA-RS and MDA increased. The contents of TBA-RS were approximately 1.6 times higher than those of MDA. Correlation between the increase in formed MDA and the decrease in tocopherols was observed. When the oxidation of the oil was initiated using 2,2'-azobis-(2,4-dimethylvaleronitrile) at 40 degrees C, TBA-RS dramatically increased during the initial stage and reached plateau. Thereafter, little increase was observed. The relative ratio of MDA to TBA-RS was much lower in the reaction performed at 40 degrees C than that observed at 170 degrees C. These results indicated that the decrease of tocopherols was accompanied by the increase of MDA but TBA-RS did not correlate with the change of tocopherols.

Determination of malondialdehyde with chemical derivatization into the pyrimidine compound and HPLC.

A new HPLC method to determine malondialdehyde (MDA) was developed. Malondialdehyde was reacted with alpha-N-benzoyl-L-arginine ethyl ester and converted into alpha-N-benzoyl-delta-N-(2-pyrimidinyl)-L-ornithine ethyl ester, which is sufficiently hydrophobic to allow us its specific determination utilizing a reversed-phase C18 column at the level of 1 pmol.

Purification of an antitumor-active, branched (1----3)-beta-D-glucan from Volvariella volvacea, and elucidation of its fine structure.

A (1--3)-beta-D-glucan branched by O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body of V. volvacea, exhibited potent growth-inhibitory activity against implanted tumors in mice. It contained protein and appeared to be heterogeneous. Fractionation by DEAE-Toyopearl column chromatography yielded an unbound, protein-free glucan fraction ([alpha]D - 30 degrees in M NaOH, mol. wt. 1.5-2 x 10(6)), which showed the highest antitumor activity. The polysaccharide had a moderately branched structure, consisting of a backbone chain of beta-(1--3)-linked-D-glucose residues, one out of five or six being substituted at O-6 with single glucosyl of beta-(1--6)-linked diglucosyl groups. Digestion of the glucan with exo-(1--3)-beta-D-glucanase yielded glucose and ++gentiobiose (molar ratio, 8:2:1.0), and a highly branched (d.b. 1/3), degraded glucan. Digestion with endo-(1--3)-beta-D-glucanase gave D-glucose, laminarabiose, a trisaccharide beta-D-Glcp-(1--6)-beta-D-Glcp-(1--3)-D-Glc, a tetrasaccharide beta-D-Glcp-(1--3)-beta-D-Glcp-(1--3)-[beta-D-Glcp-(1--6)]-D-Glc, and a highly branched (d.b. 1/2), enzyme-resistant glucan. The results suggest that the Volvariella glucan is structurally heterogeneous with regard to the distribution of branches, having less branched, moderately branched, and highly branched segments.