Etsu Kishida

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.

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.

Determination of malondialdehyde with chemical derivatization into the pyrmidine 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.

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.

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.

Fractionation of Wild Rice (Zizania palustris) Polysaccharides and Characterization of the Starch Fraction.

北アメリカ原産のイネ科マコモ属の単子葉植物, ワイルドライス (Zizania palustris) は, その独特の風味が好まれている。我々はその特異なテクスチャーに関連する成分としてデンプンおよび細胞壁多糖に着目し, ワイルドライス製品の多糖成分の分画を行い, 特にデンプンの性状を調べた。1) ワイルドライス粉末からアルカリ浸漬法によりデンプンを分離し, この上清からエタノール沈澱により水溶性多糖を分離した。ふるい上の残渣を脱脂・除タンパク後再度デンプンを分別し, 不溶性残渣から細胞壁標品を得た。2) ワイルドライスからのデンプンの収量は30.5%であり, ウルチ米の半分以下であった。ワイルドライスの細胞壁標品の収量 (3.54%) は, ウルチ米 (0.12%), モチ米 (0.3%) と比較すると, 細胞壁の含量が著しく高いことを示唆している。3) デンプン画分の走査電子顕微鏡写真から, ワイルドライスのデンプン粒の形はウルチ米と類似の多面体で, サイズはやや小さく, 1.5-6μm程度の粒径であった。細胞壁標品の走査電子顕微鏡像から, ワイルドライスの細胞壁はウルチ米に比し相当厚く強固な様子が観察された。4) デンプンをイソアミラーゼおよびプルラナーゼで完全に分枝切断し, ゲルろ過により鎖長分布を調べた結果, ワイルドライスのFr. I (アミロース) の比率は23.8%で, ウルチ米よりかなり高かつた。一方, Fr. III (アミロペクチンの短鎖長, DP 10-30) とFr. II (長鎖長, DP 30-60) の比は3.5で, ウルチ米 (2.6) よりも大きかった。5) 上記の枝切りデンプンの精密鎖長分布を, 陰イオン交換高速液体クロマトグラフィーにより解析した結果, 短鎖部分の鎖長分布はワイルドライスでは11糖が最多で, またウルチ米, モチ米と比べて10-14糖部分の含量 (モル%) が高く, アミロペクチンの微細構造も米とは異なることが示唆された。