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.