Stereospecific induction of apoptosis in tumor cells via endogenous C16-ceramide and distinct transcripts

Abstract

Concentration and distribution of individual endogenous ceramide species is crucial for apoptosis induction in response to various stimuli. Exogenous ceramide analogs induce apoptosis and can in turn modify the composition/concentrations of endogenous ceramide species and associated signaling. In this study, we show here that the elevation of endogenous C16-ceramide levels is a common feature of several known apoptosis-inducing triggers like mmLDL, TNF-alpha, H2O2 and exogenous C6-ceramide. Vice versa apoptosis requires elevation of endogenous C16-ceramide levels in cells. Enantiomers of a synthetic ceramide analog HPL-1RS36N have been developed as probes and vary in their capacity to inducing apoptosis in macrophages and HT-29 cells. Apoptosis induction by the two synthetic ceramide analogs HPL-39N and HPL-1R36N correlates with generation of cellular C16-ceramide concentration. In contrast to the S-enantiomer HPL-1S36N, the R-enantiomer HPL-1R36N shows significant effects on the expression of distinct genes known to be involved in cell cycle, cell growth and cell death (CXCL10, CCL5 and TNF-alpha), similarly on apoptosis induction. Enantioselective effects on transcription induced by metabolically stable synthetic probes provide clues on molecular mechanisms of ceramide-induced signaling, as well as leads for future anti-cancer agents.

Figure 1

Effect of mmLDL (54 μg ml⁻¹) and TNF-alpha (3 ng ml⁻¹) on ceramide concentration in macrophages. (a) C₁₆-ceramide and (b) C_24:1-ceramide concentrations in macrophages after 4 h treatment are shown as mean±S.E., n=3. One-way ANOVA in combination with post hoc t-tests and Bonferroni's correction for multiple testing, P<0.0001 (one-way ANOVA); significant with P<0.05: *versus control, ** mmLDL versus TNF-alpha.

Figure 2

Natural ceramide and synthetic ceramide analogs. Chemical structures of C₆-ceramide (1), C₁₆-ceramide (2), C₂-dihydroceramide (5) and synthetic ceramide analogs HPL-39N 4-[(1R)-(E)-1-Hydroxy-3-phenyl-allyl]-(2RS,4R)-2-phenyl-thiazolidin-3-carbonic acid-t-butylester (3), HPL-38N 4-[(1RS)-(E)-1-Hydroxy-3-phenyl-allyl]-(2RS,4R)-2-phenyl-thiazolidin-3-carbonic acid-t-butyl-ester (4), HPL-1R36N (1R)-(E)-(2-Methyl-oxazol-4-yl)-hexadec-2-en-1-ol (6) and HPL-1S36N (1S)-(E)-(2-Methyl-oxazol-4-yl)-hexadec-2-en-1-ol (7), HPL-1RS36N (1RS)-(E)-(2-Methyl-oxazol-4-yl)-hexadec-2-en-1-ol (8).

Figure 3

Induction and quantification of selected individual ceramide species in human macrophages and relative C₁₆- and C_24:1-ceramide concentrations after stimulation with compounds indicated. (a) Stimulation of C₁₆- and (b) C_24:1-ceramide concentration in macrophages after 4 and 7 h treatment with 10 μM exogenous C₆-ceramide (1), 10 μM HPL-39N (3), 10 μM C₂-dihydroceramide (4), shown as mean±S.E., n=3. One-way ANOVA in combination with post hoc t-tests and Bonferroni's correction for multiple testing, P<0.0001 (one-way ANOVA); significant with P<0.05: *** versus control, ** versus C₂-dihydroceramide (4), * versus HPL-39N (3). (c) Relative ceramide concentrations after 4 h and (d) 7 h stimulation. Relative ceramide concentrations are calculated based on data derived from a and b, and Figures 4c and d. H₂O₂ data obtained in experiments resulting in a and b. Values result from the ratios obtained from the ceramide content in response to the test agent versus ceramide content of untreated control (same incubation conditions, as indicated in Figures 3a and b, and Figures 4c and d. C₆-ceramide data derived from 8 h incubation.

Figure 4

Diverging induction of apoptosis, caspase activity and ceramide concentrations of synthetic ceramide analogs in macrophages. (a) Macrophages were treated 4 h with 0.1, 0.25 and 1.0 μM HPL-1R36N (6) or HPL-1S36N (7) and stained using YO-PRO-1 iodide (apoptotic cells) and Hoechst 33342 (complete cells). (b) Caspase activity (in situ-activated caspases labeled with CaspACE-FITC-VAD-FMK) in isolated monocytes treated 16 h with 10 μM HPL-1RS36N (8), HPL-1R36N (6), HPL-1S36N (7) and exogenous C₆-ceramide (1) as positive control. Data shown as mean±S.E., n=3. One-way ANOVA in combination with post hoc t-tests and Bonferroni's correction for multiple testing, P<0.0001 (one-way ANOVA); significantly different with P<0.05: *** versus control (a and b), ** versus HPL-1RS36N (8) (a),* versus HPL-1S36N (7) (a) or exogenous C₆-ceramide (1) (b). (c) Macrophages were treated 4 h and (d) 7 h with 10 μM HPL-1R36N (6) and HPL-1S36N (7), C₁₆- and C_24:1-ceramide concentrations shown as mean±S.E., n=3. One-way ANOVA in combination with post hoc t-tests and Bonferroni's correction for multiple testing, P<0.0001 (one-way ANOVA); significant with P<0.05: * versus control, ** versus HPL-1S36N (7).

Figure 5

Synthetic ceramide analogs affect differential gene expression in HT-29 cells. HT-29 cells were treated 2, 4, 6, 8 and 24 h with 30 μM HPL-1R36N (6) and HPL-1S36N (7). (a) Differential expressed genes in total RNA of HT-29 cells. Total RNA of cells treated 4, 6, 8, 24 h with HPL-1R36N (6) and HPL-1S36N (7) were transcribed and labeled separately, however, co-hybridized on the same array. (b) Array similarity matrix of hybridized oligonucleotide arrays. Therefore, array dissimilarity in similarity matrix (Pearson’s sample correlation) shows similar behavior as differential gene expression caused by the individual enantiomers. (c) CXCL10, CCL5 and TNF-alpha: three prominent differentially expressed genes in real-time PCR (2, 4, 6, 8, 24 h) and spotted oligonucleotide microarray (4, 6, 8, 24 h) on treatment of HT-29 cells with synthetic ceramides. Significance is supposed, if numerical value of differential gene expression >1.5 fold. Not determined (n.d.), not detectable (n.det.) due to low specific mRNA concentration.