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Stereospecific induction of apoptosis in tumor cells via endogenous C 16 -ceramide and distinct transcripts

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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).
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fig2: 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).

Mentions: HPL-39N (4-[(1R)-(E)-1-Hydroxy-3-phenyl-allyl]-(2RS,4R)-2-phenyl-thiazolidin-3-carbon-säure-t-butylester) (3) (Figure 2) is an example of our novel series of bioactive conformationally stabilized ceramide analogs displaying superior pro-apoptotic and PKC-activating effects (see ref. 13 data not shown). The biologically almost inactive agent dihydroceramide was used as a control and known for hardly affecting apoptosis.14 Time-points for measurements were selected on the basis of published data obtained from Jurkat cells and U937 cells compatible with a time-delayed increase of C16-ceramide (2) in stimulated cells (with exogenous, cell permeable C6-ceramide (1)).15,16 Hence exogenous C6-ceramide (1) was used as a reference compound for synthetic, conformationally restricted ceramide analogs. HPL-39N (3) like C6-ceramide increases the total ceramide content (P<0.05, 8 h) from 0.76 pmol to 6.01 pmol (all values calculated as pmol ceramide/nmol lipid phosphate). Under these conditions applying a (under cell culture conditions) soluble control C2-dihydroceramide (5), fails to induce any significant effect on the total lipid content in macrophages (Figures 3a and b). In response to C6-ceramide, the C24:1-ceramide concentration is changed most significantly among ceramide species (Figure 3b).


Stereospecific induction of apoptosis in tumor cells via endogenous C 16 -ceramide and distinct transcripts
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).
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Related In: Results  -  Collection

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fig2: 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).
Mentions: HPL-39N (4-[(1R)-(E)-1-Hydroxy-3-phenyl-allyl]-(2RS,4R)-2-phenyl-thiazolidin-3-carbon-säure-t-butylester) (3) (Figure 2) is an example of our novel series of bioactive conformationally stabilized ceramide analogs displaying superior pro-apoptotic and PKC-activating effects (see ref. 13 data not shown). The biologically almost inactive agent dihydroceramide was used as a control and known for hardly affecting apoptosis.14 Time-points for measurements were selected on the basis of published data obtained from Jurkat cells and U937 cells compatible with a time-delayed increase of C16-ceramide (2) in stimulated cells (with exogenous, cell permeable C6-ceramide (1)).15,16 Hence exogenous C6-ceramide (1) was used as a reference compound for synthetic, conformationally restricted ceramide analogs. HPL-39N (3) like C6-ceramide increases the total ceramide content (P<0.05, 8 h) from 0.76 pmol to 6.01 pmol (all values calculated as pmol ceramide/nmol lipid phosphate). Under these conditions applying a (under cell culture conditions) soluble control C2-dihydroceramide (5), fails to induce any significant effect on the total lipid content in macrophages (Figures 3a and b). In response to C6-ceramide, the C24:1-ceramide concentration is changed most significantly among ceramide species (Figure 3b).

View Article: PubMed Central - PubMed

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.

No MeSH data available.