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. 2008 Jan 15;98(1):98-105.
doi: 10.1038/sj.bjc.6604099. Epub 2007 Nov 20.

Mitochondrially _targeted ceramide LCL-30 inhibits colorectal cancer in mice

F Dahm  1 , A Bielawska, A Nocito, P Georgiev, Z M Szulc, J Bielawski, W Jochum, D Dindo, Y A Hannun, P-A Clavien
Affiliations

Mitochondrially _targeted ceramide LCL-30 inhibits colorectal cancer in mice

F Dahm et al. Br J Cancer. .

Abstract

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial _targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.

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Figures

Figure 1
Figure 1
Cytotoxicity of LCL-30. CT-26 cells were incubated for 24 h with increasing concentrations of LCL-30, and viability was assessed by the MTT assay (IC50=10.6 μM).
Figure 2
Figure 2
Cellular uptake of LCL-30 (A) and the resulting changes in endogenous ceramide (B) and sphingosine-1-phosphate (C) CT-26 were exposed for the indicated time to 10 μM of LCL-30 and then separately analyzed by mass spectrometry for whole cells (squares) and mitochondrially enriched fractions (circles). Results are mean±s.d. of n=3 and normalised to protein content.
Figure 3
Figure 3
(A) Cellular ATP content during exposure to 20 μM FCCP (circles), 10 μM LCL-30 (squares) and 20 μM LCL-30 (triangles). Results are mean±s.d. of n=4 and normalised to protein content. (B) Ratio of cytosolic to mitochondrial cytochrome c at different durations of incubation with 10 μM LCL-30, as assessed by ELISA. (C) Caspase-3-activity after 6 h of exposure to 10 μM LCL-30.
Figure 4
Figure 4
Concentration of LCL-30 in blood after a single intraperitoneal injection of 20 mg kg−1. Results are mean±s.d. of n=4 and normalised to protein content.
Figure 5
Figure 5
(A) Progression of subcutaneous tumours in animals receiving daily injections with vehicle (open squares), doxorubicin (closed squares), LCL-30 (triangles), or LCL-30+doxorubicin (circles), *P=0.027 vs control (ANOVA); (B) Volume of explanted tumours after the end of treatment, *P<0.05, **P<0.01 vs control (ANOVA with Student–Newman-Keuls post hoc test). (C) Proliferative activity of treated tumours assessed by Ki67 immunohistochemistry, *P<0.001 vs control (ANOVA with Student–Newman-Keuls post hoc test). Results are mean±s.d. of n=8.
Figure 6
Figure 6
(A) Levels of endogenous ceramide in tumours after 1 week of treatment with the indicated regimen, *P=0.098 vs control. (B) Levels of sphingosine-1-phosphate in tumours after 1 week of treatment with the indicated regimen, *P=0.045. Results are mean±s.d. of n=4 and normalised to protein content.
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