Briefly, cell lysates or tumour homogenate selleck chemical fractions were fortified with internal standards for quantification, and lipids were extracted and analysed as previously described (Bielawski et al, 2006). Statistical analysis Data represent mean��s.d. of n independent experiments. Mann�CWhitney U-test or one-way ANOVA with Student�CNewman�CKeuls post hoc testing was used as appropriate to compare groups, using SPSS 12.0 (SPSS Inc., Chicago, USA). A P-value below 0.05 was considered to indicate statistical significance. RESULTS LCL-30 elicits cytotoxicity in vitro We previously tested the cytotoxicity of LCL-30 on a range of human and murine cancer cell lines (Dindo et al, 2006). For the present study, we focused on the colon cancer cell line CT-26, which can be used as a syngeneic in vivo model of colorectal cancer in Balb/c mice.

LCL-30 treatment of CT-26 cells was able to effectively induce cell death in vitro in a dose-dependent manner (Figure 1). A 50% inhibition of cell viability (IC50) was achieved at 10.6��M. Time course experiments with different concentrations showed a time-dependent reduction of cell viability with a steady slope (not shown). These results were confirmed by trypan blue exclusion (not shown). Figure 1 Cytotoxicity of LCL-30. CT-26 cells were incubated for 24h with increasing concentrations of LCL-30, and viability was assessed by the MTT assay (IC50=10.6��M). LCL-30 targets mitochondria Ceramide has been detected in mitochondria (Dindo et al, 2006) as well as some ceramide-metabolising enzymes such as ceramide synthase and ceramidase.

LCL-30 represents a cationic lipid designed to be enriched in the positively charged mitochondria. To investigate whether this mitochondrial accumulation occurs in CT-26, cells were treated with the IC50 concentration of LCL-30 (10��M) for up to 8h. Whole cells and mitochondrially enriched fractions were isolated at different time points and subjected to mass spectrometry, allowing a detailed detection of endogenous sphingolipids as well as LCL-30. As illustrated in Figure 2A, LCL-30 was progressively taken up into cells with levels achieving approximately 0.95pmole��g?1 protein Drug_discovery after 4h of incubation. Notably, LCL-30 was significantly enriched in the mitochondrial fraction such that its levels in isolated mitochondria reached about 6pmole��g?1 protein by 4h. Cellular and mitochondrial uptake appeared to level off after 4h. These results demonstrate significant enrichment of LCL-30 in the mitochondria of CT-26 cells.