Here, we expand our analysis to the murine colon carcinoma cell line CT-26 cells and use CT-26 cells in syngeneic Balb/c mice as an in vivo model of colorectal cancer. LCL-30 was cytotoxic for CT-26 cells in a dose- and time-dependent fashion, in analogy to other cell lines previously tested (Dindo et al, 2006). Cellular fractionation and mass spectrometric analyses showed the following site LCL-30 to be enriched in the mitochondrial fraction, in line with published data on cationic ��-pyridinium analogues of C6 and C16 ceramide (Novgorodov et al, 2005; Dindo et al, 2006; Senkal et al, 2006). Incubation with LCL-30 led to a dose- and time-dependent decrease of cellular ATP-levels, pointing to a breakdown of mitochondrial respiration, as already described for the ��-pyridinium C6 analogue LCL-29 (Novgorodov et al, 2005).

Yet to our surprise, hallmarks of mitochondrially mediated apoptotic cell death, such as cytochrome c release or caspase activation, could not be detected. The mechanism of LCL-30-mediated cell death in CT-26 remains unclear, although ceramide has been implicated as an endogenous mediator of caspase-independent programmed cell death (Thon et al, 2005). Delineating the differences between cell lines that show caspase activation (SW403) and those without (CT-26) might help define the different mechanisms involved. Exposure to LCL-30 led to a transient depression of whole-cell ceramide levels, whereas mitochondrial ceramide levels showed a transient rise. This ceramide response is somewhat different from SW403, which show a mitochondrial decrease of ceramide levels (Dindo et al, 2006).

Importantly, the rapid and pronounced rise of mitochondrial S1P levels is comparable between both cell lines, raising the possibility of the presence of a mitochondrial sphingosine kinase (SphK). Additional experiments with isomers of LCL-30 have revealed S1P to be derived from endogenous sources and not from the breakdown of LCL-30 (Bielawska A, unpublished). S1P has been primarily regarded as a counter player of ceramide activity, although the intracellular compartmentalisation and the biological context (Ikeda et al, 2003) are important for its biological effects. Future experiments should take intracellular distribution of sphingosine kinase proteins into account.

While the activation of SphK1 leads to reduced apoptosis and improved proliferation, activation of SphK2 has been associated with enhanced cell death (Liu et al, 2003), which has been attributed to differential localisation in ER vs cytosol (Wattenberg et al, 2006). Neither SphK1 nor SphK2 has been detected in mitochondria. Nevertheless, there is evidence for additional sphingosine kinase activity Cilengitide (Fukuda et al, 2003), which might be responsible for the rise in mitochondrial levels of S1P in response to LCL-30.