In this study, a cationic water-soluble
ceramide analog L-threo-C6-pyridinium-ceramide-bromide (L-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human
head and neck squamous cell carcinoma (
HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, L-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human
squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of L-t-C6-Pyr-Cer with
gemcitabine (GMZ), a chemotherapeutic agent used in
HNSCC, was determined using isobologram studies. Then, the effects of this
ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with L-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of
HNSCC tumors in vivo. The therapeutic efficacy of L-t-C6-Pyr-Cer/GMZ combination against
HNSCC tumors was approximately 2.5-fold better than that of the combination of
5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of L-t-C6-Pyr-Cer in
HNSCC tumors were significantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this
ceramide by approximately 40%. Moreover, treatment with L-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of
telomerase activity and decrease in telomere length in vivo, which are among downstream targets of
ceramide.