In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/ mass spectroscopy. Interestingly, only one specific ceramide, C 18:0 -ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C 18 -ceramide was not detected. These data suggested the hypotheses that decreased levels of C 18 -ceramide might impart a growth advantage to HNSCC cells and that increased generation of C 18 -ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C 18 -ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C 18 -ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/ mUOG1 resulted in increased levels of only C 18:0 -ceramide by ϳ2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C 18 -ceramide by mLAG1/mUOG1 inhibited cell growth (ϳ70 -80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C 18 -ceramide in the regulation of growth of HNSCC.

It has been documented previously that defects in the generation of C(18)-ceramide, a product of ceramide synthase 1 (CerS1), also known as longevity assurance gene 1 (hLASS1), play important roles in the pathogenesis and/or progression of HNSCC. However, whether altered levels of ceramide generation in HNSCC tumors have any clinical relevance remains unknown. In this study, the levels of endogenous ceramides were measured in tumor tissues of 45 HNSCC patients as compared to their normal tissues using high-pressure liquid chromatography/mass spectrometry (LC/MS), and then possible link between ceramide levels and the clinical parameters of HNSCC were examined. The data showed that the levels of C(16)-, C(24)-, C(24:1)-ceramides were significantly elevated in the majority of tumor tissues compared to their normal tissues, while the levels of only C(18)-ceramide were significantly decreased in HNSCC tumors, especially in tumor tissues of male patients. Importantly, it was also shown here that decreased C(18)-ceramide levels in HNSCC tumor tissues were significantly associated with the higher incidences of lymphovascular invasion, and pathologic nodal metastasis. Importantly, attenuation of C(18)-ceramide was also positively linked to the higher overall stages of the primary HNSCC tumors. Therefore, these data suggest, for the first time, that the defects in the generation/accumulation of C(18)-ceramide might have important clinical roles in HNSCC, especially in lymphovascular invasion and nodal disease.

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In this study, a cationic water-soluble ceramide analog L-threo-C 6 -pyridinium-ceramide-bromide (L-t-C 6 -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 IC 50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, L-t-C 6 -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-C 6 -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-C 6 -PyrCer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of L-t-C 6 -Pyr-Cer/ GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cisplatin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of L-t-C 6 -Pyr-Cer in HNSCC tumors weresignificantly 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-C 6 -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.Human head and neck squamous cell carcinomas (HNSCCs) are among the five most common cancers in the world. Global occurrence of HNSCC is high, and it is estimated that approximately 780,000 new patients are diagnosed with HNSCC each year in the adult population. There are approximately 41,000 new HNSCC cases diagnosed annually in the United States in 2004, and the overall 5-year survival of patients with stage III and IV disease remains less than 50% (Her, 2001;Jemal et al., 2004).Historically, chemotherapy did not play a curative role in the treatment of HNSCC but was reserved for palliative therapy. Surgery and radiation therapy remained the primary curative options; however, complications of these ther-

This study is the first epidemiologic study of oral mucosal lesions in adolescents in Turkey. Angular cheilitis was the only oral mucosal lesion that had a significant correlation with anemia.