Relaxation Effect of Synthetic Ceramide Analogues in Cat Esophageal Smooth Muscle Cells

Ceramide has emerged as a novel second messenger for intracellular signalling. It is produced from sphingomyelin and is involved in the control of cell differntiation, proliferation, and apoptosis. C2- ceramide, short chain ceramide, plays a role in mediating contraction of cat esophageal smooth muscle cells. We examined the effect of synthesized ceramide analogues on the C2-ceramide and ACh-induced contraction in esophageal smooth muscle cells isolated with collagenase. CY3523, CY3525, or CY3723 inhibited C2-ceramide induced contraction, in a time dependent manne. Each analogue also inhibited the contraction in concentration dependent manners. CY 3523, CY 3525, and CY 3723 had no effect to the contraction induced by PMA. The inhibition with CY3523, CY3525 and CY3723 on the C2- ceramide induced contraction was recovered by PMA. These analogues decreased the density of MAPK bands (p44/42 or p38) in the western blot. These results suggest that ceramide analogues can inhibit C2-ceramide induced contraction via PKC and MAPK dependent pathway.

Growth Inhibitory Effects and Mechanisms of Ceramides in Renal Cell Carcinoma Cells / 대한비뇨기과학회지

PURPOSE: Because metastatic renal cell carcinoma responds to various forms of therapy with low remission rates, safe therapeutic agents is urgently needed. Ceramide is a potent and specific suppressor of cell growth and an inducer of apoptosis via an intracellular mediation of the sphingomyelin cycle. The present study was designed to assess the growth inhibitory effects and their mechanisms of C2-ceramide and C6-ceramide in renal cell carcinoma cells. MATERIALS AND METHODS: A standard microculture tetrazolium(MTT) assay was used to measure the cytotoxicity of C2-ceramide and C6-ceramide in renal cell carcinoma cell line A498. Apoptosis was confirmed by DNA fragmentation assay using agarose gel and TdT-mediated biotin-dUTP nicked-end labelling(TUNEL) technique. C2-ceramide and C6-ceramide were injected to the A498 tumor which was formed after A498 cells were implanted subcutaneously in athymic mice. Growth inhibitory effects of ceramides were examined biweekly. RESULTS: The survival fractions of A498 cells were 92.6+/-6.0, 82.8+/-14.0, 66.4+/-11.3, 41.8+/-9.6 and 24.3+/-6.3% for the concentrations of C2-ceramide 2, 4, 6, 8 and 10microM, repectively. IC50 of C2-ceramide was approximately 6.7microM. The survival fractions of A498 cells were 60.9+/-5.0, 23.4+/-3.0, 8.7+/-2.1, 5.0+/-1.2 and 3.3+/-0.6% for the concentrations of C6-ceramide 2, 4, 6, 8 and 10microM, respectively. IC50 of C6-ceramide was about 2.3microM. There were DNA fragmentations in A498 cells treated with C2-ceramide or C6-ceramide on the agarose gel and apoptotic tumor cells were also identified after treatment of C2-ceramide and C6-ceramide in TUNEL method. In in vivo study using athymic mice, the growth of A498 tumors was significantly suppressed by C2-ceramide and C6-ceramide. In vivo tumor suppressive effect was more prominent with C6-ceramide than with C2-ceramide. There`s no toxicity-related death of ceramide-treated athmic mice for 3 months. CONCLUSIONS: C2-ceramide and C6-ceramide have the growth inhibitory effects in human renal cell carcinoma cell line A498 by apoptosis mechanism in vitro and they have the in vivo tumor suppressive effects in athymic mice. C6-ceramide was more effective than C2-ceramide in both in vitro cytotoxicity test and in vivo animal experiment of growth inhibition. Therefore, ceramides may be used to treat metastatic renal cell carcinoma in the future.