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

Kim, Choung Soo; Hong, Jun Hyuk; Park, Hyung keun; Hong, Ji Hee; Hwang, Onyou; Park, Taehan

Abstract

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.