Sphingoid Bases and Ceramide Induce Apoptosis in HT-29 and HCT-116 Human Colon Cancer Cells

Abstract

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C2-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C2-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C2-ceramide inhibited growth and caused death of colon cancer cells in time-and concentration-dependent manners, whereas C2-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C2-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C2-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by Inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C2-ceramide increased the number of cells in the A0 peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G2/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis In colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.

Involvement of Dihydroceramide Desaturase in Cell Cycle Progression in Human Neuroblastoma Cells

“…Previous studies on the biological activity of dhCers using their short chain analogs concluded that dhCers were inactive sphingolipids (12)(13)(14). This study with dhCCPS analogs suggests a novel biologic function for dhCers.…”

“…Some of the limitations of these earlier studies were due to the method used for the quantitation of ceramide levels since ceramide was measured by enzymatic or labeling methods where it was difficult to differentiate Cers from dhCers. Given our experimental data, it is likely that dhCers were the sphingolipids that were elevated and not Cers in the previously published studies.Previous studies on the biological activity of dhCers using their short chain analogs concluded that dhCers were inactive sphingolipids (12)(13)(14). This study with dhCCPS analogs suggests a novel biologic function for dhCers.…”

“…In most cell studies, exogenous dhCers behave as "biological inactive compounds" compared to exogenous ceramides (12)(13)(14). In order to evaluate the biological roles of loss of DEGS-1 (which results in elevation of endogenous dhCers), we examined its effects on cell growth using a trypan blue exclusion method.…”

“…Treatment of cells with exogenous short chain dhCers has failed to inhibit cell growth and induce apoptosis (12)(13)(14).…”

“…Treatment of cells with exogenous short chain dhCers has failed to inhibit cell growth and induce apoptosis (12)(13)(14). In the present study on dihydroceramide desaturase in SMS-KCNR cells, we used a novel insitu assay to measure the activity of this enzyme using cell-permeable dihydroceramidoids (dhCCPS analogs) (15).…”