dihydroceramide and Colonic-Neoplasms

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit the development of colon cancer. This protective role may be the result of the conversion of complex sphingolipids to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. In the current study, we evaluated the significance of the 4,5-trans double bond by comparing the effects of sphingosine and the cell permeable short-chain ceramide analog C(2)-ceramide to those of sphinganine and C(2)-dihydroceramide, which lack this structural feature. The effects of the sphingoid bases, C(2)-ceramide, and C(2)-dihydroceramide on apoptosis were determined by detecting 200-bp DNA ladders or hypo-diploid areas (sub-G(0)/G(1)), indicative of apoptosis, in HCT-116 human colon cancer cells. In addition, the effects of the sphingoid bases at an apoptotic concentration for 12 hours on cell cycle distribution were determined by flow cytometry. The results indicated that the sphingoid bases and C(2)-ceramide induced apoptosis, whereas C(2)-dihydroceramide had no effects. Sphingoid bases arrested the cell cycle at the G(2)/M phase. The present study provides evidence that the 4,5-trans double bond is necessary for the apoptotic effect of C(2)-ceramide, but not for that of sphingoid bases.

Topics: Apoptosis; Cell Cycle; Cell Division; Ceramides; Colonic Neoplasms; G2 Phase; HCT116 Cells; HT29 Cells; Humans; Sphingosine

Mutations of the adenomatous polyposis coli (APC) and p53 genes are commonly found in colorectal cancers. We therefore analyzed the relative roles of APC and p53 in the induction of apoptosis of colon cancer cells by comparing the effects of the natural chemopreventive agent, C(2)-ceramide, on different human colon cancer cell lines with and without wild-type p53 and APC genes.. We studied the effect of C(2)-ceramide and C(2)-dihydroceramide on proliferation and/or apoptosis of colon cancer cell lines in vitro and determined the role of p53 and APC proteins in these processes. The protein and mRNA levels in colon cancer cell lines with and without treatments were determined by Western and Northern blot analysis, respectively. The cell cycle and apoptosis profiles were determined by FACS analysis and PARP-1 cleavage.. Our findings indicate that C(2)-ceramide can induce apoptosis independently of the p53/p21(Waf-1/Cip-1) pathway. In addition, the C(2)-ceramide induction of apoptosis showed a correlation with a reduction in the levels of the APC protein and mRNA. Moreover, the C(2)-ceramide-induced apoptosis was blocked by pre-treatment with ZnCl(2), which stabilizes APC protein levels.. These results suggest that C(2)-ceramide treatment reduces the levels of APC protein and that the reduction in the levels of this protein plays a key role in the ability of C(2)-ceramide to induce apoptosis of colon cancer cells.

Topics: Adenomatous Polyposis Coli Protein; Apoptosis; Cell Cycle; Cell Proliferation; Ceramides; Chlorides; Colonic Neoplasms; Genes, p53; Humans; Sphingosine; Tumor Cells, Cultured; Zinc Compounds

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 C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-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 C(2)-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 C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/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.

Topics: Apoptosis; Cell Cycle; Cell Division; Ceramides; Colonic Neoplasms; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; HT29 Cells; Humans; Microscopy, Fluorescence; Sphingosine; Structure-Activity Relationship; Tumor Cells, Cultured