dihydroceramide and Liver-Neoplasms

Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ceramides; Hep G2 Cells; Humans; Liver Neoplasms; Protein Kinase Inhibitors; Sorafenib; Sphingolipids

Ceramide is formed through sphingomyelin hydrolysis or de novo synthesis and may play a key role in cell growth, differentiation and apoptosis. To clarify which pathway tumor cells use to form ceramide and how its formation is regulated, we determined the levels of dihydroceramide and ceramide in mice inoculated with Sarcoma 180, B16 melanoma or Lewis lung carcinoma cells. The levels in these tumor masses were very high compared to those in other healthy tissues. The high levels were significantly reduced by a single administration of the dihydroceramide synthase inhibitor fumonisin B(1), but not by a sphingomyelinase inhibitor, sphingomyelin analog-1 (SMA-1), suggesting that the tumor cells have a very effective means of synthesizing dihydroceramide and ceramide. To investigate the characteristics of dihydroceramide synthase, we prepared microsomes from Sarcoma 180 tumor masses and healthy mouse liver cells, and compared their catalytic activities on dihydroceramide formation. A kinetic analysis using sphinganine and palmitoyl CoA as substrates revealed that the enzyme present in the tumor formed dihydroceramide 3 times more efficiently than that in healthy liver cells. Partial purification of dihydroceramide synthase from bovine liver microsomes revealed that the enzyme was present in healthy tissues as a 333 kDa form constructed of 47 kDa subunit proteins. However, gel filtration of the enzyme solubilized from the Sarcoma 180 tumor masses demonstrated that its molecular weight was 1300 kDa. These results suggest that malignant transformation causes the cell to produce a form of dihydroceramide synthase with a larger than normal molecular mass; the increased molecular mass may account for the enzyme's increased catalytic efficiency.

Topics: Amides; Animals; Apoptosis; Catalysis; Cattle; Ceramides; Chromatography, Gel; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fumonisins; Gene Expression Regulation, Enzymologic; Kinetics; Liver; Liver Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Microsomes; Microsomes, Liver; Organophosphonates; Oxidoreductases; Tumor Cells, Cultured