On the role of bioactive sphingolipids and their metabolizing enzymes in cancer

Ceramide is a family of closely related molecules, which are presumed to be in the center of sphingolipid metabolism. Among sphingolipid metabolites, several ceramide subspecies and sphingosine induce apoptosis, cell cycle arrest and death, whereas sphingosine 1-phosphate (S1P) mediates cell proliferation, invasion, angiogenesis and metastasis. Cell fate is largely dependent on the balance of ceramide and sphingosine versus S1P. The sphingosine kinases (SKs) are responsible for maintaining this balance, which leans towards S1P in many cancers. Thereby SKs have been suggested as targets for cancer therapy. The overall aim of this thesis was to target SKs in order to enhance the effects of anti-cancer agents in hepatocellular carcinoma (HCC) and bladder cancer cells.

In study I, we have treated HCC cell lines with selenite in combination with pharmacological inhibitors of sphingolipid-metabolizing enzymes. Selenite treatment induced the activity of neutral sphingomyelinase and increased the levels of long chain ceramides. Moreover, the sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI-II) sensitized HCC cells to selenite treatment by increasing the levels of ceramide subspecies, inducing reactive oxygen species formation and apoptosis, and inhibiting cell cycle progression and cell viability.

In study II, HCC cell lines were co-treated with the multi-tyrosine kinase inhibitor sorafenib and the sphingosine agonist FTY720 in order to improve the efficacy of sorafenib treatment. We have shown that a marginally toxic dose of FTY720 synergistically increased the cytotoxicity of sorafenib towards the Huh7 and HepG2 cell lines. Combined treatment with FTY720 and sorafenib mediated cell cycle arrest, caspase-dependent and –independent apoptosis, autophagy blockage and cell death in Huh7 cells.

In study III, the treatment effects of supernatant from Bacillus Calmette-Guérin -activated macrophages (SupBCG) and SKI-II on murine bladder cancer cell lines were studied. Combined treatment with SKI-II and SupBCG mediated a decrease in cell viability compared to SupBCG treatment alone. There was a transient increase in Sphk1 mRNA level following SupBCG treatment, but the SK1 protein level was unaffected. SupBCG and SKI-II individually induced PARP-cleavage. The level of dihydro C16-ceramide was increased following SKI-II treatment alone, and was further enhanced after combined treatment with SupBCG.

In our studies we have observed that inhibition of SKs enhanced the cytotoxicity of treatment with selenite or sorafenib in HCC cell lines, and of BCG in bladder cancer cell lines. Therefore, we conclude that targeting these enzymes could potentiate other treatment effects in HCC and bladder cancer cells.

List of scientific papers

I. V. Chatzakos, A. K. Rundlöf, D. Ahmed, P. J. de Verdier, J. Flygare. Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite. Biochemical Pharmacology. 84 (2012) 712– 721.
https://doi.org/10.1016/j.bcp.2012.06.009

II. Dilruba Ahmed, Petra J. de Verdier, Charlotta Ryk, Oscar Lunqe, Per Stål & Jenny Flygare. FTY720 (Fingolimod) sensitizes hepatocellular carcinoma cells to sorafenibmediated cytotoxicity. Pharma Res Per. 3(5), 2015, e00171.
https://doi.org/10.1002/prp2.171

III. Dilruba Ahmed, Petra J. de Verdier and Jenny Flygare. Effects of treatment with Bacillus Calmette-Guérin and an inhibitor of sphingosine kinase 1 in urothelial bladder-cancer cells. [Manuscript]