Characterization of Isoenzyme-Selective Inhibitors of Human Sphingosine Kinases

Abstract

Sphingosine kinases (SKs) are promising new therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate. The functions of the two SK isoenzymes, SK1 and SK2, are not redundant, with genetic ablation of SK2 having more pronounced anticancer effects than removal of SK1. Although several small molecule inhibitors of SKs have been described in the literature, detailed characterization of their molecular and cellular pharmacology, particularly their activities against human SK1 and SK2, have not been completed. Computational modeling of the putative active sites of SK1 and SK2 suggests structural differences that might allow isozyme-selective inhibitors. Therefore, we characterized several SK-inhibitory compounds which revealed differential inhibitory effects on SK1 and SK2 as follows: SKI-II and ABC294735 are SK1/2-dual inhibitors; CB5468139 is a SK1-selective inhibitor; and ABC294640 is a SK2-selective inhibitor. We examined the effects of the SK inhibitors on several biochemical and phenotypic processes in A498 kidney adenocarcinoma cells. The SK2-selective inhibitor ABC294640 demonstrated the most pronounced effects on SK1 and SK2 mRNA expression, decrease of S1P levels, elevation of ceramide levels, cell cycle arrest, and inhibition of proliferation, migration and invasion. ABC294640 also down-regulated the expression or activation of several signaling proteins, including STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to responses to the SK1/2-dual inhibitors. Overall, these results suggest that inhibition of SK2 results in stronger anticancer effects than does inhibition of SK1 or both SK1 and SK2.

Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II

“…These results suggest that conversion into SM is a fi rst response of cells to the deleterious increase in cytotoxic So, while glycosylation is activated as a sustained response. Similar effects were observed by Gao et al ( 15 ) for SKI II in A498 kidney adenocarcinoma cells. Compared with the control group, the levels of intracellular S1P were decreased by over 90% after treatment with SKI II, while total Cer levels increased and amounts of So were dramatically decreased.…”

“…Similar effects on cell cycle, namely accumulation of the G0/G1 population, were also found with DMS in rat intestinal epithelial cells ( 78 ) and by SK1 silencing in MCF-7 breast cancer ( 79 ), A498 ovarian cancer ( 80 ), and U-1242 MG and U-87 MG glioblastoma cell lines ( 81 ). In apparent contrast, Gao et al ( 15 ) reported that SKI II arrested A498 cells in S phase with a concomitant decrease in the G2/M phase, while downregulation of SK1 or SK2 in U87MG cells under hypoxia arrests the cell cycle at G2/M ( 82 ). Whether the effect on cell cycle is due to SK or Des1 inhibition cannot be concluded with the available experimental data.…”

“…Of note, treatment with SKI II alone signifi cantly elevated cellular dhCer levels, but not Cer levels, although this increase was much lower than that observed in fenretinide and fenretinide/ SKI II treatments. In another report ( 15 ), SKI II was also found to produce an increase in N -hexadecanoyldihydrosphingosine (C16dhCer ) in A498 kidney adenocarcinoma cells. Surprisingly, none of the articles addressed and discussed these observations.…”

Recent advances in the development of sphingosine kinase inhibitors

“…While SKI-II ([2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole]; also known as SKi) was initially reported, and widely used, as an SK1 inhibitor [63], recent studies have demonstrated its ability to also inhibit SK2 [48,[64][65][66][67]. Indeed, the reported K i values of 16 and 8 μM for SK1 and SK2, respectively [66] (Table 1), suggests it is a slightly more potent inhibitor of SK2 than SK1.…”

Role of sphingolipids in oestrogen signalling in breast cancer cells: an update

“…Generated from two distinct genes, human Sphk1 and SphK2 vary considerably in size, because SphK2 is almost twice longer than SphK1 (Neubauer & Pitson 2013). Although both SphK1 and SphK2 can be found in cytoplasm where the enzymes catalyse sphingosine to produce intracellular S1P, these two isoforms differ in substrate binding pockets (Gao et al 2012, Neubauer & Pitson 2013, can translocate to different intracellular compartments and appear to possess distinct biological functions , Aoyagi et al 2012. For instance, SphK1 is a potent anti-apoptotic and growth-promoting signalling enzyme, whereas SphK2 has been shown to initiate proapoptotic cascades (Liu et al 2003, Neubauer & Pitson 2013.…”