SKI II

SKI II potently inhibits endogenous SK activities in the breast cancer cell line MDA-MB-231. SKI II demonstrates significantly antiproliferative effects in human cancer cell lines including T-24, MCF-7, MCF-7/VP, NCI/ADR with IC50 of 4.6 μM , 1.2 μM, 0.9 μM and 1.3 μM, respectively. SKI II also induces apoptosis of T24 cells consistent with the hypothesized consequence of reducing S1P levels. ^[1] As demonstrated previously in MDA-MB-231 cells, SKI II decreases S1P formation in JC cells in a concentration-dependent manner, with IC50 of 12 μM. ^[2]

SKI II (50 mg/kg) after intraperitoneal or oral administration significantly decreases tumor growth a syngeneic Balb/c mouse solid tumor model that uses JC mammary adenocarcinoma cells relative to control groups with no overt toxicity or weight loss.^[2] SKI-II (50 mg/kg ip) ameliorates antigen-induced bronchial smooth muscle hyperresponsiveness in mice by endogenously inhibiting generation of S1P. ^[4]

• SK Assay

  A medium-throughput assay suitable for screening for inhibitors of recombinant human SK has been established. Briefly, 5 μg of purified GST-SK fusion protein are combined with 12 nM sphingosine, which contains a 100-fold dilution of [3-³H]sphingosine (20 Ci/mmol), 1 mM ATP, 1 mM magnesium chloride, and 200 μL of assay buffer [20 mM Tris HCl (pH 7.4), 20% glycerol, 1 mM beta-mercaptoethanol, 1 mM EDTA, 20 mM zinc chloride, 1 mM sodium orthovanadate, 15 mM sodium fluoride, and 0.5 mM 4-deoxypyridoxine]. Assays are run for 30 min at 25°C with shaking and contains either 1% DMSO or 5 g/mL test compound, which corresponds to concentrations of 10–25 μM. The reactions are terminated with 50 μL of concentrated ammonium hydroxide, followed by extraction of the assay mixture with chloroform:methanol (2:1). The aqueous portion is transferred to scintillation vials and radioactivity is quantified as a measure of [³H]]S1P formation using a Beckman LS 3801 Scintillation Counter. The intra-assay coefficient of variation is ~10%, whereas interassay variation is ~20%.

• Cell lines

  T-24, MCF-7, MCF-7/VP, NCI/ADR cell lines

• Concentrations

  ~50 μM

• Incubation Time

  48 hours

• Method

  T24, MCF-7, MCF-7/VP, and NCI/ADR cells are plated into 96-well tissue culture plates at ~15% confluency. After 24 hours, cells are treated with various concentrations of inhibitors. After an additional 48 hours, cell survival is assayed using the sulforhodamine B assay.

• Animal Models

  JC xenografts are established in mice

• Dosages

  ~100 mg/kg

• Administration

  Intraperitoneal administration or oral administration