The link of hedgehog (Hh) signaling activation to human cancer and

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Krt6a-cre: mice, which were generated as described previously[20], were maintained and mated under pathogen-free husbandry conditions. The offspring was screened using PCR to determine their transgenic status according to the instruction from the vendors. All animal studies were approved by Institutional Animal Care and Use Committee at Indiana University. Hh inhibitors Cyc was isolated and purified as previously described[21]. 3-Keto-N- (aminoethyl-aminocaproyl-dihydro-cinnamoyl) (KAAD)-Cyc[17] was purchased from Toronto Natural Products, Inc. (Toronto, ON M5R 2G3, Canada). CycT (Physique 1) was generated by reacting 1 mole of tartaric acid with 2 moles of Cyc. The mixture was heated until the solution volume decreased to one third, after which diethyl ether was added. The solution was then cooled, filtered, and precipitated. The purity of CycT was Guanosine IC50 examined by high-pressure liquid chromatography. Open in a separate window Physique 1. A diagram of the cyclopamine tartrate (CycT) salt structure. Assessment of acute toxicity The acute toxicity and Guanosine IC50 LD50 of Cyc and CycT were evaluated using 129S1/SvlmJ mice, weighing 18 to 22 g (stock number 002448, Jackson Laboratory, Bar Harbor, ME, USA). Cyc and CycT were dissolved in 100% ethanol, diluted in saline buffer to a final concentration Guanosine IC50 of 5% ethanol, and intraperitoneally administered to mice at different doses, with 10 mice per dose. An additional 10 mice were treated with the same volume of 5% Guanosine IC50 ethanol in saline buffer (control). The end-point was death or survival 7 days after treatment. Analysis of Cyc and CycT in mouse blood samples After Cyc or CycT administration, blood was drawn from the mouse tail vein at different time points (0, 0.5, 1, 3, 4, 8, 16, 24, 28 h) and kept at C20C. For analysis, samples were thawed at room heat and centrifuged at 12 000 rpm for 5 min in a Beckman benchtop centrifuge. Centrifuged samples were mixed with an equal volume of acetonitrile (Sigma, St. Louis, MO, USA), vortexed for 30 s, and then centrifuged again, as above. The transparent liquid was removed and placed into a micro autosample vial and again centrifuged as above. Samples were then analyzed for Cyc and CycT by liquid chromatography-mass spectrometry using a Thermo Fisher LCQ mass spectrometer equipped with a Surveyor autosampler, MS solvent pump, electrospray ionization source, and Betasil C18 (5 , 100 mm 2.1 mm) column (Thermo Fisher, Waltham, MA 02454, USA). Samples were eluted with 0.1% formic acid and acetonitrile at a flow rate of 0.300 mL/min as follows: 20% acetonitrile (0C1 min), linear gradient increase from 20% to 60% acetonitrile (1C2 min), isocratic flow of 60% acetonitrile (2C10 min), and returned to 20% acetonitrile (10C11 min), followed by column re-equilibration for 5 min before the next injection. The mass spectrometer was operated in the MS/MS mode scanning a parent ion range of (412.3 1) and 10 mice for Krt14-cre: values of < 0.05 indicating statistically significant difference. Power analysis for animal studies was performed with the Statistical Power Calculator from DSS Research ( With 6to 10 mice per group, the power of the study was 90 or higher, with a confidence interval of 90%. Results Assessment of properties of CycT and Cyc Solubility of CycT and Cyc was examined by dissolving them in deionized water at different concentrations. CycT could be dissolved in water at 5-10 mg/mL, whereas Cyc was water insoluble. The formation of the Cyc tartrate salt is predicted to alter Cyc conformation, which may result in changes in bioavailability, biological efficacy, etc. As shown in Physique 2, CycT exhibited a lower acute toxicity (LD50 = 62.5 mg/kg Rabbit Polyclonal to mGluR2/3 body weight for CycT vs. 43.5 mg/kg body weight for Cyc). Even considering the molecular weight of tartaric acid (150 Da), the difference between Cyc (411 Da) and CycT was still statistically significant (< 0.05), suggesting that mice are more tolerable to CycT. The plasma T1/2 for CycT and Cyc varies from animal to animal, which prevented us to accurately differentiate the two. The plasma T1/2 of CycT ranges from 1 to 7.8 h, whereas that of Cyc varies from 1 to 4 h (Determine 2 shows the average value from one experiment with more than 6 mice at each time point). Open in a separate window Physique 2. The median lethal dose (LD50) and plasma half-life (T1/2) of CycT and cyclopamine (Cyc).The LD50 of CycT (A) and Cyc (B) were determined by GraphPad Prism analyses after obtaining the survival data on 129S1/SvlmJ mice injected with different amounts of CycT or Cyc. Unpaired Student's <0.05). The plasma T1/2 values were calculated with GraphPad Prism using the values of plasma CycT Guanosine IC50 (C) or Cyc (D) at different time points following intraperitoneal injection of the compounds into 3-week aged mice (6 mice/dose). We observed significant variations.