SGI-1776 

SACC-83 and SACC-LM cells of 0 μM, 2.5 μM and 5 μM groups after SGI-1776 exposure are harvested. 6 samples of SACC cells are diluted in Kinase buffer and pipetted into the wells which is pre-coated with a substrate corresponding to recombinant p21waf1. It contains threonine residues that can be efficiently phosphorylated by Pim-1. After undergoing the procedure, measure absorbance in each well is quantitated by spectrophotometry at dual wavelengths of 450/540 nm. It reflects the relative amount of Pim-1 activity in the 6 groups of SACC cells.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are seeded in a 96-well plate at a density of 5 000 cells/well. After incubation for 24 h, different concentrations of SGI-1776 (0.625, 1.25, 2.5, 5, 10, 20, 40 µM) are added to each well and cultured for 48 h. The medium is removed and then incubated with 5 mg/L MTT for 4 h. Next, the supernatant is removed after centrifugation. Finally, 100 µL of DMSO is added and an absorbance at 570 nm wavelength (A570) is measured by enzyme-labeling instrument. Relative cell proliferation inhibition rate (IR)=(1-average A570 of the experimental group/average A570 of the control group)×100%.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

The conditions for animal room environment and photoperiod are 20-25°C, 40%-70% humidity, and 12 hours of light/12 hours of dark cycle. Each mouse is inoculated subcutaneously at the right flank with MV-4-11 tumor cells (5×10⁶). The treatments start when the tumor size reach 80-150 mm³. Mice are randomized to treatment groups based on their tumor sizes; tumor size is measured in 2 dimensions using a caliper, and the volume is expressed in mm³ using the formula: V = 0.5 a × b² where a and b are the long and short diameters of the tumor, respectively. Pretreatment randomization ensures that each group has approximately the same mean tumor size. Mice are treated with vehicle (5% dextrose), SGI-1776 or cytarabine (ara-C). SGI-1776 and ara-C are formulated in 5% dextrose. SGI-1776 is administered by oral gavage (PO) on a daily × 5/week or twice/week schedule; ara-C is administered by intraperitoneal injection 3 times/week for 3 consecutive weeks. Animals are euthanized when their measured tumor size is greater than 3000 mm³ or when they lose ≥ 20% initial body weight; if the body weight loss ≥ 15%, treatment is stopped at first until mice regain body weight. Mice are euthanized when body weight loss is still ≥ 20% even after stopping treatment. T/C value (in %) is an indication of antitumor efficacy, where T and C are the mean tumor volume of the treated and control groups, respectively, on a given day. The differences between the mean tumor sizes for comparing groups is analyzed using the ANOVA test, where P ≤ 0.05 is considered to be statistically significant.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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