Cell experiment:

The cells are plated into 96-well plates and treated with KW-2478. After 72 hours of cultivation, cell viability is determined using WST-1. WST reagent is added to the wells, followed by incubation for 4 hours at 37℃. After that, the absorbance at 450 nm with reference at 650 nm is measured with a microplate spectrophotometer[1].

Animal experiment:

Mice[1]Severe combined immunodeficient (SCID) mice are intraperitoneally injected with anti-asialo GM1 antibody. The next day, all mice are subcutaneously inoculated with NCI-H929 cells (1×107 cells) suspended in PBS containing 50% of Matrigel. After 10 days, tumor volume is measured using the Antitumor test system II, a computer-operated system including software and instruments. SCID mice with tumors (190 to 290 mm3) are selected. After randomly grouping, saline (vehicle) or KW-2478 is intravenously administered to mice once or twice daily for 5 days. 17-AAG is intravenously administered to mice. Tumor volume is calculated by the Anti-tumor test system II as follows: Tumor volume=DL×DS×DS×1/2. Fourteen days after the initial administration, blood samples of each mouse are obtained, followed by measurement of serum M protein (Ig kappa chain) with Human Kappa-b&f ELISA Quantitation Kit. The statistical analysis is performed using SAS software[1].

KW-2478 is a novel and potent nonansamycin inhibitor of heat shock protein 90 (Hsp90) to overcome the limitations, including low water solubility and hepatotoxicity, of 17-allylamino-17-demethoxygeldanamycin (17-AAG). KW-2478 exerts a strong antitumor activity against multiple myeloma (MM) cells with various chromosormal translocation, in which KW-2478 inhibits cell growth and apoptosis associated with Hsp90 client protein degradation and the decreasing of IgH translocation products including FGFR3, c-Maf and cyclin D1. Recent study results have revealed that KW-2478 is able to deplete Hsp90 client Cdk9 and the phosphorylated 4E-BP1, a transcriptional kinase and a transcription inhibitor respectively, leading to the reduced expression of c-Maf and cyclin D1.

Reference

[1].Nakashima T, Ishii T, Tagaya H, Seike T, Nakagawa H, Kanda Y, Akinaga S, Soga S, Shiotsu Y. New molecular and biological mechanism of antitumor activities of KW-2478, a novel nonansamycin heat shock protein 90 inhibitor, in multiple myeloma cells. Clin Cancer Res. 2010; 16(10): 2792-802.