In vitro activity: In intact cells, PPP efficiently inhibits IGF-1-stimulated IGF-1R, Akt (Ser 473) and Erk1/2 phosphorylation. Picropodophyllin specifically inhibits cell growth, and induces apoptosis in cultured IGF-1R-positive tumor cells. Picropodophyllin synergistically sensitizes HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Picropodophyllin and sorafenib synergistically suppress the proliferation and motility of hepatocellular carcinoma cells.

Kinase Assay: Assay of IGF-1R-catalyzed substrate phosphorylation of pTG, using a 96-well plate tyrosine kinase assay kit, is performed. We use recombinant epidermal growth factor receptor, immunoprecipitated IR from HEPG2, immunoprecipitated IGF-1R from P6 cells, and IGF-1R immunodepleted supernatant from P6 (representing “non-IGF-1R tyrosine kinases”). After 30-min treatment of the receptors with the desired compounds in the kinase buffer [50 mM HEPES buffer (pH 7.4), 20 mM MgCl2, 0.1 MnCl2, and 0.2 Na3VO4], the kinase reaction is activated by addition of ATP. The phosphorylated polymer substrate is probed with a phosphotyrosine-specific monoclonal antibody conjugated to horseradish peroxidase, clone PT-66. Color is developed with horseradish peroxidase chromogenic substrate O-phenylenediamine dihydrochloride and quantitated by spectrophotometry (ELISA reader). IGF-1R tyrosine autophosphorylation is analyzed by a sandwich ELISA assay. Briefly, 96-well plates are coated overnight at 4°C with 1 μg/well of an antibody to IGF-1R β-subunit. The plates are blocked with 1% BSA in PBS Tween for 1 h, and then 80 μg/well of total protein lysate from the P6 cell line is added. As a negative control we use total protein lysate from the R- cell line. The investigated compounds are added in tyrosine kinase buffer without ATP at room temperature for 30 min before kinase activation with ATP. Kinase assay is performed using the Sigma kit (see above). After spectrophotometry the IC50 values of inhibitors are determined using the REGRESSION function of Statistica program.

Cell Assay: The determinations are performed using the Cell proliferation kit II, which is based on colorimetric change of the yellow tetrazolium salt 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt in orange formazan dye by the respiratory chain of viable cell. All of the standards and experiments are performed in triplicates.Cells: Melanoma cells (FM 55, SK-MEL-28, SK-MEL-5, C8161, DFB, DFW and AA), sarcoma cells (RD-ES), breast carcinoma cells (MCF 7), prostate carcinoma cells (PC3), hepatoma cells (HepG2) and embryonic mouse fibroblasts (P6 and R-)

Cancer Res. 2004 Jan 1;64(1):236-42; Blood. 2006 Jan 15;107(2):655-60.