OSU-03012 (AR-12)

OSU-03012 (AR-12) induces apoptotic death in PC-3 cells with IC50 of 5 µM and reduces the activity of immunoprecipitated p70^S6K. It completely suppresses cell growth in a diverse range of tumor cell lines at concentrations of 3–5 μm, as compared with the concentration of at least 50 μm. ^[1] This compound promotes cell killing to a greater extent in glioma cells than in nontransformed astrocytes. It causes a dose-dependent induction of cell death that is not altered by p53 mutation, expression of ERBB1 VIII, or loss of phosphatase and tensin function due to a homolog deletion on chromosome 10. OSU-03012 and ionizing radiation cause an additive, caspase-independent elevation in cell killing. Its lethality as a single agent or when combined with signaling modulators is not modified in cells lacking expression of BIM or of BAX/BAK. It promotes the release of cathepsin B from the lysosomal compartment and that of AIF from mitochondria. The lethality of this compound is attenuated in protein kinase R-like endoplasmic reticulum kinase-/- cells, which correlated with the reduced cleavage of BID and suppression of cathepsin B and AIF release into the cytosol. ^[2] It inhibits thyroid cancer cell (NPA, WRO, and ARO cells) proliferation, migration and induces apoptosis, which results in an increase of cells in the S phase without an increase of cells in G2. OSU-03012 is an ATP-competitive inhibitor of PAK activity and suppresses the phosphorylation of AKT in thyroid cancer cells. ^[3] It inhibits cell growth of hepatocellular carcinoma cell lines including Huh7, Hep3B and HepG2 cells with IC50 values below 1 μM. This compound does not suppress PDK1 or AKT activity or induce cellular apoptosis but induces autophagy in Huh7 cells. Moreover, accumulation of reactive oxygen species (ROS) is detected after its treatment. ^[4] A recent study shows that it could enhance the susceptibility of (Bcr)-Abl mutant cell lines to -induced apoptosis. ^[5]

The effect of OSU-03012 (AR-12) on PC-3 cell viability is assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells are grown in 10% FBS- supplemented RPMI 1640 in 96-well, flat-bottomed plates for 24 hours. They are exposed to various concentrations of this compound (0-10 μM) dissolved in DMSO (final concentration ≤0.1%) in 1% serum-containing RPMI 1640 for different time intervals (~72 hours). Controls receive DMSO vehicle at a concentration equal to that in OSU-03012-treated cells. The medium is removed and replaced by 200 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640. The cells are incubated in the CO₂ incubator at 37 °C for 2 hours. Supernatants are removed from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye is solubilized in 200 μL DMSO per well. Absorbance at 570 nm is determined by using a plate reader.

OSU-03012 (AR-12) suppresses tumor growth by 57.59% and increases cleaved LC3 in Huh7 tumor xenografts at 200 mg/kg. ^[4] This compound remarkably decreases expression of EGFR protein in the tumors by 48% compared with vehicle controls and also prevents YB-1 from binding to the EGFR promoter in MDA-MB-435/LCC6 xenografts. ^[6] It is well tolerated and inhibits the growth of HMS-97 schwannoma xenografts by 55% after oral administration. ^[7]