Kinase experiment:

Recombinant PLK1 (10 ng) is incubated with different concentrations of Rigosertib in a 15 μL reaction mixture (50 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 2 mM Dithiothreitol, 0.01% NP-40 [pH 7.5]) for 30 min at room temperature. Kinase reactions are performed for 20 min at 30℃ in a volume of 20 μL (15 μL enzyme + inhibitor, 2 μL 1 mM ATP), 2 μL of γ32P-ATP (40 μCi), and 1 μL of recombinant Cdc25C (100 ng) or casein (1 μg) substrates. Reactions are terminated by boiling for 2 min in 20 μL of 2× Laemmli buffer. Phosphorylated substrates are separated by 18% SDS-PAGE. The gels are dried and exposed to X-ray film for 3-10 min.

Cell experiment:

Tumor cells are plated into six-well dishes at a density of 1×105 cells/mL/well, and Rigosertib is added 24 hours later at various concentrations. Cell counts are determined from duplicate wells after 96-hour of treatment. The total number of viable cells is determined by trypan blue exclusion.

Animal experiment:

Bel-7402 tumor models: twenty female athymic (NCR-nu/nu) nude mice are injected with 1 × 107 Bel-7402 tumor cells subcutaneously, and 10-14 days later, when the tumor volumes reach 200-250 mm, the mice are divided into four groups such that each group harbors tumors of the same volume. Rigosertib (ON01910, 250 mg/kg) dissolved in PBS is administered alone or in combination with NSC 266046 (100 mg/kg) intraperitonially on alternate days. Tumor measurements are done two times/week using traceable digital vernier calipers. Body weight is determined during each measurement. The animals are observed for signs of toxicity[1].

Rigosertib is a dual inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (PLK1) [1].

In vitro studies show that rigosertib inhibits the PI3K/AKT pathway, down-regulates cyclin D1, induces NOXA and BIM and activates the JNK pathway in human leukemic cells. Rigosertib induces apoptosis of a variety of human tumor cell lines including breast, prostate, ovarian, pancreatic, SCLC, colorectal, melanoma and et al. For instant, it shows anti-tumor efficacy in BT20, MCF-7, BT474, OV-CAR-3, A549 and HCT-116 with IC50 values of 80nM, 75nM, 50nM, 75nM, 90nM and 75nM, respectively. In addition, rigosertib is also effective against the drug resistant tumor cell lines. It potently inhibits tumor growth with IC50 values of 100nM and 50nM against MES-SA/DX5 and CEM/C2, respectively. Moreover, it is reported that rigosertib can affect the cell cycle of both normal cells and tumor cells. Rigosertib leads to a blockade of cell cycle progression in the G1 and G2/M phases in normal diploid human fetal lung cells. When treated with DU145 cells, rigosertib induces cell cycle arrest in G2/M phase [2].

References:
[1] Anderson R T, Keysar S B, Bowles D W, et al. The Dual Pathway Inhibitor Rigosertib Is Effective in Direct Patient Tumor Xenografts of Head and Neck Squamous Cell Carcinomas. Molecular cancer therapeutics, 2013, 12(10): 1994-2005.
[2] Reddy M V R, Venkatapuram P, Mallireddigari M R, et al. Discovery of a clinical stage multi-kinase inhibitor sodium (E)-2-{2-Methoxy-5-[(2′, 4′, 6′-trimethoxystyrylsulfonyl) methyl] phenylamino} acetate (ON 01910. Na): synthesis, structure–activity relationship, and biological activity. Journal of medicinal chemistry, 2011, 54(18): 6254-6276.