OTS514

Cell experiment:

CD34+ HSCs are cultured in RPMI supplemented with 20% fetal bovine serum and 1× StemSpan CC100. Cells are treated with OTS514 (20 or 40 nM) or OTS-964 (100 or 200 nM) for 48 hours. Collected cells are washed with phosphate-buffered saline (PBS) and resuspended in 100 μL of PBS followed by staining with CD41a antibody for 20 min at room temperature. Finally, the cells are washed with PBS again and then analyzed for CD41a staining by flow cytometry on the BD FACSCalibur. Expression of STAT5 is examined by Western blot with an anti-STAT5 antibody[1].

Animal experiment:

Mice[1]A549 (1×107 cells) or LU-99 cells (5×106 or 1×107 cells) are injected subcutaneously in the left flank of female BALB/cSLC-nu/nu mice. When A549 xenografts have reached an average volume of 200 mm3 or when LU-99 xenografts have reached an average volume of 150 or 200 mm3, animals are randomized into groups of six mice. The in vivo antitumor effect of OTS514 is first investigated in a xenograft model of A549 cells (TOPK-positive lung cancer cells). OTS514 is administered to mice bearing A549 cells after the tumor size reaches about 200 mm3. Mice are intravenously treated with OTS514 (1, 2.5, and 5 mg/kg) once a day for 2 weeks. The tumor size is measured as a surrogate marker of drug response, and the percentage of tumor growth inhibition (TGI) is calculated. The antitumor effect of OTS514 is further investigated tin another lung cancer xenograft model of LU-99 cells. OTS514 is administered to mice bearing LU-99 cancer cells after the tumor size reaches about 200 mm3. Mice are intravenously treated with OTS514 (5 mg/kg) once a day for 2 weeks. Tumor volumes are determined using a caliper. The weight of the mice is determined as an indicator of tolerability on the same days[1].

References:

[1]. Matsuo Y, et al. TOPK inhibitor induces complete tumor regression in xenograft models of human cancer through inhibition of cytokinesis. Sci Transl Med. 2014 Oct 22;6(259):259ra145.