Cell experiment:

HCT116 and HL60 cells are incubated with either 5 μmol/L Reversine or DMSO 0.01%. Cells are harvested and fixed in 70% ethanol overnight. After double washing with PBS, cells are labeled with cell cycle staining reagent PBS, 0.1% Triton X-100, 200 μg/mL DNase-free RNase, and 25 μg/mL propidium iodide and incubated at room temperature in the dark for 30 min. DNA content is analyzed using FACSCalibur. Cell viability of different tumor cell lines is assessed using ATPlite 1step. Briefly, 2×104 cells for each well are plated in a 96-well plate in presence of crescent quantity of Reversine. After 72 h, the plates are recovered and 100 μL ATPlite solution is added to each well. The plates are shaken for 2 min at 700 rpm and luminescence is measured using EnVision Multilabel plate reader. Each sample is analyzed in triplicate[1].

Animal experiment:

Mice[2] Female athymic 6-8 weeks old BALB/c nude mice are used. U14 cell suspension (5×106 cells in 100 μL of RPMI-1640 medium) is injected subcutaneously. The purpose of developing cervical tumors is to generate histological intact tumors for drug therapy. When the diameter of tumors reached up to about 1 cm, Reversine, aspirin or their combinations are administrated by intraperitoneal injection per 3 days, twenty-five of these mice are randomly assigned into one of the following five groups: (a) mice treated with RPMI-1640 medium, (b) mice treated with DMSO, (c) mice treated with Reversine (10 mg/kg), (d) mice treated with aspirin (1 μg/kg) and (e) mice treated with a Reversine and aspirin combination. Body weight and tumor size at the site of inoculation are measured three times a week. Tumor size is measured every 3 days from two diameters, tumor volume is estimated using the formula L×S2/2(L as the longest diameter, S as the shortest diameter).

IC50: 150, 500 and 400 nM for Aurora Kinase A, B and C respectively

Reversine is a novel Aurora kinases inhibitor. Aurora kinases are serine/threonine kinases playing a key role in mitotic regulation. Aurora A kinase resides at spindle poles during mitosis and is implicated in the control of centrosome maturation, duplication, and separation. Aurora B is part of a chromosome passenger complex and is implicated directly in the control of microtubule kinetochore attachment. Aurora kinases have emerged as valuable targets in cancer therapy.

In vitro: Reversine could be used to induce dedifferentiation of murine myoblasts. Previous reports also showed that reversine had a role in regeneration. Moreover, a recent report indicated reversine had anti-tumor capabilities for a myeloma cell line, as demonstrated by that reversine could suppress the expression of cell cycle related proteins Aurora kinase A and Aurora kinase B [1].

In vivo: The effects of reversine on tumor weight and volume were assessed using a murine model of cervical cancer with U14 cells, separately or combined with aspirin. The inhibition rate of cells in the combination group significantly increased; moreover, such combination could synergistically inhibit the proliferation of five cervical cancer cell lines. In the mouse model, tumor weight and volume of cervical cancer bearing mice were more reduced [1].

Clinical trial: N/A

Reference:
[1] Qin HX,Yang J,Cui HK,Li SP,Zhang W,Ding XL,Xia YH.  Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo. Cytotechnology.2013 Aug;65(4):643-53.