Ombrabulin (AC-7700)

Cell experiment:

The capacity of Ombrabulin (AC-7700) to modulate MMEC and HeyA8 cell cycle as well as apoptosis is analyzed by flow cytometry. In all assays, 3×106 tumor cells are seeded into Petri dishes and allowed to adhere overnight. The cultures are then washed with PBS and treated with regular medium (negative control) or medium containing Docetaxel, Ombrabulin (AC-7700) (HeyA8, 20 nM; MMEC 10 nM), or Ombrabulin plus Docetaxel. For cell cycle analyses, tumor cells are collected by trypsinization and pooled with the cells floating in the medium. The cell suspensions are centrifuged for 5 min at 1,500 rpm at room temperature, then washed and fixed with ethanol. For apoptosis analysis, cells are incubated overnight in 50 μL of DNA labeling solution (10 μL of reaction buffer, 0.75 μL of TdT enzyme, 8 μL of FITC-dUTP, and 32.25 μL of distilled water) at room temperature. Following the addition of rinse buffer, samples are centrifuged, washed, and fixed in ethanol. All samples are then washed with PBS, then resuspended in propidium iodide (50 μg/mL) and RNase A (20 μg/mL) in PBS for 30 min at room temperature. Stained cells are analyzed on an EPICS XL flow cytometer. The low-level gate is set at the base of the G1 peak and the percentages of cells within the G1 and G2-M phases of the cell cycle are determined by analysis with Multicycle[1].

Animal experiment:

Mice[1]Female athymic nude mice (6-8 weeks old) are used. For in vivo injection, tumor cells are trypsinized, centrifuged at 1,000 rpm ×7 min at 4°C, washed twice, and resuspended in serum-free HBSS at a concentration of 5×106 cells/mL (SKOV3ip1 and HeyA8-MDR) and 1.25×106 cells/mL (HeyA8). Tumors are established by i.p. injection of cells. Ombrabulin therapy is initiated 7 or 17 days after cell line injection. Mice (n=10 per group) are randomly assigned to the following treatment groups: (a) PBS 200 μL, i.p. weekly; (b) Ombrabulin 30 mg/kg [dissolved in PBS (pH 5)] i.p. twice weekly; (c) Docetaxel 2 mg/kg (HeyA8 and HeyA8-MDR) or 1.4 mg/kg (SKOV3ip1) i.p. weekly; (d) Ombrabulin plus Docetaxel (both drugs given at the doses and frequency described above for each drug alone). The dose of Ombrabulin used in these experiments is optimized from dose-escalation studies against tumor growth. Mice are monitored for signs of adverse effects and tumors are harvested after treatment (range 2-5 weeks). The mouse weight, tumor weight, number of tumor nodules, and volume of ascites are recorded at necropsy.

References:

[1]. Kim TJ, et al. Antitumor and antivascular effects of AVE8062 in ovarian carcinoma. Cancer Res. 2007 Oct 1;67(19):9337-45.