Cell experiment:

For cytotoxicity assays, the cells are plated at 1×104 cells/mL grown in 100 μL volume per well of 96-well tissue culture plates. The cells are left to adhere overnight and thereafter incubated with different concentrations of Treosulfan alone or in combination with gemcitabine. The drug combination is added to the cell cultures simultaneously or sequentially (the second drug added 12 h after the first). After 72 h of incubation, Alamar Blue(R) solution is added to the wells prior to further overnight incubation. Absorbance is then measured on a spectrophotometer and cell proliferation and cytotoxicity of drugs are calculated. In some experiments, proliferation and cytotoxicity are also determined by using trypan blue exclusion and cell counting with an improved Neubauer hemocytometer and cell viability assessed by staining the cells with 7-amino-actinomycin D (final concentration 200 μg/mL) and Annexin-V and analyzing via flow cytometry using a FACS Scan flow cytometer[1].

Animal experiment:

Mice[3]Female BALB/c mice are 10 to 12 weeks old and weighed approximately 20 g. Animals are fed with standard pelleted food and water ad libitum. They are housed in a climatized chamber with a dark/light cycle of 12 hours. They are divided into four groups: one group is given Treosulfan (1.5 g/kg/day) for 3 consecutive days, one group receives cyclophosphamide (0.1 g/kg/day) for 2 consecutive days, one group is treated with liposomal busulfan (37 mg/kg/day) for 4 consecutive days, and there is a control group with no treatment. Cyclophosphamide, busulfan, and Treosulfan doses are given at sublethal doses to maintain survival of the animals without bone marrow support. Animals are sacrificed on days 1, 3, 6, 9, and 12, after the last dose of treatment, and the spleen and femurs are removed. Six animals are included in each time point for the treated animals and two control animals[3].

Treosulfan (NSC 39069;Treosulphan) is an alkylating agent with activity in ovarian cancer and other solid tumor types.

Treosulfan is an alkylating agent. Treosulfan inhibits several cancer cell lines, such as Panc-1, Miapaca-2 and Capan-2 cells, with IC50s of 3.6 μg/mL, 1.8 μg/mL and 2.1 μg/mL respectively, and shows nearly 100% cytotoxicity on these cell lines at 100 μg/mL. Treosulfan (0.1-100 μg/mL) in combination with gemcitabine exhibits enhanced activity against cancer cells. However, Treosulfan (1, 2.5, 5 μg/ml) combined with 5-fluorouracil (5-FU; 0.1, 0.25, 0.5 μg/ml) has antagonistic effect on Panc-1 cells at intermediate and high concentrations, and on Miapaca-2 cells at all doses[1]. Treosulfan (800 µg/mL) dramatically reduces erythrocyte forward scatter, increases the percentage of annexin-V-binding cells, [Ca2+]i, and ROS. Removal of extracellular Ca2+ abrogates the effect of Treosulfan on annexin-V-binding[2].

Treosulfan (1.5 g/kg/day) induces a rapid myeloablation, depletes the splenic B and T cells in mice. Treosulfan (1.5 g/kg/day) causes olny interleukin-2 production in spleen cells for a short time and without obvious significant effect on synthesis of tumor necrosis factor-α and/or interferon-γ in mice[3].

[1]. Nitsch E, et al. Synergistic cytotoxic activity of treosulfan and gemcitabine in pancreatic cancer cell lines. Anticancer Res. 2014 Apr;34(4):1779-84. [2]. Peter T, et al. Programmed erythrocyte death following in vitro Treosulfan treatment. Cell Physiol Biochem. 2015;35(4):1372-80. [3]. Sj?? F, et al. Myeloablative and immunosuppressive properties of treosulfan in mice. Exp Hematol. 2006 Jan;34(1):115-21.