Inhibitory activities

For activity assays, the reaction was carried out in a total volume of 150 μl of buffer [60 mM Tris (pH 7.4) containing 30% glycerol] containing 2 μl of cell extract and, where used, 2 μl of PXD101. The reaction was started by the addition of 2 μl of [3H]labeled substrate (acetylated histone H4 peptide corresponding to the 20 NH2-terminal residues). Samples were incubated at 37℃ for 45 min, and the reaction stopped by the addition of HCl and acetic acid (0.72 and 0.12 M final concentrations, respectively). Released [3H]acetate was extracted into 750 μl of ethyl acetate, and samples were centrifuged at 12,000×g for 5 min. The upper phase (600 μl) was transferred to 3 ml of scintillation fluid and counted.

Cell lines

The human urinary bladder carcinoma cell lines 5637, T24, J82 and RT4.

Preparation method

Soluble in DMSO > 10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

1-5 μM; 48 h.

Applications

In human urinary bladder carcinoma cell lines, belinostat (PXD101) inhibits cell proliferation in a dose dependent way with IC50 values of 1.0, 3.5, 6.0 and 10.0 μM in 5637, T24, J82 and RT4 cell lines, respectively. Belinostat (PXD101) (5 μM) decreases in cell growth and proliferation by 71%, 51%, 41% and 23% in 5637, T24, J82 and RT4 cell lines, respectively. Also, belinostat reduces cells in the S phase and increases cells in the G0-G1 phase.

Animal models

UPII-Ha-ras transgenic mice.

Dosage form

100 mg/kg; 5 days each week for 3 weeks; intraperitoneal (IP) injections.

Preparation method

Dissolved in L-Arginine to give a final concentration of 20 mg/ml.

Applications

In UPII-Ha-ras transgenic mice, belinostat reduces the weights of Ras-expressing bladders of the male and female transgenic mice by 50% and 36%, respectively. Belinostat inhibits progression of bladder disease. Belinostat shows no detectable toxicity as evaluated by weight.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Plumb JA, Finn PW, Williams RJ, et al. Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histone deacetylase inhibitor PXD101. Mol Cancer Ther, 2003, 2(8): 721-728.

[2]. Buckley MT, Yoon J, Yee H, et al. The histone deacetylase inhibitor belinostat (PXD101) suppresses bladder cancer cell growth in vitro and in vivo. J Transl Med, 2007, 5: 49.