product name Belinostat (PXD101)
Description: Belinostat (also known as PXD101) is a novel HDAC inhibitor with IC50 of 27 nM in a cell-free assay, it also demonstrated activity in cisplatin-resistant tumors. Belinostat has been found to significantly increase the acetylation of histones H3 and H4 and exerts cytotoxicity in a wide range of tumor cell lines. In the treatment of urothelial carcinoma cell lines, belinostat dose-dependently inhibits proliferation in 5637, T24, J82 and RT4 cells with IC50 values of 1.0 μM, 3.5 μM, 6.0 μM and 10 μM respectively; while, in prostate cancer cell lines, it inhibits cancer cell growth with IC50 values ranging from 0.5 to 2.5 μM.
References: Mol Cancer Ther. 2003;2(8):721-8; Mol Cancer Ther. 2006;5(8):2086-95; Mol Cancer Ther. 2007;6(1):37-50.
318.35
Formula
C15H14N2O4S
CAS No.
414864-00-9
Storage
-20℃ for 3 years in powder form
-80℃ for 2 years in solvent
Solubility (In vitro)
DMSO: 64 mg/mL (201.0 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Solubility (In vivo)
2% DMSO+30% PEG 300+ddH2O: 10mg/mL
Synonyms
NSC726630, PX-105684
other peoduct :References PubMed ID：:http://www.ncbi.nlm.nih.gov/pubmed/19424345
| In Vitro |
In vitro activity: Belinostat inhibits the growth of tumor cells (A2780, HCT116, HT29, WIL, CALU-3, MCF7, PC3 and HS852) with IC50 from 0.2-0.66 μM. PD101 shows low activity in A2780/cp70 and 2780AD cells, which are cisplatin and doxorubicin-resistant derivatives of A2780 cells. Belinostat could induce apoptosis through PARP cleavage and acetylation of histones H3/H4. Belinostat inhibits bladder cancer cell growth, especially in 5637 cells, which shows accumulation of G0-G1 phase, decrease in S phase and increase in G2-M phase. The growth inhibitory activity of belinostat on cell lines is not strongly influenced by the multidrug-resistant phenotype, whereas the activity of docetaxel is clearly affected. Belinostat could enhance the growth inhibitory activity of docetaxel or carboplatin in OVCAR-3 and A2780 cells. Belinostat also shows enhanced tubulin acetylation in ovarian cancer cell lines. A recent study shows that Belinostat activates protein kinase A in a TGF-β signaling-dependent mechanism and decreases survivin mRNA. Kinase Assay: Subconfluent cultures are harvested and washed twice in ice cold PBS and pelleted by centrifugation at 200 × g for 5 min. The cell pellet is resuspended in two volumes of lysis buffer [60 mM Tris buffer (pH 7.4) containing 30% glycerol and 450 mM NaCl] and lysed by three freeze (dry ice) thaw (30 °C water bath) cycles. Cell debris is removed by centrifugation at 1.2 × 104 g for 5 min, and the supernatant is stored at −80 °C. Histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRK corresponding to the 20 NH2-terminal residues) is acetylated by a recombinant protein containing the hypoxanthine-aminopterin-thymidine domain of p300, using [3H]acetyl CoA as a source of acetate. H4 peptide (100 μg) is mixed with hypoxanthine-aminopterin-thymidine buffer (50 mM Tris HCl pH 8.0, 5% glycerol, 50 mM KCl, and 0.1 mM EDTA), 1 mM DTT, 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 1 × complete protease inhibitors, 50 μL of purified p300, and 1.85 m [3H]acetyl CoA (4.50Ci/mmol) in a final volume of 300 μL and incubated at 30 °C for 45 min. The p300 protein is removed by incubation with 20 μL of 50% Ni-agaroase beads for 1 hour at 4 °C and centrifugation. The supernatant is applied to a 2 mL Sephadex G15 column, and the flow through is collected. One milliliter of distilled H2O is gently applied, and three drop fractions are collected; this is repeated until 4–5 mL of distilled H2O has been added, and ∼40 fractions are collected. Three microliters of each fraction are diluted in 2 mL of scintillation fluid and counted in a scintillation counter to identify the fractions containing the labeled peptide. These fractions are pooled, and 1 μL of the combined sample is measured to assess the radioactivity in every peptide batch (3-7×103 cpm/μL). For activity assays, the reaction is 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 belinostat. The reaction is started by the addition of 2 μL of [3H] labeled substrate (acetylated histone H4 peptide corresponding to the 20 NH2-terminal residues). Samples are incubated at 37 °C 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 is extracted into 750 μL of ethyl acetate, and samples are centrifuged at 1.2× 104 g for 5 min. The upper phase (600 μL) is transferred to 3 mL of scintillation fluid and counted. Cell Assay: Tumor cell lines are seeded in 5 mL of medium at a density of 8 × 104 cells/25 cm2 flask and incubated for 48 hours. Cells are exposed to Belinostat (0.016 to 10 μM) for 24 hours. The medium is removed, and 1 mL of trypsin/EDTA is added to each flask. Once the cells have detached, 1 mL of medium is added, the cells are resuspended, and those from the control untreated flask are counted. Cells are diluted and plated into 6-cm Petri dishes (three per flask) at a density of 0.5-2× 103 cells/dish depending on the cell line. Cells from the drug-treated flasks are diluted and plated as for the control flasks. Dishes are incubated for 10–15 days at 37 °C. Cells are washed with PBS, fixed in methanol, and stained with crystal violet, and colonies that contained ≥50 cells counted. Sensitivity is expressed as the IC50 defined as the concentration of belinostat required to reduce the number of colonies to 50% of that of the control untreated cells. |
|---|---|
| In Vivo | Belinostat indicates significant tumor growth delay in A2780 and A2780/cp70 xenograft at a dose of 10mg/kg with no effects on the body weight. Belinostat also induces p21WAF1, HDAC core and cell communication genes in mouse bladder tumors. Belinostat monotherapy induces dose-proportional antitumor effects with TGI of 47% at a dose of 100mg/kg in A2780 xenograft. The combination of Belinostat (100 mg/kg) with carboplatin (40 mg/kg) could delay tumor growth from 18.6 days to 22.5 days. Combining with bortezomib, Belinostat results in great tumor inhibition and gastrointestinal toxicity in mice with bortezomib-resistant UMSCC-11A xenograft |
| Animal model | A2780, A2780/cp70 and HCT116 cells are injected s.c. into the right flank of CD1 nu/nu mice. |
| Formulation & Dosage | Dissolved in DMSO and then diluted in water to give a final concentration of DMSO of 10%; ≤40 mg/kg; i.p. injection. |
| References | Mol Cancer Ther. 2003 Aug;2(8):721-8; Mol Cancer Ther. 2006 Aug;5(8):2086-95; Mol Cancer Ther. 2007 Jan;6(1):37-50. |
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