Belinostat

Belinostat is a potent HDAC inhibitor with an IC50 of 27 nM in HeLa cell extracts.

Signaling Pathways >> Autophagy >> Autophagy

Signaling Pathways >> Cell Cycle/DNA Damage >> HDAC

Signaling Pathways >> Epigenetics >> HDAC

Research Areas >> Cancer

HDAC6:82 nM (IC50)

HDAC:27 nM (IC50, Hela cell)

Belinostat (PXD101) induces a concentration-dependent (0.2-5 μM) increase in acetylation of histone H4 in tumor cell lines. Belinostat is cytotoxic in vitro in a number of tumor cell lines with IC50s in the range 0.2-3.4 μM as determined by a clonogenic assay and induces apoptosis. Belinostat inhibits the growth of a number of human tumor cell lines in vitro with IC50s determined by a clonogenic assay in the range 0.2-3.4 μM[1]. Belinostat (PXD101) is a potent histone deacetylase (HDAC) inhibitor, potently inhibits the enzymatic activity of purified recombinant HDAC6 (IC50 of 82 nM)[2].

Treatment of nude mice bearing human ovarian and colon tumor xenografts with Belinostat (10-40 mg/kg/day i.p.) daily for 7 days causes a significant dose-dependent growth delay with no obvious signs of toxicity to the mice. Growth delay is also observed for xenografts of cisplatin-resistant ovarian tumor cells. A marked increase in acetylation of H4 is detected in blood and tumor of mice 3 h after treatment with Belinostat (PXD101). The inhibition of growth of human tumor xenografts in mice, with no apparent toxicity[1]. Belinostat (PXD101) displays single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which is enhanced via combination therapy with Carboplatin[2].

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 12,000× g for 5 min. The upper phase (600 μL) is transferred to 3 mL of scintillation fluid and counted[1].

The human ovarian cell line A2780 and Cisplatin (A2780/cp70) and Doxorubicin (2780AD) resistant derivatives are grown in RPMI 1640 supplemented with glutamine (2 mM) and FCS (10%). The human colon (HCT116 and HT29), melanoma (HS852), prostate (PC3), lung (CALU-3), and breast (MCF7) cell lines are grown in RPMI 1640 and the rest in DMEM supplemented as above. The human non-small cell lung cancer cell line WIL is grown in DMEM supplemented as above. Drug sensitivity is determined by a clonogenic assay. Briefly, cells are plated in 5 mL of medium at a density of 8×104 cells/25 cm2 flask and allowed to attach and grow for 48 h. Cells are exposed to Belinostat (five concentrations from 0.016 to 10 μM) for 24 h. 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 500-2000 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 (mean±SE of three experiments) defined as the concentration of drug required to reduce the number of colonies to 50% of that of the control untreated cells[1].

Mice[1] For the human tumor xenograft studies, monolayer cultures are harvested with trypsin/EDTA (0.25%/1 mM in PBS) and resuspended in PBS. About 107 cells are injected s.c. into the right flank of athymic nude mice (CD1 nu/nu mice). After 10-15 days when the mean tumor diameter is ≥0.5 cm, animals are randomized into groups of six for experiments. Belinostat is dissolved in DMSO and then diluted in water to give a final concentration of DMSO of 10% and is administered i.p. at the times specified. This formulation gives sufficient solubility for doses of ≤ 40 mg/kg. Mice are weighed daily, and tumor volumes are estimated by caliper measurements assuming spherical geometry (volume=d3×π/6).

[1]. Plumb JA, et al. Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histonedeacetylase inhibitor PXD101. Mol Cancer Ther. 2003 Aug;2(8):721-8.

[2]. Qian X, et al. Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies. Mol Cancer Ther. 2006 Aug;5(8):2086-95.

[3]. Chia S, et al. Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time. Nat Commun. 2017 Sep 5;8(1):435.

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