| 説明 |
Entinostat (MS-275) (MS-275) is an inhibitor of HDACs that inhibits HDAC1 and HDAC3 (IC50s: 0.18/0.74 μM). |
| ターゲット＆IC50 |
HDAC1:0.18 μM(cell free), HDAC3:0.74 μM(cell free)
|
| In vitro |
MS275 is partial inhibitors of HeLa nuclear extract HDAC activity, however, showing full and potent inhibition of HDAC1 and 3 isoenzymes. MS275 display a substrate competitive inhibition of HDAC1 (Ki: 1.3 μM) [1]. MS-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil [2]. MS-275 displayed dose-dependent effects in human leukemia and lymphoma cells and primary acute myelogenous leukemia blasts in relation to differentiation and apoptosis. When administered at a low concentration (e.g., 1 micro M), MS-275 exhibited potent antiproliferative activity, inducing p21(CIP1/WAF1)-mediated growth arrest and expression of differentiation markers (CD11b) in U937 cells [3].
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| In vivo |
MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil [2]. MS-275 administration (3.5 mg/kg i.p.) to Experimental autoimmune neuritis (EAN) rats once daily from the appearance of first neurological signs greatly reduced the severity and duration of EAN and attenuated local accumulation of macrophages, T cells and B cells, and demyelination of sciatic nerves [4].
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| キナーゼ試験 |
The HDAC enzyme activity assay was done as described. Briefly, 40 μl HeLa cell nuclear extract, 29 μl enzyme buffer [15 mM Tris HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% (v/v) glycerol]; for recombinant HDAC isoenzymes, 0.1 mg/ml bovine serum albumin (BSA was added) and 1 μl compound were added per well of a microtiter plate. The reaction was started by addition of 30 μl substrate (Ac-NH-GGK(Ac)-AMC final 25 μM). After incubation for 90 min at 30°C, reaction was terminated by adding 25 μl stop solution (50 mM Tris HCl pH 8, 100 mM NaCl, 0.5 mg/ml trypsin, 2 μM TSA). After 40 min incubation at room temperature, fluorescence was measured using a Wallac Victor 1420 multilabel counter (Excitation 355 nm, Emission 460 nm). The HDAC1, 3, 6 and 8 assays were done with slight modifications. About 14 ng/well HDAC1, 2 ng/well HDAC3 or 10 ng/well HDAC6 were incubated with 6, 25 or 10 lM Ac-NH-GGK(Ac)-AMC, respectively, for 2 or 3 hr at 30°C. In contrast, 100 ng/well HDAC8 were incubated with 50 μM Ac-NH-RHK(Ac)K(Ac)-AMC for 3 hr at 30°C. Termination of the reaction and all further steps were done as described earlier for HeLa cell nuclear extracts. For the enzyme kinetic studies with HDAC1, selected HDAC inhibitor (around IC50 value), as well as Ac-NH-GGK(Ac)-AMC substrate (up to 100 μM) concentrations, were evaluated under standard conditions as described earlier [1]. |
| 細胞研究 |
Cancer cells (5 × 10^3) were seeded into each well of 96-well plates and were cultured with graded concentrations of the drugs for 3 days. The cells were stained with 0.1 mg/ml neutral red for 1 h in a CO2-incubator, and, after aspiration of the medium, OD540 of the neutral red solubilized with 50 μl of ethanol and 150 μl of 0.1 M Na2HPO4 was measured. The IC50 value was determined by plotting growth inhibition of the cells against the logarithm of the drug concentration [2]. |
| 動物実験 |
A2780 cells (9 × 10^6) grown in vitro were suspended in PBS and were injected subcutaneously into the flank of nude mouse. For the other tumor lines, KB-3-1, HCT-15, 4-1St, Calu-3, St-4, Capan-1, and HT-29, tumors were passaged several times before starting in vivo antitumor testing, and a tumor lump (2–3 mm in diameter) was transplanted subcutaneously into the flank of a nude mouse by using a trocar needle. Treatment (four or five mice in each experimental group) with the drugs was started after the tumors were confirmed to have grown in the body (tumor size, 20–100 mm3). MS-27-275 and compound 2, both dissolved with 0.05 N HCl, 0.1% Tween 80, and 5-fluorouracil (5-FU) and diluted with physiological saline, were administered orally once daily 5 days per week for 4 weeks. Tumor length and width were monitored twice weekly, and tumor volume was calculated as described [2]. |
参考文献
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