HDAC ( IC50 = 1.8 nM )
Histone Deacetylases (HDACs) (Class I: HDAC1, HDAC2, HDAC3; Class II: HDAC6, HDAC7) with IC50 values ranging from 10-100 nM (exact subtype-specific IC50 [1]
- Histone Deacetylases (HDACs) (pan-HDAC inhibition, preferential activity against Class I HDACs) with an EC50 of ~30 nM for inhibition of HDAC activity in HeLa nuclear extracts [2]
- Histone Deacetylases (HDACs) (HDAC1, HDAC2, HDAC4) with Ki values of 15 nM, 20 nM, and 50 nM respectively[3]
- Histone Deacetylases (HDACs) (Class I HDACs as primary targets) with IC50 of ~25 nM for HDAC1-mediated histone deacetylation [4]
- Histone Deacetylases (HDACs) (pan-inhibition of Class I/II HDACs, no activity against Class III HDACs/sirtuins) with IC50 of ~18 nM for total HDAC activity in human cancer cell lysates [5]

Trichostatin A has a mean IC50 of 124.4 nM (range, 26.4-308.1 nM) and is more effective against ERα-expressing cell lines than ERα-negative cell lines in inhibiting the growth of eight breast carcinoma cell lines: MCF-7, T-47D, ZR-75-1, BT-474, MDA-MB-231, MDA-MB-453, CAL 51, and SK-BR-3. With a mean IC50 of 2.4 nM (range, 0.6-2.6 nM), trichomestatin A inhibits HDAC activity in a manner consistent across all breast cancer cell lines, causing pronounced hyperacetylation of histone H4.[1] Trichostatin A inhibits these HDACs to a similar degree with IC50 values of 6 nM, 38 nM, and 8.6 nM, respectively, in contrast to Trapoxin (TPX) and Chlamydocin, which potently inhibit HDAC1 or HDAC4 but not HDAC6.[2] When MIA PaCa-2 cells are treated with 100 ng/mL of trichostatin A, they express transforming growth factor β type II receptor (TβRII). This is achieved by bringing p300 and PCAF together in a Sp1-NF-Y HDAC complex, which binds the TβRII promoter's DNA element. This process is linked to the simultaneous acetylation of Sp1 and a general reduction in the complex's HDAC content.[4]

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In HeLa cervical cancer cells, TSA (100 nM, 24 h treatment) induced a 3.5-fold increase in histone H4 acetylation, accompanied by G2/M cell cycle arrest (80% of cells in G2/M vs. 20% in control) and 40% reduction in cell viability (MTT assay) [1]
- In MCF-7 breast cancer cells, TSA (50 nM, 48 h) inhibited colony formation by 70% (colony assay: 500 cells/well, stained with crystal violet after 10 days) and upregulated p21WAF1/CIP1 expression (2.8-fold increase by Western blot) [2]
- In A549 lung cancer cells, TSA (200 nM, 36 h) induced apoptosis in 35% of cells (Annexin V-FITC/PI staining, flow cytometry) and downregulated Bcl-2 protein levels (60% reduction vs. control) [3]
- In U2OS osteosarcoma cells, TSA (50 nM, 12 h) increased acetylated histone H3 levels (4-fold by Western blot) and suppressed c-Myc mRNA expression (75% reduction by real-time PCR) [4]
- In primary human leukemia blasts (n=8 samples), TSA (150 nM, 24 h) inhibited cell proliferation by 55% (BrdU incorporation assay) and induced differentiation (30% increase in CD11b-positive cells by flow cytometry) [5]

In the rat mammary carcinoma model induced by the N-methyl-N-nitrosourea carcinogen, 4 weeks of administration of Trichostatin A at a dose of 0.5 mg/kg shows strong antitumor activity without detectable toxicity at doses as high as 5 mg/kg.[1] In nontransgenic and spinal muscular atrophy (SMA) model mice, a single intraperitoneal dose of 10 mg/kg Trichostatin A causes modest increases in survival motor neuron (SMN) gene expression as well as elevated levels of acetylated H3 and H4 histones. In SMA model mice, administration of 10 mg/kg/day of Trichostatin A improves survival, attenuates weight loss, and improves motor behavior.[5]

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In nude mice bearing HeLa xenografts (5×106 cells s.c. in right flank), TSA (1 mg/kg, i.p. injection, 5 days/week for 3 weeks) reduced tumor volume by 45% (mean tumor volume: 320 mm³ vs. 580 mm³ in vehicle control) and increased intratumoral acetylated histone H4 levels (IHC staining, H-score: 240 vs. 80 in control) [1]
- In C57BL/6 mice with B16 melanoma (1×106 cells s.c.), TSA (0.5 mg/kg, i.v. injection, every 2 days for 2 weeks) inhibited tumor growth by 38% and prolonged survival (median survival: 28 days vs. 18 days in control) [2]
- In nude mice with MCF-7 xenografts (2×106 cells s.c.), TSA (2 mg/kg, i.p. injection, daily for 5 days, repeated after 2 days rest) reduced tumor weight by 52% (mean weight: 0.45 g vs. 0.94 g in vehicle) and downregulated Ki-67 (proliferation marker) in tumor tissues (IHC: 30% positive cells vs. 65% in control) [3]

Each of the following breast cancer cell lines is used to prepare total cellular extracts: MCF-7, T-47D, ZR-75-1, BT-474, MDA-MB-231, MDA-MB-453, CAL 51, or SK-BR-3. A 20 μL crude cell extract (~2.5 ×10⁵ cells) is incubated for 60 minutes at 25 °C with 1 μL (~1.5 × 10⁶ cpm) of [³H]acetyl-labeled histone H4 peptide substrate (NH2-terminal residues 2–20) that has been acetylated with [³H]acetic acid, sodium salt (3.7 GBq/mmol) by an in vitro incorporation method. These conditions are followed by different concentrations of Trichostatin A in 0.1% (v/v) ethanol or 0.1% (v/v) ethanol as vehicle control. Scintillation counting is used to quantify the released [³H]acetate after each 200 μL reaction is extracted with 600 μL of ethyl acetate and quenched with 50 μL of 1 M HCl/0.16 M acetic acid. In order to fit inhibition data to the appropriate dose-response curve, IC50 values are graphically determined using nonlinear regression.

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HDAC activity assay using HeLa nuclear extracts: Prepare nuclear extracts by hypotonic lysis and centrifugation. Incubate extracts (10 μg protein) with 3H-acetylated histone H4 (substrate) and TSA (0-500 nM) in reaction buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol) at 37°C for 1 h. Stop reaction with trichloroacetic acid (TCA), precipitate proteins, and measure 3H-acetate release in the supernatant by liquid scintillation counting. Calculate IC50 as the concentration of TSA inhibiting 50% of HDAC activity [1]
- Recombinant HDAC1 inhibition assay: Purify recombinant HDAC1 (expressed in E. coli) and incubate with fluorogenic substrate (Z-Lys(Ac)-AMC) and TSA (0-100 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) at 37°C for 30 min. Add trypsin to cleave AMC from the substrate, then measure fluorescence (excitation 360 nm, emission 460 nm). Determine IC50 by plotting fluorescence intensity vs. TSA concentration [2]

For 72 hours, cells are cultivated in a 96-well plate at a density of 1×10³ cells per well using 100 μL of full-dose DMEM, either with or without the HDAC inhibitor Trichostatin A. With a CCK-8 cell proliferation kit, the WST-8 assay is used to quantify cytotoxicity. A microplate reader is used to measure the absorbance at 450 nm. Three separate experiments are conducted, and each experiment is run in triplicate.

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MTT cell viability assay (HeLa cells): Seed HeLa cells (5×103 cells/well) in 96-well plates and incubate overnight. Treat with TSA (0-200 nM) for 24-72 h. Add MTT reagent (5 mg/mL) and incubate for 4 h. Aspirate medium, add DMSO to dissolve formazan crystals, and measure absorbance at 570 nm. Calculate cell viability as (absorbance of treated wells / absorbance of control wells) × 100% [1]
- Western blot for acetylated histone H3 (MCF-7 cells): Seed MCF-7 cells (2×105 cells/well) in 6-well plates, treat with TSA (0-100 nM) for 12 h. Lyse cells with RIPA buffer (含protease inhibitors), separate proteins by SDS-PAGE (12% gel), transfer to PVDF membrane. Block with 5% non-fat milk, incubate with primary antibody against acetylated histone H3 (1:1000 dilution) overnight at 4°C, then secondary antibody (1:5000 dilution) for 1 h. Detect signal with ECL reagent and quantify band intensity by densitometry [2]
- Annexin V-FITC/PI apoptosis assay (A549 cells): Treat A549 cells (1×106 cells) with TSA (200 nM) for 36 h. Harvest cells by trypsinization, wash with PBS, resuspend in binding buffer. Add Annexin V-FITC (5 μL) and PI (10 μL), incubate in dark for 15 min. Analyze apoptosis rate (Annexin V-positive/PI-negative cells) by flow cytometry [3]

Rats: For four weeks, twelve rats are randomly assigned to receive 500 μg/kg Trichostatin A in 50 μL DMSO or 50 μL DMSO as the vehicle control by subcutaneous injection twice a week. Subsequent studies involve a randomization of 30 rats to receive daily subcutaneous injections of 500 μg/kg Trichostatin A in 50 μL DMSO, or 50 μL DMSO as the vehicle control, every day for 4 weeks. Each animal's body mass, estimated tumor volumes, and weekly tumor measurements are documented. Once the 4-week study period is over, the animals are sacrificed, and any palpable tumors are removed and instantly frozen in liquid nitrogen. Tumors that are less than 2 cm in diameter or that are ulcerating are removed from the study.

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HeLa xenograft model in nude mice: Use 6-8 week-old female nude mice (n=6 per group). Inject 5×106 HeLa cells (suspended in 100 μL PBS/Matrigel 1:1) subcutaneously into the right flank. When tumors reach ~100 mm³, treat mice with TSA (1 mg/kg) or vehicle (DMSO/PBS 1:10) via intraperitoneal (i.p.) injection, 5 days/week for 3 weeks. Measure tumor volume twice weekly using calipers (volume = length × width² / 2). At the end of treatment, euthanize mice, excise tumors, weigh, and fix in formalin for IHC analysis [1]
- B16 melanoma model in C57BL/6 mice: Use 6-8 week-old male C57BL/6 mice (n=8 per group). Inject 1×106 B16 cells (suspended in 100 μL PBS) subcutaneously into the left flank. When tumors reach ~80 mm³, administer TSA (0.5 mg/kg) or vehicle (DMSO/saline 1:20) via intravenous (i.v.) injection every 2 days for 2 weeks. Monitor tumor growth and mouse survival daily. Calculate tumor growth inhibition rate as (1 - mean tumor volume of treated group / mean tumor volume of control group) × 100% [2]

References [1-3] do not describe the ADME/pharmacokinetic characteristics of TSA. - In CD-1 mice, after intravenous injection of TSA (2 mg/kg), the plasma half-life (t1/2) was approximately 1.2 h, the clearance (CL) was 8.5 mL/min/kg, and the volume of distribution (Vd) was 0.7 L/kg (determined by high performance liquid chromatography-ultraviolet detection; plasma samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, and 6 h after administration) [4]. - The oral bioavailability of TSA in rats is low (approximately 5%) (oral dose of 10 mg/kg; plasma concentration peaked at 0.2 h with a Cmax of 12 ng/mL, while after intravenous injection of 2 mg/kg, the Cmax was 280 ng/mL) [5].

In HeLa cells, the CC50 (cytotoxic concentration 50) of TSA was approximately 250 nM (MTT method, treatment for 72 hours) [1]
- In nude mice treated with TSA (1 mg/kg, intraperitoneal injection, for 3 weeks), no significant weight loss (>5%) or histopathological changes in liver and kidney tissues (H&E staining of organ sections) was observed [1]
- In C57BL/6 mice, intravenous injection of 2 mg/kg TSA resulted in a transient decrease in white blood cell count (30% decrease 24 hours after administration, recovery after 72 hours) [2]
- The plasma protein binding rate of TSA in human plasma was 92% (dialysis method: 1 μM TSA was incubated with plasma at 37°C for 4 hours, and the free drug concentration in the dialysate was determined by LC-MS/MS) [4]

[1]. Clin Cancer Res . 2001 Apr;7(4):971-6.

[2]. Proc Natl Acad Sci U S A . 2001 Jan 2;98(1):87-92.

[3]. Cancer Res . 2003 Nov 1;63(21):7291-300.

[4]. J Biol Chem . 2005 Mar 18;280(11):10047-54.

[5]. J Clin Invest . 2007 Mar;117(3):659-71.

Trichostatin A is an antibiotic, antifungal agent, and a compound of trichostatin and hydroxamic acid. It functions as a bacterial metabolite, an anti-aging agent, and an EC 3.5.1.98 (histone deacetylase) inhibitor. Its function is related to (R)-trichostatin acid. Trichostatin A has been reported to be found in Streptomyces, Streptomyces seoincarnata, and other organisms with relevant data. Trichostatin A is a natural derivative of diene hydroxamic acid isolated from Streptomyces bacteria. Trichostatin A (TSA) reversibly and specifically inhibits histone deacetylase, leading to excessive acetylation of core histones, thereby regulating chromatin structure. Increased histone acetylation promotes selective gene transcription and inhibits tumor growth. This drug effectively induces growth arrest, differentiation, and apoptosis in various in vitro cultured transformed cells and tumor cells in tumor-bearing animals. (NCI04)

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TSA is a natural product isolated from Streptomyces hygroscopicus. It was initially identified as an antifungal agent, but later it was found to have HDAC inhibitory activity[1]
- TSA can enhance the antitumor efficacy of cisplatin in MCF-7 xenograft tumors (TCA 1 mg/kg intraperitoneal injection combined with cisplatin 5 mg/kg intraperitoneal injection can reduce tumor volume by 68%, while TSA and cisplatin alone can only reduce it by 45% and 32%, respectively)[2]
- TSA induces epigenetic reactivation of tumor suppressor genes (such as p16INK4a) by increasing the level of histone acetylation at gene promoters (ChIP experiment: after treating A549 cells with 100 nM TSA for 24 hours, the binding of acetylated H3 to the p16INK4a promoter increased by 3 times)[3]

C17H22N2O3

302.4

302.163

C, 67.53; H, 7.33; N, 9.26; O, 15.87

58880-19-6

122292-85-7 (S-isomer); 58880-19-6 (R-isomer)

444732

White to light yellow solid powder

1.1±0.1 g/cm3

141-143ºC

1.578

2.77

2

4

6

22

447

1

O=C(C1C([H])=C([H])C(=C([H])C=1[H])N(C([H])([H])[H])C([H])([H])[H])[C@]([H])(C([H])([H])[H])/C(/[H])=C(/C(/[H])=C(\[H])/C(N([H])O[H])=O)\C([H])([H])[H]

RTKIYFITIVXBLE-QEQCGCAPSA-N

InChI=1S/C17H22N2O3/c1-12(5-10-16(20)18-22)11-13(2)17(21)14-6-8-15(9-7-14)19(3)4/h5-11,13,22H,1-4H3,(H,18,20)/b10-5+,12-11+/t13-/m1/s1

(2E,4E,6R)-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (8.27 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 2.5 mg/mL (8.27 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: 1% DMSO+30% polyethylene glycol+1% Tween 80, pH 4: 6mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)