HDAC1 0.25 μM (IC50) HDAC3 0.30 μM (IC50)
Suberoyl bis-hydroxamic acid (SBHA) inhibits histone deacetylase 1 (HDAC1) (IC50: 0.25 μM) and histone deacetylase 3 (HDAC3) (IC50: 0.30 μM) in vitro. [2]

The degree of apoptosis is improved when Suberoyl bis-hydroxamic acid (10, 20, or 50 μM; 24 hours) is combined with TRAIL. When TRAIL is paired with 20 μM SBHA, which alone causes about 10-15% apoptosis, 45–50% of the cells die[1]. The expression of the proteins Bcl-xL and Mcl-1 is not significantly affected by suberoyl bis-hydroxamic acid (20–50 μM; 10–20 hours); Bax is slightly affected, though. It increases the ratio of relative protein expression of Bcl-xL and Bax when combined with TRAIL, whereas in MM-BI and Ist-Mes2 cells, the ratio of Mcl-1 and Bax changes later[1]. MEL cells accumulate acetylated histone H4 in response to 30 μM of suberoyl bis-hydroximac acid over a 6-hour duration[2].

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Suberoyl bis-hydroxamic acid (SBHA) (10-100 μM) sensitized MM-BI and Ist-Mes2 human malignant mesothelioma cells to TRAIL (30 ng/ml)-induced apoptosis. SBHA at 20 μM alone caused only 10-15% apoptosis, but when combined with TRAIL, apoptosis reached 45-50% after 24 hours. [1]
Suberoyl bis-hydroxamic acid (SBHA) (20 μM) combined with TRAIL (30 ng/ml) caused early and significant down-regulation of Bcl-xL protein expression in MM-BI and Ist-Mes2 cells at 6, 12, and 24 hours, while SBHA alone had little effect on Bcl-xL expression. Mcl-1 down-regulation was delayed, and Bax expression showed mild changes. [1]
Suberoyl bis-hydroxamic acid (SBHA) (20 μM) combined with TRAIL (30 ng/ml) induced caspase-3 activation in MM-BI cells as assessed by flow cytometry using an antibody specific for the activated cleaved form of caspase-3. Over-expression of wild-type Bcl-xL but not ΔBcl-xL (lacking mitochondrial-docking C-terminus) suppressed apoptosis induced by SBHA plus TRAIL. Knockdown of caspase-8 or caspase-9 by siRNA antagonized apoptosis induction by SBHA plus TRAIL. [1]
Suberoyl bis-hydroxamic acid (SBHA) at 30 μM induced 94% hemoglobinized cells (differentiation) in MEL cells. [2]
Suberoyl bis-hydroxamic acid (SBHA) (30 μM) caused accumulation of acetylated histone H4 in MEL cells after 6 hours of culture as detected by acid urea/Triton X-100 (AUT) gel electrophoresis. [2]
Suberoyl bis-hydroxamic acid (SBHA) inhibited HDAC1 and HDAC3 activity in a dose-dependent manner with submicromolar concentrations (ID50 for HDAC1: 0.25 μM; for HDAC3: 0.30 μM). [2]
Suberoyl bis-hydroxamic acid (SBHA) (2.5 μM) induced accumulation of hyperacetylated histone H4 in MEL cells at 6, 24, and 48 hours, similar to TSA. HMBA (5 mM) had no detectable effect. [2]
Suberoyl bis-hydroxamic acid (SBHA) (200 mg/kg every other day for 12 days) increased acetyl-histone H4 (Lys12) levels in TT cell xenograft tumors. [3]
Suberoyl bis-hydroxamic acid (SBHA) induced the active form of Notch1 (NICD) in TT MTC cells in vivo, with concomitant decrease in achaete-scute complex-like 1 (ASCL1), a downstream target of Notch1 signaling, and the neuroendocrine tumor marker chromogranin A (CgA). [3]
Suberoyl bis-hydroxamic acid (SBHA) increased protein levels of p21CIP1/WAF1 and p27KIP1 and decreased cyclin D1 and cyclin B1 in MTC xenograft tumors. [3]
Suberoyl bis-hydroxamic acid (SBHA) increased cleaved caspase-9, cleaved caspase-3, and cleaved PARP in MTC xenograft tumors. It downregulated Bcl-2 and Bcl-XL, and upregulated Bax, Bad, and Bmf. [3]

The administration of suberoyl bis-hydroxamic acid (200 mg/kg intraperitoneally every 2 days for 12 days) results in a significant rise in the active form of Notch1 (NICD) and a corresponding decrease in ASCL1. It slows the growth of MTC tumors[3].

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Suberoyl bis-hydroxamic acid (SBHA) (200 mg/kg, intraperitoneal injection every other day for 12 days) inhibited tumor growth in nude mice bearing human medullary thyroid carcinoma (TT cells) xenografts, resulting in an average 55% inhibition of tumor growth compared to DMSO-treated controls (P < 0.05). Animal weight remained stable and no adverse effects were observed. [3]
Suberoyl bis-hydroxamic acid (SBHA) treatment in vivo increased acetyl-histone H4 (Lys12) levels in tumor tissues, verifying its activity as an HDAC inhibitor. [3]
Suberoyl bis-hydroxamic acid (SBHA) treatment in vivo induced the active form of Notch1 (NICD), decreased ASCL1 (a downstream target of Notch1), and decreased chromogranin A (CgA) levels in MTC xenograft tumors. [3]
Suberoyl bis-hydroxamic acid (SBHA) treatment in vivo increased p21CIP1/WAF1 and p27KIP1, decreased cyclin B1 and cyclin D1, increased cleaved caspase-9, cleaved caspase-3, and cleaved PARP, downregulated Bcl-2 and Bcl-XL, and upregulated Bax, Bad, and Bmf in MTC xenograft tumors. [3]

For HDAC activity assays, Jurkat cells were lysed in buffer containing Tris-HCl, NaCl, EDTA, and Nonidet P-40 with protease inhibitors. The lysate was cleared by centrifugation and precleared with protein G-Sepharose. Immunoprecipitation was performed using polyclonal antisera against HDAC1 or HDAC3 (raised against carboxyl-terminal peptides) or preimmune serum as control. Immune complexes were collected with protein G-Sepharose, washed, and resuspended in HDAC buffer. HDAC activity was measured using a 3H-acetylated peptide corresponding to amino acids 1-24 of histone H4 as substrate. Released 3H acetic acid was quantified by scintillation counting. For inhibition studies, immunoprecipitated complexes were preincubated with different drugs for 30 minutes at 4°C. SBHA inhibited HDAC1 and HDAC3 activity with ID50 values of 0.25 μM and 0.30 μM, respectively. HMBA and EMBA did not inhibit either HDAC1 or HDAC3 activity at concentrations sufficient to induce differentiation. [2]

Apoptosis Analysis[1]
Cell Types: MM-BI and Ist-Mes2 cells
Tested Concentrations: 10 μM, 20 μM or 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: demonstrated a cooperative effect in cell apoptosis.

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For apoptosis detection, MM-BI and Ist-Mes2 cells were plated at 105 per well in 24-well plates, treated with hrTRAIL (30 ng/ml) and/or SBHA (10-100 μM) for 24 hours. Floating and attached cells were combined, washed with PBS, resuspended in binding buffer (Hepes, NaCl, CaCl2, pH 7.4), incubated with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Cell death was quantified as percentage of cells with high annexin V binding. [1]
For protein expression assessment, cells were treated, harvested by trypsinization, fixed with formalin in PBS, permeabilized with saponin in PBS containing FBS, then reacted with primary antibodies against Bcl-2, Bcl-xL, Mcl-1, Bax, Bak, Bad, and Bik, followed by FITC-conjugated secondary antibody. Protein expression was estimated by flow cytometry and expressed as mean fluorescence intensity after subtraction of nonspecific background. [1]
For caspase-3 activation assay, cells were harvested, fixed, permeabilized, incubated with specific anti-caspase-3 antibody recognizing the activated cleaved form, followed by FITC-conjugated secondary antibody. The population of cells with activated caspase-3 was determined by high green fluorescence using flow cytometry. [1]
For siRNA treatment, RNA oligonucleotides with 3'-dTdT overhangs specific for caspase-8 and caspase-9 (or scrambled control) were used. Cells were seeded in 12-well plates, transfected with 60 pmol siRNA per well using Oligofectamine, and 48 hours later assessed for expression of pro-caspase-8 and -9 using specific antibodies, then used in experiments. [1]
For transfections, cells were seeded in 6-well plates, washed, incubated with 1 μg plasmid DNA (Bcl-xL-EGFP or ΔBcl-xL-EGFP) pre-mixed with Lipofectamine-2000 and OptiMEM. After 2-3 hours, cells were washed and incubated in complete DMEM for 24 hours, then placed in selection medium with G418. After five passages, cells were considered stably transfected. Efficacy of over-expression was assessed by fluorescence microscopy. [1]
For histone acetylation analysis in MEL cells, cells were cultured with SBHA (30 μM) for 6 hours. Histones were extracted and subjected to acid urea/Triton X-100 (AUT) gel electrophoresis at 170V for 24 hours at 4°C. Gels were stained with Coomassie brilliant blue R-250, dried, and photographed. The degree of histone H4 acetylation (Ac0, Ac1, Ac2, Ac3, Ac4) was determined. [2]
For differentiation commitment assay in MEL cells, cells were cultured with SBHA (2.5 μM) for 48 hours in suspension culture, then washed with SBHA-free medium and plated in methylcellulose without SBHA. After 5 days, cells were scored as committed to terminal differentiation if they formed small colonies (<32 cells) and expressed hemoglobin as determined by benzidine staining. [2]
For Western blot analysis of MTC cells and xenograft tissues, total tissue proteins were isolated, denatured by boiling, separated by SDS-PAGE (8%, 10%, or 12%), transferred onto nitrocellulose membranes, blocked in milk with Tween 20 in PBS, and exposed to primary antibodies (Notch1, ASCL1, G3PDH, acetyl-histone H4 Lys12, p21CIP1/WAF1, p27KIP1, PARP, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bcl-XL, Bad, Bmf, Bax, cyclin B1, cyclin D1, chromogranin A) overnight at 4°C. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies, then developed using chemiluminescence substrates. [3]

Animal/Disease Models: Nude mice injected with human MTC cells[3]
Doses: 200 mg/kg
Route of Administration: intraperitoneal (ip)injection; every 2 days; 12 days
Experimental Results: Resulted in an average 55% inhibition of tumor growth in SBHA treatment group.

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For xenograft model in nude mice (male nu/nu), mice received subcutaneous injections of human MTC TT cells (106 cells) into the right flank. Mice with palpable tumors were randomly divided into two groups. Suberoyl bis-hydroxamic acid (SBHA) was dissolved in DMSO at a stock concentration of 500 mg/mL and stored at -20°C. Fresh dilutions in PBS were made for each injection. The treatment group received SBHA at 200 mg/kg every two days by intraperitoneal injection for 12 days. The control group was injected with vehicle (DMSO diluted in PBS). Mice were weighed three times during the experimental period to assess toxicity. Tumors were measured every four days using calipers. Tumor volume was calculated using the formula: tumor volume = length × (width)2 × π/6. On the final day, all mice were sacrificed by carbon dioxide inhalation, and subcutaneous tumors were removed and snap frozen in liquid nitrogen. [3]

Suberoyl bis-hydroxamic acid (SBHA) treatment (200 mg/kg every other day for 12 days) in nude mice showed no signs or symptoms of toxicity; animal weight remained stable over the course of the experiments and all animals survived until the study endpoints. [3]

[1]. Sensitization of Mesothelioma to TRAIL Apoptosis by Inhibition of Histone Deacetylase: Role of Bcl-xL Down-Regulation. Biochem Biophys Res Commun. 2004 Jan 30;314(1):186-91.

[2]. A Class of Hybrid Polar Inducers of Transformed Cell Differentiation Inhibits Histone Deacetylases.Proc Natl Acad Sci U S A.

[3]. Suberoyl Bishydroxamic Acid Activates notch1 Signaling and Suppresses Tumor Progression in an Animal Model of Medullary Thyroid Carcinoma. Ann Surg Oncol. 2008 Sep;15(9):2600-5.

N,N'-Dihydroxyoctanediamide is a hydroxamic acid.

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Malignant mesothelioma (MM) is a fatal neoplastic disease with no current cure, typically associated with occupational asbestos exposure. MM cells are highly malignant and resistant to apoptosis, including resistance to TRAIL. Suberoyl bis-hydroxamic acid (SBHA) sensitizes MM cells to TRAIL apoptosis through down-regulation of Bcl-xL, and this process involves cross-talk between proximal (receptor) and distal (mitochondrial) apoptotic pathways as confirmed by caspase-8 and caspase-9 knockdown studies. [1]
Hybrid polar compounds (HPCs) such as SBHA induce terminal differentiation and/or apoptosis in transformed cells. SBHA is a second-generation HPC that is 100 times more potent than HMBA on a molar basis for inducing differentiation of MEL cells. Unlike HMBA and EMBA, SBHA inhibits HDAC activity and causes accumulation of hyperacetylated histone H4. MEL cells selected for resistance to SBHA are cross-resistant to TSA but not to HMBA, suggesting that HMBA and second-generation HPCs induce differentiation by different pathways. [2]
Suberoyl bis-hydroxamic acid (SBHA) activates Notch1 signaling, leading to reduction of neuroendocrine tumor markers (ASCL1 and chromogranin A) in medullary thyroid carcinoma. The antitumor effects of SBHA in MTC are due to both cell cycle arrest (via p21CIP1/WAF1, p27KIP1 induction and cyclin B1/D1 reduction) and caspase-dependent apoptosis (via Bcl-2/Bcl-XL downregulation, Bax/Bad/Bmf upregulation, and caspase-9, caspase-3, PARP cleavage). [3]
A phase II trial has been initiated for suberoylanilide hydroxamic acid (SAHA), a similar drug to SBHA, in patients with recurrent/metastatic head and neck cancer, showing modest single agent activity and tolerability at 400 mg qd. Nearly 40 SAHA clinical trials are being performed for various cancers. However, no early clinical data on SBHA in human cancer exists yet, although SBHA has in vivo activity with little or no toxicity in experimental models. [3]

C8H16N2O4

204.22

204.111

38937-66-5

5173

White to off-white solid powder

1.2±0.1 g/cm3

153-155ºC(lit.)

1.502

-1.81

4

4

7

14

164

0

C(CCCC(=O)NO)CCC(=O)NO

IDQPVOFTURLJPT-UHFFFAOYSA-N

InChI=1S/C8H16N2O4/c11-7(9-13)5-3-1-2-4-6-8(12)10-14/h13-14H,1-6H2,(H,9,11)(H,10,12)

N,N'-dihydroxyoctanediamide

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)

DMSO : 50 mg/mL (244.83 mM)
H2O : 8.33 mg/mL (40.79 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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 (12.24 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 (12.24 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (24.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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