4SC-202, a novel class I HDAC inhibitor (HDACi), potently inhibited survival and proliferation of primary human colon cancer cells and established CRC lines (HT-29, HCT-116, HT-15, and DLD-1). Yet, the same 4SC-202 treatment was non-cytotoxic to colon epithelial cells where HDAC-1/-2 expressions were extremely low. 4SC-202 provoked apoptosis activation in CRC cells, while caspase inhibitors (z-VAD-CHO and z-DVED-CHO) significantly alleviated 4SC-202-exerted cytotoxicity in CRC cells. Meanwhile, 4SC-202 induced dramatic G2-M arrest in CRC cells. Further studies showed that AKT activation might be an important resistance factor of 4SC-202. 4SC-202-induced cytotoxicity was dramatically potentiated with serum starvation, AKT inhibition (by perifosine or MK-2206), or AKT1-shRNA knockdown in CRC cells. On the other hand, exogenous expression of constitutively active AKT1 (CA-AKT1) decreased the sensitivity by 4SC-202 in HT-29 cells. [1]

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We determined dose response curves of 4SC-202 by MTT assay in seven UC cell lines with distinct HDAC1, HDAC2 and HDAC3 expression profiles. Cellular effects were further analyzed in VM-CUB1 and UM-UC-3 cells by colony forming assay, caspase-3/7 assay, flow cytometry, senescence assay, LDH release assay, and immunofluorescence staining. Response markers were followed by quantitative real-time PCR and western blotting. Treatment with the class I HDAC specific inhibitor SAHA (vorinostat) served as a general control.Results,4SC-202 significantly reduced proliferation of all epithelial and mesenchymal UC cell lines (IC50 0.15-0.51 μM), inhibited clonogenic growth and induced caspase activity. Flow cytometry revealed increased G2/M and subG1 fractions in VM-CUB1 and UM-UC-3 cells. Both effects were stronger than with SAHA treatment.[2]

4SC-202, a novel class I HDAC inhibitor (HDACi), potently inhibited survival and proliferation of primary human colon cancer cells and established CRC lines (HT-29, HCT-116, HT-15, and DLD-1). Yet, the same 4SC-202 treatment was non-cytotoxic to colon epithelial cells where HDAC-1/-2 expressions were extremely low. 4SC-202 provoked apoptosis activation in CRC cells, while caspase inhibitors (z-VAD-CHO and z-DVED-CHO) significantly alleviated 4SC-202-exerted cytotoxicity in CRC cells. Meanwhile, 4SC-202 induced dramatic G2-M arrest in CRC cells. Further studies showed that AKT activation might be an important resistance factor of 4SC-202. 4SC-202-induced cytotoxicity was dramatically potentiated with serum starvation, AKT inhibition (by perifosine or MK-2206), or AKT1-shRNA knockdown in CRC cells. On the other hand, exogenous expression of constitutively active AKT1 (CA-AKT1) decreased the sensitivity by 4SC-202 in HT-29 cells. [1]

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We determined dose response curves of 4SC-202 by MTT assay in seven UC cell lines with distinct HDAC1, HDAC2 and HDAC3 expression profiles. Cellular effects were further analyzed in VM-CUB1 and UM-UC-3 cells by colony forming assay, caspase-3/7 assay, flow cytometry, senescence assay, LDH release assay, and immunofluorescence staining. Response markers were followed by quantitative real-time PCR and western blotting. Treatment with the class I HDAC specific inhibitor SAHA (vorinostat) served as a general control.Results,4SC-202 significantly reduced proliferation of all epithelial and mesenchymal UC cell lines (IC50 0.15-0.51 μM), inhibited clonogenic growth and induced caspase activity. Flow cytometry revealed increased G2/M and subG1 fractions in VM-CUB1 and UM-UC-3 cells. Both effects were stronger than with SAHA treatment.[2]

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4SC-202 (referred to as Domatinostat in the query) significantly reduced the proliferation of seven urothelial carcinoma (UC) cell lines with varying phenotypes and HDAC expression profiles, with IC50 values ranging from 0.15 µM to 0.51 µM after 72-hour treatment, as determined by MTT assay. [1]
In colony-forming assays, treatment with 0.5 µM and 2.5 µM 4SC-202 for 24 or 48 hours strongly and durably inhibited the clonogenic growth of VM-CUB1 and UM-UC-3 UC cells, with effects more persistent than those of the pan-HDAC inhibitor SAHA (vorinostat). [1]
4SC-202 (0.5 and 2.5 µM) induced significant caspase-3/7 activity in UC cell lines (VM-CUB1, UM-UC-3) and control cell lines (HBLAK, HEK-293) after 24 and 48 hours, indicative of apoptosis induction. This effect was more pronounced than with SAHA treatment. [1]
Western blot analysis showed increased levels of cleaved PARP in VM-CUB1 and UM-UC-3 cells treated with 4SC-202, further confirming apoptosis induction. [1]
Treatment with 4SC-202 (2.5 µM, 48h) induced significant lactate dehydrogenase (LDH) release in UM-UC-3 cells, indicating necrotic cell death. Annexin V/PI staining in VM-CUB1 and UM-UC-3 cells revealed a mixture of apoptotic and necrotic cell death, with increases in early apoptotic, late apoptotic/necrotic, and necrotic cell populations, especially at 2.5 µM. [1]
Cell cycle analysis by flow cytometry showed that 4SC-202 (2.5 µM) induced a strong accumulation of VM-CUB1, UM-UC-3, and HEK-293 cells in the G2/M phase after 24 hours. After 48 hours, cell cycle profiles became highly irregular with an increase in cells with subG1 and aberrant DNA content. [1]
4SC-202 treatment (0.5 and 2.5 µM) increased the acetylation levels of histone H3 in VM-CUB1 and UM-UC-3 cells. Acetylation changes for histone H4 were weaker and occurred later. The compound did not affect acetylation of α-tubulin, confirming its specificity for class I HDACs over HDAC6. [1]
DAPI staining and nuclear morphology analysis revealed that 4SC-202 treatment induced a high proportion of irregular mitoses, strongly fragmented nuclei, and micronuclei in VM-CUB1 and UM-UC-3 cells after 24 and 48 hours. [1]
Immunofluorescence staining in VM-CUB1 cells showed that 4SC-202 (2.5 µM, 24h) strongly increased pan-nuclear γH2A.X staining, with some focal induction colocalizing with 53-BP1, indicating DNA damage response activation. [1]
Combination experiments comparing 4SC-202 with the separate use of a class I HDAC inhibitor (romidepsin) and an LSD1 inhibitor (SP2509) suggested that the dual inhibitory activity of 4SC-202 contributes to its effects on viability and clonogenicity, but its unique cell cycle and morphological impact may not be fully recapitulated by the combination. [1]

4SC-202, at a low concentration, enhanced oxaliplatin-induced in vitro anti-CRC activity. In vivo, we showed that oral gavage of 4SC-202 inhibited HT-29 xenograft growth in nude mice, and when combined with oxaliplatin, its activity was further strengthened. Together, these pre-clinical results indicate that 4SC-202 may be further investigated as a valuable anti-CRC agent/chemo-adjuvant.

For cell viability/proliferation assays (MTT assay), urothelial carcinoma cell lines and control cell lines were seeded in 96-well plates. Cells were treated with a range of concentrations of 4SC-202 or DMSO control for 72 hours. Following incubation, the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added. The formazan crystals produced by metabolically active cells were dissolved, and the absorbance was measured at a specific wavelength to determine cell viability. IC50 values were calculated using non-linear regression analysis. [1]
For clonogenic assays, cells were plated at low density (500-1500 cells per plate) in culture dishes. After 24 or 48 hours of treatment with 4SC-202, SAHA, or DMSO, the drug-containing medium was replaced with fresh medium. Cells were allowed to grow for two weeks to form colonies. Colonies were then washed, fixed with methanol, stained with Giemsa stain, and counted. [1]
For apoptosis assessment (Caspase-Glo 3/7 assay), cells were treated with 4SC-202 or controls in multiwell plates for 24 or 48 hours. An aliquot of the cell suspension was transferred to a 96-well plate. The Caspase-Glo 3/7 reagent was added, and after incubation, luminescence was measured. Results were normalized to cell viability determined in parallel using the CellTiter-Glo assay. [1]
For necrosis assessment (LDH release assay), cells were treated with 4SC-202 or controls. After the incubation period, the culture supernatant was collected. The LDH activity in the supernatant was measured using a commercial cytotoxicity assay kit according to the manufacturer's instructions, which involves a coupled enzymatic reaction that results in a colorimetric product, and absorbance was measured. [1]
For cell cycle analysis by flow cytometry, treated cells (both attached and in supernatant) were collected, washed, and stained with a propidium iodide (PI) buffer containing Triton X-100 and sodium citrate. The stained cells were analyzed by flow cytometry to determine DNA content and cell cycle distribution. [1]
For Annexin V/PI apoptosis/necrosis analysis by flow cytometry, treated cells were collected, washed, and resuspended in Annexin V binding buffer. Cells were then stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry to distinguish viable, early apoptotic, late apoptotic/necrotic, and necrotic cells. [1]
For immunofluorescence staining (e.g., γH2A.X, 53-BP1), cells grown on coverslips were treated, fixed with formaldehyde, permeabilized, and blocked. They were then incubated with primary antibodies overnight at 4°C, followed by incubation with fluorescently labeled secondary antibodies. Nuclei were counterstained with DAPI. Coverslips were mounted and images were acquired using a fluorescence microscope. [1]

[1]. Tumour Biol . 2016 Aug;37(8):10257-67.

[2]. Target Oncol . 2016 Dec;11(6):783-798.

4SC-202 is a novel oral benzamide isoform-specific histone deacetylase (HDAC) inhibitor. It selectively inhibits class I HDACs (HDAC1, 2, 3) and histone demethylase LSD1 (KDM1A). [1] 4SC-202 exhibits antitumor activity against urothelial carcinomas through the combined induction of apoptosis and necrosis, likely due to severe cell cycle disruption, particularly mitotic impairment and DNA damage, ultimately leading to mitotic catastrophe. [1] 4SC-202 is more effective in inhibiting urothelial carcinoma colony formation than the pan-HDAC inhibitor SAHA (vorinostat). [1] This preclinical study suggests that 4SC-202, in combination with the inhibition of HDAC1, HDAC2, and HDAC3 (and potentially LSD1), may be an effective strategy for treating urothelial carcinoma. [1] 4SC-202 has been tested in a phase I clinical trial (TOPAS) for advanced hematologic malignancies. [1]

1186222-89-8; 910462-43-0 (free)

Typically exists as solid at room temperature

Domatinostat HCl

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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