NSC 74859 (S3I-201)

S3I-201 inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3 by inhibiting Stat3·Stat3 complex formation and Stat3 DNA-binding and transcriptional activitie. Moreover, S3I-201 also inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin. ^[1] S3I-201 inhibits breast carcinoma MDA-MB-435, MDA-MB-453 and MDA-MB-231 cell lines with IC50 of 100 μM. In addition, the cells with impaired TGF-β signaling are four times as sensitive to the STAT3 inhibitor S3I-201. ^[2] A recent study shows that S3I-201 potentiates the antiproliferative effect in HepG2 and Huh-7 cells via the STAT3 signalling pathway. ^[3]

S3I-201 (5 mg/kg, i.v. every 2 or every 3 days) shows the antitumor efficacy in mouse models with human breast tumor xenografts that harbor constitutively active Stat3. ^[1] S3I-201 treatment reduces Varicella-zoster virus (VZV) replication on the basis of the bioluminescence signal and the number of positive skin xenografts compared with DMSO-treated mice by inhibiting STAT3 phosphorylation. ^[4]

• In vitro Stat3 DNA-binding assay and EMSA analysis

  Briefly, 100 mL of biotinyl-e-Ac-EPQpYEEIEL-OH (in 50 mM Tris/150 mM NaCl, pH 7.5) is added to each well of streptavidin-coated 96-well microtiter plates and incubated with shaking at 4 °C overnight. Then plates are rinsed with PBS/Tween 20 and then two times with 200 mL of BSA-T-PBS (0.2% BSA/0.1% Tween 20/PBS). Then 50 mL of Lck-SH2-GST fusion protein (6.4 ng/ml in BSA-T-PBS) is added to each well of the 96-well plate in the presence and absence of 50 mL of S3I-201 (for 30 and 100 mM final concentrations), and the plate is shaken at room temperature for 4 hours. After solutions are removed, each well is rinsed four times with BSA-T-PBS (200 mL), and 100 mL of polyclonal rabbit anti-GST antibody (100 ng/mL in BSA-T-PBS) is added to each well and incubated at 4 °C overnight. After washing with BSA-T-PBS, 100 mL of 200 ng/mL BSA-T-PBS horseradish peroxidase-conjugated mouse anti-rabbit antibody is added to each well and incubated for 45 minutes at room temperature. After four washing steps with BSA-T-PBS and three washing steps with PBS-T, 100 mL of peroxidase substrate is added to each well and incubated for 5-15 minutes. The peroxidase reaction is stopped by adding 100 mL of 1 M sulfuric acid solution, and absorbance is read at 450 nm with an ELISA plate rea

• Cell lines

  MDA-MB-435, MDA-MB-453 and MDA-MB-231 cells lines

• Concentrations

  ~ 250 μM

• Incubation Time

  72 hours

• Method

  The MTT assay is based on the conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. The MTT assay provides a quantitative determination of viable cells. Cells are seeded in 96-well microplates in complete culture medium in the absence or presence of increasing serial dosages of S3I-201 as indicated. At 72 hours after culture, the number of viable cells is measured by adding 100 μL/well of 2 mg/mL MTT solution. After 2 hours, the medium is removed. The absorbance is read at 590 nm with an enzyme-linked immunosorbent assay reader. Each treatment point is performed in 10 wells or sextuplicate.

• Animal Models

  Human breast cancer MDA-MB-231 cells are injected s.c. into the left flank of athymic nu/nu mice.

• Dosages

  ≤5 mg/kg

• Administration

  Administered via i.v.