Ectopic expression of the SH2 domain in cells was sufficient to counter the STAT3 inhibitory effects of SH4-54. prostate cancer cells and v-Src-transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates fof further development as cancer therapeutics. at 10-20 M and antitumor effects Gemifloxacin (mesylate) in pre-clinical models of breast and non-small cell lung cancers [15]. Towards further improving the potency of the salicylic acid, BP-1-102 [15], we have synthesized and evaluated the hydroxamic acid, SH5-07 and benzoic acid, SH4-54, analogs, which show improved inhibitory activities at 1-8 M. Structural data suggests these agents interact with the Stat3 SH2 and DNA-binding domains. Further, both agents inhibit growth of human glioma and breast cancer xenografts that harbor aberrantly-active Stat3. Materials and Methods Chemical synthesis of SH4-54 and SH5-07 Synthesis and detailed Gemifloxacin (mesylate) characterization of agents are described in Supplementary Materials, Methods. Cells and reagents Normal mouse fibroblasts (NIH3T3), counterparts transformed by v-Src (NIH3T3/v-Src) or overexpressing the human epidermal growth factor (EGF) receptor (NIH3T3/hEGFR), and the human breast (MDA-MB-231 and MCF-7), pancreatic (Panc-1) and prostate (DU145) cancer cells have all been reported [15,21,24,25] [26,27]. Stat3 null mouse embryonic fibroblast line (MEF/ST3KO) and ovarian cancer cells (A2780S) were kind gifts of Drs. Valeria Poli, University of Gemifloxacin (mesylate) Turin, Italy and Jin Cheng, Moffitt Cancer Center, Tampa, FL, respectively. The human glioma lines, U251MG, U373MG and U87MG (Sigma-Aldrich Corporation, St. Louis, MO), and SF-295 (Division of Cancer Treatment and Diagnosis Tumor Repository of the National Cancer Institute, Frederick, MD) were obtained from the designated sources and cultured in Roswell Park Memorial Institute medium-1640 supplemented with 1% nonessential amino acids (Corning Inc., Corning, NY) and containing 10% heat-inactivated fetal bovine serum (FBS). All other cells were grown in Dulbecco’s modified Eagle’s medium plus 10% heat-inactivated FBS. Except where designated, all antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Plasmids and molecular cloning The Stat3-dependent luciferase reporter, pLucTKS3, and the Stat3-independent reporter, pLucSRE, have been previously reported [28,29]. The pLucTKS3 reporter contains seven copies of the Stat3-specific binding sequence in the C-reactive protein gene promoter driving firefly luciferase expression, while the Stat3-independent, pLucSRE reporter is driven by the serum response element (SRE) of the c-promoter. More details of the reporters and the Stat3 SH2 and DNA-binding domain constructs are provided in Supplementary Materials, Methods. Transient transfection of expression Igf2r vectors and luciferase reporter plasmids and reporter assay Transient transfection using Lipofectamine 3000 (Life Technologies, Grand Island, NY) and luciferase assays were performed as previously reported [28,29]. Details are provided in Supplementary Materials, Methods. siRNA transfection using Dharmacon SMARTpool The ON-TARGETplus human Stat3 siRNA SMARTpool (L-003544), and the control (ON-TARGETplus Non-targeting Pool, D-001810-10-20) were both purchased from GE Dharmacon Inc (Lafayette, CO). Cells were transiently transfected with siRNA (25 nM) using Lipofactamine 3000 (Life Technologies) according to the manufacturers instructions. Forty-eight hours after transfection, Stat3 and its downstream genes were assayed in a pool of cells by Western blotting, and another pool of transfected cells was cultured in 96-well plates for additional 72 h and subjected to CyUANT cell proliferation assay (Life Technologies). Nuclear extract preparation and gel shift assays Nuclear extract preparation and DNA-binding/electrophoretic mobility shift assay (EMSA) were Gemifloxacin (mesylate) performed as previously described [24,29]. Details are provided in Supplementary Materials, Methods. Surface plasmon resonance analysis Studies were performed as previously reported [14,15]. Purified Stat3 (50 g/ml) was injected onto the HisCap Sensor Chip for immobilization. Various concentrations of agents in running buffer (1X PBS, 0.5% DMSO) were passed over the chip to produce response signals. The association and dissociation rate constants were calculated using the Qdat software. The ratio of the association and dissociation rate constants was determined.