Journal: PLoS ONE

Article Title: Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

doi: 10.1371/journal.pone.0018552

Figure Lengend Snippet: (A) Client protein downregulation in NSCLC. NCI-H1975 cells were dosed with the indicated concentrations of ganetespib for 24 h and their cell lysates analyzed to determine JAK/STAT and Hsp90 client protein levels using the antibodies indicated. (B) Ganetespib blocks IL-6 induced and constitutive STAT3 activity in NSCLC cells. HCC827 lung cancer cells were treated with increasing concentrations of ganetespib or P6 for 24 h followed by a 15 min stimulation with or without 50 ng/ml human recombinant IL-6. The levels of JAK2, total and phospho-STAT3, and PIM2 were analyzed by western blot. GAPDH is included as a loading control. (C) Client protein degradation in prostate cancer cells. DU145 cells were dosed with graded concentrations of ganetespib for 24 h and cell lysates subject to western blot to determine JAK/STAT and target protein levels using the antibodies indicated. (D) Functional Hsp90 is required for JAK2, but not JAK1, stability in DU145 cells. DU145 cells were treated with DMSO (control, C), 15 nM, 60 nM or 240 nM ganetespib for either 24 or 48 h and lysates probed by western blot with the indicated antibodies.

Article Snippet: Hsp90 inhibitors ganetespib and 17-AAG (both synthesized at Synta Pharmaceuticals, Inc.) and the JAK inhibitor Pyridone 6 (Calbiochem, Darmstadt, Germany) were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −20°C.

Techniques: Activity Assay, Recombinant, Western Blot, Control, Functional Assay

Journal: PLoS ONE

Article Title: Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

doi: 10.1371/journal.pone.0018552

Figure Lengend Snippet: Ganetespib exerts potent antitumor effects through perturbation of multiple signaling cascades, including the JAK/STAT signaling axis and cell cycle mediators. (A) The JAK/STAT pathway is a principal signaling mechanism for a wide array of cytokines and growth factors. Hyperactivation of this pathway, through ligand activated receptor tyrosine kinases (EGFR or FLT3), cytokine receptor mediated activation of JAK2, or activating mutations such as JAK2 V617F , is often associated with oncogenesis. Regulation of the cell cycle involves a number of highly coordinated and essential processes, including checkpoint control and detection/repair of genetic damage, critical for correct progression and cell division. (B) Inactivation of Hsp90 by ganetespib results in the proteasome-mediated degradation of the upstream signaling components (indicated in grey) critical for STAT, MAPK and AKT activation, thereby resulting in growth inhibition. In addition, concomitant downregulation of key cell-cycle regulatory genes induced by ganetespib (as shown in ) results in cell cycle arrest in the G 1 and G 2 /M phases of the cell cycle, and subsequent loss of S phase.

Article Snippet: Hsp90 inhibitors ganetespib and 17-AAG (both synthesized at Synta Pharmaceuticals, Inc.) and the JAK inhibitor Pyridone 6 (Calbiochem, Darmstadt, Germany) were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −20°C.

Techniques: Activation Assay, Control, Inhibition