Advanced tumors produce an excessive amount of transforming growth factor b (TGFb), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFb therapeutics for cancer. We synthesized a novel small-molecule TGFb receptor I kinase (activin receptorlike kinase 5) inhibitor termed N- [[4-([1,2,4], and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 inhibited Smad/ TGFb signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopicgrafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFb-treated breast cancer cells and 4T1 orthotopic-grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic-grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumorbearing mice. In summary, EW-7197 showed potent in vivo antimetastatic activity, indicating its potential for use as an anticancer therapy.

The pathophysiological mechanism underlying migraine-associated peripheral hypersensitivity remains unclear. Acid-sensing ion channels (ASICs) and transient receptor potential ankyrin 1 (TRPA1) are known to be causative pathogenic factors of mechanical and cold allodynia, respectively. Here, we sought to investigate their involvement in cold and mechanical allodynia of the face and hindpaws, respectively, in a mouse model of repetitive nitroglycerin (NTG)-induced migraine. NTG (10 mg/kg) was administered to the mice every other day for 9 days, followed 90 min later by HC-030031 (a TRPA1 blocker) or amiloride (a non-selective ASIC blocker). Mechanical or cold sensitivity of the hindpaw and facial regions was quantified using von-Frey filaments or acetone solution, respectively. Immunohistochemistry revealed that c-Fos expression was significantly increased in the trigeminal nucleus caudalis region but not in the spinal cord. Amiloride treatment only reduced NTG-induced hindpaw mechanical allodynia, whereas HC-030031 treatment only improved facial cold allodynia. Interestingly, the number of c-Fos positive cells decreased to a similar level in each drug treatment group. These findings demonstrate that facial cold allodynia and hindpaw mechanical allodynia are differentially mediated by activation of TRPA1 and ASIC, respectively, in mice with repetitive NTG-induced hypersensitivity.

<p>PDF - 356K, (A~C) Breast cancer model #1, described in the Material and Methods. (A) Effect of EW-7197 on lung metastasis of breast cancer in MMTV-c/Neu mice. Representative images of H&E stained lungs (total magnification: x 12.5 or 100, scale bar: 800 μm or 100 μm). Total tumor volume (B) and body weight (C) in MMTV/c-Neu mice. Data represent the mean (plus or minus) SE (Veh: n=7, EW: n=10). (D and E) Body weight in breast cancer model #2 (D) and in breast cancer model #3 (E) (described in the Material and Methods). Data represent the mean (plus or minus) SE (n=10/group in Model #2 or n=6~8/group in Model #3). Veh, LY and EW indicate artificial gastric fluid, LY-2157299 and EW-7197, respectively. (F) In breast cancer model #3 (described in the Material and Methods), on day 28, two mice of each group were selected and treated with the indicated concentration of EW-7197. One mouse from each group was injected with TGF-beta1 (50 ng/mouse) through i.v. after 30 min and another was not injected. 90 min after TGF-beta1 injection, mice were sacrificed and lysates from primary mammary tumors were analyzed by western blotting as described in the Material and Methods.</p>

<div>Abstract<p>Advanced tumors produce an excessive amount of transforming growth factor β (TGFβ), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFβ therapeutics for cancer. We synthesized a novel small-molecule TGFβ receptor I kinase (activin receptor–like kinase 5) inhibitor termed <i>N</i>-[[4-([1,2,4]triazolo[1,5-<i>a</i>]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1<i>H</i>-imidazol-2-yl]methyl]-2-fluoroaniline (EW-7197), and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic–grafted mice. EW-7197 inhibited Smad/TGFβ signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic–grafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFβ-treated breast cancer cells and 4T1 orthotopic–grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic–grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumor–bearing mice. In summary, EW-7197 showed potent <i>in vivo</i> antimetastatic activity, indicating its potential for use as an anticancer therapy. <i>Mol Cancer Ther; 13(7); 1704–16. ©2014 AACR</i>.</p></div>