RT Journal Article SR Electronic T1 Small molecule–mediated reprogramming of the epithelial–mesenchymal transition prevents fibrosis JF bioRxiv FD Cold Spring Harbor Laboratory SP 106591 DO 10.1101/106591 A1 Han-Soo Kim A1 Jun-Hwan Kim A1 Ji Yong Lee A1 Yoon Young-Min A1 Kim Ik-Hwan A1 Ho-Sup Yoon A1 Byung-Soo Youn YR 2017 UL http://biorxiv.org/content/early/2017/02/16/106591.abstract AB Fibrosis is an intractable human disease for which no curative drugs have yet been developed. Recent work revealed that renal fibrosis is a manifestation of the epithelial–mesenchymal transition (EMT). Chromones and chromone derivatives (CDs) are compounds based on a chemical scaffold found ubiquitously throughout the plant kingdom. We recently showed that eupatilin, a CD from an Artemisia species, efficiently shuts down osteoclastogenesis, concomitant with global downregulation of EMT-associated genes. For this study, we established the mouse hepatic stellate cell (HSC) line ONGHEPA1. Real-time imaging revealed that upon stimulation with TGFβ, ONGHEPA1 cells uniformly differentiated into myofibroblasts, but fibrosis was completely inhibited by 50 μM eupatilin. Concomitant with these anti-fibrotic effects, multiple EMT-inducible genes, such as Col11a1, periostin and Slit3,were significantly downregulated. Although pretreatment with eupatilin did not affect the anti-fibrotic effect by TGFβ, stimulation with eupatilin following pretreatment with TGFβ completely blocked fibrosis, suggesting that the anti-fibrotic capacity of eupatilin is tightly coupled to TGFβ signaling components or TGFβ-inducible molecules. This observation led us to hypothesize that eupatilin could reverse fibrosis in ONGHEPA1 cells by reprogramming the EMT. When ONGHEPA1 cells were sequentially stimulated and differentiated into fibroblasts with TGFβ and eupatilin or selected CDs, we observed dramatic morphological changes along with reversal of the EMT. RNA-Seq analyses and interactome studies revealed that multiple genes involved in the EMT were induced by TGFβ, but repressed by eupatilin. We identified 103 genes whose expression reached the highest levels following stimulation with TGFβ, but dropped to basal levels in cells treated with eupatilin. The majority of these genes were EMT-associated. A defined set of 34 CDs was tested in the fibrosis assay, leading to the identification of two additional CDs capable of blocking the EMT as effectively as eupatilin. The methoxy residue at the sixth carbon (C6) on the chromone scaffold is essential for anti-fibrotic effects, and the hydroxy residue at C7 is also important. However, the presence of any functional groups at C2 significantly decreased anti-fibrotic activity. Replacement of the chemical residues of the remaining carbons (C1, C3, and C4) with methoxy groups led to toxicity or apoptosis. To evaluate the suitability of eupatilin for treating human idiopathic pulmonary fibrosis (IPF), we used a mouse model of bleomycin-induced lung fibrosis. From a histopathological perspective, eupatilin significantly decreased disease severity, reducing infiltration of lymphocytes as well as the extent of extracellular matrix deposits. Taken together, our findings reveal a novel and potent EMT-inhibitory activity associated with the chromone scaffold, which is capable of reversing fulminant fibrosis to the normal state.