YC-1 Antagonizes Wnt/ß-Catenin Signaling Through the EBP1 p42 Isoform in Hepatocellular Carcinoma.

Novel drugs targeting Wnt signaling are gradually being developed for hepatocellular carcinoma (HCC) treatment. In this study, we used a Wnt-responsive Super-TOPflash (STF) luciferase reporter assay to screen a new compound targeting Wnt signaling. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was identified as a small molecule inhibitor of the Wnt/ß-catenin pathway. Our coimmunoprecipitation (co-IP) data showed that YC-1 did not affect the ß-catenin/TCF interaction. Then, by mass spectrometry, we identified the ErbB3 receptor-binding protein 1 (EBP1) interaction with the ß-catenin/TCF complex upon YC-1 treatment. EBP1 encodes two splice isoforms, p42 and p48. We further demonstrated that YC-1 enhances p42 isoform binding to the ß-catenin/TCF complex and reduces the transcriptional activity of the complex. The suppression of colony formation by YC-1 was significantly reversed after knockdown of both isoforms (p48 and p42); however, the inhibition of colony formation was maintained when only EBP1 p48 was silenced. Taken together, these results suggest that YC-1 treatment results in a reduction in Wnt-regulated transcription through EBP1 p42 and leads to the inhibition of tumor cell proliferation. These data imply that YC-1 is a drug that antagonizes Wnt/ß-catenin signaling in HCC.

PEDF promotes self-renewal of limbal stem cell and accelerates corneal epithelial wound healing.

Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelial-derived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of ΔNp63α, Bmi-1, and ABCG2 expression. BrdU pulse-labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air-lift culture of limbal equivalent, PEDF was capable of increasing the numbers of ΔNp63α-positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44-mer PEDF (residues 78-121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44-mer PEDF facilitated corneal wound healing. Microscopically, 44-mer PEDF advanced the early proliferative response in limbus, increased the proliferation of ΔNp63α-positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44-mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound.

Pigment epithelium-derived factor is an intrinsic antifibrosis factor targeting hepatic stellate cells.

The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.

Pigment epithelium-derived factor induces interleukin-10 expression in human macrophages by induction of PPAR gamma.

AIM: In search for the anti-inflammation mechanism of PEDF, we investigate whether pigment epithelium-derived factor (PEDF) induces the gene expression of interleukin (IL)-10 in human macrophages and determine the molecular basis of this induction. MAIN METHODS: Human macrophages derived from a monocytic cell line, THP-1, and peripheral monocytes were treated with PEDF. IL-10 expression was assessed by quantitative real-time PCR, enzyme-linked immunosorbent assay, semi-quantitative reverse transcriptase (RT)-PCR, and promoter-reporter assay. Activity of extracellular signal-regulated kinase 2 (ERK2) and p38 mitogen-activated protein kinase (MAPK) was assessed by immunoblotting using antibodies targeting phosphorylated kinases forms. Elk-1 and ATF-2 phosphorylation was determined as well. Pharmacological inhibitors were used to examine the involvement of ERK, p38 MAPK, and peroxisome proliferator-activated receptor gamma (PPARgamma) on the IL-10 expression induced by PEDF. KEY FINDINGS: PEDF increased the levels of IL-10 mRNA and protein in THP-1 cells and human macrophages derived from peripheral monocytes. Blockade of activity of ERK or p38 MAPK attenuated PEDF effects on induction of PPARgamma and IL-10. PEDF increased the transcriptional activity of IL-10 promoter. The effect was synergistically augmented by PPARgamma agonist, but attenuated by inhibitors of PPARgamma, ERK or p38 MAPK. These results showed that PEDF promotes IL-10 expression at transcriptional level, and that this is achieved through the ERK2/p38MAPK-dependent PPARgamma expression. SIGNIFICANCE: The anti-inflammatory property of PEDF may in part through the induction of IL-10 in macrophages. Our study supports the therapeutic potential of PEDF and PPARgamma agonists in inflammatory diseases.

Photoreceptor protection against light damage by AAV-mediated overexpression of heme oxygenase-1.

PURPOSE: To investigate whether overexpression of the cytoprotective gene heme oxygenase-1 (HO-1) in photoreceptors by gene delivery attenuates cellular injury caused by intense light damage and to document the possible mechanisms of protection. METHODS: Recombinant adeno-associated virus type 5 (rAAV5) expressing the mouse HO-1 gene (mHO-1) was delivered to cyclic-light reared Sprague-Dawley (SD) rats by subretinal injection. Three weeks after transfer of HO-1 gene, animals were subjected to 2-hour intense light exposure then were returned to darkness. Expression of HO-1, p53, p38, and cellular FLICE inhibitory protein (c-FLIP) at different times after intense light damage was evaluated by Western blot analysis. HO-1 transgene expression, along with expression of c-fos and bcl-2, was analyzed by immunohistochemistry. In addition, the protective effects of HO-1 were evaluated by determining the morphology of the retina. Finally, apoptosis in photoreceptors was measured using TdT-dUTP terminal nick-end labeling (TUNEL) 24 hours after photic injury. RESULTS: Exogenous administration of HO-1 by gene transfer led to HO-1 transgene expression in photoreceptors. Protection of retina by HO-1 overexpression is evident from the partially preserved retina structure and attenuated apoptosis in photoreceptors after photic injury. Concurrently, overexpression of HO-1 was associated with a decrease in the expression of c-fos and p53, an increase in the activation of p38 and bcl-2, and preserved the expression of c-FLIP. CONCLUSIONS: Overexpression of HO-1 in photoreceptors protected themselves from subsequent cellular damage caused by intense light exposure. The anti-apoptotic mechanisms of HO-1 may be related to the induction of p38, bcl-2, and c-FLIP and to the suppression of c-fos and p53.

Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line.

BACKGROUND: Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration. METHODS: Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPARgamma expression was blocked by PPARgamma small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry. RESULTS: TGZ dose- and time-dependently impaired cell migration through a PPARgamma independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK), and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase). At concentration higher than 10 muM, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation. CONCLUSION: These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.