Elsevier

Biochemical Pharmacology

Volume 72, Issue 10, 15 November 2006, Pages 1322-1333
Biochemical Pharmacology

2′,4′,6′-Tris(methoxymethoxy) chalcone attenuates hepatic stellate cell proliferation by a heme oxygenase-dependent pathway

https://doi.org/10.1016/j.bcp.2006.08.004Get rights and content

Abstract

Proliferation of hepatic stellate cells (HSCs) is central for the development of fibrosis during liver injury. We have shown previously that butein (3,4,2′,4′-tetrahydroxychalcone) suppresses myofibroblastic differentiation of rat HSCs. Our aim in this study was to determine whether a new synthetic chalcone derivative, 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC) inhibits HSC proliferation induced by serum- or platelet-derived growth factor (PDGF). TMMC significantly inhibited growth factor-induced HSC proliferation. The inhibition of PDGF-induced proliferation by TMMC was associated with the phosphatidylinositol 3-kinase-Akt-p70S6K pathways. TMMC induced the expression of heme oxygenase 1 (HO-1) in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of TMMC on PDGF-induced proliferation is mediated by HO-1. Glutathione (GSH) depletion produced by TMMC activated extracellular signal-regulated kinase (ERK), which led to c-Fos expression and transactivation of activator protein 1 (AP-1) and HO-1 gene expression in the HSCs. These results demonstrate that TMMC preferentially activates ERK and that this activation leads to the transcriptional activation of AP-1 and consequently to HO-1 expression. HO-1 expression might be responsible for the antiproliferative effect of TMMC on HSCs.

Introduction

Liver fibrosis is a common response to chronic liver injury and is characterized by increased deposition of extracellular matrix [1], [2]. Hepatic stellate cells (HSCs) are the primary cells producing the extracellular matrix that contributes to hepatic fibrosis [3], [4]. During liver fibrosis, HSCs become activated and transform into proliferating fibroblast-like cells, which produce large amounts of collagen. To prevent the progression of hepatic fibrosis, various types of compounds that interfere with HSC proliferation and activation have been developed as antifibrogenic agents [5], [6].

We have shown previously that butein (3,4,2′,4′-tetrahydroxychalcone) suppresses myofibroblastic differentiation of rat HSCs [7]. However, pharmacokinetic studies have reported a low oral bioavailability of native butein [8], [9]. That is, after administration of butein, large amounts of butein metabolites such as conjugated butein are found in the systemic circulation and are excreted into bile and urine. We have tried to chemically modify chalcone to improve the bioavailability of butein. In the process of synthesizing the chalcone derivatives, we found that 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC) [10] displays a potent antiproliferative effect, which is mediated by induction of heme oxygenase 1 (HO-1) in HSCs.

Glutathione (GSH) is a major cellular sulfhydryl source in nonprotein matter and plays a pivotal role in redox control in mammalian cells. It is also involved in the phase II drug metabolizing reaction as a cofactor of GSH S-transferase. Since GSH S-transferase is one of the abundantly expressed cytosolic proteins in most cells, the enzyme consumes GSH when a mass of the substrate fluxed resulting in its depletion and eventually cells to commit into the oxidative stress. It has been reported that GSH S-transferase substrates such as phorone and diethyl maleate (DEM) induce a decrease in GSH contents and a concomitant increase in HO activity in the liver of rats [11], [12].

HO is the enzyme that catalyzes the degradation of the heme group to produce carbon monoxide, biliverdin, and free iron. Biliverdin is converted to bilirubin by biliverdin reductase, and free iron is used in metabolism or is sequestered by ferritin [13]. So far, three isoforms of HO have been fully characterized. HO-2 and HO-3 are constitutive isozymes [14], [15], whereas HO-1 is induced by a variety of stimuli in various types of cells, including HSCs [16]. The exact functional role of HO-1 is not fully understood, although growing evidence indicates that HO-1 has antiproliferative effects [17], [18], [19]. Recent studies show that HO-1 is expressed in HSCs, is induced during chronic liver injury, and is part of an important pathway to limit progression of fibrosis during chronic liver diseases [16], [20], [21]. In light of the antifibrogenic role of HO-1, the specific activation of HO-1 gene expression by pharmacological modulation may represent a novel target for therapeutic intervention.

In this study, we demonstrated the effects of TMMC on growth factor-induced proliferation and the main intracellular signaling pathways elicited by platelet-derived factor (PDGF) in HSCs. We also show that the inhibitory effect of TMMC on cell proliferation is mediated by induction of HO-1 and that its molecular mechanisms involves the regulation of HO-1.

Section snippets

Reagents and cell isolation

TMMC was synthesized as described previously [22]. All reagents were from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise indicated. Tin protoporphyrin (SnPP), an inhibitor of HO activity, was from Porphyrin Products, Inc. (Logan, UT, USA). The inhibitors of mitogen-activated protein kinase (MAPK), PD98059, U0126, SP600125, and SB203580 were from Calbiochem (San Diego, CA, USA). Rat HSCs were isolated from the livers of Sprague-Dawley rats as described previously [23], [24], [25].

Effect of TMMC on serum-stimulated and PDGF-induced cell proliferation

To evaluate the effect of TMMC on cell proliferation of cultured HSCs, we examined the incorporation of BrdU into chromosomal DNA. Serum-deprived HSC were either maintained in serum-rich media (5% FBS) or incubated in 10 ng/ml PDGF, with or without TMMC at the indicated concentrations for 24 h. As shown in Fig. 1, compared with control, 5% FBS increased the incorporation of BrdU into chromosomal DNA of cultured HSC by six times and 10 ng/ml PDGF increased BrdU incorporation by three times. TMMC

Discussion

HSCs play a central role in the development of liver fibrosis. During this process, HSCs proliferate, accumulate in the diseased liver, and secrete fibrosis components [3], [4]. Blocking HSC proliferation is considered an important target for the development of antifibrotic drugs. We have shown previously that butein, a type of chalcone derivative, has beneficial effects, including antifibrogenic effects, in the liver [7], [45] and anti-inflammatory activity [28]. However, pharmacokinetic

Acknowledgment

This work was supported by the Korea Research Foundation Grant (KRF-2004-005-E00093).

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