Analysis of molecular mechanisms of 5-fluorouracil-induced steatosis and inflammation in vitro and in mice.

Judith Sommer,Wolfgang E Thasler,Claus Hellerbrand,Anja K Bosserhoff,Andreas Teufel,Kim Freese,Fulya Suzan Kuecuekoktay,Martina Müller,Tobias S Schiergens,Abdo Mahli

doi:10.18632/oncotarget.14371

Abstract

Chemotherapy-associated steatohepatitis is attracting increasing attention because it heralds an increased risk of morbidity and mortality in patients undergoing surgery because of liver metastases. The aim of this study was to develop in vitro and in vivo models to analyze the pathogenesis of 5-fluorouracil (5-FU)-induced steatohepatitis.Therefore, primary human hepatocytes and HepG2 hepatoma cells were incubated with 5-FU at non-toxic concentrations up to 24 h. Furthermore, hepatic tissue of C57BL/6N mice was analyzed 24 h after application of a single 5-FU dose (200 mg/kg body weight). In vitro, incubation with 5-FU induced a significant increase of hepatocellular triglyceride levels. This was paralleled by an impairment of mitochondrial function and a dose- and time-dependently increased expression of fatty acid acyl-CoA oxidase 1 (ACOX1), which catalyzes the initial step for peroxisomal β-oxidation. The latter is known to generate reactive oxygen species, and consequently, expression of the antioxidant enzyme heme oxygenase 1 (HMOX1) was significantly upregulated in 5-FU-treated cells, indicative for oxidative stress. Furthermore, 5-FU significantly induced c-Jun N-terminal kinase (JNK) activation and the expression of pro-inflammatory genes IL-8 and ICAM-1. Also in vivo, 5-FU significantly induced hepatic ACOX1 and HMOX1 expression as well as JNK-activation, pro-inflammatory gene expression and immune cell infiltration. In summary, we identified molecular mechanisms by which 5-FU induces hepatocellular lipid accumulation and inflammation. Our newly developed models can be used to gain further insight into the pathogenesis of 5-FU-induced steatohepatitis and to develop therapeutic strategies to inhibit its development and progression.

Highlights

5-fluorouracil (5-FU) is a uracil analogue and widely used antimetabolite agent for the treatment of various cancer types, including colorectal, breast, and head and neck cancer [1, 2]. 5-FU does not exhibit cytotoxic properties prior to intracellular conversion to one of its active metabolites, namely 5-fluorodeoxyuridine monophosphate (5-FdUMP), 5-fluorodeoxyuridine triphosphate (5-FdUTP) or 5-fluorouridine triphosphate (5-FUTP) [1]. 5-FdUMP is a suicide inhibitor of thymidylate synthase (TYMS), the only cellular de novo- source of deoxythymidine monophosphate [1]
It has been described that expression of genes encoding enzymes relevant to 5-flurouracil metabolism correlates with histologically diagnosed chemotherapy-induced hepatic injury [25]
We established an in vitro model applying HepG2 cells. This cell line has been most extensively employed in many studies analyzing drug metabolism and toxicity, since the cells retain a large part of cellular functions similar to those of normal hepatocytes [41, 42]

Summary

Introduction

5-fluorouracil (5-FU) is a uracil analogue and widely used antimetabolite agent for the treatment of various cancer types, including colorectal, breast, and head and neck cancer [1, 2]. 5-FU does not exhibit cytotoxic properties prior to intracellular conversion to one of its active metabolites, namely 5-fluorodeoxyuridine monophosphate (5-FdUMP), 5-fluorodeoxyuridine triphosphate (5-FdUTP) or 5-fluorouridine triphosphate (5-FUTP) [1]. 5-FdUMP is a suicide inhibitor of thymidylate synthase (TYMS), the only cellular de novo- source of deoxythymidine monophosphate (dTMP) [1]. NAFLD is characterized by hepatic steatosis, which is the most documented liver pathology observed in association with 5-FU administration [14, 16, 17, 20]. Hepatic steatosis is characterized by intravesicular accumulation of fat in the form of triglycerides within hepatocytes [18, 19]. This is due to an imbalance between lipid uptake on the one hand and their combustion or secretion on the other hand [18]. In NAFLD, “simple” steatosis can progress to non-alcoholic steatohepatitis (NASH), a more serious liver condition characterized by death of hepatocytes and liver inflammation with and without hepatic fibrosis [18, 19]. The aim of this study was to develop in vitro and in vivo models for 5-FU-induced steatohepatitis and to identify the underlying mechanisms for the induction of steatosis and its progression to inflammation

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