Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron

Kris V Kowdley,Donald J Messner

doi:10.1186/1471-230x-8-2

Kris V Kowdley, Donald J Messner

Open Access

https://doi.org/10.1186/1471-230x-8-2

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Abstract

BackgroundIron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies.MethodsT51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot.ResultsT51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 μM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 μM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 μM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.ConclusionThese results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.

Highlights

Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans
As there is no significant excretion of iron, excess uptake may be accompanied by severe liver damage that progresses to liver failure or hepatocellular carcinoma (HCC) [9]
Non-transferrin bound iron uptake in T51B cells T51B is a non-neoplastic liver epithelial cell line used for transformation and tumor promotion studies [37,38,44]

Summary

Introduction

Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. Most body iron stores are sequestered in a non-toxic form through high affinity binding to transport and storage proteins including transferrin and ferritin. Intracellular free iron is a necessary intermediate between iron storage depots and biosynthetic pathways that utilize iron It mediates translational control of iron homeostasis by binding to iron regulatory proteins. As there is no significant excretion of iron, excess uptake may be accompanied by severe liver damage that progresses to liver failure or hepatocellular carcinoma (HCC) [9]. This occurs in diseases of iron overload, including hereditary hemochromatosis. Elevated liver iron is associated with increased HCC in other liver diseases, including biliary cirrhosis and hepatitis C [10]

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