Cancer Letters

Cancer Letters

Volume 221, Issue 2, 28 April 2005, Pages 135-143
Cancer Letters

Silencing of glutathione S-transferase P1 by promoter hypermethylation and its relationship to environmental chemical carcinogens in hepatocellular carcinoma

https://doi.org/10.1016/j.canlet.2004.08.028Get rights and content

Abstract

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. GSTπ is encoded by the GSTP1 gene. GSTP1 null mice show an increased risk of skin tumorigenesis induced by carcinogens. GSTP1 is transcriptionally silenced by promoter hypermethylation in several human cancers including hepatocellular carcinoma (HCC). Methylation-specific PCR (MSP) was used to analyze the GSTP1 promoter hypermethylation status of 83 hepatocellular carcinoma tissues from Taiwan. Hypermethylation was detected in 38 of 83 (46%) tumors. GSTP1 expression by immunohistochemical staining of HCC tissue samples was significantly associated with methylation status. The relationship between methylation status and clinical parameters and tumor markers including environmental exposure to aflatoxin B1(AFB1) and polycyclic aromatic hydrocarbons (PAH), measured as DNA adducts, was also investigated. A statistically significant association was found between GSTP1 promoter hypermethylation and the level of AFB1–DNA adducts in tumor tissue (OR 2.81, 95% CI 1.03–7.70); a marginally significant association was found for adjacent non-tumor tissue (OR 2.57, 95% CI 0.97–6.80). There was no association between GSTP1 hypermethylation and PAH–DNA adducts in tumor or adjacent non-tumor tissues. These results suggest that epigenetic inactivation of GSTP1 plays an important role in the development of HCC and exposure to environmental carcinogens may be related to altered methylation of genes involved in hepatocarcinogenesis. The mechanism by which environmental exposures induce epigenetic changes in HCC needs further analysis.

Introduction

Hepatocellular carcinoma (HCC) is the most common cancer in the world with rates that show considerable geographic variation. Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections have been well documented as important HCC risk factors. Several environmental factors, including aflatoxin B1 (AFB1) and polycyclic aromatic hydrocarbon (PAHs) exposure are associated with HCC incidence [1], [2], [3]. As with other cancers, the development of HCC is a complex, multistep process [4]. The accumulation of genetic changes and epigenetic alterations including promoter hypermethylation involved in the transcriptional repression of tumor suppressor, DNA repair and metastasis inhibitor genes [5], [6] may also play an important role in the development of HCC.

The GSTs are a family of enzymes implicated in the detoxification of a wide range of xenobiotics and chemotherapeutic agents [7]. GSTs catalyze the conjugation of glutathione with electrophilic compounds including carcinogens and exogenous drugs [8], [9], [10]. GSTs are dimeric enzymes with subunit polypeptides encoding a wide array of genes organized into several gene families: α, μ, π, and θ [11]. GSTP1, encoding glutathione S-transferase-π, has been proposed to be a caretaker gene, protecting cells against genome damage mediated by oxidants and electrophiles from inflammation or environmental exposure [12], [13]. GSTP1 may help defend normal hepatocytes against a variety of potentially promutagenic stresses, including reactive oxygen species associated with chronic hepatic inflammation and reactive electrophilic compounds associated with the hepatic metabolism of dietary and other carcinogens [9], [10], [11], [14].

GSTπ is expressed in normal tissues at various levels in different cell types; abnormal GSTπ activity and expression have been reported in many tumors [15], [16], [17], [18]. In rat models of HCC, after exposure to an initiating carcinogen, hyperplastic nodules containing liver cells displayed increased expression of the π-class (GST-P) [19], [20], [21]. In human studies, it was found that there was an overall reduction in expression of several different GST isoenzymes in HCC tissues [22]. A recent study demonstrated silencing of GSTP1 by CpG island DNA hypermethylation in HBV-associated HCC [23].

In the present study, we explored the possible role of inactivation of GSTP1 in the development of HCC in Taiwan using MSP to detect promoter hypermethylation. We had previously analyzed these samples for DNA damage levels caused by environmental chemical carcinogens, p53 alterations, HBV status and other biomarkers [3], [24]. Recently, we reported a high frequency of promoter hypermethylation of RASSF1A, p16 and MGMT [25], [26]. Thus, we also investigated the relationship between carcinogen exposure and GSTP1 hypermethylation.

Section snippets

Subjects, clinical parameters and biomarkers

The study population consisted of subjects with hepatocellular carcinoma identified at the National Taiwan University Hospital between 1989 and 1995. Informed consent was obtained from patients, and the study was approved by the appropriate institutional review committee. All cases were histologically confirmed. Demographic data are presented in Table 1. Clinicopathological characteristics and smoking status were obtained from hospital charts. Data on AFB1–DNA adducts and mutant p53 were

GSTP1 promoter hypermethylation

Methylation of the promoter region of GSTP1, was determined by MSP. To confirm the specificity of this approach, the human HCC cell line Hep3B, previously reported to have GSTP1 methylation, was used as a positive control for MSP with every assay [23]. Representative results of the gel analysis of bisulfite-treated DNA samples amplified with methylated- and, as a control for the bisulfite modification process, nonmethylated-specific primers are shown in Fig. 1. All tissue samples were found to

Discussion

Promoter CpG island hypermethylation is an important mechanism for loss of function of tumor suppressor and DNA repair genes in human cancers. A growing number of genes have been reported to undergo CpG island hypermethylation in HCC, which indicates its potential role in hepatocarcinogenesis [32]. The π class of GSTs has been associated with preneoplastic and neoplastic changes and is often inversely correlated with prognosis or survival [33]. It has been suggested that loss of GSTP1 leads to

Acknowledgements

This work was supported by NIH/NIEHS grants ES05116 and ES09098.

References (44)

  • N. Kondoh et al.

    Genetic and epigenetic events in human hepatocarcinogenesis

    Int. J. Oncol.

    (2001)
  • P.A. Jones et al.

    Cancer epigenetics comes of age

    Nat. Genet.

    (1999)
  • B. Mannervik et al.

    Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties

    Proc. Natl. Acad. Sci. USA

    (1985)
  • C.B. Pickett et al.

    Glutathione S-transferases: gene structure, regulation, and biological function

    Annu. Rev. Biochem.

    (1989)
  • B. Coles et al.

    The role of glutathione and glutathione transferases in chemical carcinogenesis

    Crit. Rev. Biochem. Mol. Biol.

    (1990)
  • J.D. Hayes et al.

    The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance

    Crit. Rev. Biochem. Mol. Biol.

    (1995)
  • W.H. Lee et al.

    Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis

    Proc. Natl. Acad. Sci. USA

    (1994)
  • W.G. Nelson et al.

    Prostate cancer

    N. Engl. J. Med.

    (2003)
  • S. Tsuchida et al.

    Glutathione transferases and cancer

    Crit. Rev. Biochem. Mol. Biol.

    (1992)
  • C. Di Ilio et al.

    Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue

    Carcinogenesis

    (1991)
  • J. Cairns et al.

    Immunohistochemical demonstration of glutathione S-transferases in primary human breast carcinomas

    J. Pathol.

    (1992)
  • L. Gilbert et al.

    A pilot study of pi-class glutathione S-transferase expression in breast cancer: correlation with estrogen receptor expression and prognosis in node-negative breast cancer

    J. Clin. Oncol.

    (1993)
  • Cited by (0)

    View full text