Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma

Journal of Hepatology 42 (2005) 511–519 www.elsevier.com/locate/jhep

Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma Motohisa Tada1, Osamu Yokosuka1,*, Kenichi Fukai1, Tetsuhiro Chiba1, Fumio Imazeki1, Takeshi Tokuhisa2, Hiromitsu Saisho1 1

Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan 2 Department of Developmental Genetics, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, Japan

Background/Aims: NAD(P)H: quinone oxydoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) belong to phase II xenobiotic-metabolizing enzymes. GSTP1 inactivation via CpG island hypermethylation in hepatocellular carcinoma (HCC) was previously reported, but the involvement of NQO1 in HCC is not well known. In this study, we assessed the transcription and status of methylation of NQO1 gene in human hepatoma cells and primary human HCC tissues. Methods: NQO1 transcription and DNA hypermethylation in hepatoma cells with or without 5-aza-deoxycytidine (5Aza-CdR) treatment were investigated by reverse-transcription PCR (RT-PCR), sodium bisulfite sequencing and methylation-specific PCR (MSP). The methylation status of NQO1 and GSTP1, and NQO1 mRNA in 44 HCC cases was also analyzed by MSP and real-time PCR, respectively. Results: NQO1 transcription was down-regulated and the CpG island DNA was hypermethylated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored and CpG island DNA was demethylated in these cells. MSP analysis revealed that NQO1 hypermethylation occurred in 50.0% of HCC. All of the tumors that exhibited lesser amounts of NQO1 mRNA than corresponding non-tumorous tissues showed NQO1 hypermethylation. Conclusions: NQO1 transcription might be inappropriately suppressed by promoter hypermethylation in a subset of HCC, as well as GSTP1 gene. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Detoxification; Phase II xenobiotic-metabolizing enzyme; Reactive oxygen species; Hepatocellular carcinoma; Methylation; Acetylation

1. Introduction

Received 20 May 2004; received in revised form 5 November 2004; accepted 12 November 2004; available online 28 November 2004 * Corresponding author. Tel.: C81 43 226 2083; fax: C81 43 226 2088. E-mail address: [email protected] (O. Yokosuka). Abbreviations: NQO1, NAD(P)H: quinone oxidoreductase 1; GSTP1, glutathione S-transferase P1; HCC, hepatocellular carcinoma; 5-Aza-CdR, 5-aza-deoxycytidine; RT-PCR, reverse transcriptase polymerase chain reaction; MSP, methylation-specific polymerase chain reaction; ChIP, chromatin immunoprecipitation; ROS, reactive oxygen species; CYPs, cytochrome P450s; GSTs, glutathione S-transferases; GSTp, pclass glutathione S-transferase; TSA, trichostatin A; MMC, mitomycin C; HDAC, histone deacetylase.

The incidence of hepatocellular carcinoma (HCC) is increasing worldwide. Liver cancer is the fifth most common cancer in the world and the third most common cause of cancer-related death [1]. Most HCC cases arise in the setting of chronic hepatitis virus infection [2]. Dietary carcinogens, such as aflatoxin B1, also contribute to hepatic carcinogens [3]. Metabolic activation of carcinogens may also be directly promoted by oxidative stress, i.e., sustained generation of reactive oxygen species (ROS) [2]. Excessive ROS may induce toxicity, mutations and ultimately boost the development of cancer in various human tissues [4,5].

0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.11.024

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Several enzyme systems including cytochrome P450s (CYPs), conjugation enzymes and enzymes involved in detoxification of ROS cooperate to protect the organism against reactive agents that are potentially carcinogenic and stress-inducing [6]. Glutathione S-transferases (GSTs) are also involved in the so-called phase II metabolism and a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents [7]. Among GSTs, GSTp, which is encoded by the GSTP1 gene, is of particular interest in the study of cancer biology [8]. Abnormal GSTp activity and expression have been reported in many tumors, and genetic alteration such as polymorphism and epigenetic alteration such as DNA hypermethylation of GSTP1 gene have been reported in several tumors, including lung, prostate and liver [7,9,10]. NAD(P)H: quinone oxidoreductase1 (NQO1), previously known as DT diaphorase, is a flavoprotein that catalyzes the reduction of different quinines [11]. Several lines of evidence have indicated that NQO1 may have a protective effect against carcinogenicity, mutagenicity and other toxicities caused by quinones and their metabolic precursors [12–14]. This protection would most likely result from the two-electron reduction catalyzed by NQO1, which would compete with the formation of free radicals and toxic oxygen metabolites that may be generated as a result of a one-electron reduction catalyzed by enzymes such as NADPH-cytochrome P450 oxidoreductase [15]. As stated above, NQO1 is also involved in phase II metabolism and is classified as a detoxification enzyme. So, we focused on NQO1 gene, a detoxification enzyme like GSTP1 gene, and assessed the status of its expression, 5 0 CpG island methylation, and acetylation in HCC cell lines.

samples and corresponding non-tumorous liver tissues obtained by surgical resection were immediately frozen at K80 8C. All patients had given informed consent for their participation, and the ethics committee approved these studies.

2.3. Conventional and real-time RT-PCR Three mg of total RNA from 5-Aza-CdR-treated or untreated cell lines was subjected to RT reaction by using random oligonucleotide primers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Two ml of the RT reaction product was then amplified by PCR using the primer set listed in Table 2. After an initial denaturation at 95 8C for 2 min, PCR was performed in a 25-ml reaction volume for 22 cycles under the following conditions: 95 8C for 30 s, 59 8C for 30 s, 72 8C for 60 s, and finally an extension at 72 8C for 5 min. Five microlitres of the PCR product was run on a 3% agarose gel and visualized by SYBR Green (FMC, Rockland, ME) staining. RNA samples were also amplified using b-actin gene primers as control. For real-time PCR, NQO1 was amplified on the same plate with the reference, b-actin, using the TaqMan Universal PCR Master Mix and the ABI Prism 7000 Sequence Detection Systems (Applied Biosystems Japan, Tokyo, Japan), and the relative mRNA amounts were determined. Briefly, we normalized each set of samples using the difference in threshold cycles (DCT) between NQO1 and b-actin gene (DCTsampleZDCTNQO1KDCTb-actin). cDNA from the HLE cell line, which clearly expressed the mRNA of NQO1 and b-actin gene, was used as calibration sample (DCTcalibration). Relative mRNA levels were calculated by the expression 2KDDCT, where DDCTZDCTsampleK DCTcalibration. The primer set and probe used in real-time PCR are listed in Table 2.

2.4. Sodium bisulfite DNA sequencing Genomic DNA extracted from each hepatoma cell line was modified by sodium bisulfite using a CpGenome DNA Modification Kit (Intergen, Purchase, NY.) according to the manufacturer’s instructions. The bisulfitemodified DNA was amplified by nested PCR using the specific primers listed in Table 2. PCR products were then subcloned into the pCR2.1TOPO vector using a TA cloning kit (Invitrogen) according to the manufacturer’s instructions. To determine the CpG methylation status of the 5 0 CpG island of NQO1 gene, 10 clones from each cell line were sequenced using an ABI PRISM Dye Deoxy Terminator Cycle Sequencing Kit and analyzed on an ABI 377 DNA Sequencer (Applied Biosystems Japan).

2.5. Methylation-specific PCR (MSP) 2. Materials and methods 2.1. Cell culture and 5-Aza-CdR treatment The human hepatoma cell lines HLE, HuH7, HepG2, HuH6 and PLC/PRF/5 were obtained from the Health Science Research Resources Bank (Osaka, Japan), and Hep3B from the Cell Resource Center for Biomedical Research (Sendai, Japan). The cell lines were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin. For demethylation experiments, cells were plated at a density of 5.0!105 cells/100-mm dish and cultured for 24 h followed by 96 h culture with 1 mM of 5-Aza-CdR (Sigma Chemical Co., St Louis, MO).

2.2. Patients Liver tissues were obtained from 44 HCC patients (40 men and four women; mean age, 62.3G11.0 year) who underwent surgical resection. Among these patients, 13 were positive for hepatitis B surface antigen and 23 for hepatitis C virus antibody, and the remaining eight lacked evidence of either viral infection. Based on the histological findings, 44 HCC tumors were classified as follows: nine were well differentiated, 25 were moderately differentiated, and 10 were poorly differentiated. More than half of the tumors (25/44) developed in cirrhotic livers (Table 1). HCC

For detection of aberrant methylation of NQO1 and GSTP1 gene, modified DNA was amplified using primers specific for the methylated sequences (Table 2). For quality control of the bisulfite modification process, the bisulfite-modified DNA was also amplified using primers specific for the unmethylated sequence of each gene (Table 2). A hotstarted PCR was performed in a 25-ml reaction-volume using AmpliTaq Gold DNA Polymerase (Applied Biosystems Japan). Five ml of the products was run on a 3% agarose gel and visualized by SYBR Green (FMC) staining.

2.6. Chromatin immunoprecipitation (ChIP) assay For ChIP assay, cells were plated at a density of 1!106 cells/100-mm dish and cultured for 24 h followed by 24 h of culture with 0.67 mM of trichostatin A (TSA), a histone deacetylase inhibitor. Formaldehyde was added to the cells, and the cells were then incubated at 37 8C for 10 min. The cells were suspended in 10 ml of PBS, pelleted, resuspended in 0.2 ml of SDS lysis buffer (1% SDS/10 mM EDTA/50 mM Tris–HCl, pH 8.1), and incubated on ice for 10 min. Lysates were sonicated and debris was removed from samples by centrifugation at 13,000 rpm at 4 8C for 10 min. Supernatants were diluted 5-fold in immunoprecipitation buffer (0.01%SDS/1.1% TritonX-100/1.2 mM EDTA/16.7 mM Tris–HCl, pH 8.1/167 mM NaCl), and 80 ml of Salmon Sperm DNA/Protein A Agarose-50% Slurry (Upstate Biotechnology Inc., Lake Placid, NY) was

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Table 1 Methylation status of NQO1 and GSTP1 gene in the 44 HCC cases Case no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Age

74 70 55 42 59 55 63 65 73 69 64 73 62 68 57 65 71 71 50 67 60 72 64 69 64 53 28 70 65 70 56 81 71 57 43 53 78 52 40 51 60 65 81 63

Sex

M M M M M M M F M M M M M M M M M F M M M M M M M M F M M M M F M F M M M M M M M M M F

Etiology

B B C B B B C C C C C C C NBNC NBNC NBNC NBNC NBNC B C B C C B C C B C NBNC NBNC NBNC C C C C B C C C B B B C C

Surrounding liver

Differentiation

Maximum size (cm)

NQO1 DNA methylation

GSTP1 DNA methylation

T

NT

T

NT

LC CH CH LC LC LC CH LC LC LC LC LC CH CH CH CH LC CH LC CH CH CH LC LC LC CH CH CH LC CH CH LC LC LC LC LC CH CH LC LC LC CH LC LC

Moderately Moderately Moderately Moderately Moderately Well Poorly Poorly Moderately Moderately Well Moderately Well Moderately Moderately Moderately Moderately Well Moderately Well Moderately Poorly Poorly Poorly Moderately Well Poorly Moderately Moderately Moderately Poorly Moderately Poorly Well Moderately Moderately Moderately Poorly Well Moderately Poorly Well Moderately Moderately

2.8 2.5 3.0 10 5.7 1.3 2.5 4.0 4.0 5.5 2.5 3.7 12 12 4.0 9.0 6.0 2.7 2.5 4.0 8.0 3.0 3.0 4.0 6.0 4.0 5.5 3.0 5.0 3.0 4.0 1.4 10 2.5 2.0 4.5 10 12 5.5 4.5 3.5 4.0 7.0 2.6

C K C K K K K C C C K K C C C C K C K K K K K C K K K K K K C C C C C C C K C K C K C C

K K K K K K K K K K K K K C K K K K K K K K K K K K K K K K K K K K K K K K K C K K K C

C C K C K C K C C C K C K C K C K C K K C K C C K K C K K K K K K C C C C C C K K K C K

K K K K C K K K K K C K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K

CH, chronic hepatitis; LC, liver cirrhosis; T, tumor; NT, corresponding non-tumorous tissues; C, positive; K, negative.

added and incubated, rocking for 30 min at 4 8C. Beads were pelleted by centrifugation, and supernatants were placed in fresh tubes with 5 mg of antiacetylated histone H3 and H4 antibodies, or normal rabbit IgG (Upstate Biotechnology Inc.), and incubated overnight at 4 8C. Sixty ml of Salmon Sperm DNA/Protein A Agarose-50% Slurry was added, and samples were rocked for 1 h at 4 8C. Protein A complexes were centrifuged and washed five times for 5 min each, according to the manufacturer’s protocol. Immune complexes were eluted twice with 250 ml of elution buffer (1% SDS/0.1 M NaHCO3) for 15 min at room temperature. Twenty ml of 5 M NaCl was added to the combined eluates, and the samples were incubated at 65 8C for 4 h. EDTA, Tris–HCl, pH 6.5, and proteinase K were then added to the samples at final concentrations of 10, 40 and 40 mg/ml, respectively, and the samples were incubated at 45 8C for 1 h. Immunoprecipitated DNA

was recovered by phenol/chloroform extraction and ethanol precipitation and analyzed by PCR. The PCR amplification protocol consisted of 35 cycles at 95 8C for 30 s, 55 8C for 30 s and 72 8C for 30 s using the primer set listed in Table 2. Five ml of the PCR product was run on a 3% agarose gel and visualized by SYBR Green (FMC) staining.

2.7. Statistical analysis The Mann–Whitney U-test was used for comparisons of the expression levels of NQO1 mRNA in the tumor and corresponding non-tumorous tissues. Differences in frequencies were evaluated by Fisher’s exact test. A P value of !0.05 was considered statistically significant.

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Table 2 PCR primer sequences for RT-PCR (conventional and real-time), bisulfite sequencing, ChIP assay and MSP Primer set

Sequence

Product size

Annealing temperature (8C)

Cycles

RT-PCR NQO1-sense NQO1-antisense Probe

5 0 -AGGCTGGTTTGAGCGAGTTC-3 0 5 0 -ATTGAATTCGGGCGTCTGCTG-3 0 5 0 -FAM-AGAGCCATGGTCGGCAGAAGAGCA-NFQ-3 0

269 bp

59

22

5 0 -TGTATTTTAGGGTATAGTGTGTAGATGGGTTG-3 0 5 0 -AAACTCATCCCAAATCCCTAATCTCTTCCC-3 0

587 bp

50

35

5 0 -TTGGAGTTGTAGTTTTAGTA-3 0 5 0 -AAACCCCTACAACCTCCTCC-3 0

332 bp

50

40

5 0 -ACTTTTCTTGACTTCCACCAG-3 0 5 0 -TTGTGGGAGTCGCGTGTGTA-3 0

224 bp

55

35

5 0 -AGTTTCGGTTAGGGTCGTTC-3 0 5 0 -CCAATACTCGAAAAACGACCG-3 0 5 0 -AGTTTTGGTTAGGGTTGTTTT-3 0 5 0 -CCAATACTCAAAAAACAACCA-3 0 5 0 -TTCGGGGTGTAGCGGTCGTC-3 0 5 0 -GCCCCAATACTAAATCACGACG-3 0 5 0 -GATGTTTGGGGTGTAGTGGTTGTT-3 0 5 0 -CCACCCCAATACTAAATCACAACA-3 0

125 bp

50

43

125 bp

47

43

91 bp

55

43

97 bp

55

43

Bisulfite sequencing 1st PCR NQO1-sense NQO1-antisense 2nd PCR NQO1-sense NQO1-antisense ChIP assay NQO1-sense NQO1-antisense MSP NQO1-M-sense NQO1-M-antisense NQO1-U-sense NQO1-U-antisense GSTP1-M-sense GSTP1-M-antisense GSTP1-U-sense GSTP1-U-antisense

M, methylated; U, unmethylated; FAM, 6-carboxyfluorescein; NFQ, nonfluorescent quencher.

3. Results 3.1. Conventional RT-PCR analysis of NQO1 transcription in hepatoma cells RT-PCR using primers that specifically amplified the NQO1 gene revealed that the NQO1 transcript was faint in Hep3B cells and undetectable in HuH6 cells before treatment with 5-Aza-CdR, while it was abundant in HLE, HuH7, HepG2 and PLC/PRF/5 cells. The treatment with 5-Aza-CdR then restored the NQO1 mRNA expression in Hep3B and HuH6 cells. HLE, HuH7, HepG2 and PLC/PRF/5, with high initial expression of NQO1, exhibited no additional induction of NQO1 expression after treatment with 5-Aza-CdR (Fig. 1).

3.2. Methylation status of CpG island in 5 0 region of NQO1 gene in hepatoma cells by sodium bisulfite DNA sequencing and MSP The methylation status of the 24 CpG dinucleotides encompassing the promoter and exon 1 is shown in Fig. 2. The 5 0 CpG island of NQO1 was densely methylated in each clone of HuH6 and in five of 10 clones of Hep3B in which the expression of NQO1 was up-regulated by 5-AzaCdR, while in HLE, HepG2 and PLC/PRF/5, few CpG dinucleotides were methylated. In HuH7, no CpG dinucleotide was methylated. MSP analysis was performed to assess the methylation status of the 5 0 CpG island of NQO1 in these cells after treatment with demethylating agent. The primer set for MSP was designed on the basis of the CpG methylation status determined by bisulfite sequencing

Fig. 1. RT-PCR analysis of NQO1 expression in hepatoma cell lines. (A) Without treatment with 5-Aza-CdR, NQO1 mRNA expression was downregulated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored in Hep3B and HuH6 cells. (B) RNA samples were also amplified using b-actin gene primers as control. C, 5-Aza-CdR-treated; K, untreated.

M. Tada et al. / Journal of Hepatology 42 (2005) 511–519 Table 3 NQO1 mRNA expression in HCC cases Case no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

NQO1 DNA methylation

NQO1 mRNA expression

T

NT

T

NT

C K C K K K K C C C K K C C C C K C K K K K K C K K K K K K C C C C C C C K C K C K C C

K K K K K K K K K K K K K C K K K K K K K K K K K K K K K K K K K K K C K K K C K K K C

0.171943 0.238192 1.635804 0.061214 2.281527 0.074842 1.741101 0.001575 0.460094 0.098755 0.109576 0.001953 0.11744 0.000174 2.602684 0.029360 0.095391 0.002762 1.071773 0.006992 1 2.361985 0.203063 0.004371 0.131215 0.888843 0.013230 1.741101 5.81589 0.177013 0.175556 0.009820 0.01323 1.547565 0.216134 0.033726 0.011924 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

1.071773 0.002355 0.066064 0.000355 0.012604 0.002595 0.004375 0.00198 0.112656 0.228458 0.003521 0.0002153 0.055553 0.006524 7.412704 0.079660 0.003933 0.007041 0.01278 0.002455 0.003173 0.013415 0.026645 0.029564 0.006087 0.0056874 0.002455 0.048027 0.027584 0.001391 0.015093 0.026278 0.007189 2.07053 0.068869 0.020905 0.007546 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

T/NT

0.160428 101.1427 24.76104 172.4459 181.0161 28.84001 397.9659 0.795536 4.084049 0.432269 31.12496 9.071063 2.114017 0.026645 0.351111 0.368567 24.25147 0.392292 83.86518 2.8481 315.173 176.0694 7.621104 0.1478787 172.4459 156.2828 5.388934 36.25228 210.8393 127.2559 11.63178 0.373712 1.840375 0.747425 3.138336 1.613284 1.580083 K K K K K K K

T, tumor; NT, corresponding non-tumorous tissues; NQO1 mRNA expression, NQO1 mRNA expression normalized to that of b-actin; T/NT, ratio of NQO1 mRNA expression in the tumor to that in corresponding nontumorous liver tissues; C, positive; K, negative.

(Table 2). The 5 0 CpG island of NQO1 was hypermethylated in Hep3B and HuH6 cells without treatment with 5-AzaCdR, and it was demethylated in these cells after 5-AzaCdR treatment (Fig. 3). 3.3. Status of histone acetylation of NQO1 gene in hepatoma cells ChIP assay was performed to determine the relation of CpG methylation with the acetylation of histones H3 and H4

515

associated with the NQO1 gene promoter. In Hep3B and HuH6 cells, in which the promoter hypermethylation of NQO1 gene was detectable, acetylated histone H3 was undetectable, but it was abundant in HLE, HuH7, HepG2 and PLC/PRF/5 cells (Fig. 4A). Treatment with TSA did not alter the acetylation status of histone H3 and H4 in Hep3B and HuH6 cells (Fig. 4A and B). 3.4. Methylation status of CpG island in 5 0 region of NQO1 and GSTP1 genes in hepatocellular carcinoma cases The occurrence of NQO1 hypermethylation in 44 primary HCC tumors was analyzed by using MSP assay, and GSTP1, a family of phase II detoxification enzymes like NQO1, was also analyzed (Table 1). The 5 0 CpG island of NQO1 gene was demonstrated to be hypermethylated in 22 (50.0%) out of 44 tumors, whereas in the corresponding non-tumorous liver tissues, NQO1 hypermethylation was detected in four (9.1%) out of 44 samples (P!0.01). The 5 0 CpG island of GSTP1 gene was demonstrated to be hypermethylated in 22 (50.0%) out of 44 tumors, whereas in the corresponding non-tumorous liver tissues, GSTP1 hypermethylation was detected in two (4.5%) samples (P!0.01). In approximately one-third of the HCC tissues, both NQO1 and GSTP1 genes were hypermethylated. In 30 of 44 (68.2%) HCC tissues, the promoter hypermethylation of either NQO1 or GSTP1 gene was observed (Table 4, Fig. 5). Any correlation between the occurrence of NQO1 or GSTP1 hypermethylation and parameters such as age, tumor size, etiology, the status of surrounding non-tumorous liver tissues, and differentiation of HCC was not apparent in the current study (Tables 5 and 6). 3.5. Quantitative real-time PCR analysis of NQO1 transcription in hepatocellular carcinoma cases Of the 44 HCC cases, 37 were subjected to quantitative NQO1 mRNA expression analysis by real-time PCR. In 10 of the 37 tumors, the transcription level of NQO1 was less than that in corresponding non-tumorous tissues (0.3796G 0.2446, meanGSD). All of these 10 tumors exhibited NQO1 hypermethylation. In the remaining 27 tumors, the NQO1 transcription level was higher than in corresponding non-tumorous tissues (Tables 3 and 7). Among the 27 cases, the ratio of mRNA expression in the tumor to that in the non-tumorous tissues was significantly lower in NQO1 hypermethylated cases (6.345G8.157) than in NQO1 unmethylated cases (117.889G111.332) (P!0.01).

4. Discussion Endogenous oxidants generated by multiple intracellular pathways are an important class of naturally occurring carcinogens [16,17]. ROS are endogenous oxygen-containing molecules formed as normal products during aerobic

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Fig. 2. Methylation status of 24 CpG dinucleotides located between K113 and C219 in the promoter and exon 1 of NQO1 gene. Bisulfite sequencing was performed to determine the methylation status of the 5 0 CpG island of NQO1 gene in each cell line indicated. Each circle indicates a CpG site in the primary DNA sequence, and each line of circles represents the analysis of a single cloned allele. Closed circles, methylated CpG dinucleotides; open circles, unmethylated CpG dinucleotides. The 5 0 CpG island of NQO1 was densely methylated in each clone of HuH6 cells and in five of 10 clones of Hep3B cells, while few nucleotides were methylated in HLE, HepG2 and PLC/PRF/5, and no CpG dinucleotide was methylated in HuH7.

Fig. 3. MSP analysis performed to assess the methylation status of the 5 0 CpG island of NQO1 in hepatoma cells. (A) Without treatment with 5-AzaCdR. (B) Treatment with 5-Aza-CdR. MSP analysis revealed that the 5 0 CpG island of NQO1 was hypermethylated in Hep3B and HuH6 cells without treatment with 5-Aza-CdR. After treatment with 5-Aza-CdR, the 5 0 CpG island of NQO1 was demethylated in Hep3B and HuH6 cells. M, methylated; U, unmethylated.

Fig. 4. ChIP assay. Crosslinked chromatin from each cell line was incubated with acetylated histones H3 (A) and H4 (B). Immnoprecipitates from each antibody were aliquotted and analyzed by PCR with primer specific NQO1 promoter. Acetylated histone H3 and H4 were undetectable in Hep3B and HuH6 cells without treatment with trichostatin A, while they were abundant in HLE, HuH7, HepG2 and PLC/PRF/5 cells. But histones H3 and H4 were also deacetylated in Hep3B and HuH6 cells after treatment with trichostatin A. C, TSA-treated; K, untreated.

M. Tada et al. / Journal of Hepatology 42 (2005) 511–519 Table 4 Hypermethylation of NQO1 and GSTP1 genes in the 44 HCC cases analyzed by MSP

NQO1 GSTP1 NQO1 and GSTP1 NQO1and/or GSTP1

T (nZ44)

NT (nZ44)

Pa

22/44 (50.0%) 22/44 (50.0%) 14/44 (31.8%) 30/44 (68.2%)

4/44 (9.1%) 2/44 (4.5%) 0/44 (0%) 6/44 (13.6%)

!0.01 !0.01 !0.01 !0.01

a

P for Fisher’s exact test; T, tumor; NT, corresponding non-tumorous liver tissues.

metabolism [18]. ROS can induce genetic mutations as well as chromosomal alterations and thus contribute to cancer development in multistep carcinogenesis [19,20]. Most HCC cases arise in the setting of chronic hepatitis virus infection, and ROS generated during persistent inflammation induces continuous cell death and regeneration. This leads to genetic damage and may result in the initiation of HCC. On the other hand, dietary carcinogens, such as aflatoxin B1, also contribute to hepatic carcinogens [3]. Many dietary constituents modify the metabolism of carcinogens by inductions of enzymes involved in xenobiotic metabolism, and this is one well-established mechanism for modulating the risk of cancer [21]. That is to say, a major mechanism regulating neoplasia is the balance between phase I enzymes such as CYPs, which initiate biotransformation of compounds, do not possess functional groups suitable for conjugation, and can thereby generate toxic and mutagenic/carcinogenic intermediates [22], and phase II enzymes such as NQO1 and GSTP1, which follow CYPs-dependent oxidative activation and generally decrease the reactivity of these intermediates by linkage reactions facilitating their excretion from the body [6]. Thus, disruption of the detoxification enzyme may cause excessive ROS and result in the initiation of HCC. Indeed, Iizuka et al., based on the oligonucleotide microarray method, reported that blockade of the detoxification system was a common pathway during carcinogenesis and/or progression of B-type and C-type HCCs [23]. As for the GSTP1 gene, its polymorphism and promoter hypermethylation are reported to be associated with HCC

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[7,9,24]. In particular, it has been reported that epigenetic silencing of GSTP1 gene expression by CpG island DNA hypermethylation was common in HCC, and somatic GSTP1 inactivation via CpG island hypermethylation might contribute to the pathogenesis of HCC [7,9]. But involvement of NQO1, which belongs to phase II enzymes like GSTP1, in HCC is not well known. NQO1 is an important component of the chemopreventive activities in the cell because of its capacity to promote obligately two-electron reduction of quinones, preventing their participation in redox cycling, oxidative stress and neoplasia [25]. Moreover, there have been interesting reports of the bioreductive activation of mitomycin C (MMC) by NQO1 enzyme. That is to say, NQO1 enzyme activity is required for effective cytotoxicity of MMC in colon and gastric carcinoma cells [26]. Therefore, the studies on the regulation mechanism of the NQO1 gene are of considerable interest and significance. Then, we concentrated on the NQO1 gene, examining its transcription in human hepatoma cell lines. Conventional RT-PCR analysis showed that NQO1 transcription was down regulated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, it was restored in these cells. On the other hand, sodium bisulfite DNA sequencing and MSP showed that NQO1 hypermethylation occurred in Hep3B and HuH6 cells, and after treatment with 5-Aza-CdR, the 5 0 CpG island of NQO1 was revealed to be demethylated in these cells. Based on these results, we considered that NQO1 might be transcriptionally repressed by promoter hypermethylation, as with GSTP1. Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of under-acetylated histones [27–29]. MeCP2, a methyl-CpGbinding protein, can form transcriptionally repressive chromatin on methylated promoter in vitro, and this process can be reversed by TSA [30–32]. Concordant with these reports, ChIP assay revealed that before treatment with trichostatin A, histones H3 and H4 of NQO1 were hypoacetylated in Hep3B and HuH6 cells in which NQO1 transcription was down-regulated due to NQO1 hypermethylation, whereas they were hyperacetylated in HLE,

Fig. 5. Representative results of MSP analysis of NQO1 and GSTP1 hypermethylation in primary human HCC tumors and corresponding nontumorous liver tissues. (A) Analysis of NQO1 hypermethylation. (B) Analysis of GSTP1 hypermethylation. Case 1 and Case 3 were hypermethylated at NQO1 gene and Case 1 and Case 2 were hypermethylated at GSTP1 gene. Corresponding non-tumorous liver tissues were neither methylated at NQO1 gene nor GSTP1 gene in the three cases. M, methylation; U, unmethylation.

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Table 5 Results of MSP analysis of NQO1 gene in the 44 HCC cases Methylation (nZ22)

Unmethylation (nZ22)

Pa

Etiology B C NBNC

4/22 (18.2%) 13/22 (59.1%) 5/22 (22.7%)

9/22 (40.9%) 10/22 (45.5%) 3/22 (13.6%)

0.099 0.273 0.349

Surrounding liver CH LC

8/22 (36.4%) 14/22 (63.6%)

11/22 (50.0%) 11/22 (50.0%)

0.272 0.272

Differentiation Poorly Moderately Well

4/22 (18.2%) 14/22 (63.6%) 4/22 (18.2%)

5/22 (22.7%) 12/22 (54.6%) 5/22 (22.7%)

0.500 0.380 0.500

a

P for Fishers’ exact test; CH, chronic hepatitis; LC, liver cirrhosis.

HuH7, HepG2 and PLC/PRF/5 cells that showed NQO1 DNA unmethylation in this study. In the primary HCC tissues, 22 of 44 cases showed NQO1 hypermethylation. To investigate the relationship between NQO1 hypermethylation and transcription, quantitative real-time PCR was performed in 37 cases. Of these, the majority of HCCs showed higher NQO1 mRNA expression than the corresponding non-tumorous tissues, as also reported by Cresteil et al. [15] and Ernster et al. [33]. Of the 27 cases with higher NQO1 mRNA expression in the tumors, 19 showed NQO1 DNA unmethylation and eight showed NQO1 DNA hypermethylation. We do not know the reason(s) for the higher NQO1 mRNA expression in HCC in the cases with NQO1 DNA unmethylation and in some of the cases with hypermethylation. Interestingly, all 10 cases with lower NQO1 expression in HCC showed NQO1 hypermethylation. There was a significant correlation between NQO1 hypermethylation and the lower NQO1 mRNA expression in HCC than in non-tumorous tissues (P!0.01, Fisher’s exact test, Table 7). In addition, the ratio of NQO1 transcription in HCC and non-tumorous tissue tended to be lower in cases with NQO1 hypermethylation than in those with unmethylation. Thus, in the subset Table 6 Results of MSP analysis of GSTP1 gene in the 44 HCC cases Methylation (nZ22)

Unmethylation (nZ22)

Pa

Etiology B C NBNC

8/22 (36.4%) 11/22 (50.0%) 3/22 (13.6%)

5/22 (22.7%) 12/22 (54.6%) 5/22 (22.7%)

0.255 0.500 0.349

Surrounding liver CH LC

8/22 (36.4%) 14/22 (63.6%)

11/22 (50%) 11/22 (50%)

0.272 0.272

Differentiation Poorly Moderately Well

5/22 (22.7%) 13/22 (59.1%) 4/22 (18.2%)

5/22 (22.7%) 12/22 (54.6%) 5/22 (22.7%)

0.640 0.500 0.500

a

P for Fisher’s exact test; CH, chronic hepatitis; LC, liver cirrhosis.

Table 7 Relation of methylation status of NQO1 gene and NQO1 mRNA expression level in the HCC cases

T/NTO1 T/NT!1

Methylation

Unmethylation

8 10

19 0

T/NTO1, T/NT!1: ratio of NQO1 mRNA expression in the tumor to that in corresponding non-tumorous liver tissues above 1 or below 1. All 10 cases exhibiting lower expression in the tumor showed NQO1 hypermethylation, and no case exhibiting NQO1 unmethylation showed lower expression in the tumor (P!0.01, by Fisher’s exact test).

of primary HCC tissues, NQO1 might be transcriptionally repressed due to promoter hypermethylation as well as in hepatoma cell lines. In conclusion, several enzymes involved in phase II of the detoxification system have been reported to be related to HCC. However, almost all of them are associated with HCC through a decrease in their reactivity due to their genetic polymorphism. The relation of epigenetic alteration, such as DNA hypermethylation, with HCC has only been reported in terms of the GSTP1 gene [7,9]. In this study, we showed that the disruption of the detoxification process due to NQO1 DNA hypermethylation might be involved in the pathogenesis of human HCC, like GSTP1.

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