Epigenetic Regulation of Cytochrome P450 Enzymes and Clinical ...

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Epigenetic Regulation of Cytochrome P450 Enzymes and Clinical Implication Xiaojing Tang and Shuqing Chen* Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, China Abstract: CYPs are a large and diverse group of drug-metabolizing enzymes, which govern the metabolism of the majority of xenobiotic substances as well as endogenous components. The high inter-subject variability of CYP bioactivity has been largely attributed to gene polymorphism until the rapid development in epigenetics in the last decade that revealed another aspect of regulatory mechanism of drug-related genes. Epigenetics is the study of changes in gene expression or cellular phenotype that are not caused by changes in the underlying DNA sequence. The modification of histone proteins, together with DNA methylation and miRNAs, is the most extensively studied epigenetic mechanism in mammals. Recently, it has been demonstrated that alterations in epigenetic regulation occur during multiple pathological processes, especially carcinogenesis. As CYPs play an important role in carcinogen and anti-cancer drug biotransformation, epigenetic changes in CYP genes would lead to interindividual differences in drug responses. In this review, we provide an up-to-date summary of epigenetic studies on human CYPs, and discuss how such information could be integrated with clinical application.

Keywords: Adverse drug reactions, cytochrome P450, DNA methylation, epigenetics, histone modification, microRNA, personalized medicine. INTRODUCTION In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the human genome, announcing the primary success of Human Genome Project [1]. Since then, the international collaboration has been working to interpret this draft into a genome sequence with high accuracy and coverage. During this process, however, it became increasingly apparent that the secret of life lies largely outside DNA sequence. Epigenetics, which is a rapidly growing research field that investigates heritable changes in gene expression or cellular phenotype that are caused by mechanisms other than changes in the DNA sequence [2], is expected to answer a series of questions beyond genetics. DNA methylation, histone modification and miRNAs are the most studied epigenetic mechanisms in mammals. DNA methylation is a biochemical process whereby a methyl group is added to the C5 position of the cytosine ring within a CpG dinucleotide via the function of DNA methyltransferases, with DNMT3A and DNMT3B functioning as de novo methyltransferases and DNMT1 being responsible for the maintenance of methylation status. CpG dinucleotides exist in the genome as either sporadic sites or CpG islands with an unusual high CpG density. In normal tissues, a high percentage of single CpG sites is methylated, while the majority of CpG islands stay unmethylated. Aberrant methylation status of CpG islands could exert great influence on the activity of genes since approximately 60% of all promoters colocalize with CpG islands [3]. Alterations in the cancer epigenome are generally associated with loss of global DNA methylation and gain of methylation in specific gene promoters. DNA methylation is tightly linked with transcriptional repression in the following ways. First, the *Address correspondence to this author at the Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, China; Tel: +86 571 88208411; Fax: +86 571 88208410; E-mail: [email protected]

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methyl group forms a steric hindrance which directly blocks transcriptional activators from binding to the genes [4, 5]. Secondly, MBPs recognize methylated DNA and recruit co-repressors, leading to chromatin remodeling, which finally results in hindered transcriptional initiation or inhibited mRNA elongation (Fig. 1) [6, 7]. In nucleosomes, the amino terminal tails of the core histones (H2A, H2B, H3, and H4) extend from the globular region, making them accessible to multiple types of modifications, which include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, etc. Acetylation and methylation of lysines at histone tails are the two most common post-translational modifications with distinct distributions along both euchromatin and heterochromatin, contributing to the “open” or “closed” state of certain genes [8]. It is surprising that there are only ~20 000 protein-coding genes in the human genome, whereas ~90% of the genome is transcribed into ncRNAs, which is a large family including transfer RNA, ribosomal RNA, small nuclear RNA and a variety of regulatory RNAs. Among the regulatory ncRNAs, miRNAs are the most widely studied. miRNAs are a class of posttranscriptional regulators containing ~22 nucleotide RNA sequences that bind to complementary sequences in the 3’ UTR of target mRNAs, usually resulting in gene silencing by way of translation inhibition or mRNA degradation. It has been confirmed that miRNAs target ~60% of all genes, comprising an important part of gene regulation system [9]. In addition to its classical functions, miRNAs have also been reported to play a role in promoter targeting and translational activation [10, 11]. Until now, more than 700 miRNAs have been identified in humans and over 800 more are predicted to exist [12], suggesting that tremendous efforts are expected to explore the mysterious world of miRNAs. With the progress in researches into the field of epigenetics, it is becoming evident that these various mechanisms are not working independently. For example, DNMT enzymes, in addition to their function as DNA methyltransferase, interact with HDAC [13, 14] © 2015 Bentham Science Publishers

Epigenetics of Cytochrome P450 Enzymes

Current Drug Metabolism, 2015, Vol. 16, No. 2

tion between DNA methylation and histone modification has been comprehensively reviewed previously [16]. Furthermore, the miRNAs-regulated genes include a number of epigenetic enzymes, whereas the expression of miRNAs in turn is affected by DNA methylation and histone modification [17]. Therefore, these epigenetic mechanisms, along with genetic factors, interact with each other, constituting an intricate network of gene regulation. This network is likely to get further complicated with more and more novel mechanisms being discovered (Fig. 2). A recent discovery involves 5hmC, the oxidative product of 5mC catalyzed by teneleven translocation enzymes, which is thought to be an intermediate product of the demethylation procedure of 5mC [18, 19]. Contrary to the repressive role of 5mC, 5hmC seems to be linked with the active state of genes [20]. Alterations in 5hmC level are associated with organ development (e.g., liver) and age-related neurodegenerative diseases (e.g., Alzheimer's disease) [20, 21]. Besides, non-CpG methylation is found to be prevalent in embryonic stem cells [22, 23] and neurons, suggesting that it might be related with mammalian brain development [24]. These findings reshaped the classical concept of epigenetic markers, indicating a pivotal role that epigenetic mechanisms play in gene control, cell differentiation, individual development and pathological processes.

TF

X

TSS

X

A

B

C

Co-repressor

TSS

MBP

87

TSS

X

MBP

X

Fig. (1). Mechanisms of transcriptional repression mediated by DNA methylation. Black circles indicate methylated CpG sites. A. The methyl groups directly block transcriptional factors from binding to the targeted DNA sequence. B. MBPs recognize methylated CpG sites and recruit corepressors which results in gene silence. Chromatin remodeling is also involved in this process. C. Methylated CpG sites within gene body can also recruit MBPs and hinder the elongation of transcript.

and HMT [15]. In this case, DNMT enzymes themselves are coupled to transcriptional repression and chromatin remodeling. Histone-modifying enzymes can also recruit DNA methyltransferases to mediate DNA methylation at certain genomic loci. The interrela-

Historically, the study of epigenetics mainly focused on its relationship with multiple disease processing and tumorigenesis. Interestingly, reports on epigenetic regulation of CYPs began to emerge since 2000. The pioneer work performed by Magnus Ingelman-Sundberg and his colleagues has laid the foundation of pharmacoepigenetics, the study of epigenetic regulation of ADME genes, and its potential role in interindividual variations in drug response [25-29]. In the present review, we intend to give a detailed update of progress made in epigenetic regulation of human CYP genes and discuss how epigenetic mechanisms could serve clinical application in the future. EPIGENETIC REGULATION OF CYPS AND ITS ROLE IN DISEASE Cytochrome P450 is a family of ~60 different enzymes found in all human tissues, responsible for phase I reactions involved in biotransformation of a vast majority of drugs, dietary constituents

miRNA coding gene

iRNA miRNA

Histone modifying enzymes

Target gene

DNA modifying enzymes

Co-activator/repressor

Histone modification

Fig. (2). The cross-working epigenetic mechanisms of gene regulation.

DNA modification

88 Current Drug Metabolism, 2015, Vol. 16, No. 2

and endogenous chemicals. The bioactivity of CYPs is affected by transcriptional factors, genetic variability, and CYP inducers. Luke O Dannenberg [30] reported that HepG2 treated with 5-aza-dC and/or TSA exhibited altered expression of a large number of CYPs, suggesting a crucial role of epigenetic factors in the regulation of this superfamily. However, this study is incomprehensive as a large number of CYPs are not present in HepG2 cell line. There are 57 putatively functional genes in CYP450 family, until now, only around 20 of them are reported to be under epigenetic control (Table 1). Some other members, such as CYP2C19 and CYP2B6, are likely to be involved, but currently there is no data confirming it [31]. DNA methylation and histone modification participate in the regulation of CYPs via targeting either their promoter region or upstream transcriptional factors such as PXR and VDR, while miRNAs have also been confirmed to modulate the level of several CYP transcripts [32]. Each individual owns a rather unique epigenomic profiling, thus contributing to the interindividual differences in disease susceptibility and drug response. CYP1A1/1A2/1B1 The CYP1 family, which includes CYP1A1, CYP1A2, and CYP1B1, is most important as CYP1A1 and CYP1B1 are particularly effective at activating polycyclic aromatic hydrocarbons with possible carcinogenic activities, and CYP1A2 accounts for nearly 4% of total CYPs metabolizing drugs, including acetaminophen, caffeine, clozapine, phenacetin and tacrine [43]. All of the three genes are regulated by AhR/ARNT, a heterodimeric transcription factor [38]. It has been documented that epigenetic mechanism is also involved in the regulation of CYP1 family genes. TCDD is a compound that can induce CYP1A1, CYP1A2 and CYP1B1 effectively in MCF-7 but not in Hela cells. However, pretreatment with 5-aza-dC and TSA in Hela cells increased the levels of CYP1 family proteins induced by TCDD, which suggested that the lower inducibility of TCDD in Hela cells results partly from DNA methylation and histone acetylation [35]. In addition, AhR is negatively regulated by miR-203 in response to TCDD or BaP treatment, indicating that the CYP1 family is indirectly targeted by miRNAs [44]. Epigenetic change on CYP1A1 was demonstrated to play a remarkable role in prostate cancer. The high methylation status of TCDD-responsive CYP1A1 enhancer in cancerous LNCaP cells prevented the inducibility of TCDD of CYP1A1 when compared with the noncancerous PWR-1E and RWPE-1 cells which are less methylated. The possible mechanism is that the methyl groups inhibited the binding of AhR/ARNT complex to the XRE sites. Furthermore, Chip experiments revealed a loss of H3K4, a mark of active genes, on the CYP1A1 regulatory region in LNCaP cells in comparison with PWR-1E and RWPE-1 cells. CYP1A1 enhancer methylation is not detected in normal prostate tissues, whereas hypermethylation occurs in a number of prostate cancer samples [34]. Another research revealed that in utero tobacco exposure modifies placental CYP1A1 expression by way of decreasing the methylation level of CpG sites immediately proximal to the XRE sites. Therefore, maternal smoking transfers epigenetic markers to the offspring, leading to smoking-related growth restrictions and discrepancies in disease susceptibility in adulthood [45]. CYP1A2 acts on 5-10% of drugs in current clinical practice, including clozapine, imipramine, caffeine, fluvoxamine, paracetamol, phenacetin and others. Moreover, CYP1A2 activates several aromatic amines and thus is a key enzyme in chemical carcinogenesis [36]. The bioactivity of CYP1A2 is highly variable among indi-

Tang and Chen

viduals. Epigenetic regulation of CYP1A2 was firstly discovered in 2001 by Hammons. They reported that the methylation status of the CCGG site (bp –2759) located adjacent to an AP-1 site in the 5’flanking region of the gene was inversely correlated with CYP1A2 expression in liver samples [46]. Other inverse correlations with CYP1A2 expression is the methylation extent of a CpG island close to the translation start site in the CYP1A2 exon 2 [45], and a putative GC box located on the promoter region. This finding suggests that methylation might play an essential role in the tissue-specific expression of CYP1A2 [47]. A complex epigenetic regulation mechanism of CYP1B1 was proposed in vitro as CYP1B1 was inducible by dioxin in MCF-7 cells but not in HEPG2 cells [38].In MCF-7 cells, dioxin induced the recruitment of AhR and the transcriptional coactivators p300 and p300/cAMP response element-binding PCAF to the CYP1B1 enhancer and induced recruitment of pol II or the TBP and acetylations of H3 and H4 or methylation of H3 at the promoter. However, the recruitments of p300 and AhR were not sufficient for eliciting the above responses to dioxin in HEPG2 cells, because the CpG dinucleotides within the XREs at the enhancer were partially methylated, while those at the promoter were fully methylated, preventing the recruitment of TBP and pol II. Treatment of HepG2 cells with 5-aza-dC led to partial demethylation of the promoter thus restored CYP1B1 inducibility. Aberrant methylation status is shown to be related with the altered expression of CYP1B1 in colorectal cancers, nodular goiter and breast cancer [37, 48, 49]. Additionally, CYP1B1 is a target of miR-27b. The expression level of miR-27b is decreased in breast cancer tissues, accompanied by a high level of CYP1B1 expression [50]. This mechanism was also shown in colorectal cancer, in which CYP1B1 was overexpressed due to the decreased expression of miR-27b. Further in depth study revealed that IL-6, a pre-inflammatory cytokine which can promote colorectal carcinogenesis, participated in this epigenetic regulation by inducing methylation in the CpG island located close to miR27b through phosphorylation of DNMT1 [51]. This finding described a long pathway of epigenetic regulation and gave an example of how different epigenetic mechanisms interact with one another. CYP2E1 Ethanol, a substrate of CYP2E1, is an auto-inducer of this enzyme. Studies have shown that acetaldehyde, the intermediate metabolite of ethanol, may be the critical factor involved in the carcinogenic effect of ethanol by directly altering normal levels of DNA methylation and indirectly damaging DNA repair systems [52]. The methylation of dinucleotide CpG residues located in the 5’ end of the CYP2E1 gene has been demonstrated to play an important role in liver development and parkinsonism [53], as well as the tissue-specific expression of CYP2E1 [54]. However, hepatocellular carcinoma cell lines treated with 5-azacytidine did not show a corresponding increase of CYP2E1 mRNA with the reduced DNA methylation, assessed both at genomic and gene level [55]. TSAmediated up-regulation of CYP2E1 expression is associated with histone H3 acetylation and the recruitment of HNF-1 and HNF-3 on the proximal promoter, which is involved in reactive oxygen species generation and apoptosis in HepG2 cells [56]. It has been elucidated that human CYP2E1 expression is regulated by miR378, mainly via translational repression [57]. CYP2E1 is also known to activate a variety of environment chemicals and carcinogens, therefore participating in the process of tumorigenesis.


Table 1.


Cytochrome P450s significantly affected by 5-aza-dC and/or TSA in cell lines [30, 33-42]

Gene

Involvement

Cell Line

Treatment

Regulation

CYP1A1

Metabolism of polycyclic aromatic hydrocarbons

MCF-7, Hela, LNCaP

5-aza-dC

Up

MCF-7, Hela

TSA

Up

Metabolism of polycyclic aromatic hydrocarbons and other xenobiotics include caffeine, aflatoxin B1, and acetaminophen

MCF-7, Hela, B16A2

5-aza-dC

Up

MCF-7, Hela

TSA

Up

Metabolism of procarcinogens such as polycyclic aromatic hydrocarbons and 17beta-estradiol

MCF-7, Hela, HepG2

5-aza-dC

Up

MCF-7, Hela

TSA

Up

MCF-7

5-aza-dC/TSA

Up

HepG2

5-aza-dC

Up

5-aza-dC/TSA

Up

HepG2, BAFs, MCF-7

5-aza-dC

Up

HepG2

TSA

Up

5-aza-dC/TSA

Up

CYP1A2

CYP1B1

CYP17A1

CYP19A1

Steroidogenic pathway

Estrogen biosynthesis

CYP2A13

Metabolism of metabolize 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone

NCI-H441

5-aza-dC/TSA

Up

CYP20A1

Not clear

HepG2

5-aza-dC

Down

CYP24A1

Degradation of 1,25-dihydroxyvitamin D3

NCI-H460, SK-LU-1,HT-29, Coga1A, PC3, LNCaP

5-aza-dC

Up

NCI-H460, SK-LU-1, PC3, LNCaP

TSA

Up

NCI-H460, SK-LU-1, PNT-2, HT-29, PC3, LNCaP

5-aza-dC/TSA

Up

CYP27A1

Bile synthesis pathway

HepG2

5-aza-dC/TSA

Up

CYP27B1

Synthesis of 1alpha,25-dihydroxyvitamin D3

PNT-2, DU-145

5-aza-dC/TSA

Up

CYP2C9

Metabolism of many xenobiotics, including phenytoin, tolbutamide, and S-warfarin

HepG2

TSA

Up

CYP2R1

Transformation of vitamin D into the active ligand for the vitamin D receptor

HepG2

5-aza-dC

Up

5-aza-dC/TSA

Up

CYP2W1

Not clear

HepG2, B16A2

5-aza-dC

Down

CYP3A4

Metabolism of half drugs in use and also other steroids and carcinogens

HepG2, HT29, Caco2, SW48, HCT116, MCF-7

5-aza-dC

Up

HepG2

5-aza-dC/TSA

Up

HepG2

TSA

Up

5-aza-dC/TSA

Up

5-aza-dC

Up

5-aza-dC/TSA

Up

5-aza-dC

Up

5-aza-dC/TSA

Up

CYP3A43

CYP3A5

CYP3A7

Not clear

Metabolism of drugs as well as the steroid hormones testosterone and progesterone

HepG2

Hydroxylation of testosterone and dehydroepiandrosterone 3-sulphate

HepG2

89


Tang and Chen

Table (1) contd….

Gene

Involvement

Cell Line

Treatment

Regulation

CYP4F3

Inactivation and degradation of leukotriene B4

HepG2

5-aza-dC

Up

5-aza-dC/TSA

Up

CYP4V2

Fatty acid synthesis

HepG2

5-aza-dC

Down

CYP51A1

Cholesterol synthesis

HepG2

5-aza-dC

Down

TSA

Down

5-aza-dC/TSA

Down

5-aza-dC

Up

5-aza-dC/TSA

Up

CYP8B1

Bile acid synthesis

CYP3A4 CYP3A4, which is the most abundant CYP in human liver, has been well-studied for its function in the metabolism of a wide range of endogenous steroids and xenobiotics. It is responsible for the biotransformation of more than 50% of currently marketed drugs [58]. Only up to 25% of the CYP3A4 variability could be explained by the unique complexity of the upstream regulatory region [59] and the genetic polymorphisms in the promoter region together with genotypes of different transcription factors including PXR, HNF-4 and HNF-3 [60]. It has been gradually discovered that several miRNAs participate in the regulation of CYP3A4 at both the transcriptional and posttranscriptional level. For example, miR-148a posttranscriptionally regulates human PXR, which alters the inducible or constitutive levels of CYP3A4 in the human liver [61]. Furthermore, miR-27b and mmu-miR-298 may control CYP3A4 expression by targeting the 3’UTR of CYP3A4 directly and the 3’UTR of VDR indirectly [62]. Based on a mathematical model, Wei et al proposed that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly down-regulate the translation efficiency of CYP3A4 mRNA in the human liver [63]. Histone modification and DNA methylation are also involved in the epigenetic regulation of CYP3A4 [33, 64]. PRMT1 is a major histone methyltransferase associated with PXR. In response to the PXR agonist, PRMT1 is recruited to the regulatory region of CYP3A4 with a concomitant methylation of H4R3, which then facilitates H4 acetylation, indicating the transcriptional activation of CYP3A4 [65]. DNA methylation on the promoter of PXR is involved in the regulation of CYP3A4 in colon cancer cell lines, and the methylation level is inversely correlated with CYP3A4 expression in primary colorectal cancers. Furthermore, the PXR promoter methylation was not associated with the profile of microsatellite instability or other methylated genes, which suggested that the altered PXR methylation was accumulated during colorectal tumorigenesis [33]. CYP2R1/CYP24A1/CYP27B1 CYP2R1, CYP24A1 and CYP27B1 are key enzymes involved in the metabolism of vitamin D. CYP2R1 is a vitamin D hydroxylase that converts vitamin D into 25-hydroxyvitamin D3, which is the major circulatory form of the vitamin. CYP27B1 catalyzes the synthesis of 25-hydroxyvitamin D3 to the active hormone 1, 25dihydroxyvitamin D3, while CYP24A1 initiates the degradation of 1, 25-dihydroxyvitamin D3 by hydroxylation of the side chain to form calcitroic acid [66, 67]. Extensive studies have demonstrated

HepG2

that epigenetic regulation of these enzymes plays a vital role in the vitamin D metabolizing system and multiple diseases. There are CpG islands spanning the promoters or gene body of CYP2R1, CYP24A1 and CYP27B1. Therefore, alterations of DNA methylation and histone modifications in these regions may lead to chromatin remodeling and transcriptional repression/activation of these genes. Genome wide association studies found increased CYP2R1 and decreased CYP24A1 promoter methylation in leukocyte DNA from individuals with severe vitamin D deficiency compared with control group in African American adolescents [68]. This finding reinforced the key role of CYP2R1 and CYP24A1 in vitamin D deficiency. In another study, 446 white postmenopausal women were randomized to a calcium and vitamin D intervention for at least 12 months. From these subjects, 18 with the highest 12-month increase in serum 25-hydroxyvitamin D3 were selected as responders, while 18 with the lowest 12-month increase were selected as nonresponders. Compared to non-responders, responders showed significantly lower baseline DNA methylation levels in the promoter region of CYP2R1 (8% in the responders vs. 30% in the nonresponders, P=0.004) and CYP24A1 (13% in the responders vs. 32% in the non-responders, P=0.001). A subsequent study was conducted in another group of 145 white postmenopausal women. Results showed 12-month increases in serum 25-hydroxyvitamin D3 were negatively associated with baseline DNA methylation levels at eight CpG sites for CYP2R1 and two CpG sites for CYP24A1. Taken together, these findings indicated that baseline DNA methylation levels of CYP2R1 and CYP24A1 might be potential biomarkers predicting vitamin D response variation [69]. CYP24A1 is primarily expressed in the kidneys, while lower expression is found in many other vitamin D target tissues [70]. A research on the placenta-specific methylation of vitamin D related enzymes revealed that the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue, whereas no methylation was detected in any somatic human tissue tested. Gene expression analysis and in vitro reporter assay demonstrated that promoter methylation directly down-regulates basal promoter activity, and confirmed a reduced capacity of CYP24A1 response to 1, 25-D3-mediated transcription, most likely due to reduced binding affinity of the VDR to the methylated VDRE in the promoter. The study indicated that 1, 25-dihydroxyvitamin D3 plays an important role during pregnancy progression, and methylation of the core CYP24A1 promoter is


essential in maximizing active vitamin D bioavailability at the fetomaternal interface [71]. Other studies demonstrated that DNA methylation and histone modifications can partly contribute to the overexpression of CYP24A1 in cancers including lung adenocarcinoma [40], colorectal tumors [72, 73] and prostate tumors [74], which limited the antimitotic efficacy of 1, 25-dihydroxyvitamin D3. Expression of CYP27B1 is negatively regulated by 1, 25dihydroxyvitamin D3[ 75].It is reported that a bHLH-type activator VDIR can directly bind to the negative VDRE located in the CYP27B1 promoter, thus activating the transcription in the kidney cells. However, ligand-induced combination of VDR and VDIR results in transrepression of the CYP27B1 gene, due to the switching of VDIR from a co-activator complex to a co-repressor complex by recruiting the HDACs instead of HATs [76]. The histone deacetylation by recruited HDACs to VDR/VDIR at the VDRE is a critical step for chromatin remodeling at the CYP27B1 gene promoter in VDR-mediated transrepression. Furthermore, a DNA methyltransferase was identified as a part of this co-repressor complex, which indicated that DNA methylation is also involved in the ligand-induced transrepression of CYP27B1. However, this negative feedback loop is frequently lost in malignant cells [76]. It has also been proven in several cell lines that the aberrant CYP27B1 expression correlates with epigenetic events. In breast cancer cells MDA-MB-231, CYP27B1 hypermethylation resulted in gene silencing, which is reversible when treated with deoxyC [77]. In prostate cancer cell lines, combination of 5-aza-dC and TSA increased the activity of CYP27B1[78]. In the choriocarcinoma cell lines BeWo and JAR, the promoter of CYP27B1 was found to be densely methylated [71]. Moreover, the methylation level of CYP27B1 was markedly higher in primary lymphoma and leukemia cells than in normal peripheral blood lymphocytes [79]. CYP19A1 CYP19A1, also named aromatase, is an enzyme responsible for a key step in aromatization of androgens to estrogens. The entire CYP19 gene spans approximately 123 kb with a rather huge 5’flanking region of 93kb. This region contains multiple promoters, and is controlled in a tissue-specific manner. The most proximal gonad-specific promoters I.3/II contain a c-AMP response element, which is normally methylated in human skin fibroblasts. Methylation of this element can contribute to the low responsive ability of CRE to c-AMP stimulation through inhibiting CREB binding [80]. However, this regulation mechanism does not operate in all tissues. Primary breast adipose fibroblasts treated with 5-aza-dC showed a 40-fold increase of CYP19A1 expression driven by promoter I.4, surprisingly not due to the demethylation within the promoter itself [81]. In addition, up-regulation of CYP19A1 mRNA expression was demonstrated in endometriotic tissues, but methylation analysis failed to identify the correlated CpG islands in CYP19A1 gene [82]. A study into the correlation between promoter I.4 methylation and CYP19A1 expression revealed that percentage methylation at the I.4.1 and I.4.2 CpG sites was inversely correlated with CYP19A1 expression in omental adipocytes, while percentage DNA methylation at the I.4.3 and I.4.5 sites was positively correlated with CYP19A1 expression in subcutaneous adipocytes [83]. These findings suggest a tissue-specific epigenetic regulation of the CYP19A1 gene, whereas the underlying mechanisms and its physiological role still need further investigation. Histone modification is also involved in the epigenetic regulation of CYP19A1. MDA-MB-231 cells treated with the histone deacetylase inhibitor entinostat re-


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expressed CYP19A1 with an increased recruitment of acetyl histone H3 [84]. Human placenta syncytiotrophoblasts treated with cortisol, FSK and db cAMP increased the binding of Sp1 and pol II to CYP19A1 promoter flanking the 5’ end of exon I.1, while the acetylation of associated H3K9 increased and the methylation of H3K9 decreased [85]. Another study identified an miRNA, let-7f, which can target CYP19A1 mRNA. CYP19A1 inhibitors are widely used as adjuvant therapy in postmenopausal women with hormone receptor-positive breast cancer. It was demonstrated that letrozole, a CYP19A1 inhibitor, may exert tumor-suppressing effects upon breast cancer cells by repressing CYP19A1 gene activity via elevated expression of let-7f [86]. CYP2W1 CYP2W1 is a novel enzyme identified recently, which is only expressed in colon during fetal stages, and is re-expressed in several tumors, including bladder, breast, liver, pancreas, stomach and thyroid, with the highest expression in colon cancer [39]. Studies into the tumor-specific expression of CYP2W1 revealed that DNA methylation is involved. The first exon and first intron of CYP2W1 gene are found to contain extensive CpG sites. Methylation analysis used PCR of genomic DNA after Hpa II digestion. Results showed that in B16A2 cells, where CYP2W1 is not expressed, the CpG-rich segment is hypermethylated, while in HEPG2 cells where CYP2W1 is detected at a rather high level, it is hypomethylated. The CYP2W1 expression ability was restored after treatment with 5aza-dC in B16A2 cells[39]. A later study demonstrated that the methylation level is negatively related with CYP2W1 expression in different types of tumor [87]. CYP2A13 CYP2A13, which is selectively expressed in the respiratory tract, plays an indispensable role in chemical carcinogenesis, especially tobacco-specific nitrosamines [88]. The expression of CYP2A13 is elevated in lung cancer compared with normal lung tissues. In NCI-H441 human lung cancer cells, treatment with a combination of 5-aza-dC and TSA resulted in increased CYP2A13, indicating a cooperation of DNA methylation and histone deacetylation in the activation of CYP2A13 gene, however, exact molecular mechanism is unclear [41]. The methylation status is analyzed in head and neck cancer, however, no significant association was found [89]. Multiple researches were conducted during the recent years on the epigenetic regulation of other important CYPs. For example, CYP7B1 expression is higher in the tumor area compared with the adjacent non-tumor tissues in prostatic adenocarcinoma, which might be a result of aberrant local methylation of CYP7B1 promoter region [90]. Recent studies revealed that miRNAs also play a crucial role in the variability of drug metabolizing enzymes in the human liver. Association analysis with CYP gene profiles suggested several significant correlations at protein level, such as CYPs 1A1 with miR-142-3p, miR-200a, miR-200, CYP2A6 with miR-142-3p, CYP2C19 with miR-34a, miR-185 [32], and CYP2C8 with miR-103, miR-107 [91]. These miRNAs might alter the posttranscriptional expression of CYPs by directly targeting transcribed CYP mRNAs or indirectly affecting the upstream transcriptional factors such as HNF-4 with miR-24a, miR-34, GR with miR-18, miR-124a [92, 93]. Although the miRNA database of microrna.org displayed a total number of 56 CYPs that are potential targets of a large number of miRNAs, it remains a huge task to experimentally confirm the validity since a false positive rate of >30% is claimed


based on the algorithm [94]. Moreover, the functions of many CYPs, such as CYP2W1 and CYP2S1, are not fully understood yet, which makes it more challenging to elucidate the implication of their miRNA regulation. In addition to the relation with cancers, epigenetic alterations are also found in other complex diseases, such as Alzheimer’s disease and schizophrenia, with which CYPs are also tightly associated [95, 96]. FUTURE PERSPECTIVES As aberrant epigenetic mechanisms of CYPs are welldocumented in various cancers, extensive investigations have been undertaken to understand how these markers could be integrated into clinical application. Nowadays, three aspects are mainly emphasized. EPIGENETICS AND ALTERED DRUG RESPONSE Different epigenetic profiles result in variability of enzyme activity, thus leading to different pharmacokinetics and drug response among individuals [26]. As CYP enzymes are involved in the activation or detoxification of many xenobiotics, altered CYP activity may exert profound impact on chemotherapy regimens. For example, CYP2B6 and CYP2C19 are involved in the activation of cyclophosphamide, which is an alkylating agent that inhibits cell proliferation by interfering with DNA replication [97], therefore, reduced expression of these enzymes is a potential mechanism of drug resistance. Enhanced cyclophosphamide activation was observed together with increased CYP expression in primary human hepatocytes following the treatment of phenobarbital, rifampin, or dexamethasone, which suggested that cyclophosphamide pharmacokinetics might be altered when combined with these CYP inducers in clinical application [98]. CYP3A4 is responsible for the inactivation of docetaxel, a chemotherapeutic agent effective in various solid tumors. Breast cancer patients with low CYP3A4 mRNA levels exhibited a significantly (p < 0.01) higher response rate (71%) to docetaxel treatment than those with high CYP3A4 mRNA levels (11%), which showed that the expression level of CYP3A4 in breast tumors could be a possible predictor of individual response to docetaxel [99]. In cancer patients who received a combined treatment of docetaxel and ketoconazole, a CYP3A4 inhibitor, a decreased clearance of docetaxel was observed [100]. Similarly, some dietary constituents are also important factors that modify drug metabolism. Piperine is a dietary plant alkaloid/amide which has been shown to inhibit CYP3A4 activity. Study demonstrated that treatment with piperine inhibited hepatic CYP3A4 activity in vivo which correlated with an increased AUC, half-life and maximum plasma concentration of docetaxel in an animal model of castration-resistant prostate cancer [101]. CYP variability among individuals is affected by multiple factors including genetic polymorphisms, age, sex and disease states. Over the past 60 years, numerous studies have been carried out on genetic factors that contribute to individual variability. Some of the achievements have already been applied in clinical practice. For example, polymorphisms predominate in determining the activity of CYP2C19 enzyme, which is known to metabolize many important xenobiotics. CYP2C19*2, CYP2C19*3 and CYP2C19*17 can account for the poor metabolizing ability of CYP2C19 in most cases [90]. These polymorphisms are suggested to be tested in order to help adjusting the dosage of CYP2C19 substrates such as mephenytoin, diazepam and clopidogrel [102]. However, the exact relation between epigenetic profiles and enzyme activity is not easily eluci-

Tang and Chen

dated. Unlike stable genetic polymorphisms, epigenetic modification is a dynamic procedure which can be altered along with changes in diet, stress and environment exposures [103]. Furthermore, the modification at certain gene loci of a specific tissue is difficult to be detected by non-invasive methods. Reasons mentioned above can partly explain why the practicability of these fundamental researches into epigenetics is still limited. EPIGENETICS IN CANCER DIAGNOSIS AND PROGNOSIS As DNA methylation has been mostly reported to be associated with multiple disease processes, a breakthrough is likely to be made in this field. Actually, important achievements are heralding the dawn as more and more representative DNA methylation alterations are discovered in tumors. Endometriosis is a benign estrogendependent disease, which has been demonstrated to be related to a series of DNA methylation alterations in genes such as progesterone receptor-B, E-cadherin, HOXA10, ER, steroidogenic factor-1 and CYP19A1 [104]. Menstrual blood is a possible material for DNA methylation detection since it contains DNA shed from endometrial cells. It has been confirmed by a recent study showing that ER gene is hypomethylated in endometriosis using menstrual blood, which is in accordance with the previous report [105]. Another study revealed a strong correlation between demethylation of sporadic AHRR CpG dinucleotides and serum cotinine levels among a cohort of 22-year-old smoking African American men, using genomic DNA extracted from lymphocyte cell pellets [106]. Furthermore, emerging evidences show that circulating DNA in body fluid is indicating a new way for non-invasive and convenient detection for DNA methylation in early stage tumorigenesis, which might solve the problem caused by epigenetic pattern variations from tissue to tissue [107]. In relation to CYPs, a correlation between CYP19A1 promoter methylation detected in saliva and pubertal timing in urban girls is discovered [108]. Although aberrant methylation patterns are widely demonstrated in CYP genes in pathological processes, there is still a long way to go to facilitate clinical practice. Similar to DNA methylation profiling, accumulating evidences are indicating an altered profiling of miRNA in a variety of malignancies including colorectal cancer, breast cancer and hepatocellular carcinoma. For example, miR-214 is one of the most upregulated miRNAs in osteosarcoma. Further investigation into a 92 children cohort with primary osteosarcoma showed that miR-214 expression is positively correlated with large tumor size (P=0.01), positive metastasis (P=0.001) and poor response to pre-operative chemotherapy (P=0.006). Moreover, poor overall (P