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Green tea consumption: A potential chemopreventive measure for hepatocellular carcinoma? - Editorial
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Hepatology 14 November 2017
AFP alfa-fetoprotein
CI confidence interval
γ-OHPdG gamma-hydroxy-1,N2-propanodeoxyguanosine
HCC hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is a major global health problem.[1] HCC is the sixth-most common incident cancer and the fourth-leading cause of cancer death in 2015.[2] HCC is more common in men; 1 in 45 men and 1 in 113 women develop liver cancer before age 79 years in the world.[2] HCC is a highly lethal cancer given that a majority of patents with HCC are diagnosed in noncurative stages.[3] Although patients with early-stage HCC are eligible for potentially curative treatment, up to 70% of patients who receive curative resection or local ablation eventually develop recurrent tumors within 5 years.[1] There are no effective chemopreventive strategies for these patients and those at risk for HCC development.
In order to decrease the burden of HCC mortality, it is crucial to identify high-risk groups of patients and provide interventions to decrease the risk of tumor development and recurrence after curative treatment. Prognostic biomarkers should provide information to identify such high-risk patients. Serum alfa-fetoprotein (AFP) is a well-established diagnostic and prognostic biomarker for HCC.[4] Although highly elevated serum AFP predicts a poor clinical outcome, therapeutic implications of an elevated AFP in HCC patients remain unclear. Similarly, several other proteomic or genomic biomarkers aid in diagnosis and correlate with prognosis; however, they remain underutilized because of lack of wide availability. For example, with recent technical advances in genomics and transcriptomics, researchers used genome-wide expression profiling of liver cancer and adjacent benign tissues and showed that the gene expression pattern of tissue adjacent to the tumor predicts clinical outcome in HCC patients after surgical resection.[5] Although this gene expression signature provides prognostic information, it does not provide biological information amenable to interventions that may improve clinical outcomes. More recently, sorafenib was tested as an adjuvant treatment, but failed to prolong recurrent free survival following curative surgical resection or local ablation in a large, phase 3, double-blind, placebo-controlled trial.[6] Identification of mechanism-based prognostic biomarkers that can be intervened to improve the clinical outcomes is a critical unmet need in HCC research.
In the current issue of Hepatology, Fu et al. have published their research examining the utility of an exocyclic deoxyguanosine DNA adduct, gamma-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), in three mouse models of hepatocarcinogenesis and its utility as a prognostic biomarker with further validation in human HCC samples.[7] γ-OHPdG is one of the most common lipid peroxidation-derived DNA adducts that can modify DNA bases, including p53 genes, and it frequently causes G to T and G to A mutations. It is a well-established mutagen in smoking-induced lung cancer. However, the role of γ-OHPdG in HCC has not been investigated in the past. The investigators showed that γ-OHPdG levels increase with age in nucleotide excision repair-deficient mice (Xpa-/-) and the whole-liver levels correlated with HCC development. With whole-exome sequencing of tumors and adjacent benign tissues, they determined that GC>TA mutation is the dominant alteration in Xpa-/- mice, implying that γ-OHPdG may play a role in HCC-specific mutagenesis. Next, investigators showed that suppression of γ-OHPdG formation in the mouse liver led to inhibition of hepatocarcinogenesis. Three antioxidants (Theaphenon E, α-lipoic acid, and α-tocopherol) were tested. Theaphenon E, which is a potent antioxidative green tea extract, was shown to lower the level of γ-OHPdG the most. Furthermore, Theaphenon E prevented tumor formation and decreased tumor number and size in diethylnitrosamine-injected mice (Fig. 1). Last, the investigators examined the levels of γ-OHPdG in HCC patients who had undergone a liver resection and stratified them by γ-OHPdG expression level. Higher levels of γ-OHPdG were strongly associated with shorter overall survival and recurrence-free survival, confirming the role of γ-OHPdG as a prognostic biomarker in human HCC.
Figure 1.
Green tea for the chemoprevention of HCC. γ-OHPdG, a mutagenic lipid peroxidation-induced DNA adduct, correlates with HCC incidence in experimental murine models of HCC. Theaphenon E administration reduced the HCC tumor burden and γ-OHPdG levels, suggesting that the chemopreventive effect of green tea might be mediated by Theaphenon E by decreasing γ-OHPdG in liver DNA and consequent DNA mutations.


The current study findings provide biological evidence of the chemopreventive effect of Theaphenon E for HCC. A population-based case-control study from China on 204 HCC cases and 415 healthy controls showed that green tea consumption was inversely associated with the risk of HCC. Individuals who drank green tea longer than 30 years were at lowest risk compared with nondrinkers (adjusted odds ratio, 0.44; 95% confidence interval [CI], 0.19-0.96).[8] A recent meta-analysis of nine prospective cohort studies (465,274 subjects and 3,694 cases) again confirmed the association between green tea consumption and reduced risk for liver cancer (relative risk, 0.88; 95% CI, 0.19-0.96).[9] Whereas the vast majority of epidemiological evidence supporting the protective effect of green tea is from Asia, a recent European study confirmed a preventative effect of tea on HCC: Tea intake was associated with a 59% reduction in the risk of HCC development among 486,799 subjects after a median follow-up of 11 years.[10] However type of tea was not available in that European study.
An important limitation of the current study is the small number of human HCC tissues analyzed. It remains unclear whether γ-OHPdG levels are independently associated with clinical outcomes after adjusting for known prognostic factors, such as tumor size, number of lesions, AFP, vascular invasion, and metastasis. For the same reason, it has not been investigated whether γ-OHPdG is implicated in hepatocarcinogenesis in an etiology-specific manner. Nonetheless, the investigators reported a mechanistically relevant prognostic biomarker that can help select a subgroup of HCC patients who may benefit from Theaphenon E/green tea consumption.
Should we recommend green tea consumption in patients who are at increased risk for HCC development or recurrence? The current study provides a biological mechanism for the protective effect of green tea on HCC, which has been demonstrated in previous epidemiological studies. Therefore, we may encourage high-risk patients to drink green tea. In order to clearly measure the biological impact of Theaphenon E/green tea, the associations between green tea consumption, the level of γ-OHPdG in tumor and adjacent benign tissues DNA, and HCC development and recurrence should be externally validated, especially in Western countries where it has been under studied. Last, it is worth mentioning that green tea catechins exert chemopreventive properties by modulating signal transduction and metabolic pathways, hence inhibiting metabolic syndrome-related carcinogenesis in addition to antioxidant and anti-inflammatory activities.[11] This might provide a different level of preventive effect in nonalcoholic steatohepatitis, which is emerging as a major etiology of HCC. The etiology-specific mutagenic role of γ-OHPdG in hepatocarcinogenesis should be further investigated in the future, and this will help toward identifying patients who may get the most benefit from an antioxidant effect of green tea in the era of precision and individualized medicine.
Ju Dong Yang, M.D., M.Sc. - Harmeet Malhi, M.B.B.S.
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN
Original Article 2018
An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, mainly because of its poor prognosis. A valid mechanism-based prognostic biomarker is urgently needed. γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ-OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine-injected mice, both prone to HCC development. γ-OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ-OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ-OHPdG and survival or recurrence-free survival. γ-OHPdG levels in liver DNA showed an age-dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ-OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine-injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ-OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence-free survival (P = 0.007). Conclusion: These results support γ-OHPdG as a mechanism-based, biologically relevant biomarker for predicting the risk of HCC and its recurrence.
Liver cancer is a type of cancer that evolves over the course of decades and is extremely difficult to treat once diagnosed. Therefore, developing a mechanism-based, biologically relevant biomarker is extremely important for liver cancer intervention. The role of γ-OHPdG, an endogenous promutagenic DNA lesion preferentially formed at p53 mutation hot spots in human cancer,[23] has not been investigated. In vitro studies showed that it is repaired by NER pathways and that it causes GC>TA and GC>AT mutations.[21, 22] In this work, we provided evidence in vivo that γ-OHPdG is repaired by NER in animals as its levels are significantly higher in Xpa-/- mice compared to WT. We also found that γ-OHPdG levels increase age-dependently in Xpa-/- mice, indicating that the endogenous lesions accumulate over time. A similar trend was observed in LEC rats and DEN-injected mice.
DEN-induced reactive oxygen species accumulation, which are known to induce HCC development, may play a role in the increase of γ-OHPdG in DEN-injected mice.[41] Such an increase of γ-OHPdG was not observed in the lungs of Xpa-/- mice, suggesting a tissue-specific accumulation in the liver, possibly reflecting its relatively high lipid content in the liver and inhibition of NER by acrolein.[42]
The potential importance of γ-OHPdG in HCC is also supported by observations that there is an overwhelming representation of GC>TA mutation in Xpa-/- mouse liver tumors. Except for lung cancer, human HCC is the only solid cancer to show a relatively high rate of GC>TA mutation.[1] The mutation signature in Xpa-/- mice coincides with the signature of mutational processes of human HCC with a prevalence at 12.1%,[2] linking DNA lesions on the G:C pair with HCC. When comparing the mutations in human HCC, we found that a number of genes overlapped; for example, up to 121 mutations per nodule are detected with a high frequency of mutations on the G:C pair.[1] This was explained primarily by the methylated cytosine.[1] It is known that methylation at CpG sites enhances γ-OHPdG formation at these sites; moreover, CpG methylation greatly increases γ-OHPdG-induced GC>AT and GC>TA mutation frequency.[43] Although other DNA lesions associated with oxidative stress may also induce GC>TA, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine, data from these studies together implicate γ-OHPdG as a significant lesion that causes the high-frequency mutations at CpG sites found in human HCC.
A cohort study in Ohsaki, Japan, showed that consumption of green tea is associated with a reduced risk of human liver cancer incidence.[44] Other possible explanations for tumor inhibitory effects of Theaphenon E come from evidence showing that tea polyphenols, especially epigallocatechin gallate, can alter DNA methylation patterns of genes and the nuclear factor erythroid 2-related factor 2 pathway.[45] Tea polyphenols may possess multiple functional roles; however, we focused in this study on their ability to block the formation of an endogenous DNA adduct as a critical mechanism. Theaphenon E, a green tea extract, is a formulation containing a well-defined composition of polyphenols, mostly epigallocatechin gallate, the most abundant and potent antioxidative catechin in green tea (Supporting Table S5). α-Lipoic acid is an organosulfur antioxidant. Unlike α-lipoic acid and Theaphenon E, α-tocopherol is a lipid-soluble antioxidant. In fact, α-tocopherol has been associated with an increased risk of prostate cancer in healthy men.[27] In contrast, the α-tocopherol, β-carotene trial showed that α-tocopherol reduces prostate cancer incidence of heavy smokers. These conflicting clinical outcomes are plausibly due to the lack of valid biomarkers and may highlight the importance of identifying the high-risk population suitable for intervention trials.
Oxidative stress has emerged as a crucial factor in HCC development under various pathological conditions.[46, 47] Moreover, it has been shown to be involved in migration, invasion, and metastasis of HCC.[48] Serum quantification of derivatives of reactive oxygen metabolite levels, a simple method for measuring hydrogen peroxide, is found to predict the risk of HCC recurrence.[49] These findings highlight the potential of developing oxidative stress-related biomarkers as prognostic tools for HCC and its recurrence.
Our studies demonstrated that the LPO-derived γ-OHPdG is closely associated with liver carcinogenesis; it serves as a mechanistically relevant biomarker for HCC. Its levels in HCC, but not in normal adjacent liver, are highly reliable at predicting survival and recurrence-free survival in HCC patients (Supporting Fig. S9). More importantly, γ-OHPdG is independent of other known prognostic biomarkers such as microvascular invasion and tumor differentiation (Supporting Fig. S10).[50] The clinical applications of γ-OHPdG as a prognostic biomarker for HCC in guiding future intervention trials warrant study.

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