Methylation Frequency of Each Gene in CG, IM, GA, and GC 
The field of epigenetics describes information transmission through cell divisions of heritable changes of gene transcription activity without DNA sequence changes. Epigenetic information is biologically important for tissue or organ development and cell differentiation. Alteration of epigenetic information is involved in the development of cancers and other diseases. DNA methylation, histone modifications, and transmitted chromatin structure are the underlying mechanisms for epigenetic transmission. Aberrant DNA methylation is found in two distinct forms, hypermethylation and hypomethylation. Global hypomethylation and regional hypermethylation are characterized as two features of human cancer cells [1,2].
The regional hypermethylation involves CpG islands located in the promoter and 5’-exon(s). DNA methylation, the incorporation of a methyl group to the C-5 position of the cytosine ring in the context of 5’-CpG-3’ dinucleotides, which leads to the formation of 5-methylcytosine (5-mC), is the most studied epigenetic change to date in gastric carcinogenesis. Hypermethylation of CpG islands recruits methyl DNA binding proteins, and subsequently histone deacetylases. Deacetylation of the histone tails makes the DNA structure of the promoter into a closed chromatin structure that is inaccessible to transcription factors leading to transcriptional silencing of tumor suppressor genes, mimicking their genetic mutations. Thus, aberrant hypermethylation of these CpG islands acts as an alternative way to genetic changes for the inactivation of tumor suppressor genes. Global hypomethylation at repetitive sequences causes genomic instability. Both types of DNA methylation changes were implicated in the development and progression of cancers [1-3].
It is interesting to note that site-specific gene hypermethylation is an early event in
2. Accumulation of DNA methylation changes in progressions of gastritis, intestinal metaplasia, and dysplasia to GC
As early as in the 19th century, a German pathologist von Waldeyer stated that ‘cancer, in particular gastric cancer, may be considered an adaptive response to an adverse environment, characterized by a progressive phenotypic alteration.’ Currently, it is well accepted that the phenotypic transformation preceding GC, that is, superficial gastritis, chronic atrophic gastritis (CAG), intestinal metaplasia, and dysplasia/adenoma, are the consequences of an accumulation of molecular alterations triggered by a chronic inflammatory process [5,6]. Therefore, the progressive phenotypic alteration could at times represent an altered epigenome, which is the result of host adaptive responses to environmental exposure. If hypermethylation of promoter CpG islands of some genes plays an important role in the malignant transformation of gastric epithelial cells, it’s reasonable to assume that the pattern of hypermethylation could be found in premalignant lesions in the stomach. Kang
|CG(N=74) (%)||IM(N=57) (%)||GA(N=79) (%)||GC(N=80) (%)||p value|
|APC||48 (64.9)||46 (80.7)||57 (72.2)||62 (77.5)||NSa|
|COX2||1 (2.2)||5 (8.8)||3 (3.8)||37 (46.3)||< 0.001|
|DAP-K||26 (35.1)||28 (49.1)||27 (34.2)||45 (56.3)||0.012|
|E-cadherin||63 (85.1)||41 (71.9)||46 (58.2)||53 (67.5)||0.003|
|GSTP1||0||0||0||13 (16.3)||< 0.001|
|hMLH1||0||4 (7)||7 (8.9)||16 (20)||< 0.001|
|MGMT||11 (14.9)||5 (8.8)||8 (10.1)||17 (21.3)||NS|
|p14||22 (29.7)||18 (31.6)||60 (75.9)||50 (62.5)||< 0.001|
|p16||2 (2.7)||4 (7)||9 (11.4)||35 (43.8)||< 0.001|
|THBS1||13 (17.6)||28 (49.1)||27 (34.2)||45 (56.3)||< 0.001|
|TIMP3||17 (23)||25 (43.9)||22 (27.8)||52 (65)||< 0.001|
|Average number of methylated genes||2.7b||3.6c,d||3.4b,c||5.2d|
During multistep gastric carcinogenesis, there is a steep rise in the number of methylated genes from chronic gastritis to intestinal metaplasia, which was a consistent finding in a series of studies. Intestinal metaplasia is a precancerous lesion with the typical characteristics of trans-differentiation of gastric progenitor cells into those committed to intestinal cell lineage, which normally present in intestinal mucosa . Regardless of the status of H. pylori infection, the number of methylated genes in chronic gastritis with intestinal metaplasia was significantly higher than that in chronic gastritis without intestinal metaplasia. This suggests that intestinal metaplasia is an epigenetically altered lesion. Hypermethylation of promoter CpG island in chronic gastritis without intestinal metaplasia occurs in association with
Although the loss of DNA methylation was the first epigenetic alteration identified in cancer, global hypomethylation has been overlooked in favor of gene promoter associated hypermethylation. Global DNA hypomethylation is associated with hypomethylation of normally methylated repetitive sequences, such as LINE1, Alu, and Satα, as well as centromeres and microsatellite DNA [15, 16]. In a recent study, using the immunohistochemical evaluation of 5-mC, Compare
Indeed, hypomethylation and hypermethylation of CpG islands in the gene promoter region may activate proto-oncogenes or inactivate tumour suppressor genes that confer selective growth advantage leading ultimately to cell hyperproliferation and cancerous growth [19,20].
3. Dynamic DNA methylation related to progression of gastritis
The stomach is one of the organs frequently showing aberrant methylation of CpG islands in epithelial cells because it is directly contacted with chemical and biological toxic agents daily. DNA methylation changes accumulate continuously during progression of gastritis, particularly during the establishment of the methylation pattern. Dynamic changes of methylation pattern occur consistently for gastric mucosae cells adapting to the environmental causal factors such as
3.1. Association between methylation of CpG islands and environmental/dietary factors, lifestyle, aging in gastric carcinogenesis
Despite strong evidences from observational epidemiology data and experimental animal studies suggesting that environmental/dietary factors, lifestyle, and aging are risk factors of gastric cancer, there has been limited understanding of the mechanisms through which such exposures have their effects on the molecular steps in tumorigenesis. It is now becoming apparent that altered epigenetic marks may play a fundamental role in determining not only susceptibility to cancer, but also contribute to promote neoplastic pathogenesis. Alternatively, the altered epigenome could at times represent adaptive responses to environmental exposure.
Environmental factors known to play crucial roles in the etiology of human cancer include chemical carcinogens (such as those found in cigarette smoke), microorganism infections, dietary contaminants (such as N-nitroso compounds), and lifestyles (such as alcohol consumption, excess intake of salt), deficiency of nutritional regimes. Stress may also contribute to the development of gastric cancer. Environmental and dietary factors in animals and humans inevitably affect epigenetic patterns, although a clear-cut causal relationship has yet to be established. The major obstacle in establishing such relationship is the fact that environmental and dietary factors induce changes are most likely subtle and cumulative, and culminate into a quantitative manifestation over a long period of time.
While animal and human studies have linked dietary factors to epigenetic regulation, it has been challenging to determine the exact mechanisms that nutrients may systemically affect epigenetic changes. The most studied and best-understood fact is the relationship between dietary methyl donors (including vitamins B6 and B12, methionine, and folate) and DNA methylation . As an essential amino acid, methionine plays the central role in the epigenetic regulation by serving as a methyl donor for methylation reactions. In the process of cytosine methylation, DNMT enzyme converts the donor S-adenosyl-L-methionine (SAM) to S-adenosylhomocysteine (SAH), and transfers a methyl group from the donor SAM to the C-5 cytosine carbon atom. Therefore, an optimal supply of SAM or removal of SAH is essential for a normal establishment of genome-wide DNA methylation patterns. When methyl groups are in short supply, there is a competition for the limited resources. Perturbations in this system may be caused by dietary imbalances affecting the supply of methyl donors such as folate.
Because humans tend to consume foods and nutrients that are highly interrelated, study of dietary patterns may have improved the power of detecting the effect of diet on DNA methylation. In humans, studies on diet and DNA methylation have yielded inconsistent findings.Animal studies have provided direct evidence that dietary factors induce changes in DNA methylation patterns. Bai
|Chronic atrophic gastritis||3/20(15.0)||1/4(25.0)||4/24(16.7)|
Promoter methylation is also present in non-neoplastic cells as an age-related tissue-specific phenomenon. Aging was first revealed to be an methylation inducing factor of
3.2. Induction of aberrant DNA methylation in gastric mucosa by H. pylori infection
Up to 80% of GC patients have a current or past
The fraction of methylated DNA molecules was quantified for eight regions of seven genes using DNA from the antral noncancerous gastric mucosae. Methylation levels increased in individuals with
Our group studied the
||Methylation frequency of
The dynamic and reversible nature of methylation profile could be explained by an old concept that stem cells may be the cellular origin of cancer. Although it is unknown which methylation changes in the genome are reversible or not, one of the possible mechanisms for the diverse fates of methylation of CpG islands is that the fates of methylation is dependent on the fates of host cells in which the methylation occurs. The aberrant methylations are likely temporary events if they occur in fully or partially differentiated epithelial cells, because most gastric epithelial cells will quickly drop off from the gastric epithelium through regular regeneration within several days. The methylation changes are likely permanent if they occur in tissue stem cells, because these cells will remain for life-time in the stomach and even benefit from these changes. It is reported that the temporary component is the one that disappears after eradicating the infection [34-36]. Further works have demonstrated the existence of gene specificity in the DNA hypermethylation induced by infection. Methylation of specific genes that occurs in a significant number of cells in the mucosa establishes an ‘epigenetic field for cancerisation’ or ‘epigenetic field defect’, characterizing a site with high risk for subsequent malignant transformation [33, 37].
Although the mechanistic understanding of the
3.3. Spread of
de novo methylation of p16 CpG island from normal mucosae, gastritis, to gastric carcinoma
It has been reported that
4. DNA methylation as a clinical biomarker for risk assessment, and anticancer drugs target
Epigenetic changes provide a potential explanation for how environmental factors can modify the risk for common diseases among individuals. The growing interest in cancer epigenetics stems from the fact that epigenetic changes are implicated in virtually every step of the development and progression of cancers. This is supported by the studies demonstrating that epigenetic changes including DNA hypermethylation are an early event in carcinogenesis. A distinguishing feature of epigenetic changes in comparison with genetic changes is that epigenetic changes are reversible. Therefore, aberrant DNA methylation, histone acetylation and methylation are attractive targets for cancer prevention and the epigenetic therapy. There are some evidences that methylation-based tests could predict which individuals are at high risk for different types of cancers, so that screening tests and prophylactic treatment can be applied more effectively. A pharmacological modification of the epigenetic status may be a potent strategy for the prevention of gastric cancers, based on the fact that methylation was a frequent event, particularly in
Aberrant DNA methylation is more frequently present in gastric cancers than mutations , so we can take advantage of the frequent presence of aberrant methylation in cancers by using it as a clue to identify tumor markers for cancer screening purposes and carcinogenic risk assessment. One advantage of using aberrant DNA methylation as a biomarker instead of mutations/expression is that it is possible to detect even a single aberrantly methylated DNA molecule embedded in 1000 unmethylated DNA molecules . The high sensitivity and specificity of measurements has particular application when it is well recognized that stem cells in precancerous or cancer tissues should contribute to cancer development, metastasis, recurrence, and formation of drug resistance. In contrast, using regular gene expression assays, such as immunostaining, Western blot, RT-PCR, and Northern blot, detection of alterations of gene expression in limited number of stem cells is very difficult. Additionally, DNA methylation can be remained in various samples stored at wide-range conditions (fresh/frozen or paraffin- embedded tissue blocks, free DNA in plasma, detached cells in gastric juice, sputum, urine, stool samples, and other body fluids). For example, we observed that methylation of
Currently, the greatest hopes are associated with clinical applications of several promising epigenetic modification inhibitors target for anticancer therapy, which inhibits DNA methylation and leads to the demethylation of the genome, thereby restoring expression of methylated genes. Such as DNMT inhibitor 5-aza-deoxycytosine and histone deacetylase inhibitors vorinostat or romidepsin, have recently approved by FDA for patients with myelodysplasia syndrome and cutaneous or peripheral T-cell lymphoma, respectively [3,53]. However, the potential pleiotropic effects of such an intervention as a result of induced genome hypomethylation (leading to, e.g., genome instability) need to be carefully considered. Therefore, the basic challenge in designing potential anticancer drugs functioning at the DNA methylation level is the specific recognition of molecular targets.
In conclusion, aberrant CpG islands methylation occurs in the early stages of gastric carcinogenesis and tends to increase as the multistep process advances. It is highly plausible that
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