A Pilot Study Of Molecular Basis Of Gastrocarcinogenesis In Dalian, A Gastric Cancer At-risk Region

Backgrounds and Objectives:Gastric cancer (GC) is one of the commonest gastric-intestinal malignancies in China with higher morbidity and mortality in the world. Due to lack of early diagnosis, prevention and treatments, GC remains the second killer malignancy among clinical patients around the world. Its development is a multi-factor, multi-step process in which many genes and several signaling pathways are involved. Thus, it is very important to further study the molecular mechanism of GC occurrence, development and metastasis in order to establish effective measures of early diagnosis and treatment of GC.Dalian belongs to GC at-risk regions in China. Its special geographical and physiognomy characteristics (a coastal and mountainous area located at the far south of Northeast China), ecological environment, the particular diet habits of its native residents, and relatively close genetic backgrounds play key roles in the development of GC. So Epidemiologic studies about this area seem significant to further understand the molecular mechanism of GC development and take effective measures of early prevention, diagnosis and treatment of GC.Epidemiologic studies have demonstrated that people in this area were accustomed to eating toasted or salted seafood and meat. These foods are rich in gastrocarcinogens such as heterocyclic amines, polycyclic aromatic hydrocarbons, and the well-known DNA methylators such as nitrite and benzo(a)pyrene which may enhance the risk of genetic and epigenetic alterations.Many chemicals in the environment are indirect pro-carcinogens and can be catalyzed to activated forms before playing their carcinogenic roles in the target cells. Cytochrome P450 (CYP450) enzymes are a group of oxidases that mainly distribute in smooth endoplasmic reticulum of cells and can activate or inactivate many types of exogenous compounds. Some activated products can play their carcinogenic roles by impairing biomacromolecules and/or forming DNA adducts in the target cells. It has been recognized that most of the known environmental carcinogens are the substrate of CYP450 enzymes.CYP1A1 is a major member of CYP450 superfamily and can be induced by many kinds of endogenous or exogenous compounds such as benzo(a)pyrene. The product of CYP1A1 gene is a kind of aryl hydrocarbon hydroylase which involves in tumorigenesis by activating polycyclic aromatic hydrocarbons (PAHs). The studies so far performed on the role CYP1A1 expression in stepwise gastrocarcinogenesis is lesser known. Since the people in Dalian region are used to eating some food that contain an overdose of procarcinogens, the status of CYP1A1 expression in Dalian gastric cancers and its potential link with tumor formation were investigated. Meanwhile, the studies about the modulation mechanism of CYP1A1 expression and CYP1A1 polymorphism distribution in the population of Dalian area were performed.Our previous work demonstrated that abnormal methylation of the COX-2 promoter could result in infrequent expression of COX-2 in Dalian GCs, as was inconsistent with many viewpoints of that time. However, it strongly suggested us that the mechanism of Dalian GCs development might be different from that of other areas, which was very important. So we speculated that the development of Dalian GCs might depend on epigenetic modulation of DNA abnormal methylation which could be chief criminal of Dalian GCs. The epigenetic modulation could be closely related with special environmental factors and genetic backgrounds of Dalian residents.Epigenetic changes influence gene transcription without alterations in DNA sequence including DNA methylation and chemical modification of histone. DNA methylation is a major category of epigenetic change often occurring in the cytosine of CpG islands located in gene promoter regions, which is thought to be closely correlated with tumorigenesis. The inducibility and reversibility of DNA methylation provide us a new thought about tumor development and treatment. Since the Epidemiologic characteristics and the traditional diet habits (food containing many methylating agents) in this region , it is significantl to compare DNA methylation of multi-loci between normal population and Dalian GC patients (also called CpG island methylator phenotype, CIMP). This study not only can explore the CIMP profiling of Dalian GCs but also can estimate the GC risk of normal people by measuring their methylation susceptibility. Thus it can provide certain theory basis for prevention, early diagnosis and treatment of GCs in this region.Now our study aims at exploring the role of genetic and environmental factors in tumorigenesis and the relation between them through testing genetic and epigenetic changes of GC tissues, premalignant tissues, GC surrounding tissues and noncancerous mucosa, which can provide some new evidences and thoughts for early diagnosis, prevention, and treatment of clinical GCs.Materials and methods:All specimens in this study were selected from the Human Frozen Gastric Tissue Bank of the Cancer Institute, Liaoning Laboratory of Cancer Genomics, Dalian Medical University. By the methods of frozen embedded tissue array immunohisto- chemistry, RT-PCR, EROD, Western blotting, SSCP, RFLP-PCR and MS-PCR, the expression pattern of CYP1A1 in different gastric tissues, the modulation machinery of this gene expression (concurrent expression of AhR-Aryl hydrocarbon receptor and nuclear translocation) and the correlation between its MspⅠand Exon7 polymorphism and tumor susceptibility were explored. Meanwhile, a paralleled study about five CpG island methylator phenotype (CIMP)-associated genes (p16, hMLH1, MINT1, MINT2, and MINT31) and the methylation statuses of the 5′-promoter region of CDH1( it was closely related with malignant phenotype of GCs) were conducted by methylation- specific polymerase chain reaction (PCR). The data were statistically analyzed by Kruskal-Wallis and Mamm Whitney with SPSS 12.0 software.Result:1. The expression status of CYP1A1 gene in different gastric mucosa tissues.The results of IHC displayed that the rates of CYP1A1 expression were 11.1%(1/9)in GC surrounding tissues, 60%(12/20)in premalignant tissues and 86.0%(37/43)in GC tissues. The difference was significant between different groups (p<0.005). The results of RT-PCR and Western blotting were almost consistent with the above ones.2. The expression pattern and nuclear translocation of AhR in different gastric tissues.The positive staining of AhR was found in 94.9%(37/39)of GCs and 50% (3/6) of non-cancerous mucosa. Furthermore, the incidences of AhR nuclear translocation were 94.9% (37/39) in GCs and 16.67% (1/6) in non-cancerous mucosa. Statistical analysis showed that the incidences and levels of AhR expression increased significantly in abnormal gastric tissues (including GC and premalignant tissues) in comparison with that of non-cancerous ones (p<0.05) and AhR nuclear translocation was positively correlated with CYP1A1 up-regulation in GC tissues (rs=0.437,p<0.01).3. The analysis of CYP1A1 gene polymorphism.3.1 The analysis of MspI polymorphismThe frequencies of m1m1, m1m2 and m2m2 genotypes in GC group were 43.3%, 45% and 11.7% and those of control group were 45.6%, 49.1% and 5.3%, respectively. Statistical analyses revealed no significant difference of genotype frequencies between cancer and control groups. So MspI polymorphism of CYP1A1 gene was not significantly correlated with the susceptibility of GCs.3.2 The analysis of Exon 7 polymorphismThe rates of Ile/Ile, Ile/Val and Val/Val genotypes in GC group were 26.7%, 50% and 23.3% and those of control group were 52.6%, 28.1% and 19.3%, respectively. Statistical analyses revealed no significant difference of genotype frequencies between cancer and control groups.3.3 The relation between the polymorphism of CYP1A1 gene and its expression.The results of RT-PCR and IHC displayed that the expression rate of CYP1A1 in the population with genotypes m2m2 and Val/Val was100% while 54-72% in the population with genotypes m1m1 and Ile/Ile. Statistical analysis showed significant difference between the two group(p<0.05),which revealed that the polymorphism of CYP1A1 gene could greatly influence its expression level and enzyme activity.4. The distribution pattern of CIMP in evolutional Dalian GCs.The frequencies of CIMP-H were 51.1% (24/47) in GC group, 23.8% (5/21) in premalignant lesions, and 0% (0/16) in noncancerous mucosa. CIMP-L were 38.3% (18/47) in GCs, 71.4% (15/21) in premalignant, and 75% (12/16) in noncancerous tissues, respectively. CIMP-N were 10.6% (5/47) in GCs, 4.8% (1/21) in premalignant, and 25% (4/16) in noncancerous tissues. Significant statistical difference existed in different stages of GCs (p=0.001), which revealed that the methylation of gene multi-site was related with the development of GCs. With the evolution of GCs, the number and intensity of gene methylation increased. Among the 31 gastric biopsies obtained from GC-free patients, None exhibited CIMP-H, whereas 12/31 (38.7%) were found with CIMP-L. In comparison with the data obtained from the noncancerous mucosa of GC-bearing patients, the frequencies of hMLH1, MINT1, MINT2 and MINT31 methylation were significantly lower (p=0.000–0.008) except that of p16 methylation.5. The methylation status of CDH1 promoter.The methylation frequency of CDH1 promoter was 48.9%(23/47)in GC mucosa tissues, which was closely related with CIMP, development, age and lymph node metastasis of GCs.6. The correlation analysis between CIMP distribution and CYP1A1 expression in Dalian GCsThe expression frequencies of CYP1A1 were 100% (20/20) in CIMP-H group and 62.5% (10/16) in CIMP-L/N group. There was significant difference between the two groups(p<0.01).Conclusions:1. The expression of CYP1A1 gene is an important molecular event throughout the whole gastric tumorigenesis in Dalian area whose expression level varies with the different development stages of GCs. In this regard, expression of CYP1A1 may become a potential biomarker in exploring the influence of environmental chemical carcinogen on health and judging the degree of Dalian GCs;2. The high level of CYP1A1 expression is induced by exogenous chemicals through AhR pathway;3. No significant correlation is found between the polymorphism of CYP1A1gene and the susceptibility of Dalian GCs. The expression level and enzymatic activity of CYP1A1 vary in different genotype individuals. CYP1A1 level and enzymatic activity are relatively high in susceptible genotype individuals. However, because of strong environmental carcinogens in the region, regardless of susceptible and not susceptible genotype individuals, they may be susceptible to GC.4. The distribution of CIMP is greatly different between GC group and control group, which reveal the high risk of Dalian GCs consists in a kind of abnormal methylation pathway. The abnormal methylation is an early molecular event in the development of GCs and its intensity increase with the evolution of disease.5. The strong susceptibility of methylation and/or CIMP in GC high-risk Dalian population may be closely related with the first stage metabolic activity of certain environmental methylators. In summary, there are many environmental mutagenic agents in Dalian. They may bring about a series of genetic and epigenetic changes which are closely linked with tumor formation. Therefore, it is highly possible to reduce the exposure of the population in this area to environmental chemical factors by giving people some guidance (for example, change the diet habits of native residents or adopt demethylators), which is in urgent need and would be essential to interfere with GC formation and to decrease disease risk and mortality.