Biological Study On Etiopathogenesis Of Xinjiang Kazakh's Esophageal Carcinoma And Intervene Experiment On Esophageal Carcinoma Cell Line

Objective: Esophageal cancer (EC) is a common disease in several areas of central Asia,including Xinjiang Uigur Autonomous Region, north of China. The incidence of Kazakh's EC has been considered as the highest among population in Xinjiang,and its age-adjusted mortality rate up to 91/100 000 reported by epidemiological study. The Kazakh population was estimated to be as 13 million around the world including 10 million Kazakh distributed in Kazakhstan and 2 million in Xinjiang, north of China. The population in the current study was a Kazakh isolated community, which located at the Northwest of Xinjiang. The genetic homogeneity and geography stability of the population, along with shared exposure to common environmental variables, may provide an excellent opportunity for the study on genetic influence for EC. These cancers are mostly squamous cell carcinoma (SCO.The series studies have suggested that a number of risk factors be involved in the carcinogenesis for Kazakh's SCC, including deficiencies in vitamins and minerals, consumption of pickled foods and environmental exposure to specificnitrosamines, genetic susceptibility, etc.The high incidence in special areas indicated the importance of environmental factors in esophageal etio-carcinogenesis. Only a small part of individuals in the high-risk area for EC developed into EC although all the residents in that area may share very similar environment-related risk factors and lifestyle, which suggested that the host susceptibility factors, such as the polymorphisms of phase I (CYP2E1) and phase II enzymes (GSTM1), may play an important role in increased risk for EC. Generally, phase I enzymes can activate the carcinogen directly and produce more active metabolites. Phase II enzymes can detoxify and process the activated metabolites for final breakdown or excretion. Therefore, the genotypes with high phase I enzyme activity and low phase II enzyme level can be considered to pose a high risk to cancer development.Thus, the current study was undertaken to assess the genetic polymorphisms of CYP2E1 and GSTM1 between Kazakh's ESCC and control group from Xinjiang in correlation to these genetic polymorphisms and susceptibility to esophageal carcinogenesis.Besides the factor of gene polymorphisms in metabolic enzymes, viral infections, in particular HPV infection, have been reported in EC from China. HPV-16 encodes E6 protein, which may bind to cellular tumor-suppresser protein p53 and directs degradation through the ubiquitin pathway. To our knowledge, p53 polymorphism has apparently not been documented in Kazakh's ESCC.EC could be considered as a multifactorial disease,and no single agent has been identified thus far as the cause of EC. Until now, the genes related to Kazakh's EC has not been elucidated. To understand the molecular basis for Kazakh's EC, the relevant subsets of differentially expressed genes associated with carcinogenesis must be identified, suppression subtraction hybridization ( SSH) was a powerful approachto identify and isolate complementary DNAs (cDNAs) of differentially expressed genes.Using suppression subtractive hybridization (SSH) to construct a subtractive library containing differentially expressed fragments of gene to be cloned and identified in the differentially expressed genes in relation to Kazakh's EC.In additional, the use of natural products as anticancer agents had a long history and has been incorporated into traditional and allopathic medicine through years. Most of the research performance today focused on the development of new products for the treatment of cancer, as well as viral and microbial infections. The aims for present study was to provide basis on the anticancer potential of black currant from Xinjiang in ESCC cell line.Methods: ?The genotypes of cytochromes P450 (CYP) 2E1 and glutathione S-transferase (GST) Ml were investigated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) following PCR in 104 Kazakh's patients with ESCC and 104 non-cancer controls.?Encoding regions of p53 codon 72 and HPV-16 E6 were amplified by PCR-RFLP and PCR methods using pairs of primary ESCC tissues and corresponding normal mucosa, which were collected from 104 Kazakh patients in Xinjiang, China.(g)Using suppression subtractive hybridization ( SSH ) to construct a subtractive library containing differentially expressed the fragments of genes,cloning and identifying these special genes, and homology of cDNA fragments from SSH was analysed by Blastn through Genbank.(4)The tumor cell lines were treated with 10'VmK 10'2 g/mh 10"3 g/ml of the water extracts black currant for 24h ,and the survival cells and protein synthesis were determined by MTT and Bradford assays. Moreover, the cell-DNA ploidy distribution and apoptotic rate were measured by flow cytometry (FCM) and morphological observation.Results: (T)The frequency of CYP2E1 cl/cl genotype wassignificantly higher in patients with ESCC (77.9%) than in control subjects (24.0%)(p<0.05,OR,l 1.13,95%CI,5.84-21.22). The difference of GSTMl null were significantly more frequent in the ESCC (34.6%) versus the control group(3.8%)(p<0.05,OR, 13.24,95%CI, 4.50-38.89). On the other hand, the combination of GSTMl presence and CYP2Elcl /cl genotypes increased the risk for ESCC (p<0.05, OR, 13.42, 95%CI, 6.29- 28.3). ?The arginine allele was detected in 70.1%( 39/55) of HPV -16-E6- positive cases but only in 40.8% (20/49) of HPV-16- E6-negative cases(p<0.05,OR,3.53,95% CI,1.57-7.98). In contrast, such a significant correlation between p53 polymorphism and HPV infection was not evident in corresponding normal mucosae. The allele frequency of Arg allele in ESCC cases (0.68) was higher than that in normal mucosa samples(0.54) (p<0.05,OR,l.80,95% CI, 1.21-2.69). ?The differentially expressed gene cDNA library for Kazakh's ESCC was successfully constructed.Through Blastn analysis, sequences of 6 positive clones showed that they were homologus with the genes related transmembrane helix receptor> breast cancer putative transcription factor> spliced protein gene ^ chromosome 8,clone RP11-27N21 , clone RP11-301G21 on chromosome 1 published in GenBank. ?There was a significant growth inhibition of the cell survival and protein synthesis of ESCC cell at a dose of lO^g/ml of the water extracts, In the normal rat liver cells,the cell survival was not affected. An apoptosis peak appeared before diploid peak in FCM and apoptotic rate was 49.6% with significant morphological change for their apoptotic cells.Conclusions: ?The current result suggested that CYP2Elcl/cl, GSTMl deletion genotypes were genetic susceptibility biomarkers for ESCC in Kazakh's population. Individuals with allele cl of Rsa I polymorphic locus for CYP2E1 may increase risk for ESCC. Moreover, CYP2E1 wild type (cl/cl) increased the susceptibility to ESCC risk in Kazakh's individual with GSTMl presence genotype.(Dp53 codon 72 Arghomozygous genotype has been considered one of the high-risk genetic factors and pathogenetic events for HPV-associated ESCC of Kazakh. Individuals with Arg allele compared to those with Pro allele had an increased risk for ESCC.?The differentially expressed gene cDNA library from Kazakh's ESCC was successfully constructed and it would lay the foundation for further screening full-length genes in Kazakh's ESCC.?Black currant may inhibit growth of human EcalO9 line through inducing apoptosis.