The Impact Of Iodine-125Seed Irradiation On The Gastric Cell Proliferation, Apoptosis And Gene Expression In Carcinoma Xenografts

ObjectiveIodine125(125I) seed irradiation has been used as an important supplementary treatment for unresectable advanced gastric cancer.however, its molecular biology mechanism is still unclear. Well-differentiated gastric cancer cell line NCI-N87was used to establish tumor-bearing nude mouse model. Iodine125seed was planted into the tumor for brachetherapy. The situation of the tumor’s growth was recorded to compute tumor inhibition rate and draw tumor growth curve. Nude mice were sacrificed28days. then the expression status of tumor cells proliferating cell nuclear antigen (PCNA) was evaluated by quantitative morphological analysis and the tumor cell apoptosis index was detected by the plain end fluorescence-dUTP transferase-mediated nick end labeling (TUNEL) analysis. For each group.12tumor specimens were randomly selected to analyze the expression of the genes related to cell proliferation, apoptosis and cell cycle arrest of gastric tumor by using NimbleGen gene expression chip. The differentially expressed genes were validated by real-time quantitative PCR. The global methylation expression profile of gastric tumors was detected by coupling the methylated DNA immunization co-precipitation (MeDIP) with Nimblegen CpG promoter microarray experiment.The methylation status of differential genes was further validated by MeDIP-PCR. Through these analyses, we aim to comprehensively elucidate the inhibitory effects and the biological effects induced by125I seed irradiation in human gastric cancer xenograft model, and provide the clinical theoretical basis for the125I brachetherapy to the well-differenciated gastric cancer.MethodsPart1The NCI-N87gastric cancer xenograft model in nude mice was constructed. The48mice bearing NCI-N87gastric cancer xenografts were randomly separated into2groups:sham seeds (O mCi) were implanted into the control group (n□=□24);125I seeds (0.9mCi) were implanted into the treatment group (n□=□24). After implantation, the long diameter and short diameter of the tumor were measured every three days to calculate the tumor volume and to map out the tumor growth curve. Nude mice were killed after28days, and dissected their tumor tissue to weigh and produce tumor tissue samples for routine pathological examination. The mitotic index and apoptotic index were evaluated by quantitative morphometric analysis of the expression of proliferating cell nuclear antigen (PCNA) and in. situ terminal transferase-mediated fluorescein deoxy-UTP nick end labelling (TUNEL), respectively.Part2Nude mice were killed after28days, and their tumor tissues were dissected to extract DNA and RNA for further molecular biology examination. Global gene expression changes induced by125I seed irradiation were analyzed by using Nimblegen Human gene expression array. The cell cycle-and apoptosis-related differential genes selected by microarray screen were further validated with quantitative real time PCR. DNA methylation profile in the tumors from the control group was investigated with methylated DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays. The changes in the methylation status of selected genes were further investigated by using MeDIP-PCR.ResultsPart1 The growth curves of the tumors indicated that the gastric cancer xenograft growth of tumor-bearing nude mice in the125I treatment group was slow down, and had the tendency to shrink; while the tumor of control group grew rapidly. The average inhibition rate was38.4%by measuring tumor weight. In situ apoptosis analysis through TUNEL method indicated that the apoptosis index in the125I treatment group and control group were23.2%and8.1%, respectively. The difference of apoptosis index between the two groups were significant (p<0.05). Through Immunohistochemical staining method, the expression level of PCNA in the experiment group was found to be significantly lower than that in the control group (p <0.05).Part2Gene expression profiles revealed that the expression levels of544genes, many of which were associated with apoptosis or cell cycle arrest, including BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A, were upregulated following125I seed irradiation. Through RT-PCR detection, the mRNA expression levels of BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A in the125I treatment group were increased as compared to that in the control group, and the difference between them was statistically significant. Furthermore,1954genes with promoter hypermethylation in the non-irradiated tumors were identified with MeDIP-chip analysis, and some of these genes with promoter hypermethylation were upregulated after receiving125I irradiation. Through MeDIP-PCR and quantitative RT-PCR validation, the expression levels of BNIP3and WNT9A were found to be regulated by irradiation-induced DNA demethylation.Conclusions1. Iodine-125seed implantation brachytherapy had caused the tumor-bearing nude mice xenograft’s growth was inhibited coming from NCI-N87well-differentiated gastric cancer cell line.2. By the immunohistochemical methods PCNA and TUNEL, we revealed that both inhibition of cell proliferation and induction of apoptosis contributed to the125I-induced tumor suppression in nude mouse model, P value>0.05,the difference was statistically significant.3.125I seed irradiation could significantly induce the up-regulation of apoptosis-and cell cycle-related genes in human gastric cancer xenografts. 4. Some of the up-regulation might be attributed to125I-irradiation induced demethylation in gene promoter regions. Collectively, these findings provided evidence for the efficacy of this modality for the treatment of gastric cancer.