Investigation Of DNA Hypermethylation And Protien Expression In Pancreatic Carcinoma

Objective: To investigate aberrant methylation status of E-cad and TIMP-3 gene in pancreatic carcinoma. Relationship between methylation statues and clinicopathological characteristics are also discussed. Methods: The CpG island methylation of E-cadherin and TIMP-3 in 27 cases of surgically resected pancreatic carcinoma(PC),6 cases of normal pancreatic tissues adjacent to cancer,3 cases of pancreatitis, 1 case of pancreatic serouss cystadenoma were detected by methylation specific PCR (MSP). Immunohistochemistry staining was used to detect E-cad protein expression. Relationship between methylation and clinicopathological features were statistically analzyed.Results: l).No aberrant methylation of E-cad and TIMP-3 genes were detected in 11 cases of normal pancreatic tissue and benign pancreatic tumor. 2).Aberrant methylated E-cad was detected in 9/27 cases of pancreatic cancer. Among of them, 6 were completely methylated, 3 were hemimethylation. The methylation positive rates of TIMP-3 gene in pancreatic cancer was 7/27, 3 of 7 cases were completely methylated, 4 cases were hemimethylation. 3).E-cad protein expressed on ductal andacinus cells with typical membranous staining. 6 cases of normal specimens and 9 cases of cancer tissue were positive staining. 18 cases of pancreatic carcinoma had loss and decrease expression of E-cad(66.7%).6 of 10 cancer tissues with E-cad protein lossed completely were methylated. Of 9 cancer tissues with E-cad staining positive, only 1 had DNA methylation. Downregulation of E-cad expression was found to be related to promoter hypermethy- lation(P<0.05). 4).A total of 40.74% of PC had methylation of at least one of these genes. 18.52% of PC had methylation in CpG island of both E-cad and TIMP-3 gene. There was a positive relationship between the methylation of E-cad and TIMP-3. 5).No significant relationship was found between methylation of E-cad or TIMP-3 and clinicopathological characteristicals including age, sex, tumor size.But the methylation of E-cad was found to be associated with histological differentiation. Conclusion: l).Our results showed that DNA aberrant methylation of E-cad and TIMP-3 is an frequent event in PC. The methylation of E-cad was accordant with methylation of TIMP-3. DNA methylation was an important biomarker of carcinoma. 2). Frequent loss of E-cad expression in PC was a consequences of DNA hypermethylation and some alternative mechanism.Objectives To explor the posibility of induction of genetic re-expression silenced by DNA methylation, which is a common epigenetic modification in carcinogensis. 5-Azacytidine, an inhibitor of DNA methylation, was used to determine the effects of the expression of tumor-suppressor gene E-cadherin in tumor cells. We then investigate whether DNA methylation and the invasive phenotype of tumor cells were associated. Tumor cellular growth supression, adhesiveness to extracellular matrix and invasive potential in vitro were also observed.Methods Methylation specific PCR(MSP) was utilized to examine methylation status of E-cad gene on breast carcinoma cell line MDA-MB-435 and pancreas carcinoma cell lineBxPc-3 before and after the treatment with 5-Aza. Immunohistochemistry was used to test the re-expression of E-cad protein. Semi-quantitative RT-PCR method was used to detect the changes in mRNA of E-cad. MTT colorimetric assay was performed to test the inhibition on cellular growth. Transwel] chambers in vitro invasion assay was used to examine the effects of inhibiting their invasive abilities.Results 1). The E-cad methylation was positive and its unmethylation was negative on MDA-MB-435 cell before the treament with 5-Aza. Aftertreated with 5.0umol/L 5-Aza for 3 days, methylation was changed negatively and unmethylation positive bands(97bp) was detected. On BxPc-3 cell,the E-cad methylation was negative and unmethylation was positive,which remained unchanged before and after the treatment with 5-Aza. 2). The E-cad protein expression was not detected by immunohistochemical(IHC)on MDA-MB-435 cell before 5-Aza treament. While E-cad staining positive on the cell membrane after the treament, indicated the re- expression of the E-cad protein. 3). The E-cad mRNA was failed to be amplified in MDA-MB-435 cell before treament. Following the treament with 0.5 umol/L, 1.0 umol/L, 2.0 umol/L and 5.0umol/L 5-Aza for 3 days,respectively, E-cad mRNA expression was detected on the fourth day with a dose dependent manner.Correlationship between the mRNA expression level and the agent concentration was observed. But no significant changes of E-cad mRNA expression level on BxPc-3 cell was found before and after the treatment with varied concentrations of 5-Aza. 4). The MDA-MB-435 cell and BxPc-3 cell that had been treated with 5-Aza of varied concentrations grew at a lower rate than those did in the control group. The inhibitive effects on the cells climbed as the agent concentration grew. 5). After treatment with varied concentrations of 5-Aza ,the adhesiveness to extracellular matrix of MDA-MB-435 cell decreased in all groups (PO.05). But the change in the adhesiveness of BxPc-3 cell was statistically insignificant after the treatment. 6). It was demonstrated in the Transwell charmbers in vitro invasion assay that the ability of MDA-MB-435 cell to invade the reconstituted basement membrane was suppresed significantly by the treatment with variedconcentrations of 5-Aza(P<0.05), but such suppression for invasion of BxPc-3 cell were not effective.Conclusions The demethylation agent effectively reverses the aberrant E-cad methylation in breast carcinoma cell line MDA-MB-435 and re-express E-cad mRNA and protein. The cell growth is significantly inhibited, the adhesiveness to extracellular matrix and in vitro invasive ability to artificial basement membrane are suppressed; the inhibitive effection increased as the agent concentration grows. However, such suppression for BxPc-3 cell is not effective. We concluded that 5-Aza might be a valuable candidate agent for anti-cancer therapy, but the application of the demethylation agent is patient-selective.