Relationship Between The COX-2, HMLH1, HMSH2 Protein Expression, Their Gene Regulation And Microsatellite Instability In Gastric Carcinoma

Objective: In China, gastric carcinoma is a severe disease that threatens people health. Research had revealed that the absence or reduction of mismatch repair enzyme (MMR) had close relationship with tumorigenesis. MMR is an important enzyme in DNA repair system. Its function was to correct mispaired nucleotides during DNA synthesis. There were nine mismatch repair enzymes in MMR family. Among them, human mut-l homologue 1 (hMLH1) protein and human mut-s homologue 2 (hMSH2) protein, which has been found absent or reduced in many tumors, are the most important enzymes. When the expression of MMR was reduced or diminished, the replication errors of microsatellite (MS) could not be repaired and these errors give rise to microsatellite instability (MSI). MS is simple repetitive DNA sequences which were composed of 1 to 6 nucleotide repeat units and prone to produce replication error of DNA. MSI means the increase or decrease of simple nucleotide repeat units in the result of replication error. MSI could increase DNA genome instability and random mutation frequency, giving rise to the changes of a series of tumor-related genes and subsequently leading to tumor occurrence. Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis. Over the past several years, a large body of evidence had showed that COX-2 protein, which did not express in normal tissues, had positive expression in digestive tract cancer and accelerated the development of cancer. However, some studies revealed that COX-2 protein expression in MSI-type gastric carcinoma was lower than that in MSS type gastric carcinoma, and the low expression of COX-2 protein was probably related to methylation of COX-2 gene promoter CpG islands. In addition, studies had showed that the low expression of hMLH1 protein due to methylation of gene promoter CpG islands was also observed in MSI type gastric cancer. Kim's study found that methylation of hMSH2 gene promoter was accompanied with the low protein expression in MSI type colorectal cancer. It suggested that gastric tumorigenesis via different pathway. Western blot, polymerase chain reaction (PCR) and methylation specific PCR (MSP) methods were employed in this study in order to investigate the expression of COX-2, hMLH1 and hMSH2 protein, the promoter methylation of these genes and MSI frequency in human gastric carcinoma and normal tissues, and to elucidate the relationship between them and occurrence of gastric carcinoma. However, little attention has been paid to the collaborating study about the relationship between COX-2, hMLH1, hMSH2, MSI and occurrence ofgastric carcinoma. Therefore, this study was designed to provide theoretic basis for further exploring mechanism of gastric molecular tumorigenesis and for clinical diagnosis and right therapy of gastric carcinoma. Materials and methods 1 Materials: 43 cases surgically resected gastric carcinoma and normal gastric mucosa (>10cm from carcinoma, next called nomal tissues). Cases from January, 2003 to December, 2004 were collected from department of Gastrointestinal Surgery in the second hospital of HeBei Medical University. All the cases were verified by pathologic dignosis and all the patients had not received radiotherapy or chemotherapy before surgery. 2 Methods: Total protein and genomic DNA was extracted from gastric carcinoma and normal tissues. The expression of COX-2, hMLH1 and hMSH2 protein was detected by Western blot methods. Five microsatellite loci (BAT-25, BAT-26, D2S123, D5S346, D17S250) were analyzed by PCR methods. The methylation of COX-2, hMLH1 and hMSH2 gene promoter CpG islands was detected by methylation specific PCR (MSP) methods. 3 Statistical analysis: The relationship between protein expression, MSI and the methylation of gene promoter were evaluated by chi-square or Fisher's exact probabilities test or Pearson relatedχ~2 test. Pearson related coefficient C shows intimate degree. Statistical significances were determined at level of P < 0.05. The statistical package was SAS6.12.Results 1 MSI of gastric carcinoma Of 43 gastric carcinoma cases, the total frenquency of MSI was 48.84% (21/43). The MSI frenquency in 5 loci (BAT-25, BAT-26, D2S123, D5S346,D17S250) was (9/43, 20.93%), (14/43, 32.56%), (13/43, 30.23%), (9/43, 20.93%), (9/43, 20.93%), respectively. And no significant discrepancy was found among the 5 loci. The carcinoma was classified into MSI-High (MSI-H, ≥2 loci), MSI-Low (MSI-L, only 1 loci) and microsatellite stable (MSS). The number of MSI-H, MSI-L and MSS was 14, 7 and 22, respectively. 2 The expression of COX-2, hMLH1 and hMSH2 protein in gastric carcinoma and normal tissues βactin protein: The molecular weight is 42kD. After Western hybridization, 42kD bands were found. βactin protein was detected when COX-2, hMLH1 and hMSH2 protein were examined and as the standard for semiquantitative analysis of COX-2, hMLH1 and hMSH2 protein expression. COX-2 protein: The molecular weight is 72kD. After Western hybridization, 72kD bands were found. The bands were analyzed by gel image analyzing system. The protein expression was semiquantitative data (named IOD), and the relative IOD was used for the comparison of protein expression. Comparing to normal tissues, there were 17 cases (62.79%) with high expression of COX-2 protein in gastric carcinoma, and 16 cases showed the low expression of COX-2 protein.hMLH1 protein: The molecular weight is 85kD. After Western hybridization, 85kD bands were found. The bands analysis was same as COX-2 protein. Comparing to normal tissues, there were, there were 19 cases (44.19%) with low expression of hMLH1 protein in gastric carcinoma. hMSH2 protein: The molecular weight is 110kD. After Western hybridization, 110kD bands were found. The bands analysis was same as COX-2 protein. Comparing to normal tissues, there were 17 cases (39.53%) with low expression of hMSH2 protein in gastric carcinoma. 3 The methylation of COX-2, hMLH1 and hMSH2 gene promoter CpG islands Cases with methylation of COX-2 and hMLH1 gene promoter CpG islands in gastric carcinoma were 8 and 13, respectively, and they were not detected in normal tissues. Methylation of hMSH2 gene promoter CpG islands was not detected in gastric carcinoma or normal tissues. 4 The relationship between the expression of COX-2, hMLH1 and hMSH2 protein, their gene promoter methylation and MSI The cases with low expression of COX-2 protein mainly occurred in MSI-H and MSI-L gastric cancer, the number was 10 and 5, respectively. However, the cases with high expression of COX-2 protein mainly occurred in MSS gastric carcinoma (21/22, 95.45%). The expression of COX-2 protein in MSI gastric cancer was significantly different from that in MSS(P<0.01) (there was no difference between MSI-H and MSI-L). The low expression rate of hMLH1 protein in MSI-H and MSI-L was 92.86% (13/14) and 71.43% (5/7), significantly higher than that in MSS (1/22, 4.55%) (P<0.01). The expression of COX-2 and hMLH1 protein was strongly associated with MSI (C value was 0.57 and 0.639). The expression of hMSH2 protein was not significantly different among MSI-H, MSI-L and MSS. The methylation rate of hMLH1 gene promoter CpG islands in MSI-H was 78.57% (11/14), significantly higher than that in MSS (0/22, 0%) (P<0.01). There was no significant difference between MSI-H (11/14, 78.57%) and MSI-L (2/7, 28.57%), and the same result was found between MSI-L (2/7, 28.57%) and MSS (0/22, 0%). 8 cases with methylation of COX-2 gene promoter CpG islands only occurred in MSI-H gastric cancer, and 7 cases both with methylation of COX-2 and hMLH1 gene promoter CpG islands was observed in MSI-H. The low expression rate of hMLH1 protein had significant difference between cases with (13/13, 100%) or without (6/30, 20.00%) methylation of hMLH1 gene promoter CpG islands (P<0.01). The low expression rate of COX-2 protein had significant difference between cases with (8/8, 100%) or without (8/35, 22.86%) methylation of COX-2 gene promoter CpG islands (P<0.01). Conclusion1 There were probably two pathways in tumorigenesis of gastric carcinoma, namely, the MSI and MSS. That means that gastric cancer could be classified into MSI and MSS types. The results of this study suggest that methylation of hMLH1 and COX-2 gene promoter CpG islands (gene epigenetic modification) may both occured in the development of MSI (especially MSI-H) gastric carcinoma, giving rise to the reduced expression of hMLH1 and COX-2 protein. These indicate that MSI resulting from the absent or reduced expression of hMLH1 protein was an important mechanism of cacinogenensis of MSI gastric carcinoma, and COX-2 protein plays an unimportant role in this type of carcinoma. In MSS gastric carcinoma, the expression of COX-2 protein was high and the low expression of hMLH1 protein was not found. Those suggest that COX-2 had important function in accelerating development of MSS gastric cancer and occurrence of MSS gastric carcinoma has no association with hMLH1 protein. However, the exact mechanism of MSS pathway cancer occurrence needs further study. 2 Treatment of gastric carcinoma should take carcinoma types(MSI and MSS types) into account. COX-2 inhibitor had effect on gastric carcinoma with high expression of COX-2 protein, but alternative treatment should be taken in MSI gastric carcinoma with low expression of COX-2 protein. Demethylating agent can recover the expression of...