Relationship Of HMLH1, HMSH2 And MSI In Colon Cancer Lovo Cell Lines Be Treated With NSAIDs

Objective:There is an ample reason to study colorectal cancer(CRC):it is the fourth most common incident cancer and the second most common cause of cancer death in the world.It is a key to explore the mechanism of occurrence,and findeffective measures , early detection ,early diagnosis, early treatment of cancer will help reduce the morbidity and mortality.Research had revealed that the stability of microsatellite (MS) had close relationship with tumorigenesis. MS is simple repetitive DNA sequences which were composed of 1 to 6 nucleotide repeat units and prone to produce replication error of DNA. Experiments confirmed occurrence of tumors there may be a microsatellite instability (MSI)and microsatellite stability (MSS)in different ways. 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. Colorectal Cancer experiments confirmed part of the The mutation of mismatch repair genes (MMR),especially the tow important human mut-l homologue 1 (hMLH1) and human mut-s homologue 2 (hMSH2) have Gloss relationship wite MSI,it will lend to the deficiency of mismatch repair genes result in hereditary instability and cause the MSI happened.Lots of research has revealed that MMR is an important enzyme in DNA repair system. Its function is to repair mispaired nucleotides during DNA synthesis. There are nine mismatch repair enzymes in MMR super family. Among them, hMSH2 protein and hMLH1 protein, which have been found absent or reduced in many tumors, are the most important enzymes and lead to tumorigenesis. This experiment by selecting hMSH2 missing Lovo cell line expression so as to observe the expression and influence factors of hMLH1 gene and protein with drug. Research has found that local DNA methylation can cause chromosomal structural change, loss of heterozygosity and allelic gene deletion. And studies had showed that the low expression of hMLH1 protein due to methylation of gene promoter CpG islands was also observed in MSI type CRC. Generally, hMLH1 protein expression due to promoter hypermethylation of CpG Islands is caused by the lack of dissemination of gastrointestinal cancer main cause of MSI.Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently applied to anti inflammation and to relieve pain in clinic. Epidemiological investigation has shown that NSAIDs has the function of prevention and treatment. Some research has revealed that its effect is commonly attributed to the inhibition of COX-2, and other research has revealed that the induction of apoptosis by activating P38MAPK signaling pathway is one of the possible mechanisms. But for COX-2 low expression of MSI tumor, some studies have reported recently that Aspirin and Sulindac can significantly reduce the MSI phenotype, prevent the transformation of colorectal polyps to cancer, and even reverse the early stage of colorectal cancer after adding to the colon cancer cell lines which is defective in mismatch repair enzymes.It may also prompt us that NSAIDs prevent tumors during reducing the cell MSI phenotype and affecting protein expression of MMR system.We choose selective the non-selective COX inhibitor indometacin act on person colon cancer cell lines lovo,whose MSI is positive (hMSH2 loss of expression). We are using Western blot, RT-PCR, MSP and other methods to observe the drug on the proliferation, hMLH1 protein expression, microsa- tellite instability and CpG island methylation of DNA mismatch repair gene, in order to study the different mechanisms of NSAIDs in prevention and treatment preliminarily.We hope provide a theoretical and experimental basis on the occurrence , prevention and appropriate treatment for colorectal cancer.Methods:1 Materials: lovo (MSI is positive ,hMSH2 loss of expression)are permanent cell lines derived from colon adenocarcinoma cell line. Indometacin (COX-2 non-selective inhibitor).2 Methods:2.1 We choose methyl thiazolyl tetrazolium (MTT) to determine proliferation of cell in each group lovo and to determine the half inhibitory concentration (IC50): established of the control group (control), solvent control group (solvent)and drug group. Set up the drug concentration: 12.5μmol/L, 25μmol/L,50μmol/L,100μmol/L and 200μmol/L. Action time: 72h, measure IOD values, calculate the rate of cell growth inhibition, and determine IC50 according to the formula.2.2 Experimental groups2.2.1 Concentration of group: three different concentrations of the drug were selected according to IC50, Indometacin: 25μmol/L, 50μmol/L, 100μmol/L and acted on lovo cell lines.2.2.2 Time Group: We selected three concentrations under IC50 of the drug to act on lovo cell lines for 24 h, 48 h and 72 h , and then collected cells.2.3 Detecting index2.3.1 Western blot was used to detect the expression of hMLH1 and hMSH2 protein after using drugs for24h,48h and 72h.2.3.2 Five microsatellite loci (BAT-25, BAT-26, D2S123, D5S346, D17S250) were analyzed by PCR methods.2.3.3 The methylation of hMLH1 and hMSH2 gene promoter CpG islands was detected by methylation specific PCR (MSP) methods.Results:1 The effects of Indometacin on the proliferation in lovoThe IC50 Indometacin acting on lovo human colon cancer cell lines for 72h was 104.23μmol/L.The drugs can inhibite proliferation,and the more concentration of drug we add to,the stronger effect on inhibition is shown.Solvents show no significant effect on cell growth.2 The protein expression of hMLH1 and hMSH2 in lovo detected by Western blotThe proteins molecular weights of hMLH1 and hMSH2 were 85kD, 110kD,and the corresponding positive bands will be found position after Western blot.The hybrid stripe was analysed by Gelimaging analysis system,the protein content was shown by IOD,andβ-actin protein was chose as an internal,compared the relative integral optical density in order to show the level of protein expression.The inhibitory effect of Indometacin on hMLH1 expression increased gradually with time extending,especially in the 72h (p<0.05). HMSH2 protein in lovo cells before and after the drugs were no expression.3 MSI of lovo cell line(1)BAT-25 test resultsBAT-25 DNA advanced glycation end product size is～90bp. IOD BAT-25 DNA product value (0.70±0.01), (0.63±0.02) and (0.52±0.01) for Indometacin function to 25μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.60±0.03), (0.52±0.01) and (0.37±0.01) for Indometacin function to 50μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.47±0.02), (0.38±0.02) and (0.23±0.02) for Indometacin function to 100μmol/L after 24, 48 and 72h. All of them are lower than the control group (0.98±0.01). The difference is statistically (p<0.05).(2)BAT-26 test resultsBAT-26 DNA advanced glycation end product size is 80~100bp. IOD BAT-25 DNA product value (1.20±0.03), (1.00±0.02) and (0.77±0.03) for Indometacin function to 25μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (1.08±0.03), (0.92±0.02) and (0.75±0.03) for Indometacin func- tion to 50μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.97±0.02), (0.75±0.02) and (0.29±0.03) for Indometacin function to 100μ- mol/L after 24, 48 and 72h. All of them are lower than the control group (1.33±0.02). The difference is statistically (p<0.05).(3) D2S123 test resultsD2S123 DNA advanced glycation end product size is 197～227bp. IOD BAT-25 DNA product value (0.79±0.03), (0.65±0.03) and (0.46±0.02) for Indometacin function to 25μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.69±0.02), (0.41±0.02) and (0.30±0.02) for Indometacin func- tion to 50μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.62±0.01), (0.35±0.02) and (0.19±0.01) for Indometacin function to 100μ- mol/L after 24, 48 and 72h. All of them are lower than the control group (1.12±0.01). The difference is statistically (p<0.05).(4) D5S346 test resultsD5S346 DNA advanced glycation end product size is 96～122bp. IOD BAT-25 DNA product value (0.95±0.01), (0.87±0.02) and (0.73±0.01) for Indometacin function to 25μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.85±0.02), (0.71±0.02) and (0.63±0.01) for Indometacin func- tion to 50μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.79±0.02), (0.67±0.01) and (0.46±0.01) for Indometacin function to 100μ- mol/L after 24, 48 and 72h. All of them are lower than the control group (1.05±0.01). The difference is statistically (p<0.05).(5) D17S250 test resultsD17S250 DNA advanced glycation end product size is～150bp. IOD BAT-25 DNA product value (0.93±0.02), (0.71±0.03) and (0.44±0.03) for Indometacin function to 25μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.76±0.02), (0.68±0.02) and (0.31±0.02) for Indometacin func- tion to 50μmol/L after 24, 48 and 72h. IOD BAT-25 DNA product value (0.72±0.01), (0.54±0.02) and (0.27±0.03) for Indometacin function to 100μ- mol/L after 24, 48 and 72h. All of them are lower than the control group (1.07±0.02). The difference is statistically (p<0.05).4 The methylation of hMLH1 and hMSH2 gene promoter CpG islandsIndometacin function before and after hMLH1 gene promoter CpG islands methylation DNA products, negative unmethylation DNA products. And hMSH2 gene promoter CpG islands unmethylation DNA products, negative methylation DNA products. IOD HMLH1 gene promoter CpG islands methylation DNA product value (0.51±0.02), (0.46±0.03) and (0.38±0.02) for Indometacin function to IC50 after 24, 48 and 72h. All of them are lower than the control group (0.96±0.02). The difference is statis- tically (p<0.05).Conclusions:1 NSAIDs could inhibit the growth of lovo human colon cancer cell, the inhibition role which was dependented with time and dose.2 NSAIDs could increase the expression of hMLH1 protein.3 After colon cancer cell line with NSAIDs, hMLH1 CpG island methylation levels over time and the increase in the concentration of significantly reduced.But MSI five sites BAT-25, BAT-26, D2S123, D5S346, D17S250 tables-reduce. That's promptting us, NSAIDs may tip MSI tumor by resuming hMLH1 gene promoter CpG islands methylation inhibition and reduce MSI, to play anti-tumor effect.4 HMSH2 protein expression and its gene promoter CpG islands methylation is independent in lovo, its expression reduces causes still need further study on the molecular level.