| Background and Objective The gastric carcinoma ranks the fourth in the tumormorbidity and ranks the second in tumor mortality. Studies have shown that the5-yearsurvival rate of the patients with early stage gastric carcinoma is about95%, while itdecreased to only10%-20%in advanced gastric carcinoma patients. Chemotherapy isstill the main therapy method to advanced gastric cancer in combined modalitytreatment. As the wide application of the5-fluorouracil, taxane, irinotecan and targeteddrugs, the efficacy of chemotherapy in gastric cancer improved. However, because ofthe insensitivity to conventional chemotherapy, there is still a considerable proportion ofpatients with gastric carcinoma are not sensitive to conventional chemotherapy whichcause the poor clinical effect. Therefore, it is particularly important to explore the newclinical treatment of the gastric cancer. Aberrant methylation of tumor suppressor geneis not only involved in the occurrence and development of gastric carcinoma, but alsoclosely related to the sensitivity of gastric carcinoma cells to chemotherapeutic agents.In this study, the methylation of reprimo gene and the mRNA expression of three humangastric adenocarcinoma cell lines at different degrees of differentiation were detectedafter the stimulaation by5-aza-2-’deoxycytidine (5-Aza-dC), trichostatin A (TSA),paclitaxel and5-fluorouracil alone or in combination. Based on this, the authorpreliminarily discussed the influence of5-Aza-dC, TSA, paclitaxel and5-fluorouracilalone or in combination to the growth of gastric cancer cells and the methylation status.Materials and Methods The human gastric adenocarcinoma cell lines MGC-803(well differentiated), SGC-7901(moderately differentiated) and MKN-45(poorlydifferentiated) of were treated with three groups of drugs5-Aza-dC, TSA, paclitaxel and5-fluorouracil alone or in combination, a blank control group was also set at the sametime. The changes of the cell morphology after the treatment were observed by anoptical microscope, and the survival rate of the cells in each group was detected bytrypan blue staining method. The methylation degree of the promoter region of reprimogene in the gastric cancer cell lines treated by the drugs for72h was detected bymethylation specific PCR (MSP). The degrees of mRNA levels of reprimo gene wereassayed by RT-PCR.Results①The morphology of three different degrees of differentiation of humangastric adenocarcinoma cell lines MGC-803, SGC-7901, MKN-45are epithelioid,fusiform and some visible pseudopodia, observed under a optical microscope. Three celllines were cultured with the5-Aza-dC, TSA, paclitaxel+5-fluorouracil monotherapygroup, the5-Aza-dC+TSA,5-Aza-dC+paclitaxel+5-fluorouracil, TSA+paclitaxel+5-fluorouracil two drugs combined group,and the5-Aza-dC+TSA+paclitaxel+5-fluorouracil three-drug combination group,and after72h the cellular volume increasedwhile the nuclear and nuclear cytoplasm ratio decreased. Some cells were polygonalafter cultured for24h, after72h a suspension of a large number of adherent cellsappeared, the shape of most cells changed significantly. Single-agent treatment groupTSA acted more obviously compared with5-Aza-dC, while the paclitaxel plus5-fluorouracil effected the best. Combination of two drugs effected significantly than asingle agent. TSA combination with paclitaxel plus5-fluorouracil effected the best,while the combined treatment of the three groups had the strongest effect. Defined thenumber of cells of the control group as100%and compared with the drug-treated groupafter trypan blue staining. After cultured with the monotherapy group paclitaxel+5-fluorouracil, the number of survived cells was less than5-Aza-dC and TSA. Aftercultured with the two drugs combined group TSA combination with paclitaxel+5-fluorouracil, the number of survived cells was less compared with other two groups. After cultured with the three-drug combination group,the number of survived tumorcells was the least(P<0.05).②The MSP detection results showed that there were less methylation and moreunmethylation in reprimo gene promoter region in the drug-treated group than those incontrol group among MGC-803, SGC-7901and MKN-45cell lines. The DNAmethylation level of CpG island in reprimo gene promoter of monotherapy group hadreduced more compared with the control group, and the unmethylation level hadimproved. The CpG island methylation level of reprimo gene promoter in two-drugcombination group had reduced more and the unmethylation level had improved morecompared with that in monotherapy group. The CpG island methylation level in reprimogene promoter in three-drug combination group had improved more and theunmethylation level had reduced more compared with that in two-drug combinationgroup.③The RT-PCR detection results showed that there were more expression in reprimogene in the drug-treated groud than those in control group among MGC-803, SGC-7901,MKN-45cell lines. The eight groups of gray scale ratios of the reprimo gene inMGC-803cells line were control group (0.1364±0.0279),5-Aza-dC (0.3722±0.0259), TSA (0.3368±0.0225), paclitaxel+5-fluorouracil (0.2059±0.0266),5-Aza-dC+TSA (0.4370±0.0253),5-Aza-dC+paclitaxel+5-fluorouracil (0.4205±0.0424), TSA+paclitaxel+5-fluorouraci lgroup (0.3492±0.0152),5-Aza-dC+TSA+paclitaxel+5-fluorouracil (0.5569±0.0154). The eight groups of gray scale ratios ofthe Reprimo gene in SGC-7901cells line were control group (0.1713±0.0296),5-Aza-dC (0.4285±0.0165), TSA (0.3850±0.0103), Paclitaxel+5-fluorouracil(0.2240±0.0355),5-Aza-dC+TSA (0.4728±0.0222),5-AzadC+paclitaxel+5-fluorouracil (0.4270±0.0028), TSA+paclitaxel+5-fluorouracil group (0.3956±0.0186),5-Aza-dC+TSA+paclitaxel+5-fluorouracil (0.5737±0.0157). The eightgroups of gray scale ratios of the reprimo gene in SGC-7901cells line were controlgroup(0.2096±0.0339),5-Aza-dC (0.4564±0.0074), TSA (0.4253±0.0091), paclitaxel+5-fluorouracil (0.2325±0.0156),5-Aza-dC+TSA (0.4975±0.0136),5-Aza-dC+paclitaxel+5-fluorouracil (0.4718±0.0151), TSA+paclitaxel+5-fluorouracil group (0.4353±0.0111),5-Aza-dC+TSA+paclitaxel+5-fluorouracil(0.6097±0.0153). The mRNA expression levels of the reprimo gene in the threegroups of cells monotherapy group has improved compared with the control group(P<0.05). The mRNA expression levels of the reprimo gene in the two-drug combinationgroup has improved compared with the monotherapy group (P<0.05). The mRNAexpression levels of the reprimo gene in the three-drug combination group5-Aza-dC+TSA+paclitaxel+5-fluorouracil has improved compared with the two-drugcombination group (P<0.05).④In the model directly injected with tumor cell suspension,the tumor-take rate wasslower （P<0.05）. The methylation level of reprimo gene and the molecular expressionof Ki-67and CD31in the group of subcutaneous vaccination tumor were both higherthan those of the directly injected group（P<0.05）.Conclusion Without drug treatment, the human gastric adenocarcinoma cell linesMGC-803, SGC-7901, MKN-45are epithelioid, fusiform and have visible pseudopodia,the CpG island of reprimo gene promoter region is hypomethylated and the mRNAexpression levels were low. After treated with5-Aza-dC, TSA, paclitaxel+5-fluorouracil alone or combination, these cells show some morphological changes atvarying degrees, reduce of methylation level of reprimo gene and increase the level ofmRNA expression, in which5-Aza-dC+TSA+paclitaxel+5-fluorouracil is mostsignificantly. The above results provide new experimental basis for the clinicalcombinated therapy of gastric cancer. The growth condition of tumor in the method ofsubcutaneous vaccination tumor is better than the method of directly injected with cellsuspension. |
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