| Objective Gastric cancer (GC) is one of the most common causes of cancer related todeath worldwide. The new cases each year of GC have been accounted for nearly onehalf of global GC population in China.We have known that epigenetic changes is one ofthe most important mechanisms of gastric cancer.DNA methylation and histonemodification are the two predominant forms of epigenetic alteration. It is well acceptedthat epigenetic alteration of cancer suppressor gene is one of the most important factorsof carcinogenesis. CHFR (Checkpoint with FHA and ring finger) is a cell cycle mitoticstress checkpoint gene and one of the most important cancer suppressor genes related togastric cancer. This lab’s earlier studies have shown that in promoter area of cancersuppressor gene is closely related to the development of cancer. In this study, we aim toinvestigate the effect of5-Aza-2’-deoxycytidine (5-Aza-dC, methyltransferase inhibitor)combined with trichostatin A (TSA, histone deaceylase inhibitor) on cell proliferationand CHFR gene expression of GAC cell lines SGC-7901and MGC-803.Methods SGC-7901and MGC-803cells were cultured in vitro, and the cells inlogarithmic phase were divided into control group,5-Aza-dC (10μmol/l) group, TSA(1μmol/l) group and5-Aza-dC combined with TSA (10μmol/l+1μmol/l) group. Toobserve the time-dependent response of5-Aza-dC or TSA alone and their combinationon SGC-7901and MGC-803cells, they were treated for24,48and72hours,respectively. The cell proliferation inhibition was assayed with absorbance (A) value in450nm wavelength by cck-8method and the inhibition rate was calculated according tothe following formula: inhibition ratio=[1-(Experimental group absorbance- Absorbance blank hole)/(Controlled hole absorbance-Absorbance blank hole)]×100%. In order to assay cell apoptosis by flow cytometry, SGC-7901or MGC-803cellswere treated by5-Aza-dC or TSA alone and combination for48hours. Similarly, themRNA expression of CHFR gene treated for48hours was assayed by RT-PCR method,CHFR protein expression was assayed by western blotting.Results The inhibition rates of SGC-7901cell treated by5-Aza-dC alone were19.3%,31.8%and52.5%alone, those were16.3%,33.2%and62.0%by TSA alone, and thosein two drug combination groups were33.1%,49.7%and70.0%for24h,48h and72h,respectively. Similarly, the inhibition rates of MGC-803cell treated by5-Aza-dC alonewere22.0%,37.8%and56.2%. Those were23.4%,40.8%and66.3%by TSA alone,and those in two drug combination group were37.9%,55.6%and77.2%for24h,48hand72h, respectively. The apoptosis rate of SGC-7901cell treated by5-Aza-dC alonewas11.47%that was13.57%by TSA alone, and two drug combination group18.12%for48h. Similarly, the apoptosis rate of MGC-803cell treated by5-Aza-dC alone was17.43%, that was21.43%by TSA alone, and two drug combination groups was28.65%for48h. The relative mRNA expression of CHFR gene was0.167±0.034treated by5-Aza-dC alone, that was0.132±0.009by TSA group alone and Combination groupwas0.265±0.005in SGC-7901cell, while the control mRNA level was as0.061±0.0001. The relative protein expression of CHFR gene was0.431±0.020treated by5-Aza-dC alone that was0.290±0.061by TSA alone group and Combination groupwas0.590±0.048in SGC-7901cells while the control protein level was as0.074±0.006.Conclusion5-Aza-dC or TSA alone can inhibit the proliferation and induce apoptosisof gastric cancer cell lines SGC-7901and MGC-803, the cells MGC-803is moresensitive than cells SGC-7901to those two drug treatment, and the time-dependent, andthe two combination obtain a more significant effect on inhibiting cancer cell proliferation. By the mechanism analysis, we found that5-Aza-dC or TSA decreased thepromoter methylation, and improved the expression of mRNA and protein of CHFRgene, and the drug combination showed more. Our results have demonstrated that5-Aza-dC and TSA have a potential candidate of clinical drugs. |
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