Effects Of 5-Aza-2'-deoxycytidine On Cell Proliferation And Apoptosis In Colonic Cancer Cell Line Lovo And Expression Of Tumor Suppressor Gene RUNX3

Objective:RUNX3 is newly discovered as a tumor-suppressor and human RUNX3 gene is located at 1p36. Recent researches have revealed the tumor supperssor activity of RUNX3 is inactivated in various cancer cells. 5-Aza-2'-deoxycytidine(5-Aza-CdR), an inhibitor of DNA methyltransferase, reactivate gene expression when hypermethylation of CpG islands reduced gene expression.Methods:Human colon cancer cell line Lovo were cultured with RPMI 1640. They were randomly divided into four groups. The 3 Groups were treated with 5-Aza-CdR 0.4, 4, 40μmol/L, a specific demethylating agent, for 3 d. The control one were cultured with RPMI 1640, 5-Aza-CdR free. Medium of all 4 groups were changed correspondently every 24 h and discarded after 3 d. The cells were cultured with RPMI 1640, which containing 10% fatal cattle serum. After 5 d, activation in human colon cancer cell line Lovo were respectively observed by MTT assay before and after 5-Aza-CdR treatment. The change in expression of RUNX3 mRNA was observed by semi-quantitative RT-PCR. The apoptosis was analyzed by flow cytometry.Results:Human colon cancer cell line Lovo treated with 5-Aza-CdR 0.4, 4, 40μmol/L displayed a slowed growth rate in different degree in comparison with the control cells, and the growth rate decreased accordingly with the increase of 5-Aza-CdR concentration. RUNX3 mRNA was expressed in Lovo cells after 5-Aza-CdR treatment, but it was undetectable before the treatment. After 5-Aza-CdR 0.4, 4, 40μmol/L treatment, the level of RUNX3 mRNA expression was 0.46±0.06,0.71±0.06, and 0.84±0.07, respectively, and it was significantly correlated with the concentration of 5-Aza-CdR (F=168.4, P<0.01). The apoptotic rate Lovo cells treated with 5-Aza-CdR 0.4, 4, 40μmol/L was 10.95%±2.09%,17.61%±1.51%,26.60%±1.89%, respectively, which was markedly higher than that of the controls (2.92%±0.93%)(P<0.01). The apotosis was also notably correlated with 5-Aza-CdR concentration (F=145.7, P<0.01).Conclusion:The excessive methylation of RUNX3 probably causes colonic cancer cell line Lovo inactivation of transcription. The re-expression of RUNX3 could inhibit growth of cell and induce partly the apoptosis of cells.