The Effect And Its Mechanism Of 5'-Methylthioadenosine On Apoptosis In Human Colon Cancer Cells

Colorectal cancer is one of the most common malignancies of the gastrointestinal track.This disease is often detected at a late stage when clinical outcome is poor.Development of better prevention and therapeutic strategies is critical to improve the prognosis for patients with colorectal cancer.In the biosynthesis of polyamines,5-methylthioadenosine(MTA)is generated as an end product.Celluar Fas-associated death domain-like interleukin -1b-converting enzyme(FLICE)-like inhibitory protein(cFLIP)is an antiapoptosis gene。In this study,RKO and Hct116 human colorectal cancer cells and NCM460 cells were treated with MTA,the aim of which is to investigate whether MTA can inhibit proliferation,induce apoptosis in colon cancer cells and to explore the effect of cFLIP in these processes.Chapter 1 Effects of MTA on cell proliferation and apoptosis in human colon cancer cellsobjectivesTo explore the effect of MTA on cell proliferation and apoptosis of human colorectal cancer cells.Methods1)MTT assay were used to quantify the influence of different concentration of MTA in the proliferation of RKO,Hct116 and NCM460 cells.2)The method of Hoechst33258 staining was used to quantify the influence of different concentration of MTA inducing apoptosis in RKO, Hct116 and NCM460 cells.3)The method of Annexin V-FITC was used to compare the effects of MTA,5-FU and TRAIL inducing apoptosis in RKO cells.Results1)MTA could inhibit proliferation in RKO,Hct116 and NCM460 cells,and this role is in a concentration -dependent manner.The inhibiting effects on RKO,Hct116 cells were stronger than on NCM460 cells at the same concentration.2)MTA could induce apoptosis in RKO,Hct116 and NCM460 cells,and this role is in a concentration -dependent manner.The inducing effects on RKO,Hct116 cells were stronger than on NCM460 cells at the same concentration.3)MTA,5-FU and TRAIL could induce apoptosis in RKO cells.TRAIL combined with MTA or with 5-FU could enhance the effects of inducing apoptosis in RKO cells.Conclusions1)MTA could inhibit proliferation and induce apoptosis in RKO and Hct116 cells significantly,and these effects are stronger than its on NCM460 cells.2)MTA as well as 5-FU could enhance apoptosis induced by TRAIL.Chapter 2 The role of cFLIP in MTA-induced apoptosis in RKO and Hct116 cellsobjectivesTo explore the effects of cFLIP on the processes of MTA-induced apoptosis in RKO and Hct116 cells.Methods1)The method of realtime-PCR was used to detect the expression of cFLIP gene on mRNA level during the course of MTA-induced apoptosis in RKO and Hct116 cells.2)The method of western blot was used to detect the expression of cFLIP gene on protein level and the expression of its downstream protein,active Caspase-8.3)Th method of gene trasfection was used to study the effects of MTA-induced apoptosis in RKO cells after cFLIP being overexpressed.Results1)mRNA cFLIP was downregulated significantly after RKO or Hct116 cells being treated with MTA for 4 to 16hours. 2)Protein cFLIP was downregulated significantly after RKO cells being treated with MTA for 6 to 16hours.Active Caspase-8,cFLIP's downstream protein was also upregulated.3)Overexpression of cFLIP gene could inhibit MTA-induced apoptosis in RKO cells.ConclusionsMTA- induced apoptosis in RKO or Hct116 cells may be associated with down-regulation of the expressions of cFLIP gene,activation of caspase-8.