A Study Of Anti-tumor Effect Of TRAIL

TRAIL(TNF-related apoptosis-inducing ligand), identified by Wiley and his colleagues in 1995, is a new member of TNF family. TRAIL can bind to five different receptors, comprising TRAIL-R1/DR4, TRAIL-R2/DR5 , TRAIL-R3/DcRl , TRAIL-R4/DcR2 and OPG. TRAIL induces cell apoptosis by binding to DR4 and DR5 but not DcRl, DcR2 and OPG. TRAIL induces apoptosis in a large variety of cancer cells but not in most normal human cells. This feature makes TRAIL a potential antitumor agent. However, not all tumors respond to TRAIL, raising questions about resistance mechanisms. Therefore it is very important to investigate how to overcome the resistance to TRAIL, and to explore the new methods to increase the sensitivity to TRAIL.Based on TRAIL protein preparation, we studied the anti-tumor effects of TRAIL on colon cancer cell line SW480 and hepatocarcinoma cell line SMMC-7721. The anti-tumor effect of combination of TRAIL and 5-Fu on colon cancer cell line SW480, and combination of TRAIL and Aspirin on hepatocarcinoma cell line SMMC-7721 were also observed. The mechanism of cooperation of TRAIL and aspirin on hepatocarcinoma SMMC-7721 cells was found by detection of expression of Bcl-2 family members. The expression of Bcl-2 family members in SMMC-7721 cells which were transfected with eukaryotic expression vectors inserted antisense DNMT1 gene fragment was observed, and SMMC-7721 cells sensitivity to TRAIL was studied. The results and the methods were displayed as follows:(1) TRAIL protein was identified by Western blot, and its molecular weight, 20.1 KDa, was shown by SDS-PAGE.(2) The anti-tumor effect of TRAIL on colon cancer cell line SW480 and hepatocellular carcinoma cell line SMMC-7721 were measured by MTT assay and TUNEL method. The results demonstrate that TRAIL can obviously kill SW480 and SMMC-7721 cells, and show classic dose-effect and time-effect relationship on SW480. Cooperative effect of 5-Fu and TRAIL was also determined. The structure feature of apoptosis cell such as karyopyknosis, karyorrhexis and apoptotic body was found light-microscopically and electron- microscopically.(3) A cooperative effect of aspirin and TRAIL was found by MTT assay and flow cytometry. Aspirin could inhibit the expression of Bcl-2 but not bax in SMMC-7721. The cooperative effect of aspirin and TRAIL is related to the expression of Bcl-2.(4) SMMC-7721 cells transfected with antisense DNMT1 (DNA methyltransferase I ) gene fragment (SMMC-7721 aDNMTl) was more sensitive to TRAIL than SMMC-7721 cells transfected with sense DNMT1 gene fragment or vectors without any ectogenous gene fragment. The expression of Bax, Bad in SMMC-7721 aDNMTl cells increased, but no change of the expression of Bcl-2 was found.These results suggest: 1) TRAIL can kill SW480 and SMMC-7721 cells by inducing cells apoptosis; 2) 5-Fu and TRAIL can induce apoptosis cooperatively in SW480 cells; 3) Aspirin can synergestically enhance the effect of TRAIL on inducing apoptosis in SMMC-7721 cells, and this effect is related to the decrease of expression of Bcl-2 induced by aspirin; 4) The sensitivity of tumor cells to TRAIL can be improved by inhibition of DNMT1 with transfection of antisense DNMT1 gene fragment, which is related to the increase of expression of Bax and Bad.