Purpose: We previously isolated a newly discovered gene, hRFI, standing for human Ring Finger homologous to Inhibitor of apoptosis protein (IAP), by a two-hybrid yeast screening technique using one of two variant forms of a human homologue of l(2)tid, hTID-1L, as a bait. Then we reported that hRFI was preferentially expressed in the digestive tract regions of several cancers such as esophagus, stomach, and colorectum, and that the over-expression of hRFI exhibited a tendency to inhibit TNF-α induced apoptosis. Furthermore, in the previous AACR meeting, we demonstrated that hRFI expression effects on the response to Fluorouracil derived chemotherapeutic agents in human cancer xenografts. In this study, we sought to validate the effect of hRFI expression on the apoptosis induced by 5-FU and analyzed for molecular mechanism. Experimental Design: At first, we established HCT116 cell lines stably overexpressing hRFI at two different degrees (HCT116/RFI-high and HCT116/RFI-low) and investigated whether hRFI expression affected apoptosis induced by 5-FU for these lines and the control. We also analyzed expressions of several genes belonging to Bcl-2 and IAP families by Western blot analysis and Real-time PCR. Results: hRFI overexpression resulted in significant resistance against 5-FU-induced apoptosis with reduction of cytochrome c release from mitochondria and inactivation of caspase-3 and -9. The IC50 values toward 5-FU were also significantly higher in hRFI transfectant. Western blot analysis and Real-time PCR analysis revealed specific up-regulation of Bcl-2 and Bcl-XL in hRFI overexpressing cells. Furthermore, siRNA towards Bcl-2 or Bcl-XL increased the rate of 5-FU induced apoptosis in these cell lines, indicating that both Bcl-2 and Bcl-XL are important determinants of apoptosis sensitivity to 5-FU. Conclusions: These results clearly demonstrate the chemoresistance of hRFI is caused by the up-regulation of Bcl-2 and Bcl-XL. This evidence suggests that hRFI might be a new predictive marker for chemotherapy, and a novel therapeutic target for gene therapy in colorectal cancer.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]