Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of Apoptosis in certain types of Cancer cells. However, the role of Nogo-B in human Cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal Cancer cells. In clinical colorectal Cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal Cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in Apoptosis with insistent overexpression of cleaved Caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that Apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal Cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal Cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of Caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies. In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal Cancer cells, and may thus become a novel therapeutic target for colorectal Cancer.