| Tumor necrosis factor receptor-associated factor 6(TRAF6)possesses a unique set of biological functions and structural specificities therein.It also plays an important role in human immune systems as well as tumorigenesis and cell metastasis.TRAF6 not only regulates intracellular signal transductions of the TNFR superfamily,but also mediates IL-1/TLR superfamily signal transduction.More specifically,the RING domain of TRAF6 at the N-terminus is responsible for E3 ligase activity,which leads to activations of both AKT and TAK1 and promotes cell survivals,while the ensuing ZINC-finger domain of TRAF6 provides essential structural support for activities of the RING domain.On the other hand,the C-terminal domain of TRAF6 has been shown to be mainly involved in immune signal transductions and is critical in regulating innate immunity,adaptive immunity,stress response,and inflammatory response.Therefore,identifying small molecules that can interact specifically with TRAF6 at the N-terminal domain could lead to de novo anti-cancer targets without compromising immune functions of TRAF6.In this study,the RING domain of TRAF6 were selected as a target for developing new chemotherapeutics and exploring anti-tumor activities through both in vitro and in vivo experiments.By employing computer-assisted virtual screening,we identified naturally occurring Cinchona alkaloids that could competitively bind to the RING domain of TRAF6 with Ubc13.These natural products were found to inhibit the proliferation of tumor cells as confirmed by MTT assay,and flow cytometry further demonstrated that they could induce early apoptosis in tumor cells.Western blot and immunoprecipitation assay were used to detect TRAF6-mediated signaling pathway and validate that these compounds could inhibit the activation of AKT as well as TAK1 and subsequently suppress the expression level of pro-apoptosis protein Bcl-2 and promote the expression level of anti-apoptosis protein Bax.Furthermore,animal experiments revealed that tumor proliferation rate of compound-stimulated mice was significantly lower than that of the non-stimulated group in vivo.TUNEL staining showed that the TUNEL-positive cells in the compound-stimulated group were more than those in the unstimulated group.At the same time,detection and analysis of T lymphocyte subsets and cytokines showed that these Cinchona alkaloids could increase the secretion of TNF-α,IFN-γ,and IgG,but did not significantly affect the ratio of CD4+T/CD8+T,thereby suggesting that alkaloids deployed here do not influence the C-terminal domain of TRAF6 and could instead enhance anti-tumor immunity.Lastly,we selected cinchonine as a representative member of Cinchona family,designed and synthesized a fluorescently labeled cinchonine and a derivative of cinchonine in which the active hydroxyl group was capped.In addition,we also constructed cell lines with low expressions of TRAF6.Immunofluorescence experiments revealed that the fluorescently labeled cinchonine could bind to TRAF6 in cells,thereby verifying the binding of cinchonine itself to TRAF6.MTT assay showed that by capping the active hydroxyl group in cinchonine that is mainly responsible for binding to TRAF6,the effect on cell proliferation was indeed significantly lower than that of cinchonine itself.Finally,cells with low-expression level of TRAF6 were significantly less sensitive to these alkaloids than normal cells.These studies collectively indicate that the naturally occurring Cinchona alkaloids can block the binding of TRAF6 to Ubc13 to inhibit the growth of tumor cells and pose minimal impact on immune responses related to activities of the C-terminal domain of TRAF6.Elucidation of the TRAF6 RING-Zn domain as a possible anti-tumor target unveils a novel approach for developing new chemotherapeutic for treatment of cancers. |
