TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, can selectively kill cancer cells with little or no cytotoxicity toward normal human cells and is regarded as a potential relatively safe antitumor drug. However, some cancer cells are resistant to TRAIL-induced apoptosis. Thus, reagents that potentiate TRAIL-induced cytotoxicity are needed. Herein, we investigated whether shikonin, a natural compound from the root of Lithospermum erythrorhizon, can sensitize TRAIL-resistant cells to TRAIL-induced cytotoxicity. The viability of A549 cells, which were resistant to TRAIL, was significantly decreased after treatment with TRAIL followed by shikonin. The underlying mechanisms by which shikonin sensitizes cells to TRAIL-induced cytotoxicity were also examined. Combined treatment with shikonin and TRAIL activated the caspase and JNK pathways, inhibited the STAT3 and AKT pathways, downregulated the expression of Mcl-1, Bcl-2, Bcl-xL, c-FLIP and XIAP and upregulated the expression of Bid. In conclusion, the results indicated that shikonin sensitized resistant cancer cells to TRAIL-induced cytotoxicity via the modulation of the JNK, STAT3 and AKT pathways, the downregulation of antiapoptotic proteins and the upregulation of proapoptotic proteins.

Anti-cytokine therapeutic antibodies have been demonstrated to be effective in the treatment of several auto-immune disorders. However, The problems in antibody manufacture and the immunogenicity caused by multiple doses of antibodies inspire people to use auto-cytokine as immunogen to induce anti-cytokine antibodies. Nevertheless, the tolerance for inducing immune response against self-antigen has hindered the wide application of the strategy. To overcome the tolerance, here we proposed a strategy using the inter-species cytokine as immunogen for active immunization (TISCAI) to induce anti-cytokine antibody. As a proof of concept, an inter-species cytokine RANKL was successfully used as immunogen to induce anti-RANKL immune response. Furthermore, to prevent undesirable side-effects, the human RANKL was mutated based on the crystal structure of the complex of human RANKL and its rodent counterpart receptor RANK. We found, the antibodies produced blocked the osteoclast development in vitro and osteoporosis in OVX rat models. The results demonstrated this strategy adopted is very useful for general anti-cytokine immunotherapy for different diseases settings.

Secreted Nano-luciferase (secNluc) is a newly engineered secreted luciferase that possesses advantages of high structural stability, long half-life, and glow-type kinetics together with high light emission intensity, and thus would become one of the most valuable tools for bioluminescence assays. However, like other secreted luciferases, secNluc has to mix with the components in the conditioned medium surrounding test cells, or in the biological samples such as blood or urine after being secreted. These components may interfere with secNluc-catalyzed bioluminescence reactions and thus limit the application of the secNluc reporter system. In this study, we first examined the effects of three factors, pH, serum and residual reagents, on secNluc-catalyzed bioluminescence reactions, finding that these factors could interfere with bioluminescence reactions and result in background signal. To resolve these problems, we applied a simple affinity purification strategy in which secNluc was fused with a FLAG-tag, and anti-FLAG magnetic beads were used to catch and transfer the fusion protein to PBST, an optimal buffer for secNluc-catalyzed bioluminescence reactions that was identified in this study. The results indicated that this strategy could not only negate the interferences from serum or residual reagents and enhance the stability of light emission but also greatly increase signal intensity through enzyme enrichment. This strategy may contribute to biomedical studies that utilize secNluc and other secreted luciferases, especially those requiring superior sensitivity, low background noise and high reproducibility.