Necroptosis inhibits autophagy by regulating the formation of RIP3/p62/Keap1 complex in shikonin-induced ROS dependent cell death of human bladder cancer

Shikonin, a natural naphthoquinone compound, has a wide range of pharmacological effects, but its anti-tumor effect and underlying mechanisms in bladder cancer remain unclear.We aimed to investigate the role of shikonin in bladder cancer in vitro and in vivo in order to broaden the scope of shikonin's clinical application.We performed MTT and colony formation to detect the inhibiting effect of shikonin on bladder cancer cells. ROS staining and flow cytometry assays were performed to detect the accumulation of ROS. Western blotting, siRNA and immunoprecipitation were used to evaluate the effect of necroptosis in bladder cancer cells. Transmission electron microscopy and immunofluorescence were used to examine the effect of autophagy. Nucleoplasmic separation and other pharmacological experimental methods described were used to explore the Nrf2 signal pathway and the crosstalk with necroptosis and autophagy. We established a subcutaneously implanted tumor model and performed immunohistochemistry assays to study the effects and the underlying mechanisms of shikonin on bladder cancer cells in vivo.The results showed that shikonin has a selective inhibitory effect on bladder cancer cells and has no toxicity on normal bladder epithelial cells. Mechanically, shikonin induced necroptosis and impaired autophagic flux via ROS generation. The accumulation of autophagic biomarker p62 elevated p62/Keap1 complex and activated the Nrf2 signaling pathway to fight against ROS. Furthermore, crosstalk between necroptosis and autophagy was present, we found that RIP3 may be involved in autophagosomes and be degraded by autolysosomes. We found for the first time that shikonin-induced activation of RIP3 may disturb the autophagic flux, and inhibiting RIP3 and necroptosis could accelerate the conversion of autophagosome to autolysosome and further activate autophagy. Therefore, on the basis of RIP3/p62/Keap1 complex regulatory system, we further combined shikonin with late autophagy inhibitor(chloroquine) to treat bladder cancer and achieved a better inhibitory effect.In conclusion, shikonin could induce necroptosis and impaired autophagic flux through RIP3/p62/Keap1 complex regulatory system, necroptosis could inhibit the process of autophagy via RIP3. Combining shikonin with late autophagy inhibitor could further activate necroptosis via disturbing RIP3 degradation in bladder cancer in vitro and in vivo.