Abstract 4659: Switch between Apoptosis and Autophagy induced by 4-HPR-driven ROS: The role of DJ-1 and it's different oxidative modifications
Authors: Ji Cao, Nan Xie, Jun Zhang, Hong Zhu, Qiaojun He, Bo Yang
Journal: Cancer Research (2012)

Abstract Aims: Due to the still-unclear molecule mechanism of ROS in tumor inhibition including apoptosis and autophagy, we chose the classical ROS-mediated tumor suppressor 4-HPR as the molecule probe, to discover the role of DJ-1 and its different oxidative modifications in the switch between apoptosis and autophagy. Methods: 1) Hela cells were treated with chosen dose 4-HPR; Flow cytometry analysis was performed to detect cell apoptosis and autophagy; 2) Flow cytometry analysis and GSSG Assay Kit was performed to detect ROS and GSH level; 3) Misfold-proteins level were tested by Highly selective fluorescent turn-on probe 1 and Ellman's reagent; 4) The expressions of ERS downstream proteins were shown by Western blot; 5) si RNA and inhibitors were used to knock out JNK and p38 and confirm the effect of the two different proteins on the switch; 6) siRNA and DJ-1 stabilizer were used to test the function of ASK1 and DJ-1 in inducing apoptosis; 7) Immune precipitation and Immunofluorescence were used to test the interaction of ASK1, DJ-1, Ire1 and TRAF2 after 4-HPR treatment; 8) IEF was performed to detect the potential change after different drug concentrations treatment. Results: 1) Results showed a accumulation of LC3-II in a dose-dependent Manner; while only under 20μM 4-HPR treatment induces the activation of PARP; 2) GSH decrement was associated with ROS increase, however, amplitude of decrements under 10 or 20 μM 4-HPR shows no significant distinction compared with the change of ROS level; 3) Probe 1 and Ellman assay shows the lose of free sulfhydryl; 4) Western blot results showed parts of the ERS downstream proteins had considerable up-regulated; Indicate the activation of ERS; 5) Inhibition of p38 and p38 RNAi inhibits apoptosis, and inhibition of JNK or JNK RNAi inhibits autophagy, while induce apoptosis under low concentration; 6) ASK1 RNAi down regulation the expression both of p-JNK and p-p38, while silence DJ-1 make the occurrence of apoptosis under low concentration of 4-HPR and compound-23 inhibit the apoptosis induced by 4-HPR; 7) Immune precipitation and Immunofluorescence showed that under 10μM 4-HPR treatment the interaction of ASK1 and Ire1 or DJ-1 were increased; while 10μM and 20μM 4-HPR treatment make the increase of the ubiquitination levels of TRAF2; 8)The result of IEF verification the different oxidative modifications of DJ-1 under different drug concentrations.Conclusions: Data demonstrated that 4-HPR contribute to the ROS induced unfold protein accumulation, activate ERS mediated several pathways. Ire1 recruitments ASK1 and TRAF2, induce autophagy by keeping on activating JNK; In low concentration of 4-HPR, activated DJ-1 competitive binding to ASK1 with Ire1, partially devitalized it; and releasing ASK1 under high concentration due to its deactivation, then ASK1 activate p38 pathway and induce apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4659. doi:1538-7445.AM2012-4659