Abstract A20: Concomitant induction of autophagosome synthesis and impairment of lysosomal integrity by the redox-active anticancer drug Dp44mT converts autophagy into a detrimental process.

Abstract

Abstract Background: Autophagy functions as a cell survival mechanism against cellular stress and its up-regulation serves as a means of resistance against numerous anti-cancer agents (Nat Rev Cancer 2005;5;726-34). The redox-active thiosemicarbazone, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), developed in our laboratory has been shown to possess marked and selective anti-tumor and anti-metastatic activity in vitro and in vivo (Blood 2004;104:1450-8; PNAS 2006;103:14901-6). Interestingly, due to its lysosomotropic properties, Dp44mT has been reported to localize within the lysosomal compartment binding metal ions liberated from metalloproteins through autophagy (Cancer Res. 2011;71:5871-80). Considering that the lysosome plays an integral role in autophagy and cell death, it was important to determine the effect of Dp44mT on this process to further develop this chelator as an anti-tumor agent. Methods: Cellular proliferation was determined via the MTT assay. Autophagic activity was assessed by western blotting and immunofluorescent detection of autophagic markers (LC3 and p62). Lysosomal stability studies were implemented using the lysosomotropic fluorochrome, acridine orange, and specific lysosomal markers, cathepsin D and LAMP2. Results: An increased number of autophagosomes was shown by increased LC3-II levels after incubation with Dp44mT and this occurred concurrently to its anti-proliferative activity. By utilizing bafilomycin A1, which inhibits autophagosome breakdown, it was demonstrated that Dp44mT induced autophagosome formation. Unexpectedly, Dp44mT also blocked autophagosome degradation since an increase in p62 was observed after incubation with this agent. This finding indicated a simultaneous induction of autophagy and inhibition of autophagic flux by Dp44mT. The redox activity of Dp44mT upon binding iron and copper was found to be important in its ability of inducing autophagy as well as inhibiting autophagosome degradation. This observation was supported by the results showing decreased autophagic activity and no p62 accumulation when Dp44mT was co-incubated with the anti-oxidant, N-acetylcysteine. Lysosomal stability studies analyzing the redistribution of cathepsin D or acridine orange from the lysosomes to the cytosol indicated disruption of lysosomes by Dp44mT. Moreover, inhibition of autophagic initiation via the siRNA-mediated knockdown of autophagy regulators (ATG5/Beclin1) significantly (p<0.01) decreased Dp44mT-mediated cell death. Conclusions: This study demonstrates the anti-proliferative activity of Dp44mT involves induction of autophagy and lysosomal permeabilization. These findings have important implications in selectively targeting lysosomal function to overcome the pro-survival activity of autophagy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A20. Citation Format: Elaine Gutierrez, Sumit Sahni, Des R. Richardson, Patric J. Jansson. Concomitant induction of autophagosome synthesis and impairment of lysosomal integrity by the redox-active anticancer drug Dp44mT converts autophagy into a detrimental process. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A20.