P13-32 Bafilomycin induces mitochondrial reorganization, nuclear deformation and γH2AX increase in T24 bladder cancer cells

Abstract

Autophagy competence is essential for bladder cells, which are continuously exposed to urinary occurring xenobiotics. Bafilomycin A1 (BAFI), a commonly used autophagy inhibitor, has been previously described to have a significant effect on the mitochondrial morphology and function in T24 bladder cancer cells [1]. Yet, mitochondrial toxicity can have downstream effects on multiple cascades [2]. Untargeted proteomic profiling of T24 cells (24h, 10nM BAFI), returned multiple regulatory events, including a consistent downregulation of several NADH ubiquinone oxidoreductases. According to bioinformatics analysis this signature could be traced back to the regulation of the KEGG pathway “Chemical carcinogenesis – reactive oxygen species” [3, 4]. Starting from these data, a potential role for BAFI in the regulation of genotoxic damage in bladder cells was hypothesized. To recreate exposure scenarios in the bladder and to limit possible confounding factors related to long-term autophagy inhibition, experiments were performed privileging short incubation times (4h, 0.1–1–10nM BAFI). Live cell imaging experiments revealed a concentration-dependent rearrangement of the mitochondrial network towards the nucleus (1–10nM BAFI). These results aligned with the proteome signature (24h) showing increased perinuclear mitochondrial protein enrichment. Stemming from the mitochondrial recruitment in proximity of the DNA, a potential involvement of reactive oxygen species (ROS) was verified. DCF-assay revealed no significant increase in ROS production related to the mitochondria rearrangement. In order to assess a potential DNA damage, immunofluorescence analysis of γH2AX signal was performed. In this case, BAFI (10nM, 4h) significantly increased the detection of the biomarker for DNA double-strand breaks. This signature was consistent even in presence of the antioxidant enzyme catalase (100nM), further supporting the view that DNA damage could occur even without direct involvement of oxidative stress. Looking for molecular mechanisms of action potentially sustaining these effects, significant deformation of the nuclear morphology was observed, suggesting a possible involvement of mechanical stress as trigger of the genotoxic damage [5]. In conclusion, the data collected in this study suggest the presence of pathways of relevance for carcinogenesis related to mitochondrial rearrangement and potentially complementary to ROS insults.

Jasmin Di Franco

PhD Student

My research interests include Cell biophysics, Living matter and Soft matter physics.

Roberto Cerbino

Professor of Experimental Soft Matter Physics

My research interests include Soft matter physics, living matter, cell biophysics and quantitative microscopy.