ROS-mediated lysosomal membrane permeabilization and autophagy inhibition regulate bleomycin-induced cellular senescence.

ABSTRACT

Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects.Abbreviations AT2 cells type II alveolar epithelial cells; ATG7 autophagy related 7; bEnd.3 mouse brain microvascular endothelial cells; BNIP3L BCL2/adenovirus E1B interacting protein 3-like; CCL2 C-C motif chemokine ligand 2; CDKN1A cyclin dependent kinase inhibitor 1A; CDKN2A cyclin dependent kinase inhibitor 2A; FTH1 ferritin heavy polypeptide 1; γ-H2AX phosphorylated H2A.X variant histone; GAPDH glyceraldehyde-3-phosphate dehydrogenase; HUVEC human umbilical vein endothelial cells; HT22 hippocampal neuronal cell lines; Il interleukin; LAMP lysosomal-associated membrane protein; LMP lysosome membrane permeabilization; MTORC1 mechanistic target of rapamycin kinase complex 1; NAC N-acetylcysteine; NCOA4 nuclear receptor coactivator 4; PI3K phosphoinositide 3-kinase; ROS reactive oxygen species; RPS6KB/S6K ribosomal protein S6 kinase; SA-GLB1/ß-gal senescence-associated galactosidase, beta 1; SAHF senescence-associated heterochromatic foci; SASP senescence-associated secretory phenotype; SEC62 SEC62 homolog, preprotein translocation; SEP superecliptic pHluorin; SQSTM1/p62 sequestosome 1; TFEB transcription factor EB.