Inhibitors Of Non-receptor Tyrosine Kinase C-Abl Induce Mitochondria Damage And Suppress Mitophagy

c-Abl (cellular Abelson gene product, c-Abl),member of non-receptor tyrosine kinase superfamily Abelson, have an important role in many important signaling pathways of cell growth and survival, such as cytoskeleton remodeling, cell migration and adhesion, receptor-mediated endocytosis, autophagy, DNA damage response and apoptosis, promoting transportation of autophagy into lysosome and lysosomal components to work properly. Under oxidative stress, c-Abl move into mitochondria and mediated mitochondrial dysfunction and cell death. Under normal circumstances, the intracellular c-Abl activity is relatively low, when ionizing radiation or oxidative stress, c-Abl are activated. Chromosomal translocation made BCR-Abl fusion gene, which encodes the BCR-ABL fusion protein that is in constitutively active state, being the leading cause of chronic myelocytic leukemia (CML). c-Abl inhibitor STI571 (also known as imatinib, Gleevec) is developed in the 1990s,drugs of tyrosine kinase inhibitor, is the first small molecular targeted drugs in the world, have played an important role in treatment of BCR-Abl positive acute and chronic leukemia. The patient continued to be treated with it, turns out that its life expectancy is not significantly different from the normal population. STI571 has been reported in the literature can increase cellular level of reactive oxygen species(ROS), while the epidermal growth factor inhibitor combined with STI571 treatment synergistically to promote non-small cell lung cancer cells death.Mitochondria is an important place in eukaryotic cells for capacity, it produce ATP by the redox recation of respiration substrates and electron transfer through the respiratory chain complex collaboration on the inner membrane. In mitochondria and mitochondrial particles and sub-intact cells, cell respiration in addition to maintaining normal metabolism, would inevitably produce some reactive oxygen species such as H2O2, superoxide anion, hydrogen radicals and hydroxyl radicals. Especially when mitochondrial respiration is inhibited or dysfunction, there would be a substantial generation of reactive oxygen species. Reactive oxygen species produced by mitochondria can be toxic to cells and induce cell death. If mitochondrial is damaged, the surface protein will be ubiquitination, and fused with lysosomes to remove damaged mitochondria, the process is called mitochondrial autophagy (Mitophagy)AIF (apoptosis-inducing factor, AIF) is an integral part of the respiratory chain complex I located in the inner mitochondrial membrane, which plays a dual role of maintenance of mitochondrial homeostasis or inducing cell apoptosis. When mitochondria are in normal physiological status, AIF act as a NADH dependent oxidoreductase. When mitochondria were damaged, AIF would be cleaved by protease, detached from the inner mitochondrial membrane and translocated to the nucleus, which triggers chromatin condensation and DNA fragmentation. However, AIF’s special redox activity is still unclear. Based on the preliminary research of our laboratory that there is an interaction between the non-receptor tyrosine kinase c-Abl and AIF, we built AIF knockdown cell lines of A549 cells (Non-small cell lung cancer cells,NCLL) to study the redox activity of AIF.Early laboratory studies have demonstrated c-Abl can interact with AIF and cause AIF phosphorylation. On the basis of preliminary studies, first by immunoprecipitation and purification expressed Flag-AIF in c-Abl expressing cells, SDS-PAGE electrophoresis separation of AIF protein, cut out protein gel block containing the AIF and identified by LC-MS/MS. It was found that AIF protein in Y170 and Y253 can be phosphorylated. For further study of AIF’s Y170 and Y253 site-specific phosphorylation,, we synthesized phosphorylated peptides of both Y170 and Y253 for tyrosine phosphorylation sites, prepared by immunizing rabbits with the corresponding sites feature phosphorylation antibody, AIF-Y (170)-p-Tyr and AIF-Y (253)-p-Tyr. By immunoblotting experiments of Y (170F), Y (253F) single mutants and Y (170253F) double mutants verifying the specific phosphorylation of these sites.To study the biological significance of phosphorylation of AIF by c-Abl, we have designed and synthesized siRNA to interference AIF gene expression, after retroviral vector packaging, transfected into A549 cells, positive clones whose genomic sequence integrated with the interference siRNA were screened out with puromycin and verification by immunoblotting,which showed AIF gene expression levels is lower than 80%, stable cell line of AIF knockdown A549 was successfully constructed. Though Immunofluorescence, AIF were marked by green fluorescent FITC-labeled secondary antibody, Mitotracker red dye labeled mitochondria, we observed in the microscope that AIF knockdown cell lines showed fragmented mitochondria.Early laboratory studies have demonstrated c-Abl inhibitor STI571 can cause mitochondrial damage, further study of this phenomenon, we find c-Abl inhibitor STI571 inducing non-small cell lung cancer cells and other cell fragments of mitochondrial swelling and even serious injury is with a time and dose dependent manner,which prompt us to imagine that c-Abl inhibitor STI571 inducing damage to mitochondria may relevant with the change of the positioning of the AIF. On the other hand, found by immunoblotting, STI571 can inhibit c-Abl phosphorylation of AIF. To explore the relationship between c-Abl inhibitor STI571 caused mitochondria damage and AIF positioning, we use our previous constructed fusion protein COX-AIF-GFP with AIF gene sequence C-terminal mitochondrial localization sequence (MLS) replacement by the inner mitochondrial membrane anchor sequences COX-Ⅳ and inserting in GFP the label vector, can enable AIF anchored on the inner mitochondrial membrane without being detched from mitochondria. By exogenous expression COX-AIF-GFP, immunofluorescence showed anchored on mitochondrial AIF can ease the mitochondrial damage induced by STI571.Indicating the change of AIF position in mitochondria is a reason for c-Abl inhibitor STI571 causing mitochondrial damage.To study the effect of AIF in STI571 elevated cellular ROS level, we used flow cytometry combined with DCFH fluorescent probe to measure the average ROS value of STI571 treated AIF knockdown A549 cell line and found that STI571 still can significantly elevate ROS levels of the AIF knockdown A549 cells. Indicating that AIF may had no significant impact on STI571 induced ROS levels elevation.Mitochondria can be degraded by fusion with lysosomes. We used immunofluorescence and lysosomal marker LC3 labeled lysosomes, mitochondrial uncoupler CCCP (20μM,6h,12h,24h) time gradient process, another c-Abl inhibitor Nilotinib (AMN,25μM,24h) single treatment group and CCCP (20μM,24h) and nilotinib (25μM,24h) combined treatment of A549 cells to study c-Abl inhibitor affecting mitochondria autophagy. We found that, comparing with the control group, with the extension of CCCP treatment time, first LC3 particles gathered in the mitochondria increasing and then downward trend is shown, reflecting the formation of the mitochondrial autophagy and gradually degradation process. Compared to the CCCP (6-24h) gradient treatments, nilotinib single treatment also appeared the amount of particles LC3 mitochondria increased, indicating that it can also induce mitochondrial autophagy. But contrast with CCCP single processing (20μM,24h) that LC3 particles substantially reduced,nilotinib and CCCP 24h co-treatment group significantly appeared more LC3 particles, indicating that autophagy evolving is retained without being degraded in the lysosome, leading to autophagic flux suppresstion.In summary, we found that c-Abl inhibitor-induced mitochondrial reactive oxygen species increasing may be not associated with phosphorylation of AIF by c-Abl.c-Abl inhibitor can induce mitochondrial damage by changing AIF location and promote mitochondrial autophagy,but suppress autophagy flux.