| Objective:Autophagy is an evolutionally conserved protein degradation pathway in eukaryotes,which is essential for cell survival under nutrient-limiting conditions. During autophagy,portions of the cytoplasm are sequestered by double-membraned vesicles calledautophagosomes, and are degraded after fusion with lysosomes for subsequent recycling. Aswe all know, mitochondria are the major sites of intracellular energy metabolism, whichgenerate ATP is the main energy source for cell life activities.As an important intracellularorganelles, mitochondria play an important role in physiological activity of the cell. Manyhuman diseases are closely related with the occurrence of mitochondria. Abnoemalitymitochondrial damage is the direct cause of many human diseases, and mitochondrialautophagy is cell depletion in vivo mitochondrial homeostasis and maintaining its own as animportant regulatory mechanism. Studies have shown that if cells were defective autophagy,it will cause a lot of damage accumulation and aging mitochondria within cells, therebypromoting tumor development. Mitochondrial defects or activation of autophagy intumorigenesis or progression correlated. However, how mitochondrial autophagy egulatesautophagy is not clear yet.Topotecan (Topotecan,TPT) is known as a DNA damage anti-cancer drug.Semisynthetic-water-soluble camptothecin analog, which is a specific inhibitor oftopoisomerase I in the nucleus. ATM belongs to PI-3K family of proteins, is a direct factor of induction DNA damage. In the previous study, we found that the phosphorylation of Beclin1and ATM in tumor cells could be induced by topotecan (Topotecan,TPT), and accompaniedby mitophagy. These indicated thatATM, Beclin1was related to mitophagy.This study discuss ATM, Beclin1-mediated topotecan (Topotecan,TPT) induced tumorcell mitochondrial autophagy and its molecular mechanisms, elucidated the role of ATM andBeclin1in mitochondrial DNAdamage, mitophagy in tumor cells.This study focuses on the sinal pathway of TPT-ATM-Beclin1-mitochondrialautophagy, and to elucidate the role ofATM, Beclin1on mitophagy in cancer cells.Methods:Cultured cell lines as follows: A549strains that is human non-small cell lung cancercells, GLC-82strains that is human lung adenocarcinoma cells, HeLa strains that is humancervical carcinoma cell lines that lines, Hep3B strains that is human hepatoma cell line.Confocal microscopy were used to detecte the punctuate dots of GFP-LC3and mitochondrialautophagy. Western blotting were used to analysis the expression of phospho-ATM, LC3,P62, ATM, phospho-Beclin1-T57. The mitochondrial autophagy were detected by confocalmicroscopy when ATM activity were inhibited by Ku55933(ATM inhibitor) and RNAi-ATM.Co-immunoprecipitation assay was used to detecte Caspase-8, RIP1, FLIP combined withATG5situation, the proliferation inhibotory effects of tumor cells were detected by MTTassay.Results:1. DNA-damage drug Topotecan induced autophagy in Tumor cellsUsing Western blotting analysis, we observed LC3II type increased gradually, P62expression gradually weakened, and in a concentration-dependent manner.Asignificantaccumulation of GFP-LC3dots was observed after cancer cells were transient with GFP-LC3plasmids,which indicated induction of autophagy. 2. Topotecan induced phosphorylation ofATM and mitophagy in cancer cellsCancer cells were first treated with0.6μg/ml TPT for24h, a large number of γ-H2AXrallying point for coarse particles appeared in Hep3B nuclei was observed, indicating TPT(topotecan) can cause cancer cells to DNAdamage. Cancer cells were first treated with0.6μg/ml TPT for different time, and then visualized under confocal microscopy。When cancercells were treated with TPT24h TOM20perinuclear aggregation occurs with prolongedduration of action when48h TOM20perinuclear aggregates back to normal, andMitoTracker-labeled mitochondria and LC3, LAMP2colocalization.3. Topotecan induced phosphorylation ofATM in cancer cells and thus induce tumorautophagy and mitochondrial autophagyThe results of western blotting showed thatATM kinase phosphorylation level could beenhanced in a concentration-dependent and time-dependent manner. When tumor cells wereTransfected withATM shRNAor10μM Ku55933for the indicated periods, and untreated ortreated with0.6μg/ml topotecan for24h, The results of western blotting showed that thedecrease of LC3-II and increase of P62. The results of confocal microscopy showed that24hTOM20perinuclear aggregation occurs with prolonged duration of action when48h TOM20perinuclear aggregates back to normal, but when tumor cells were transfected withATMshRNAor10μM ku55933for the indicated periods, TOM20perinuclear can not aggregatesback to normal.4. Topotecan induced phosphorylation of Beclin1T57in cancer cellsBased on sequence analysis of Beclin1was analyzed by Scansite software,we generatedphospho-specific antibody with Beclin1T57and checked its effectiveness. The results ofwestern blotting showed that the expression of p-Beclin1T57level could be enhanced in aconcentration-dependent and time-dependent manner in cancer cells.5.ATM activation involved in TPT (topotecan) mediated phosphorylation ofBeclin1-T57Using Western blotting analysis, when tumor cells were treated with0.6μg/mltopotecan and/or10μM Ku55933and/or for transfected withATM siRNAs for the indicated periods, we observed a notable decrease of p-Beclin1-T57and p-ATM.When tumor cellswere transfected with sh-ATM for the indicated periods, we observed a notable decrease ofp-Beclin1-T57and p-ATM. It showed Beclin1-57might be involved in regulation of TPT(topotecan)-induced phosphorylation of ATM thereby affecting mitochondrial autophagy.Co-immunoprecipitation showed that Beclin1, ATM has combined, Beclin1-57mutationsaffect the binding of Beclin1, ATM. we conclude that Beclin1-T57is TPT (topotecan)induced phosphorylation of ATM downstream targets.6. ATM phosphorylation by regulating Beclin1T57affecting TPT (topotecan) inducedmitophagy and its effect on cell survival statusWhen tumor cells were transfected with Beclin1siRNAs for the indicated periods or GLC-82-Beclin1-WT, GLC-82-Beclin1-T57A cells, and untreated or treated with0.6μg/ml TPT for the indicatedperiods, the results of confocal microscopy showed that24h TOM20perinuclear aggregation occurs withprolonged duration of action when48h TOM20perinuclear aggregates back to normal, but when tumorcells were transfected with Beclin1siRNAs for the indicated periods, TOM20perinuclear can notaggregates back to normal. The results of MTT assay showed that the role of cell death of tumor cells weretransfected with Beclin1siRNAs or GLC-82-Beclin1-T57Acells were reduced.7. Mitophagy activated caspase-8and in conjunction with autophagy promote itsprecursor complexes to promote cell deathThe results of western blotting showed when cancer cells were treated with differentconcentration of topotecan for different time, the caspase-8level could be enhanced.Andwhen cancer cells were treated with0.6μg/ml topotecan for different time,the caspase-8level could be enhanced. The results of immunoprecipitation showed that endogenousATG5could be combined with Caspase-8and RIP1.The results of MTT assay showed that whenTumor cells were exposed to Necrotatin-1or3-MA, TPT, Necrotatin-1and TPT for72h. Theresults showed a certain appreciation inhibition when Necrotatin-1or3-MA, TPT were singleused on tumor cells, The effect is more obvious when Necrotatin-1or3-MAcombined withTPT. Conclusions:1. Topotecan induced autophagy, mitochondrial autophagy, and the phosphorylation of ATMin Tumor cells.2. Beclin1-T57involved in regulation of Topotecan induced the phosphorylation of ATM,mitochondrial autophagy and affeced the survival of the cells.3. Mitophagy activated caspase-8and in conjunction with autophagy promote its precursorcomplexes to promote cell death... |
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