The Effects Of Ethidium Bomide Induced Loss Of MtDNA On Biological Hehavior Of Lung Cancer Cells And The Mechanism Study Of Accompanied Mitochondiral Degradation

IntroductionMitochondria, a type of double membrane-bound compartments, are highly essentialand dedicated organelles present in all eukaryotic cells. Mitochondria function as chemicalfactories for key metabolic reactions and energy generation, and as communication site fordiverse signaling pathways. However, the role of mitochondria goes beyond the above, theyare also the major source of endogenous reactive oxygen species (ROS), the side productsof oxidative phosphorylation. Excessive ROS is potentially deleterious and may causedamage to mitochondrial proteins, mtDNA and lipids, even lead to cell death by promotingthe intrinsic apoptotic pathway. In addition, mitochondria have been implicated inmitochondrial disorders, cardiac dysfunction, aging process and other human diseases.From this point, the quality and quantity of mitochondria need to be accurately controlledfor energy metabolism homeostasis and other key essential cellular processes. To date,several lines of evidence suggest that the selective degradation of mitochondria byautophagy controls mitochondrial number and health. Clearly, the link between autophagyand mitochondria is the selective removal of superfluous and damaged mitochondria, aprocess termed mitophagy.Autophagy is primarily supposed to be a protective process for the cell. Basal levels ofautophagy play critical role in maintaining normal cellular homeostasis by recycling ofintracellular components. Today, the role of autophagy has been extended up through tohuman diseases and physiology. For example, unregulated activation of autophagy likelycontributes to a broad spectrum of pathological processes, ranging from infections,neurodegeneration, heart disease to aging and cancer. Autophagy is firstly associated withcancer through the identification and characterization of the beclin1gene, which issuggested as a tumor suppressor. Moreover, several other autophagy genes are implicated in tumorigenesis, including MAP1-LC3(ATG8homolog) and HsGSA7gene (ATG7ortholog).Accordingly, Atg proteins, the products of Atg genes, are essential factors for the process ofautophagy. LC3(microtubule associated protein light chain3), a mammalianautophagosomal ortholog of yeast Atg8, is identified to form LC3-Ⅰ by cleaving the C-terminus of newly synthesized ProLC3. Then, an E2-like enzyme Atg7cleaves22aminoacids from the C-terminus to form LC3-Ⅱ, which is recruited to form autophagosomes andserves as an autophagic marker protein.Under normal conditions, basal levels of autophagy serve to maintain cellularhomeostasis by physiologically elimination of damaged organelles and long-lived proteins,and autophagy is activated in response to metabolic stress or energy crisis, such as nutrient,growth factor, and oxygen deprivation or mitochondria damage. We know that,mitochondria plays crucial role in cellular functions, including cell growth, division, andenergy metabolism. Obviously, these functions are also necessary for cancer cellproliferation, survival and migration. According to previous study, the rate of spontaneousdepolarization of mitochondrial membrane potential is increased under serum deprivation.Then these damaged mitochondria are demonstrated to move into acidic vacuoles, besequestrated and digested in autophagosomes and autolysosomes. During our establishmentof human lung cancer cell lines lacking mtDNA, progressive depopulation of mitochondriawas indentified. What’s important, autophagy was activated in the process. In the presentstudy, we try to indentify the involvement of autophagy in mitochondrial degradation, toexplore its molecular mechanisms and the potential impacts on biological behaviors of lungcancer cells.Methods1. Establishment and identification of lung cancer cells lacking mitochondrial DNAA549, SPC-A1and H322human lung cancer cell lines were maintained in mediumsupplemented with10%FBS and100ng/ml penicillin and streptomycin. The cells werepassaged when cells were90%~95%confluent. For EtBr treatment, cells were exposed to250ng/ml ethidium bromide for7days. The medium was additionally supplemented with50μg/ml uridine and100μg/ml pyruvate. The mtDNA content and the mRNA expressionof cytochrome c oxidase subunit II (COX II) were measured by quantitative real-time PCR.2. The effects of EtBr on tumor growth in vitro and in vivo Cells treated with EtBr for1,3,5or7days were trypsinized and counted. Cellproliferation assay, clonogenic assay and cell migration assay were carried out to evaluatethe ability of cell proliferation and migration after mtDNA knocked out. Additionally, cellcycle and cell apoptosis were investigated by PI staining and Annexin V-FITC flowcytometry, respectively. Finally, In vivo analysis of tumor growth was performed.NOD/SCID mice (five mice per group) were injected subcutaneously in the left flank with-treated cells suspended in200μl phosphate-buffered saline (PBS). Tumor volume wasmeasured with calipers twice a week for6weeks, after which the mice were sacrificed.Tumors were removed and photographed.3. Investigation of mitochondrial degradation after EtBr treatmentFirstly, confocal microscopy was used to identify the mitochondrial degradation bylysosome. The treated cells were co-loaded with200nM MitoTracker Green and200nMred-fluorescing LTR for20min. After fluorescence loading, cells were washed thrice withfresh PBS. Confocal images were collected at2μm intervals. Secondly, confocalmicroscopy and flow cytometry were employed to verify loss of mitochondrial membranepotential. Lastly, mitochondrial degradation during EtBr treatment was further studied withtransmission electron microscopy.4. Mechanism study of EtBr-caused mitochondrial degradationConfocal microscopy and transmission electron microscopy were carried out toidentify the autophagic structures in EtBr treated cells. The cells were transfected with GFP-LC3, and then the distribution pattern was studied by confocal microscopy. Then autophagyrelated protein expression of Beclin-1and LC3was detected. We also examined whether3-MA, an autophagy inhibitor, could inhibit EtBr-induced autophagy or not, which could helpus understand better of the EtBr caused mitochondrial degradation pathways. Finally, weuse immuno-fluoresence to detect the expression level of Beclin-1and PINK1.Results1. Success in establishing lung cancer cells lacking mtDNAWe have succeeded in establishing the lung cancer cells lacking mitochondrial DNA.The PCR results demonstrated that these two markers significantly decreased in a time-dependent manner after EtBr treatment. Confocal of TMRM loading, used for detectingpolarized mitochondria, showed that EtBr-caused decrease of mitochondrial membrane potential (MMP) was in a time-dependent manner, which was consistent with the PCRresults. In other words, EtBr can knock down the mitochondrial gene.2. EtBr inhibits lung cancer cells growth in vitro and in vivo.In vitro cell proliferation, clonogenic and migration assays demonstrated that EtBrinhibited lung cancer cell growth and migration in a time-dependent manner, but there wasno significant increase in apoptotic events in EtBr-treated cells. The PI staining resultsdemonstrated that EtBr-treated cells underwent cell cycle arrest. In vivo, EtBr-treated cellsgrew more slowly than untreated control cells in lung cancer xenograft models.3. Confocal microscopy and TEM revealed mitochondrial degradationDual-labeled confocal microscopy revealed that LTR and MTG co-localizationstructures significantly increased in EtBr-treated lung cancer cells, in contrast to the controlgroup. Similar observation was evidenced in cells under starvation. Compared withstarvation, EtBr treatment more dramatically increased the co-localization of MTG-andLTR-positive structures. In addition, the numbers of mitochondria in cells treated with EtBrwere discovered to be less compared to the control cells with TEM.4. Mitochondrial degradation in the presence of EtBr was completed via autophagyAfter EtBr treatment, an increasing of LTR was observed with the decreasing ofmitochondria. Also, more MTG-labeled mitochondria were identified to move into the LTRstructures after EtBr treatment. Further, GFP-LC3expression level was dramaticallyincreased after the treatment, which distributed as green dots. Then the recruitment of LC3-II to autophagosomes during mitochondrial degradation was further identified by westernblot analysis, and the process could be inhibited by3-MA. Besides, an obvious increased ofBeclin-1protein was verified by WB. Besides, the expression level of Beclin-1and PINK1were shown to be increased via immuno-fluorescence.Conclusions1. We have succeeded in establishing lung cancer cells lacking mtDNA with a lowconcentration of EtBr, which was identified by PCR results of mtDNA content and mRNAexpression level of cytochrome c oxidase subunit II. EtBr treatment led to decreased cellproliferation, colony formation and migration in vitro. Tumor suppression effect of EtBrtreatment was also observed in lung cancer xenograft model. 2. Imaging method identified progressive depopulation of mitochondria during EtBrtreatment, accompanied by increased LTR uptake and co-localization of LTR-and MTG-positive structures. We conclude that autophagy is responsible for mitochondrialdegradation when exposed to a low concentration of EtBr, most likely through the PI3K-Beclin1pathway. The high expersson level of Beclin-1and PINK1detected by immuno-fluorescence also demonstrated they were involved in the process of autophagic degradationof mitochondria.