Telomerase Reverse Transcriptase Suppresses Cisplatin-dependent Apoptosis In Osteosarcoma Cells

Osteosarcoma is the most common malignant tumor derived from bone,it is prevalent in adolescent,and it causes high morbidity and mortality,the 5-year survival was only 20%when surgery is the only treatment.Since 1970s,the introduction of chemotherapy has changed the situation,survival rate of those receiving chemotherapy has improved significantly.In addition,limb salvage combined with joint prothesis preserves both the function and appearance of the involved limb.Together,the osteosarcoma patients have now obtained both longer-term survival and the quality of life.Currently,the chemoresistance of osteosarcoma patients is an enormous challenge to surgeon.First,the patient suffered from chemoresistance should be administrated with more intensive chemotherapy.Second,chemoresistance would enforce local recurrence of tumor,which would cause severe mental stress.Third,widespread lung metastasis and respiratory failure,even subsequently irreversible multi-organ failure can occur due to chemoresistance.For these patients,the 5-year survival is less than 20%.Telomere is the special structure of terminal end of chromosomes in eukaryote,with important biological functions.Telomerase is a key enzyme in maintaining telomere,the major being telomerase reverse transcriptase(TERT),its RNA partner TERC and telomerase associated protein(TP).Telomerase activity can be detected only in germ cell lines and small number of somatic cells.However,its activity is prominent in 90%of all human cancers.The relationship between telomerase and cancer has been one of the hot spots,telomerase is closely related to cell cycle and cell apoptosis in cancer and activates or inhibits oncogenes in the transcription and translation level.Cisplatin,as one of the most efficacious antimitotic drugs,mainly targets DNA,which interferes with DNA replication and induces cell apoptosis.Therefore,the relationship between telomerase and cisplatin in osteosarcoma need to be further explored.Part I TERT translocates from the nucleus to mitochondria following cisplatin treatment in osteosarcoma cellsObjective:To determine whether TERT expression and localization is altered in cisplatin-treated osteosarcoma cells.Methods:The human osteosarcoma cell lines MG63,U2OS and 143B were obtained and from China Centre for Type Culture Collection(CCTCC)and cultured in DMEM supplemented 10%FBS and 1&antibiotics(penicillin/streptomycin).TERT mRNA and protein level in cisplatin-treated(5?mol/L,24h)osteosarcoma cells compared to untreated cells were assessed by qRT-PCR and Western-blotting respectively.The localization of TERT in cisplatin-treated osteosarcoma cells to untreated cells was determined by Confocal Fluorescence Microscopy.Mitochondrial localization pattern of TERT was further detected by Western-blotting.Results:TERT was strongly expressed at both mRNA and protein levels in all three osteosarcoma cells treated with 5?mol/L cisplatin for 24 h compared to untreated cells.Osteosarcoma cells treated with cisplatin exhibited robust TERT expression in mitochondria.This mitochondrial localization pattern of TERT was further detected by Western-blotting and similar results were obtained.Conclusions:TERT expression is altered and shuttles from the nucleus to mitochondria in cisplatin-treated osteosarcoma cells.Part ? TERT protects osteosarcoma cells against cisplatin-induced apoptosis by inhibiting the mitochondrial pathway and alleviating intracellular ROS levelObjective:To study the role of TERT in cisplatin-resistant osteosarcoma cells in vitro.Methods:To generate stably transfected cell lines(TERT-wildetype,TERT-catalytically inactive,TERT-siRNA,Control),osteosarcoma cells were infected with the viral supernatant according to the manufacturer's protocol and incubated for 48 h prior to selection of stable transfectants by addition of puromycin.TERT expression were detected by Western-blotting to test and verify the transfection effect.Apoptosis rate in transfected osteosarcoma cells were analyzed by flow cytometric and immuno-fluorescence.Apoptosis related protein Bax,Bcl-2 and Bcl-xl in transfected osteosarcoma cells were analyzed by Western-blotting.Caspase-3 and cytochrome c oxidase(COX)activity were assessed in transfected osteosarcoma cells.ROS response of TERT-WT,TERT-CI and siRNA transfected cells to cisplatin treatment were assessed by flow cytometric.GSH/GSSG couple,glutathione peroxidase(GPx)and reductase(GR)were investigated in transfected osteosarcoma cells.Results:we detected TERT expression and found it increasingly expressed in the TERT-wildtype(TERT-WT)and catalytically-inactive TERT(TERT-CI)cells and decreasingly expressed in siRNA-transfected cells.TERT expression were observed as no differences between TERT-WT and TERT-CI cells.Double staining with annexin V-fluorescein isothiocyanate(FITC)and propidium iodide(PI)revealed a marked decrease in TERT-WT and TERT-CI cells.While apoptosis of TERT-siRNA transfected cells was significantly increased,compared to that in control MG63 osteosarcoma cells.Similar results were obtained with Immuno-fluorescence analysis.Moreover,no marked differences were evident between TERT-WT and TERT-CI transfected cells,suggesting that this anti-apoptotic effect does not rely on telomerase reverse transcriptase activity.In all three cell lines,Bcl-2 and Bcl-xl protein levels were significantly increased and Bax protein was decreased in TERT-WT and TERT-CI transfected groups,while opposite expression patterns were observed in TERT-siRNA transfected cells.TERT significantly inhibited caspase-3 activity in osteosarcoma cells.Cells transfected with wildtype and catalytically inactive TERT maintained higher COX activity,and conversely,TERT-siRNA transfected cells showed lower COX activity,compared with control cells.Flow cytometric analysis revealed significantly lower basal and mitochondrial ROS levels in TERT-WT and TERT-CI transfected cells,and conversely,higher ROS levels in TERT siRNA transfected cells.Glutathione assay suggested that a higher GSH/GSSG ratio was determined in TERT-WT and TERT-CI cells and a lower ratio in TERT-siRNA cells,compared with control cell group.GPx and GR assay revealed that higher GPx and GR activities were maintained in TERT-WT and TERT-CI cells,while TERT-siRNA cells showed lower activity,compared with control cells.Conclusions:TERT reduces apoptosis induced by cisplatin in osteosarcoma cells by alleviating ROS level and inhibiting mitochondrial apoptosis pathway,and this effect is independent of telomerase reverse transcriptase activity.Part ? TERT enhances cisplatin resistance of osteosarcoma cells in vivo and its underlying mechanismObjective:To study the role of TERT in cisplatin-resistant osteosarcoma cells in vivo and generate a schematic diagram.Methods:We subcutaneously inoculated tumor xenografts derived from 143B cells into nude mice(n=24).Mice were injected intraperitoneally with 5 mg/kg cisplatin at intervals of 4 days for 4 weeks.All mice were euthanized and tumor burdens assessed at the time of death.Apoptotic proteins Bax,Bcl-2 and Bcl-xl expression in xenograft tissues were assessed by Western-blotting.Results:TERT-WT and TERT-CI inoculated tumors displayed no obvious differences relative to the initially implanted size.TERT-siRNA treated tumor cells showed the slowest growth,displaying the smallest sizes after 4 weeks.TERT-WT and TERT-CI treated tumors displayed the largest volumes and burdens,while TERT-siRNA treated tumors showed smallest volumes and burdens than the control tumor group.Western-blot analysis revealed that the Bax protein level stayed the lowest in TERT-WT and TERT-CI treated tumors and highest in TERT-siRNA treated tumors,compared with control tumors.Conversely,Bcl-2 and Bcl-xl protein levels remained the highest in TERT-WT and TERT-CI tumors and lowest in TERT-siRNA inoculated tumors,compared with control tumors.Conclusions:Cisplatin-resistance of osteosarcoma cells in vivo is promoted by TERT.In cisplatin-resistant osteosarcoma cells,TERT shuttles from the nucleus to mitochondrion and alleviates intracellular ROS levels upon cisplatin treatment,which results inhibited mitochondrial apoptosis and cell survival.