The Elffect Of 2-bromopalmitate Sensitizes Osteosarcoma Cells To Adriamycin And Its Mechanism Study

Background:Osteosarcoma(OS)is the primary bone tumor that most commonly affects children,adolescents and young adults.Before 1970,treatment for OS primarily included surgical resection.The introduction of chemotherapy led to a dramatic improvement in prognosis for patients;long-term survival rates of less than 20%improved to 65%to 70%after the advent of multiagent chemotherapy regimens Methotrexate(MTX),cisplatin(DDP),adiamycin(ADM)and isosfamide(IFO)are the four first-class drugs used for OS chemotherapy.However,the treatment for OS has remained unchanged during the past decades.Thus innovative modalities are urgently needed to further prolong survival in osteosarcoma patientsAdriamycin(ADR)has been claimed to be the most effective agent for OS,which can destroy DNA tertiary structure as well as trigger endoplasmic reticulum(ER)stress Unfortunately,the clinical use for this drugs is limited by cumulative dose-related cardiotoxicity which may ultimately lead to severe cardiotoxicity.Moreover,the outcome of adriamycin chemotherapy in osteosarcoma remains unsatisfactory due to tumor recurrence,metastasis and intrinsic or acquired resistance to ADR.Therefore,it is particularly urgent to provide better chemotherapies which could dramatically raise the efficacy of adriamycin for osteosarcoma patientsPalmitoylation is a common type of post-translational lipid modification,classified into N-palmitoylation(irreversible)and S-palmitoylation(reversible).S-palmitoylation describes the attachment of a palmitate onto a cysteine residue of protein via thioester linkage rather than amide linkage,which can be mediated by Aspartate-Histidine-Histidine-Cysteine(DHHC)protein.Numerous studies suggest that palmitoylation has a significant impact on tumourigenesis and progression Therefore,palmitoylation inhibitors could be beneficial for the treatment of neoplasms Based on these findings,our study intends to evaluate 2-Bromopalmitate,a palmitoylation inhibitor,plus adriamycin synergy in OS cell apoptosis and gain insight into the underlying mechanismsObjective:The aim of this study was to investigate the effects and mechanisms of combination of 2-BP and ADR in OS cells,mainly including two parts:1)evaluate the synergistically anti-tumor effects of 2-BP and ADR in OS cells.2)elucidate the role of ROS-induced endoplasmic reticulum(ER)stress in the apoptosis of osteosarcoma cells induced by 2-BP combined with ADR;explore the changes of downstream signal of ER stress after combined treatment of 2-BP and ADRMethods:Human osteosarcoma cell lines(U20S and MG63)and human primary osteosarcoma cells(OS14.OS15,OS20 and OS21)were treated with 2-BP and ADR to study the impact of combination treatment on the proliferation and apoptosis of osteosarcoma cells.Cell survival assay was determined by Sulforhodamine B(SRB)colorimetric assay;the death of cells was detected by Propidium Iodide(PI)staining with flow cytometry;the morphology of cells and cell nuclei was observed by Diamidino-2-phenylindole(DAPI)staining assay under microscope;the apoposis of cells was detected by Annexin V/PI staining with flow cytometry;the cleavage of cell apoptosis-related proteins(PARP,Caspase 8 and Caspase 3)was detected by western blot;after cells were treated with different programmed cell death inhibitors.the death of cells was detected by PI staining with flow cytometry,then the cleavage of cell apoptosis-related proteins was detected by western blotHu,man osteosarcoma cell lines(U2OS)and human primary osteosarcoma cells(OS21)were treated with 2-BP and ADR to explore the underlying mechanisms of cell apoptosis induced by combination treatment.Cell cycle distribution was measured by PI staining with flow cytometry;expression level of DNA damage response related protein(p-ATM,p-ATR,p-CHKI and p-CHK2)was detected by western blot;after cells were pre-incubated with reactive oxygen species(ROS)scavenger N-acetyl-L-cysteine(NAC)or L-Glutathione(GSH).cell cycle distribution was determined by PI staining with flow cytometry;the production of ROS was measured by using the oxidation-sensitive fluorescent dye carboxy-DCFDA;after cells were treated with tunicamycin,the death of cells was detected by PI staining with flow cytometry;the expression level of downstream signaling pathways of unfolded protein reaction(ATF4,ATF6 and XBP1)was detected either by western blot or by semi-quantitative PCR;the mRNA level of CCAAT-enhancer-binding protein homologous protein(CHOP)was determined by quantitative real-time PCR;the protein level of CHOP was measured by western blot;after cells were treated with ROS scavenger NAC,the protein level of CHOP was measured by western blot;after cells were transfected with CHOP siRNA,the transcriptional level of CHOP was determined by quantitative real-time PCR,then the death of cells and apoptosis of cells was detected by PI staining and AV-PI staining with flow cytometry,respectively,finally the cleavage of cell apoptosis-related proteins was detected by western blot.Results:1)2-BP synergized with ADR to induce proliferation inhibition and trigger apoptosis in osteosarcoma cells2-BP increased ADR inhibitory effects on cell proliferation in osteosarcoma cell lines and primary osteosarcoma cells,which was dose-dependent;all combination index(CI)values were below 1.0 across all combination doses tested;furthermore,the addition of 2-BP lowered the IC50 of ADR in osteosarcoma cell lines and primary osteosarcoma cells.PI staining and AV-PI staining showed 2-BP synergized with ADR to induce apoptosis;DAPI staining showed that the nuclear fragmentations in the combination group were more distinguished than those in the single agent treated groups;besides,the combination treatment markedly increased the activation of caspase-8,followed by the cleavage of PARP and caspase-3 in dose-dependent manner;the addition of pan-caspase inhibitor Z-VAD-fink reversed the apoptosis induced by the combination treatment.2)Reactive oxygen species were strongly correlated with apoptosis induced by 2-BP plus ADRCompared with Adriamycin mono-treatment,the cell cycle arrest was not further enhanced during the combination treatment,and DNA damage response(DDR)associated proteins(ATM,CHK,ATR and CHK2)similarly showed no obvious change.However,ROS scavenger NAC and GSH exhibited a significant protective effect against the cytotoxicity induced by the combination treatment;ROS were moderately upregulated in osteosarcoma cells treated with ADR alone,but this effect did not further increase upon 2-BP and ADR co-treatment compared with ADR alone.3)2-BP synergized with ADR to activate CHOP signaling pathway triggered by ER stressER stress inducer tunicamycin increased the apoptosis induced by 2-BP and/or ADR;besides,ADR was strengthened by 2-BP more strongly than tunicamycin.The expression level of downstream signaling pathways of unfolded protein reaction(UPR)showed that ATF4 was highly elevated when exposed to the combination treatment,while ATF6 and spliced XBP1 showed no obvious change.Moreover,compared with 2-BP or ADR mono-treatment,the transcription level of CHOP was obviously increased in the combination treatment in a time-dependent and dose-dependent manner;the protein level of CHOP was also strongly unregulated when treated with 2-BP and ADR;NAC treatment also dramatically weakened the CHOP upregulation caused by the combination of 2-BP and ADR;besides,silencing CHOP reversed the apoptosis induced by the combination treatment:both PI staining and AV-PI staining demonstrated that CHOP knockdown cells showed markedly reduced apoptosis after ADR plus 2-BP treatment compared with control cells;similarly,decreased protein levels of cleaved caspase8.PARP and caspase3 were observed in osteosarcoma cells treated with the combination after CHOP knockdown,compared with the non-silenced group.Conclusion:Our study demonstrated the potential synergistic effects of 2-Bromopalmitate and adriamycin against human osteosarcoma cell lines and primary osteosarcoma cells Moreover,the strongly enhanced apoptosis by combination was mediated by the activation of CHOP,which was reactive oxygen species-dependent.In summary,this study estimated the synergism between 2-Bromopalmitate and adriamycin in the treatment of osteosarcoma,thus raising the possible application of this combination therapy in the clinical treatment of osteosarcoma.