Effects Of CXCR1 Knockdown On The Chemotherapy Sensitivity Of Osteosarcoma Cells

BECKGROUND:Chemotherapy resistance is one major cause to the poor prognosis of osteosarcoma patients.Tumor cells could escape chemotherapy drugs by changes of genes expression.In previous study,we have isolated and established the Saos2-lung subtype from the pulmonary metastasis Saos2 cell lines in nude mice bearing the osteosarcoma.In this study,we will investigate the chemotherapy sensitivity of Saos2-lung cells and underlying molecular mechanisms,by which to provide a new target to improve the treatment of osteosarcoma.OBJECTIVES:The aim of this study is to investigate the molecular mechanisms underlying the chemotherapy resistance of osteosarcoma cells and find a new target to improve the cells' chemotherapy sensitivity both in vitro and in vivo.METHODS:1.Analysis of chemotherapy sensitivity of osteosarcoma cells: The Saos2 cell line and the Saos2-lung subtype were treated with cisplatin or doxorubicin.The chemotherapy sensitivity of these cells were determined by CCK-8 assay and apoptosis test.The CXCR1 expression was detected by Western Blot.Finally,other two osteosarcoma cell lines,U2 OS and MG63 cells,were used to investigate their chemotherapy sensitivity and CXCR1 expression.2.Investigation of the effects of CXCR1 gene knockdown on the sensitivity of osteosarcoma cells and underlying molecular mechanisms: Saos2 and Saos2-lung cells were transfected with two shRNAs against CXCR1 sequences by lentiviral vectors.The chemotherapy sensitivity to the cisplatin with or without the stimulation of IL-8 were determined.Western blot was used to test the activity of Akt signaling pathway and expression of Caspase-3.We also transfected the shRNA against CXCR1 into U2 OS cells to investigate the changes of chemotherapy sensitivity to cisplatin.3.Investigation of sensitivity of Saos2-lung cells after CXCR1 knockdown in vivo: The luciferase labelled Saos2-lung cells with CXCR1 knockdown or control cells were injected into the bone marrow cavity of tibia of nude mice.After cisplatin treatment,the tumor size and the luciferase intensity of nude mice were detected and compared with the mice without cisplatin treatment.The expression of IL-8,CXCR1,p-Akt,Cleaved-Caspase-3 and PCNA in specimens were also determined by immunohistochemistry.RESULTS:1.The molecular characterization of chemotherapy sensitivity of osteosarcoma cells: Saos2-lung cells were more resistant to the treatment of cisplatin or doxorubicin than the parental Saos2 cells.Western Blot demonstrated that there was higher expression of CXCR1 in Saos2-lung cells than parental cells.The U2 OS cell line also was demonstrated to have higher CXCR1 expression and stronger chemotherapy resistance than the MG63 cells.2.Sensitivity of osteosarcoma cells to in vitro cisplatin treatment after CXCR1 knockdown: CXCR1 knockdown Saos2 and Saos2-lung cells had been established respectively.CXCR1 knockdown improved cell's sensitivity to the cisplatin greatly.IL-8 could reduce the chemotherapy sensitivity of osteosarcoma cells and CXCR1 knockdown could blocked this effect.Sensitivity to the cisplatin was also increased after CXCR1 knockdown in U2 OS cells.Western Blot showed that CXCR1 knockdown increased Caspase-3 expression and suppressed the Akt signal pathway activated by IL-8.3.Cisplatin sensitivity of Saos2-lung cells after CXCR1 knockdown in vivo: Compared with the control group,the tumor size and luciferase intensity in nude mice bearing CXCR1 knockdown cells was reduced greatly after cisplatin treatment.The immunohistochemistry staining indicated that the expression of IL-8,CXCR1,p-Akt and PCNA were suppressed and Cleaved-Caspase-3 expression was increased in CXCR1 knockdown groups after the treatment of cisplatin.CONCLUSIONS:Higher CXCR1 expression was related to the chemotherapy resistance of osteosarcoma cells and CXCR1 knockdown could improve the cell's sensitivity to cisplatin by blocking the IL-8/CXCR1/Akt signaling pathway.CXCR1 will be a new target for osteosarcoma treatment.