title: AMD3100 reduces CXCR4-mediated survival and metastasis of osteosarcoma by inhibiting JNK and Akt, but not p38 or Erk1/2, pathways in in vitro and mouse experiments.
abstract: Osteosarcoma (OS) has an unfavorable prognosis and tends to metastasize to lung tissue. Although the CXCL12-CXCR4 axis appears to affect progression and metastasis in numerous tumors, its mechanism and downstream pathways in OS remain unclear. We used western blotting and flow cytometry to detect CXCR4 and CXCR7 expression in two OS cell lines (LM8 and Dunn). An MTT assay was used to evaluate the effects of CXCL12 and AMD3100, a specific CXCR4 antagonist, on cell viability. Flow cytometry was utilized to analyze changes in apoptosis induced by serum deprivation following treatment with CXCL12 and AMD3100. A Transwell assay was used to assess cell migration in response to CXCL12 and AMD3100. Western blotting was performed to identify the phosphorylation of signaling molecules (JNK, c-Jun, Akt, p38 and Erk1/2) and expression of caspase-3 and -8, and PARP. Mouse models were employed to evaluate AMD3100 inhibition of primary OS growth and lung metastasis in vivo. CXCR4 expression was detected in LM8 but not Dunn cells, and neither cell line expressed CXCR7. The addition of CXCL12 induced the survival and migration of serum-starved CXCR4+ LM8 cells activating JNK and Akt pathways, which were abrogated by adding AMD3100. However, similar results were not observed in CXCR4- Dunn cells. CXCL12 protected LM8, but not Dunn cells, from apoptosis induced by serum deprivation by suppressing PARP cleavage, which was partly reversed by AMD3100. In a mouse model, AMD3100 reduced primary tumor growth and lung metastasis compared with the controls. Thus, the CXCL12-CXCR4 axis regulated OS survival and metastasis through the JNK and Akt pathways, and blocking them with AMD3100 was found to be a potential OS treatment.
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Yu-Xin
authors.initials: YX
authors.lastname: Liao
authors.affiliation: Department of Orthopaedics, Shanghai First People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200080, P.R. China.
authors.forename: Ze-Ze
authors.initials: ZZ
authors.lastname: Fu
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Cheng-Hao
authors.initials: CH
authors.lastname: Zhou
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Lian-Cheng
authors.initials: LC
authors.lastname: Shan
authors.affiliation: Department of Orthopaedics, Shanghai First People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200080, P.R. China.
authors.forename: Zhuo-Ying
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Shanghai First People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200080, P.R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Yin
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Long-Po
authors.initials: LP
authors.lastname: Zheng
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Ying-Qi
authors.initials: YQ
authors.lastname: Hua
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China.
authors.forename: Zheng-Dong
authors.initials: ZD
authors.lastname: Cai
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001596:Benzylamines; D053148:Caspase 3 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000096:biosynthesis* / Q000235:genetics; D000080027:Cyclams; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006571:Heterocyclic Compounds / Q000008:administration & dosage; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000096:biosynthesis; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D009362:Neoplasm Metastasis; D051058:Oncogene Protein v-akt / Q000096:biosynthesis; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D054387:Receptors, CXCR / Q000096:biosynthesis* / Q000235:genetics; D019718:Receptors, CXCR4 / Q000096:biosynthesis* / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis
chemical_list: C501896:ACKR3 protein, human; D001596:Benzylamines; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D000080027:Cyclams; D006571:Heterocyclic Compounds; D054387:Receptors, CXCR; D019718:Receptors, CXCR4; D051058:Oncogene Protein v-akt; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D053148:Caspase 3; C088327:plerixafor