The Role And Mechanism Of Chemokine CXCL1in Bone Cancer Pain

Objective Bone cancer pain is vey common and cancer metastasis to bone is frequently accompanied by cancer patients. Chemokines are a special class of cytokines, play an important role in mediating immune function, inflammation, wound healing and allograft rejection. However, the role of chemokines in bone cancer pain remains unclear. In this study, we investigated the expression and distribution of chemokine CXCL1and its major receptor CXCR2in the spinal cord and dorsal root ganglion (DRG) following prostate cancer cells-induced bone cancer pain in mice, and explored their role in the regulation of bone cancer pain.Methods Bone cancer pain was induced by injection of prostate cancer cells (20μl,106cells) into the right femur of mice. Mechanical allodynia, heat hyperalgesia and spontaneous pain were tested at different time points after injection of prostate cancer cells. The expression of chemokine CXCL1and CXCR2mRNA were detected by Real-time PCR. The protein expression of CXCL1and CXCR2were examined by Western blot or immunofluorescence in the DRG and L4-5spinal segments. The cellular distribution of CXCL1and CXCR2were defined by immunofluorescence double-staining. The cultured astrocytes were pretreated with BAY11-7082and then stimulated with TNF-α. CXCL1mRNA was detected by Real-time PCR and the protein level of CXCL1was examined by ELISA. The effect of CXCL1neutralizing antibody, BAY11-7082and CXCR2antagonist SB225002on prostate cancer cells-induced pain hypersensitivity were tested by pain behavior after intrathecal injection.Results (1) Unilateral injection of prostate cancer cells into the right femur of mice induced bone destruction and tumor cells invasion into the bone at day14.(2) Cancer cell inoculation also induced persistent mechanical allodynia, heat hyperalgesia and sponteneous pain. The pain hypersensitivity started from7days after injection and lasted for more than21days, suggesting the development of bone cancer pain.(3) Bone cancer induced the upregulation of CXCL1mRNA and protein in the spinal cord. The upregulation of CXCL1mRNA was shown from7days and lasted for21days. The protein increase was started from7days and maintained for21days. Immunofluorescence double staining showed CXCL1was mainly colozalized with astrocytic marker, GFAP, but not with neuronal marker, NeuN or microglial marker, CD11b.(4) In cultured astrocytes, TNF-α stimulation increased CXCL1expression; Pretreatment with NFκB inhibitor BAY11-7082decreased TNF-α-induced upregualtion of CXCL1mRNA and protein.(5) Bone cancer also increased protein expression of P-NFκB in the spinal cord. The p-NFκB protein was significantly increased at7d,14d and21d. Immunofluorescence double staining showed p-NFκB was colocalized with astrocytic marker, GFAP.(6) Bone cancer induced the mRNA and protein upregulation of CXCL1 in the DRGs. The mRNA increase was shown at7days and maintained for more than21days. Meanwhile, immunofluorescence staining showed that the protein was significant increased at7days after injection and lasted for more than21days.(7) Intrathecal injection of CXCL1neutralizing antibody or BAY11-7082partially attenuated cancer-induced mechanical allodynia and heat hyperalgesia.(8) Bone cancer also increased the mRNA and protein expression of CXCR2in the spinal cord. CXCR2mRNA was increased at7d,14d, and21d following injection of cancer cells. The CXCR2protein was also significantly increased at7d,14d and21d. Double immunofluorescent labeling showed CXCR2was colocalized with neuronal marker, NeuN, suggesting its expression in spinal cord neurons.(9) In the DRGs, the mRNA expression of CXCR2was increased at7d,14d and21d after cancer cells injection. Immunofluorescence staining also showed that CXCR2protein was significantly increased at7d,14d and21d.(10) Intrathecal injection of CXCR2antagonist SB225002partially attenuated cancer-induced mechanical allodynia and heat hyperalgesia.Conclusions Unilateral injection of prostate cancer cells into the right femur of mice produced marked bone cancer pain and upregulation of chemokine CXCL1and its receptor CXCR2in the spinal cord and DRG. In the spinal cord, CXCL1and CXCR2were involved in the regulation of bone cancer pain by astroglial-neuronal interaction. Targeting CXCL1-CXCR2may be a potential strategy for treating bone cancer pain.