SDF-1α/CXCR4Pathway Mediates Radiation-induced Invasiveness In Non-small Cell Lung Carcinoma Cells

Objectives:Non-small cell lung cancer (NSCLC) remains a major cause of cancer-related death worldwide, and radiotherapy is an important modality for the treatment of inoperable NSCLC. Recently, accumulating evidence has shown that irradiation can promote the invasion and metastasis of cancer cells during radiotherapy; however, the underlying mechanisms are not fully understood. This study aimed to investigate the molecular mechanism by which radiation enhances the invasiveness of NSCLC cells.Methods:H1299cells were exposed to2Gy X-ray irradiation. RT-PCR and Western blot analyses revealed the mRNA and protein levels after treatment. The expression and translocation of HIF-1α into the cell nucleus were evidenced by the immunofluorescence assay. The expression of HIF-1α, CXCR4and SDF-la in H1299xenografts of nude mice and human lung cancer tissues were analyzed using immunohistochemistry. Binding of HIF-la to the CXCR4promoter was analyzed by the ChIP assay. The ROS levels of H1299cells were measured by the ROS-sensitive dye DCFH2-DA. The BrdU incorporation assay and MTT assay were used to determine the proliferation rate of H1299cells. Wound healing migration and matrigel invasion assays were applied to detect the invasiveness of H1299assay.Results: We found that the protein levels of HIF-la and CXCR4were upregulated following exposure to2Gy ionizing radiation. After irradiation, HIF-1α was transported into the nucleus, where it bound to the HRE in the CXCR4promoter and promoted the transcription of CXCR4.HIF-1α, CXCR4and SDF-1α were co-localized and showed similar expression in the area of hypoxia in H1299xenografts of nude mice and in human lung cancer tissues. ChIP assay confirmed the direct binding of HIF-1α to the CXCR4promoter in cells exposed to hypoxia, suggesting that CXCR4expression was modulated by HIF-la. Administration of N-acetyl-1-cysteine (NAC), a free radical scavenger, abolished the radiation-induced upregulation of CXCR4, suggesting that ROS play a role in the expression of CXCR4. Our results also revealed that2Gy X-ray irradiation could promote the metastasis and invasiveness of H1299cells. Furthermore, when treated with SDF-la, the invasiveness of irradiated H1299cells was significantly increased. The SDF-1α/CXCR4-specific inhibitor AMD3100or CXCR4shRNA suppressed the increased invasiveness of H1299cells. We also found that the PI3K/pAkt and MAPK/pErkl/2pathways were involved in the radiation-induced matrix metalloproteinase (MMP) expression. We examined MMP-2and MMP-9protein levels after treatment with PI3K/pAkt and MAPK/pERKl/2inhibitors (LY294002and PD98059), and the reduced expression of MMPs confirmed the indispensable role of pAkt and pERKl/2in radiation-induced tumor invasion and metastasis.Conclusions:Taken together, our findings demonstrate that irradiation-induced HIF-1α and ROS regulated CXCR4expression and transduced the signals to intracellular effectors by activating the PI3K/pAkt and MAPK/pErkl/2pathways, which consequently enhanced the invasiveness of H1299cells via the regulation of MMP expression.