1. Department of Orthopedics, The Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital, Wenzhou 325027, China.
2. Zhejiang Provincial Key Laboratory of Orthopedics, The Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital, Wenzhou 325027, China.
3. Orthopedic Oncology Institute, Department of Orthopedic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
Purpose: This study aimed to investigate the role and mechanism of Hedgehog/GLI1 signaling pathway in regulating the resistance to cisplatin in osteosarcoma (OS).
Materials and methods: Immunohistochemistry, western blotting and qRT-PCR assay were performed to analyze and compare the expression of GLI1 in OS tumor tissue and normal bone tissue as well as in cisplatin sensitive and resistant cell lines (SOSP-9607 and SOSP-9607/CR). Meanwhile, the biological role of GLI1 in OS was investigated by using down-regulated expression of GLI1 and functional assays, including CCK-8, colony formation assay, flow cytometry, and wound healing assay. Moreover, the relationship between GLI1 and γ-H2AX (DNA damage protein) in cells treated with GLI1 siRNA and cisplatin was examined using western blot analysis. In addition, GANT61, a inhibitor of Hedgehog pathway was used in xenograft tumor model to further verify the effect and mechanism of GLI1 on cisplatin resistance in OS.
Results: We showed that GLI1 expression was up-regulated in OS patients and cisplatin-resistant cells. Silencing GLI1 significantly restored the sensitivity of OS to cisplatin, reduced proliferation, migration and cloning capacity of cisplatin sensitive and resistant cells, and increased the apoptosis rate in vitro. Furthermore, combined administration of GANT61 and cisplatin markedly inhibitted tumor growth in the mouse model. Mechanitic studies found that γ-H2AX is involved in the cisplatin resistance, and blockade of Hedgehog/GLI1 pathway increased the expression of γ-H2AX.
Conclusion: Abnormal activation of Hedgehog-GLI1 pathway can regulate the expression of γ-H2AX, thus affecting DNA damage and repair functions, and promoting acquired cisplatin resistance of OS.
Keywords: Hedgehog pathway, GLI1, osteosarcoma, cisplatin resistance, γ-H2AX