Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Mol Cancer. 2010 Jan 12;9:5. doi: 10.1186/1476-4598-9-5.
The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.
To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.
These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.
Hedgehog 信号通路在胚胎发育中起组织者的作用。最近的研究表明，Hedgehog 通路在各种类型的恶性肿瘤中持续激活。然而，Hedgehog 通路如何参与骨肉瘤的发病机制仍不清楚。为了探讨异常 Hedgehog 通路在骨肉瘤发病机制中的作用，我们研究了 Hedgehog 通路在骨肉瘤中的表达和激活，并研究了 SMO 抑制的作用。
为了评估 Hedgehog 通路基因的表达，我们使用骨肉瘤细胞系和骨肉瘤活检标本进行了实时 PCR 和免疫组织化学检测。为了评估 SMO 抑制的效果，我们进行了体外细胞活力、集落形成、细胞周期以及体内异种移植模型实验。实时 PCR 显示骨肉瘤细胞系过度表达 Sonic hedgehog、Indian hedgehog、PTCH1、SMO 和 GLI。实时 PCR 显示骨肉瘤活检标本中 SMO、PTCH1 和 GLI2 的过表达。这些发现表明 Hedgehog 通路在骨肉瘤中被激活。SMO 的特异性抑制剂 cyclopamine 抑制 SMO 可减缓骨肉瘤的体外生长。细胞周期分析显示 cyclopamine 促进 G1 期阻滞。cyclopamine 降低了细胞周期加速器的表达，包括 cyclin D1、cyclin E1、SKP2 和 pRb。另一方面，cyclopamine 处理上调了 p21(cip1) w 蛋白。此外，SMO shRNA 敲低可防止骨肉瘤在体外和体内的生长。
这些发现表明，SMO 的失活可能是治疗骨肉瘤患者的一种有效方法。
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