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Smoothened as a new therapeutic target for human osteosarcoma.

Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S - Mol. Cancer (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.

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Related in: MedlinePlus

Expression of activated Hh-GLI pathway molecules. Total RNA extracted from osteosarcoma cell lines were used for real-time PCR. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin, GAPDH or ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results.
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Figure 1: Expression of activated Hh-GLI pathway molecules. Total RNA extracted from osteosarcoma cell lines were used for real-time PCR. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin, GAPDH or ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results.

Mentions: To examine the role of Hh�GLI pathway in osteosarcoma, we tested for the expression of Hh in osteosarcoma cell lines. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. To extend these findings, we performed immunocytochemistry for SMO and GLI2, and found that only osteosarcoma cells expressed detectable levels of SMO and GLI2. GLI2 was located in the nuclei of osteosarcoma cells (see additional file 1). We next examined SMO expression in osteosarcoma patient' biopsy specimens. Real-time PCR revealed that 9 of 9 human biopsy specimens of osteosarcoma increased SMO 1.44- to 55.5-fold (Fig. 2). In addition, real-time PCR revealed that expression of PTCH1 was increased in 8 of 9 patients' biopsy samples 2.44- to 29.4-fold (Fig. 2). GLI2 was up-regulated 2.5-to 58.4-fold in 9 of 9 human biopsy specimens of osteosarcoma (Fig. 2). Of most importance was the finding that markers of active Hh�GLI signaling, GLI2 and PTCH1 were consistently up-regulated in the examined osteosarcoma cells, demonstrating the aberrant Hh-GLI pathway activation [12-14]. Our findings suggest that Hh-GLI signaling is active in osteosarcomas.


Smoothened as a new therapeutic target for human osteosarcoma.

Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S - Mol. Cancer (2010)

Expression of activated Hh-GLI pathway molecules. Total RNA extracted from osteosarcoma cell lines were used for real-time PCR. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin, GAPDH or ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Expression of activated Hh-GLI pathway molecules. Total RNA extracted from osteosarcoma cell lines were used for real-time PCR. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin, GAPDH or ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results.
Mentions: To examine the role of Hh�GLI pathway in osteosarcoma, we tested for the expression of Hh in osteosarcoma cell lines. Real-time PCR revealed that 4 of 5 human osteosarcoma cell lines increased Sonic Hedgehog (SHH) 2.1- to 18.8-fold (Fig. 1). In addition, 5 of 5 osteosarcoma cell lines increased Desert Hedgehog 1.3- to 24.4-fold (Fig. 1). To further examine Hh pathway molecules expression, we performed real-time PCR for Hh receptors and Hh target genes. PTCH1 was up-regulated 2.7-to 65.8-fold in 5 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. SMO was up-regulated 2.1-to 5.8-fold in 4 of 5 human osteosarcoma cell lines. GLI1 was up-regulated 2.5-to 8.9-fold in 5 of 5 human osteosarcoma cell lines. GLI2 was up-regulated 1.2-to 9.9-fold in 5 of 5 human osteosarcoma cell lines. To extend these findings, we performed immunocytochemistry for SMO and GLI2, and found that only osteosarcoma cells expressed detectable levels of SMO and GLI2. GLI2 was located in the nuclei of osteosarcoma cells (see additional file 1). We next examined SMO expression in osteosarcoma patient' biopsy specimens. Real-time PCR revealed that 9 of 9 human biopsy specimens of osteosarcoma increased SMO 1.44- to 55.5-fold (Fig. 2). In addition, real-time PCR revealed that expression of PTCH1 was increased in 8 of 9 patients' biopsy samples 2.44- to 29.4-fold (Fig. 2). GLI2 was up-regulated 2.5-to 58.4-fold in 9 of 9 human biopsy specimens of osteosarcoma (Fig. 2). Of most importance was the finding that markers of active Hh�GLI signaling, GLI2 and PTCH1 were consistently up-regulated in the examined osteosarcoma cells, demonstrating the aberrant Hh-GLI pathway activation [12-14]. Our findings suggest that Hh-GLI signaling is active in osteosarcomas.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.

Show MeSH
Related in: MedlinePlus