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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

Cyclopamine treatment promotes G1 arrest. A, HOS and143B cells were treated with 10 μM cyclopamine. After 48-hour treatment cells were collected and subjected to cell cycle analysis. When 143B cells were cultured without cyclopamine, 39.8% of cells were in G1 phase. On the other hand, when cultured with cyclopamine, 56.6% of cells were in G1 phase. In the case of HOS cells cultured without GSI, 55.4% of cells were in G1 phase, while 72.3% of cells were in G1 phase when treated with cyclopamine (error bar means S.D.). B, Real-time PCR was performed to quantify mRNAs of cell cycle related genes. Twenty-four-hour treatment with cyclopamine reduced levels of cyclin D1, Cyclin E1, SKP2, and NMYC transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. C, Western blot analysis of levels of cell cycle-related genes. Forty-eight-hour treatment with cyclopamine reduced levels of expression of cyclin D1, cyclin E1, SKP2, and phosphprylated RB (pRb) proteins. Expression of P21cip1 protein was upregulated by cyclopamine treatment. The experiment was triplicate with similar results (cyclopamine: 10 μM).
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Figure 4: Cyclopamine treatment promotes G1 arrest. A, HOS and143B cells were treated with 10 μM cyclopamine. After 48-hour treatment cells were collected and subjected to cell cycle analysis. When 143B cells were cultured without cyclopamine, 39.8% of cells were in G1 phase. On the other hand, when cultured with cyclopamine, 56.6% of cells were in G1 phase. In the case of HOS cells cultured without GSI, 55.4% of cells were in G1 phase, while 72.3% of cells were in G1 phase when treated with cyclopamine (error bar means S.D.). B, Real-time PCR was performed to quantify mRNAs of cell cycle related genes. Twenty-four-hour treatment with cyclopamine reduced levels of cyclin D1, Cyclin E1, SKP2, and NMYC transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. C, Western blot analysis of levels of cell cycle-related genes. Forty-eight-hour treatment with cyclopamine reduced levels of expression of cyclin D1, cyclin E1, SKP2, and phosphprylated RB (pRb) proteins. Expression of P21cip1 protein was upregulated by cyclopamine treatment. The experiment was triplicate with similar results (cyclopamine: 10 μM).

Mentions: We examined cell cycle characteristics by flow cytometry. Of 143B cells cultured without cyclopamine, 39.8% of cells were in G1 phase, while 56.6% of cells were in G1 phase following treatment with cyclopamine. In the case of HOS cells were cultured without cyclopamine, 55.4% cells were in G1 phase. On the other hand, when cultured with cyclopamine, 72.3% of cells were in G1 phase (Fig. 4A). These findings suggested that cyclopamine promoted G1 arrest. We then examined the transcription of cell cycle-related genes. Real-time PCR revealed that cyclopamine prevented the transcription of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and NMYC (Fig. 4B). In mammalian cells, cyclin D, cyclin E, and p21cip1 are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. We performed western blot analysis to determine protein levels, and found that cyclopamine reduced the levels of expression of cyclin D1 and cyclin E1 proteins. Cyclopamine also reduced the levels of expression of cyclin D1, cyclin E1, pRb, and SKP2 proteins (Fig. 4C). We next examined the expression of p21cip1, and found that p21cip1 protein was up-regulated by cyclopamine treatment (Fig. 4C). These findings suggested that cyclopamine promoted G1 arrest by inhibition of G1-S phase progression. These findings suggest that inhibition of SMO inhibited osteosarcoma growth via cell cycle regulation.


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)

Cyclopamine treatment promotes G1 arrest. A, HOS and143B cells were treated with 10 μM cyclopamine. After 48-hour treatment cells were collected and subjected to cell cycle analysis. When 143B cells were cultured without cyclopamine, 39.8% of cells were in G1 phase. On the other hand, when cultured with cyclopamine, 56.6% of cells were in G1 phase. In the case of HOS cells cultured without GSI, 55.4% of cells were in G1 phase, while 72.3% of cells were in G1 phase when treated with cyclopamine (error bar means S.D.). B, Real-time PCR was performed to quantify mRNAs of cell cycle related genes. Twenty-four-hour treatment with cyclopamine reduced levels of cyclin D1, Cyclin E1, SKP2, and NMYC transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. C, Western blot analysis of levels of cell cycle-related genes. Forty-eight-hour treatment with cyclopamine reduced levels of expression of cyclin D1, cyclin E1, SKP2, and phosphprylated RB (pRb) proteins. Expression of P21cip1 protein was upregulated by cyclopamine treatment. The experiment was triplicate with similar results (cyclopamine: 10 μM).
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Related In: Results  -  Collection

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Figure 4: Cyclopamine treatment promotes G1 arrest. A, HOS and143B cells were treated with 10 μM cyclopamine. After 48-hour treatment cells were collected and subjected to cell cycle analysis. When 143B cells were cultured without cyclopamine, 39.8% of cells were in G1 phase. On the other hand, when cultured with cyclopamine, 56.6% of cells were in G1 phase. In the case of HOS cells cultured without GSI, 55.4% of cells were in G1 phase, while 72.3% of cells were in G1 phase when treated with cyclopamine (error bar means S.D.). B, Real-time PCR was performed to quantify mRNAs of cell cycle related genes. Twenty-four-hour treatment with cyclopamine reduced levels of cyclin D1, Cyclin E1, SKP2, and NMYC transcription (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. C, Western blot analysis of levels of cell cycle-related genes. Forty-eight-hour treatment with cyclopamine reduced levels of expression of cyclin D1, cyclin E1, SKP2, and phosphprylated RB (pRb) proteins. Expression of P21cip1 protein was upregulated by cyclopamine treatment. The experiment was triplicate with similar results (cyclopamine: 10 μM).
Mentions: We examined cell cycle characteristics by flow cytometry. Of 143B cells cultured without cyclopamine, 39.8% of cells were in G1 phase, while 56.6% of cells were in G1 phase following treatment with cyclopamine. In the case of HOS cells were cultured without cyclopamine, 55.4% cells were in G1 phase. On the other hand, when cultured with cyclopamine, 72.3% of cells were in G1 phase (Fig. 4A). These findings suggested that cyclopamine promoted G1 arrest. We then examined the transcription of cell cycle-related genes. Real-time PCR revealed that cyclopamine prevented the transcription of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and NMYC (Fig. 4B). In mammalian cells, cyclin D, cyclin E, and p21cip1 are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. We performed western blot analysis to determine protein levels, and found that cyclopamine reduced the levels of expression of cyclin D1 and cyclin E1 proteins. Cyclopamine also reduced the levels of expression of cyclin D1, cyclin E1, pRb, and SKP2 proteins (Fig. 4C). We next examined the expression of p21cip1, and found that p21cip1 protein was up-regulated by cyclopamine treatment (Fig. 4C). These findings suggested that cyclopamine promoted G1 arrest by inhibition of G1-S phase progression. These findings suggest that inhibition of SMO inhibited osteosarcoma growth via cell cycle regulation.

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