Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of Hh pathway prevents osteosarcoma growth in vitro. A, We performed real-time PCR to determine which concentration of cyclopamine effectively inhibited Hh-GLI activity in osteosarcoma cells, and then measured the expression of the Hh-GLI pathway target PTCH1 and GLI2. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 in 143B cell (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. B, Growth of viable 143B and HOS cells over 3 days was slowed in dose-dependent fashion by cyclopamine treatment. The experiment was triplicate with similar results. C, Colony formation assay revealed cyclopamine reduced colony formation in soft agar. The experiment was triplicate with similar results. (*: P < 0.01) (error bar means S.D.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2818696&req=5

Figure 3: Inhibition of Hh pathway prevents osteosarcoma growth in vitro. A, We performed real-time PCR to determine which concentration of cyclopamine effectively inhibited Hh-GLI activity in osteosarcoma cells, and then measured the expression of the Hh-GLI pathway target PTCH1 and GLI2. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 in 143B cell (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. B, Growth of viable 143B and HOS cells over 3 days was slowed in dose-dependent fashion by cyclopamine treatment. The experiment was triplicate with similar results. C, Colony formation assay revealed cyclopamine reduced colony formation in soft agar. The experiment was triplicate with similar results. (*: P < 0.01) (error bar means S.D.)

Mentions: To determine whether activation of Hh-GLI signaling is required for osteosarcoma cell growth, we used cyclopamine, a pharmacological agent known to effectively block Hh-GLI signaling by inhibiting SMO activation [11]. We performed real-time PCR to determine whether cyclopamine effectively inhibited the expression of the GLI target gene PTCH1 and GLI2 [14]. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 and GLI2 in osteosarcoma cells by more than 60%, consistent with the expected down-regulation of Hh-GLI signaling (Fig. 3A). As cyclopamine was used to prevent cancer cells growth at 10 to 20 μM [15-17] we decided 20 μM was appropriate concentration for osteosarcoma. MTT assay showed that cyclopamine slowed the growth of HOS and 143B in dose-dependent fashion (Fig. 3B). On the other hand, MTT assay showed that proliferation of osteosarcoma cells was enhanced by SHH. We next used a clonogenic assay to determine whether cells capable of forming anchorage-independent colonies were depleted by cyclopamine. This assay revealed cyclopamine reduced colony formation in soft agar (Fig. 3C). These findings suggest that inhibition of SMO inhibited osteosarcoma growth in vitro.


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)

Inhibition of Hh pathway prevents osteosarcoma growth in vitro. A, We performed real-time PCR to determine which concentration of cyclopamine effectively inhibited Hh-GLI activity in osteosarcoma cells, and then measured the expression of the Hh-GLI pathway target PTCH1 and GLI2. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 in 143B cell (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. B, Growth of viable 143B and HOS cells over 3 days was slowed in dose-dependent fashion by cyclopamine treatment. The experiment was triplicate with similar results. C, Colony formation assay revealed cyclopamine reduced colony formation in soft agar. The experiment was triplicate with similar results. (*: P < 0.01) (error bar means S.D.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818696&req=5

Figure 3: Inhibition of Hh pathway prevents osteosarcoma growth in vitro. A, We performed real-time PCR to determine which concentration of cyclopamine effectively inhibited Hh-GLI activity in osteosarcoma cells, and then measured the expression of the Hh-GLI pathway target PTCH1 and GLI2. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 in 143B cell (error bar means S.D.). The comparative Ct (ΔΔCt) method was used to determine fold change in expression using ACTB. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results. B, Growth of viable 143B and HOS cells over 3 days was slowed in dose-dependent fashion by cyclopamine treatment. The experiment was triplicate with similar results. C, Colony formation assay revealed cyclopamine reduced colony formation in soft agar. The experiment was triplicate with similar results. (*: P < 0.01) (error bar means S.D.)
Mentions: To determine whether activation of Hh-GLI signaling is required for osteosarcoma cell growth, we used cyclopamine, a pharmacological agent known to effectively block Hh-GLI signaling by inhibiting SMO activation [11]. We performed real-time PCR to determine whether cyclopamine effectively inhibited the expression of the GLI target gene PTCH1 and GLI2 [14]. Cyclopamine at 20 μM reduced mRNA levels of PTCH1 and GLI2 in osteosarcoma cells by more than 60%, consistent with the expected down-regulation of Hh-GLI signaling (Fig. 3A). As cyclopamine was used to prevent cancer cells growth at 10 to 20 μM [15-17] we decided 20 μM was appropriate concentration for osteosarcoma. MTT assay showed that cyclopamine slowed the growth of HOS and 143B in dose-dependent fashion (Fig. 3B). On the other hand, MTT assay showed that proliferation of osteosarcoma cells was enhanced by SHH. We next used a clonogenic assay to determine whether cells capable of forming anchorage-independent colonies were depleted by cyclopamine. This assay revealed cyclopamine reduced colony formation in soft agar (Fig. 3C). These findings suggest that inhibition of SMO inhibited osteosarcoma growth in vitro.

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