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Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells.

Oue T, Uehara S, Yamanaka H, Nomura M, Usui N - Pediatr. Surg. Int. (2013)

Bottom Line: Two kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines.The number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line.Hh inhibitors may provide a new paradigm for the treatment of RMS.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Surgery, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan, ooue@pedsurg.med.osaka-u.ac.jp.

ABSTRACT

Purpose: In the treatment of rhabdomyosarcoma (RMS), invasion and metastasis remain the most critical determinants of resectability and survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in the invasion of RMS.

Methods: Two kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines. The effects of the Hh signaling inhibitors on tumor cell invasion and motility were investigated using Matrigel invasion assays and wound closure assays, respectively.

Results: The number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line. Furthermore, the wound closure assays revealed that a blockade of the Hh signaling pathway by the Hh inhibitors strongly impairs RMS cell motility, as visualized by the delayed closure of the gaps generated in the cultured cell monolayers of the three RMS cell lines.

Conclusions: Both the invasive capacity and motility of RMS cells are significantly suppressed by Hh signaling inhibitors, demonstrating that the Hh pathway plays an important role in the invasion of RMS. Hh inhibitors may provide a new paradigm for the treatment of RMS.

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Related in: MedlinePlus

Principle of the Matrigel invasion assay The Matrigel invasion chamber has an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The chambers were placed into the lower chambers filled with the medium supplemented with 5 % FBS as a chemoattractant, therefore cells will invade into BMM and move to the lower surface of the membrane through the 8 μm pores. After a 22-h incubation, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained
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Fig1: Principle of the Matrigel invasion assay The Matrigel invasion chamber has an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The chambers were placed into the lower chambers filled with the medium supplemented with 5 % FBS as a chemoattractant, therefore cells will invade into BMM and move to the lower surface of the membrane through the 8 μm pores. After a 22-h incubation, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained

Mentions: Cell invasion was evaluated using a BioCoat Matrigel invasion chamber (BD Bioscience, Bedford, MA, USA) (Fig. 1). Cell suspensions (5 × 104 cells/ml) of RMS-YM, RD and RH30 cells were prepared in serum-free culture medium in the absence (control) or presence of the Hh inhibitors, cyclopamine (10 μM; Sigma Aldrich Co., Tokyo, Japan) or forskolin (100 μM; Sigma Aldrich Co., Tokyo, Japan). 500 μl of each cell suspension was added to the Matrigel invasion chamber. The chambers have an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The upper chambers were placed into the lower chambers, which were filled with 750 ml of DMEM supplemented with 5 % FBS as a chemoattractant so that the cells would invade the BMM and move toward the lower surface of the membrane through the 8 μm pores. After 22 h of incubation in a tissue culture incubator at 37 °C, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained. The number of invading cells in six random fields was counted using bright field microscopy at 200× magnification. The experiments were performed three times using duplicate samples.Fig. 1


Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells.

Oue T, Uehara S, Yamanaka H, Nomura M, Usui N - Pediatr. Surg. Int. (2013)

Principle of the Matrigel invasion assay The Matrigel invasion chamber has an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The chambers were placed into the lower chambers filled with the medium supplemented with 5 % FBS as a chemoattractant, therefore cells will invade into BMM and move to the lower surface of the membrane through the 8 μm pores. After a 22-h incubation, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824305&req=5

Fig1: Principle of the Matrigel invasion assay The Matrigel invasion chamber has an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The chambers were placed into the lower chambers filled with the medium supplemented with 5 % FBS as a chemoattractant, therefore cells will invade into BMM and move to the lower surface of the membrane through the 8 μm pores. After a 22-h incubation, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained
Mentions: Cell invasion was evaluated using a BioCoat Matrigel invasion chamber (BD Bioscience, Bedford, MA, USA) (Fig. 1). Cell suspensions (5 × 104 cells/ml) of RMS-YM, RD and RH30 cells were prepared in serum-free culture medium in the absence (control) or presence of the Hh inhibitors, cyclopamine (10 μM; Sigma Aldrich Co., Tokyo, Japan) or forskolin (100 μM; Sigma Aldrich Co., Tokyo, Japan). 500 μl of each cell suspension was added to the Matrigel invasion chamber. The chambers have an 8 μm pore size polycarbohydrate membrane and the upper surface of the membrane is coated with a uniform basement membrane matrix (BMM). The upper chambers were placed into the lower chambers, which were filled with 750 ml of DMEM supplemented with 5 % FBS as a chemoattractant so that the cells would invade the BMM and move toward the lower surface of the membrane through the 8 μm pores. After 22 h of incubation in a tissue culture incubator at 37 °C, nonmigratory cells from the upper surface of the filter were removed and invasive cells that had passed through to the lower surface of the filter were fixed and stained. The number of invading cells in six random fields was counted using bright field microscopy at 200× magnification. The experiments were performed three times using duplicate samples.Fig. 1

Bottom Line: Two kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines.The number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line.Hh inhibitors may provide a new paradigm for the treatment of RMS.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Surgery, Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan, ooue@pedsurg.med.osaka-u.ac.jp.

ABSTRACT

Purpose: In the treatment of rhabdomyosarcoma (RMS), invasion and metastasis remain the most critical determinants of resectability and survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in the invasion of RMS.

Methods: Two kinds of specific Hh signaling inhibitors, cyclopamine and forskolin, were used to suppress activated Hh signals in three RMS cell lines. The effects of the Hh signaling inhibitors on tumor cell invasion and motility were investigated using Matrigel invasion assays and wound closure assays, respectively.

Results: The number of invaded cells counted in six random microscopic fields in the Matrigel chambers was significantly decreased by both cyclopamine and forskolin in every RMS cell line. Furthermore, the wound closure assays revealed that a blockade of the Hh signaling pathway by the Hh inhibitors strongly impairs RMS cell motility, as visualized by the delayed closure of the gaps generated in the cultured cell monolayers of the three RMS cell lines.

Conclusions: Both the invasive capacity and motility of RMS cells are significantly suppressed by Hh signaling inhibitors, demonstrating that the Hh pathway plays an important role in the invasion of RMS. Hh inhibitors may provide a new paradigm for the treatment of RMS.

Show MeSH
Related in: MedlinePlus