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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

Matrigel invasion assays RMS-YM, RH and RH30 cells were seeded onto a Matrigel invasion chamber containing either cyclopamine (10 μM) or forskolin (100 μm) for the cell invasion assay and incubated for 22 h. The cells that actively migrated to the lower surface of the filters were stained and counted using bright field microscopy at ×200 in 6 random fields. The photographs show the typical staining of the RMS-YM cells in the control group, cyclopamine (10 μM) treated group and forskolin (100 μm) treated group
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Fig2: Matrigel invasion assays RMS-YM, RH and RH30 cells were seeded onto a Matrigel invasion chamber containing either cyclopamine (10 μM) or forskolin (100 μm) for the cell invasion assay and incubated for 22 h. The cells that actively migrated to the lower surface of the filters were stained and counted using bright field microscopy at ×200 in 6 random fields. The photographs show the typical staining of the RMS-YM cells in the control group, cyclopamine (10 μM) treated group and forskolin (100 μm) treated group

Mentions: We employed cyclopamine and forskolin (specific inhibitors of the Hedgehog pathway) to block the Hh pathway in the RMS cell lines and then assessed the changes in the invasive potential of the cells. The Matrigel invasion assays indicated that RD cells exhibit the strongest invasive potential. As shown in Figs. 2 and 3, the number of invaded cells counted in six random microscopic fields in the Matrigel chamber was significantly decreased by both cyclopamine and forskolin in every RMS cell line. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RMS-YM cells was 145.2 + 55.5, 27.2 + 7.9 and 43.0 + 16.3 cells (P < 0.01)/6 random microscopic fields, respectively. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RD cells was 190.7 + 67.2, 77.3 + 29.0 and 131.5 + 22.7 cells (P < 0.05)/6 random microscopic fields, respectively. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RH30 cells was 104.3 + 14.1, 62.2 + 16.0 and 51.3 + 21.9 cells (P < 0.05)/6 random microscopic fields, respectively.Fig. 2


Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells.

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

Matrigel invasion assays RMS-YM, RH and RH30 cells were seeded onto a Matrigel invasion chamber containing either cyclopamine (10 μM) or forskolin (100 μm) for the cell invasion assay and incubated for 22 h. The cells that actively migrated to the lower surface of the filters were stained and counted using bright field microscopy at ×200 in 6 random fields. The photographs show the typical staining of the RMS-YM cells in the control group, cyclopamine (10 μM) treated group and forskolin (100 μm) treated group
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Matrigel invasion assays RMS-YM, RH and RH30 cells were seeded onto a Matrigel invasion chamber containing either cyclopamine (10 μM) or forskolin (100 μm) for the cell invasion assay and incubated for 22 h. The cells that actively migrated to the lower surface of the filters were stained and counted using bright field microscopy at ×200 in 6 random fields. The photographs show the typical staining of the RMS-YM cells in the control group, cyclopamine (10 μM) treated group and forskolin (100 μm) treated group
Mentions: We employed cyclopamine and forskolin (specific inhibitors of the Hedgehog pathway) to block the Hh pathway in the RMS cell lines and then assessed the changes in the invasive potential of the cells. The Matrigel invasion assays indicated that RD cells exhibit the strongest invasive potential. As shown in Figs. 2 and 3, the number of invaded cells counted in six random microscopic fields in the Matrigel chamber was significantly decreased by both cyclopamine and forskolin in every RMS cell line. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RMS-YM cells was 145.2 + 55.5, 27.2 + 7.9 and 43.0 + 16.3 cells (P < 0.01)/6 random microscopic fields, respectively. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RD cells was 190.7 + 67.2, 77.3 + 29.0 and 131.5 + 22.7 cells (P < 0.05)/6 random microscopic fields, respectively. The mean invasiveness for the control, cyclopamine-treated and forskolin-treated RH30 cells was 104.3 + 14.1, 62.2 + 16.0 and 51.3 + 21.9 cells (P < 0.05)/6 random microscopic fields, respectively.Fig. 2

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