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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

Wound closure assays. Confluent monolayers of RMS-YM, RH and RH30 cells were wounded by scratching with a pipette tip. The culture medium was replaced with fresh medium containing either cyclopamine (10 μM) or forskolin (100 μM) to suppress Hh signals. Wound closure was monitored using phase contrast microscopy. Photos were taken immediately (0 h) and every 4 h until the wounds closed. The experiments were repeated three times with similar results. A representative photomicrograph of RH-30 cells in each condition at ×200 magnification is shown. In the control group, the wound was closed at 12 h, whereas in the cyclopamine or forskolin-treated group, the wound was closed at 24 h or later
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Fig4: Wound closure assays. Confluent monolayers of RMS-YM, RH and RH30 cells were wounded by scratching with a pipette tip. The culture medium was replaced with fresh medium containing either cyclopamine (10 μM) or forskolin (100 μM) to suppress Hh signals. Wound closure was monitored using phase contrast microscopy. Photos were taken immediately (0 h) and every 4 h until the wounds closed. The experiments were repeated three times with similar results. A representative photomicrograph of RH-30 cells in each condition at ×200 magnification is shown. In the control group, the wound was closed at 12 h, whereas in the cyclopamine or forskolin-treated group, the wound was closed at 24 h or later

Mentions: The wound closure assays revealed that the Hh inhibitors strongly impair RMS cell motility, as visualized by the delayed closure of the gaps generated in cultured cell monolayers of the three RMS cell lines (Fig. 4). Time to wound closure for the cyclopamine-treated and forskolin-treated RMS cells was significantly delayed compared with that of the non-treated control cells. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RMS-YM cells was 24, 36 and 40 h, respectively. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RD cells was 16, 40 and 36 h, respectively. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RH30 cells was 12, 24 and 28 h, respectively (Fig. 5).Fig. 4


Hedgehog signal inhibitors suppress the invasion of human rhabdomyosarcoma cells.

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

Wound closure assays. Confluent monolayers of RMS-YM, RH and RH30 cells were wounded by scratching with a pipette tip. The culture medium was replaced with fresh medium containing either cyclopamine (10 μM) or forskolin (100 μM) to suppress Hh signals. Wound closure was monitored using phase contrast microscopy. Photos were taken immediately (0 h) and every 4 h until the wounds closed. The experiments were repeated three times with similar results. A representative photomicrograph of RH-30 cells in each condition at ×200 magnification is shown. In the control group, the wound was closed at 12 h, whereas in the cyclopamine or forskolin-treated group, the wound was closed at 24 h or later
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Wound closure assays. Confluent monolayers of RMS-YM, RH and RH30 cells were wounded by scratching with a pipette tip. The culture medium was replaced with fresh medium containing either cyclopamine (10 μM) or forskolin (100 μM) to suppress Hh signals. Wound closure was monitored using phase contrast microscopy. Photos were taken immediately (0 h) and every 4 h until the wounds closed. The experiments were repeated three times with similar results. A representative photomicrograph of RH-30 cells in each condition at ×200 magnification is shown. In the control group, the wound was closed at 12 h, whereas in the cyclopamine or forskolin-treated group, the wound was closed at 24 h or later
Mentions: The wound closure assays revealed that the Hh inhibitors strongly impair RMS cell motility, as visualized by the delayed closure of the gaps generated in cultured cell monolayers of the three RMS cell lines (Fig. 4). Time to wound closure for the cyclopamine-treated and forskolin-treated RMS cells was significantly delayed compared with that of the non-treated control cells. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RMS-YM cells was 24, 36 and 40 h, respectively. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RD cells was 16, 40 and 36 h, respectively. The blockade of the Hh signaling pathway by time to closure of the control, cyclopamine-treated and forskolin-treated RH30 cells was 12, 24 and 28 h, respectively (Fig. 5).Fig. 4

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