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Tumor Biology

, Volume 32, Issue 1, pp 113–127 | Cite as

Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors

  • Mehdi H. Shahi
  • Paula Schiapparelli
  • Mohammad Afzal
  • Subrata Sinha
  • Juan A. Rey
  • Javier S. Castresana
Research Article

Abstract

It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.

Keywords

Sonic hedgehog Neuroblastoma Methylation Cyclopamine GLI1 

Abbreviations

DMEM

Dulbeco’s modified Eagle’s medium

Hh

Hedgehog

HHIP

Hedgehog interacting protein

MCA-Meth

Melting Curve Analysis-based methylation assay

MSP

Methylation-specific PCR

qRT-PCR

Quantitative (real-time) reverse transcribed-PCR

SFRP1

Secreted frizzled-related protein 1

SHH

Sonic hedgehog

Notes

Acknowledgements

Authors are grateful to Laura Stokes for help with editing the manuscript, to Dr. Paula Lázcoz for assisting during culture of neuroblastoma cell lines, and to CIMA, Pamplona, Spain for providing FACS facility. M.H. Shahi was a fellow of AECI (Agencia Española de Cooperación Internacional), Madrid, Spain. J.S. Castresana expresses his gratitude to the Asociación Española de Pediatría, Madrid, for the VIII Premio Nutribén de Investigación Pediátrica. This research was supported in part by grants from the Departmento de Salud del Gobierno de Navarra (9/07), Caja Navarra (08/13912), and Fundación Universitaria de Navarra, Pamplona; Fondo de Investigación Sanitaria (PI081849), and Fundación Mapfre Medicina, Madrid.

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2010

Authors and Affiliations

  • Mehdi H. Shahi
    • 1
    • 2
    • 3
  • Paula Schiapparelli
    • 1
  • Mohammad Afzal
    • 2
  • Subrata Sinha
    • 3
  • Juan A. Rey
    • 4
  • Javier S. Castresana
    • 1
    • 5
  1. 1.Brain Tumor Biology Unit-CIFAUniversity of Navarra School of SciencesPamplonaSpain
  2. 2.Department of ZoologyAligarh Muslim UniversityAligarhIndia
  3. 3.Department of BiochemistryAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Research UnitLa Paz University HospitalMadridSpain
  5. 5.Unidad de Biología de Tumores Cerebrales, CIFAUniversidad de NavarraPamplonaSpain

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