sulforaphane eradicates pancreatic cancer stem cells by nf- b .2 department of molecular immunology,

Download Sulforaphane eradicates pancreatic cancer stem cells by NF- B .2 Department of Molecular Immunology,

Post on 17-Jul-2018

212 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • Georgios Kallifatidis1,3, Vanessa Rausch1,3, B. Baumann 5, A. Apel1,3, B. M. Beckermann1,3, A. Groth1,3, J. Mattern1,3, Z. Li4, A. Kolb4,P. Altevogt6, T. Wirth5, J. Werner4, Gerhard Moldenhauer2, Markus W. Bchler4, Peter Schemmer4, Alexei V. Salnikov1,2, Ingrid Herr1,3,4

    1 Molecular OncoSurgery Group, University of Heidelberg and German Cancer Research Center, Heidelberg, Germany2 Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany

    3 Department of Experimental Surgery, University of Heidelberg, Germany4 Department of General Surgery, University of Heidelberg, Heidelberg, German

    5 Institute of Physiological Chemistry, University of Ulm, Ulm, Germany6 Tumour Immunology Program, German Cancer Research Center, Heidelberg, Germany

    Sulforaphane eradicates pancreatic cancer stem cells by NF-B

    Emerging evidence suggests that cancer stem cells (CSCs) play a central role in the pathogenesis of cancer. We identified CSCs inpancreatic cancer cell lines and patient tumors by expression of CSC markers and correlation to growth on nude mice, differentiationcapacity, clonogenicity, sphere formation and therapy resistance. The chemopreventive agent sulforaphane prevented NFB bindingin CSCs, downregulated apoptosis inhibitors and induced apoptosis along with prevention of clonogenicity.

    MIA-PaCa2 cells were left untreated (CO), orwere treated with Sulforaphane (SF).Twenty-four hours later, TRAIL was addedto untreated (TR) or pre-treated cells(SF+TR) and activity of caspase 8, 9 and 3+7was analyzed by fluorochrome-linkedinhibitors of caspases (FLICA).

    Pancreatic cancer cell lines were left untreated (CO)or were treated with sulforaphane (SF), TRAIL (TR) orSF+TR for time points indicated. NF-B binding wasanalyzed by EMSA. Three different bands becamevisible corresponding to three different NF-Bsubunits binding to the oligonucleotide (1, 2, 3).

    CSC-like characteristics in four human pancreaticcancer cell lines were classified by expression ofspecific surface markers, colony and spheroid formingcapacity. We defined the CD44+/CD24- population asCSC-like phenotype in pancreatic cancer cell lines.

    Expression of CSC surface markers inpancreatic cell lines

    Cells were left untreated (CO) or were treatedwith recombinant TRAIL (5, 25, 50 ng/mL) and 24h later mitochondrial activity/viability wasmeasured by MTT assay. Only BxPC3 cells weresensitive to TRAIL-induced apoptosis.

    TRAIL-sensitivity in pancreatictumor cell lines Sensitivity of CSCs to sulforaphane

    0

    40

    80

    120

    VIA

    BIL

    ITY

    (CO

    =100

    %)

    MIAPaCaACPC-1Capan-1BxPC3

    CO 5 25 50ng/ml TRAIL

    ConclusionsCSCs are present in pancreatic tumors and are resistant towards chemotherapy. We observed specific binding of transactivationpotent c-Rel containing NF-B complexes in CSCs but not in non-CSCs. Sulforaphane prevented NF-B binding along with stronginduction of apoptosis. In a xenograft model, sulforaphane strongly blocked tumor growth and combination with TRAIL had anadditive effect without obvious cytotoxicity to normal cells. Our data suggest combination of sulforaphane with TRAIL aspromising strategy for targeting of pancreatic CSCs.

    Correspondence to: i.herr@dkfz.de

    NFB Complex

    CSChigh

    CSClow

    Complex 3

    Complex 1

    cRELcREL

    p50p50

    SF inhibits binding of transactivationcompetent NF-B dimers in CSChigh cells

    Mice s.c. xenografted with MIA-PaCa2 cellsreceived SF, TRAIL or both agents together atdays 4 to 6 after tumor cell implantation. Thetumor volumes were measured daily.

    In vivo effect of Sulforaphane CD44CD44++/CD24/CD24++ and and CD44CD44++/CD133/CD133++CSCsCSCs correlatecorrelate to to ex vivoex vivo sensitivitysensitivityof of patientpatient tumorstumors

    Isolated primary tumor cells were treated with SF TRAIL. Cells from tumor 2 were sensitivetowards SF and TRAIL, while cells from tumor 1were only sensitive towards SF. The high grade ofresistance of tumor 1 corresponded to thepresence of rare populations of CD44+/CD133+and CD44+/CD24+ cells in the tissue sections.

    Introduction

    Results1 2 3

    4 65

    Viability

Recommended

View more >