title: Hedgehog pathway signaling regulates human colon carcinoma HT-29 epithelial cell line apoptosis and cytokine secretion.
abstract: The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.
authors.affiliation: Laboratório Multidisciplinar de Pesquisa, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Agnes N
authors.initials: AN
authors.lastname: Yoshimoto
authors.forename: Claudio
authors.initials: C
authors.lastname: Bernardazzi
authors.forename: Antonio José V
authors.initials: AJ
authors.lastname: Carneiro
authors.forename: Celeste C S
authors.initials: CC
authors.lastname: Elia
authors.forename: Cesonia A
authors.initials: CA
authors.lastname: Martinusso
authors.forename: Grasiella M
authors.initials: GM
authors.lastname: Ventura
authors.forename: Morgana T L
authors.initials: MT
authors.lastname: Castelo-Branco
authors.forename: Heitor S P
authors.initials: HS
authors.lastname: de Souza
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics / Q000378:metabolism; D002087:Butyrates / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D019073:HT29 Cells; D053823:Hedgehog Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014704:Veratrum Alkaloids / Q000494:pharmacology; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: C525809:BMP4 protein, human; C525819:BMP7 protein, human; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D002087:Butyrates; D018932:Chemokine CCL2; D016207:Cytokines; C551027:GANT 61; D053823:Hedgehog Proteins; D016209:Interleukin-8; D051741:Kruppel-Like Transcription Factors; D015513:Oncogene Proteins; D011725:Pyridines; D011743:Pyrimidines; D015534:Trans-Activators; D014704:Veratrum Alkaloids; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D051176:beta Catenin; C000541:cyclopamine