title: Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.
abstract: Activation of the hedgehog (HH) pathway plays a critical role in the development and continued growth of pancreatic adenocarcinoma (PAC). Cyclopamine, a HH pathway inhibitor, has been shown to suppress PAC cell proliferation in vitro and in vivo. However, the molecular basis of response to cyclopamine has not been fully elucidated nor have genes that predict sensitivity to this compound been identified. To better understand these features of HH pathway inhibition, we evaluated the biological and molecular effects of cyclopamine in vitro. The viability of 9 human PAC cell lines following cyclopamine exposure was determined using MTS assay. Proliferation and induction of apoptosis in treated cells were examined by bromo-deoxyuridine incorporation, caspase activation, and mitochondrial membrane potential. Gene expression before and after cyclopamine treatment was determined using Taqman real-time quantitative polymerase chain reaction (RTQ-PCR) and Taqman low-density array (TLDA). Among the cell lines examined, cyclopamine IC50 values ranged from 8.79 to >30 µM. Response to cyclopamine included reduced cell proliferation and induction of apoptosis with and without mitochondrial membrane depolarization. Regression analysis revealed that GLI3 expression significantly correlated with cyclopamine resistance (r = 0.80; p = 0.0102). Knockdown of GLI3 using siRNAs increased sensitivity to cyclopamine. In addition, GLI3 siRNAs decreased PAC cell viability and reduced expression of genes involved in HH signaling (Patched 1 and GLI1) and cell proliferation, similar to cyclopamine. These effects were not observed in PAC cells with undetectable GLI3 expression. These data suggest that Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Steg
authors.forename: Hope M
authors.initials: HM
authors.lastname: Amm
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Novak
authors.forename: Andra R
authors.initials: AR
authors.lastname: Frost
authors.forename: Martin R
authors.initials: MR
authors.lastname: Johnson
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001973:Bromodeoxyuridine; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014704:Veratrum Alkaloids / Q000494:pharmacology / Q000627:therapeutic use*; D000074290:Zinc Finger Protein Gli3
chemical_list: D000970:Antineoplastic Agents; C065683:GLI3 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D014704:Veratrum Alkaloids; D000074290:Zinc Finger Protein Gli3; D020169:Caspases; D001973:Bromodeoxyuridine; C000541:cyclopamine
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