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      Sonic Hedgehog Activation Is Implicated in Diosgenin-Induced Megakaryocytic Differentiation of Human Erythroleukemia Cells

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          Abstract

          Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

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          Most cited references 53

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

           K Livak,  T Schmittgen (2001)
          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Hedgehog signaling in animal development: paradigms and principles.

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              Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened.

              The steroidal alkaloid cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to vertebrate Hedgehog signaling. We show here, using photoaffinity and fluorescent derivatives, that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                16 April 2014
                : 9
                : 4
                Affiliations
                Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, Limoges, France
                Schulze Center for Novel Therapeutics, Mayo Clinic, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LG JLB BL DYL. Performed the experiments: LG YL DYL. Analyzed the data: LG BL DYL. Contributed reagents/materials/analysis tools: YL. Wrote the paper: LG YL DYL.

                PONE-D-13-46882
                10.1371/journal.pone.0095016
                3989280
                24740159

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Pages: 10
                Funding
                This research was supported by grants from the French Ministry of Education and Research and from the Conseil Régional du Limousin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Cell biology
                Signal transduction
                Cell signaling
                Signaling cascades
                MAPK signaling cascades
                Molecular Cell Biology
                Developmental Biology
                Cell Differentiation
                Medicine and Health Sciences
                Hematology
                Hematologic Cancers and Related Disorders
                Leukemias
                Myeloid Leukemia
                Acute Myeloid Leukemia

                Uncategorized

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