title: Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.
abstract: Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bassani
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Desirèe
authors.initials: D
authors.lastname: Bartolini
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Pagani
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Principi
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.|Ceinge Biotecnologie Avanzate, Naples, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Zollo
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.|Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Noonan
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Adriana
authors.initials: A
authors.lastname: Albini
authors.affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Bruno
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D053453:Caspase 9 / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D017313:Fenretinide / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008527:Medulloblastoma / Q000473:pathology*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000473:pathology; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D060509:Wnt3A Protein / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D060509:Wnt3A Protein; D051176:beta Catenin; D017313:Fenretinide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D053453:Caspase 9