title: Off-target function of the Sonic hedgehog inhibitor cyclopamine in mediating apoptosis via nitric oxide-dependent neutral sphingomyelinase 2/ceramide induction.
abstract: Sonic hedgehog (SHh) signaling is important in the pathogenesis of various human cancers, such as medulloblastomas, and it has been identified as a valid target for anticancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramide's roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectively via induction of neutral sphingomyelin phosphodiesterase 3, SMPD3 (nSMase2) in Daoy human medulloblastoma cells. Importantly, short interfering RNA-mediated knockdown of nSMase2 prevented cyclopamine-induced ceramide generation and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic wild-type N-SMase2 caused cell death, compared with controls, which express the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a downstream signaling transcription factor of Smo, did not affect nSMase2. Mechanistically, our data showed that cyclopamine induced nSMase2 and cell death selectively via increased nitric oxide (NO) generation by neuronal-nitric oxide synthase (n-NOS) induction, in Daoy medulloblastoma, and multiple other human cancer cell lines. Knockdown of n-NOS prevented nSMase2 induction and cell death in response to cyclopamine. Accordingly, N-SMase2 activity-deficient skin fibroblasts isolated from homozygous fro/fro (fragilitas ossium) mice exhibited resistance to NO-induced cell death. Thus, our data suggest a novel off-target function of cyclopamine in inducing apoptosis, at least in part, by n-NOS/NO-dependent induction of N-SMase2/ceramide axis, independent of Smo/Gli inhibition.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: Marisa
authors.initials: M
authors.lastname: Meyers-Needham
authors.forename: Jocelyn A
authors.initials: JA
authors.lastname: Lewis
authors.forename: Salih
authors.initials: S
authors.lastname: Gencer
authors.forename: R David
authors.initials: RD
authors.lastname: Sentelle
authors.forename: Sahar A
authors.initials: SA
authors.lastname: Saddoughi
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Clarke
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
authors.forename: Haakan
authors.initials: H
authors.lastname: Norell
authors.forename: Telma Martins
authors.initials: TM
authors.lastname: da Palma
authors.forename: Michael
authors.initials: M
authors.lastname: Nishimura
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Kraveka
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Khavandgar
authors.forename: Monzur
authors.initials: M
authors.lastname: Murshed
authors.forename: M Ozgur
authors.initials: MO
authors.lastname: Cevik
authors.forename: Besim
authors.initials: B
authors.lastname: Ogretmen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D002518:Ceramides / Q000096:biosynthesis; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D018384:Oxidative Stress; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors; D000072086:Smoothened Receptor; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014704:Veratrum Alkaloids / Q000494:pharmacology*
chemical_list: D002518:Ceramides; D053823:Hedgehog Proteins; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D014704:Veratrum Alkaloids; D009569:Nitric Oxide; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase; C000541:cyclopamine