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 anti-cancer 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 N-SMase2 expression in Daoy human medulloblastoma cells. Importantly, siRNA-mediated knockdown of N-SMase2 prevented cyclopamine-induced ceramide generation, and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic expression of wild type N-SMase2 caused cell death, compared to controls, expressing the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a down-stream signaling transcription factor of Smo, did not affect N-SMase2 expression, or apoptosis. Mechanistically, our data showed cyclopamine induced N-SMase2 mRNA 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. 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 Nnos/NO-dependent induction of N-SMase2 expression/ceramide axis, independent of Smo/Gli inhibition.

SULF1/SULF2 reactivation during liver damage and tumour growth

“…To examine if Hedgehog signalling plays a role in the growth of Hep3B and PRF5 cells, we initially examined the effect of 5µM, 10µM and 20µM cyclopamine on their growth to rule out any off target effects described for certain cell lines (Meyers-Needham M1 et al 2012). All three cyclopamine concentrations showed a linear dose response although the effect was much lower at 5µM concentration and increased effect was observed at 20 µM concentration that we used in all subsequent experiments in this study.…”

ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation

“…Increases in nSMase2 expression have been reported in response to bone morphogenetic protein-2 (BMP2) (17,18), daunorubicin (19), cigarette smoke (20), confluence (6), the hedgehog signaling mediator cyclopamine (21), and all-trans retinoic acid (ATRA) (5,22,23). Notably, in the latter two cases, increased expression of nSMase2 was required for ATRA-induced growth arrest (23) and cyclopamine-induced apoptosis, respectively (21). Additionally, expression of nSMase2 was increased in mature osteoblasts compared with mesenchymal precursors, consistent with a role for nSMase2 in bone homeostasis (10).…”

“…However, despite multiple known inducers of nSMase2, to date only three transcription factors have been implicated in regulating nSMase2 expression; Sp1 and Sp3 were suggested to be important for daunorubicin and ATRA responses (19,22) while Runx2 was implicated in the BMP2 response in both osteoblasts and chondrocytes (17,18). In addition, cyclopamine induction of nSMase2 required generation of reactive nitrogen species (RNS) and was sensitive to treatment with N-acetylcysteine (NAC) (21). Finally, PKC- was implicated in ATRAmediated nSMase2 induction through mediating Sp1 phosphorylation (22).…”

“…As shown, protein was extracted in RIPA buffer, and nSMase2 levels analyzed by immunoblot using actin as loading control. to various stimuli (17)(18)(19)21). Indeed, Sp1 and Sp3 were reported to be upstream of nSMase2 in the ATRA response, although this was inferred by use of pharmacological inhibition (22).…”

“…We next turned our attention to additional pathways implicated in nSMase2 regulation, namely, ROS/RNS generation and Runx2 (17,18,21). To determine whether ROS are required for ATRA induction of nSMase2, the ROS/RNS scavenger NAC, previously shown to inhibit case when a longer (2,000 bp to +25 bp) promoter construct was utilized (data not shown).…”