Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Mol Cancer Ther. 2012 May;11(5):1092-102. doi: 10.1158/1535-7163.MCT-11-0705. Epub 2012 Mar 27.
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.
Sonic hedgehog (SHh) 信号通路在多种人类癌症的发病机制中起重要作用，如髓母细胞瘤，它已被确定为抗癌治疗的有效靶点。SHh 抑制剂环巴胺诱导细胞凋亡。生物活性神经酰胺介导细胞死亡对各种化疗药物的反应；然而，神经酰胺在环巴胺诱导的细胞凋亡中的作用/机制尚不清楚。在这里，我们报告环巴胺通过诱导中性鞘磷脂磷酸二酯酶 3（nSMase2）选择性地介导神经酰胺的产生，在 Daoy 人髓母细胞瘤细胞中。重要的是，nSMase2 的短发夹 RNA 介导的敲低可防止环巴胺诱导的神经酰胺生成，并保护 Daoy 细胞免受药物诱导的凋亡。因此，与表达无催化活性的 nSMase2 突变体的对照相比，外源性野生型 N-SMase2 导致细胞死亡。有趣的是，Smo（环巴胺的靶蛋白）或 Gli1（Smo 的下游信号转导转录因子）的敲低并不影响 nSMase2。从机制上讲，我们的数据表明，在 Daoy 髓母细胞瘤和多种其他人类癌细胞系中，环巴胺通过神经元一氧化氮合酶（n-NOS）诱导的一氧化氮（NO）生成选择性地诱导 nSMase2 和细胞死亡。n-NOS 的敲低可防止 nSMase2 诱导和对环巴胺的细胞死亡。因此，来自纯合 fro/fro（脆性骨病）小鼠的 N-SMase2 活性缺陷型皮肤成纤维细胞对 NO 诱导的细胞死亡表现出抗性。因此，我们的数据表明，环巴胺在诱导细胞凋亡方面具有新的非靶点功能，至少部分是通过 n-NOS/NO 依赖性诱导 N-SMase2/神经酰胺轴来实现的，而不依赖于 Smo/Gli 抑制。
