Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death
Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death
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DOI：
10.1093/carcin/bgn147
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发表时间：
2008-10-01
期刊：
影响因子：
4.7
通讯作者：
Westermann, Frank
中科院分区：
文献类型：
--
作者：
Sagulenko, Vitalia;Muth, Daniel;Westermann, Frank
化疗耐药的高发生率是MYCN扩增的神经母细胞瘤亚群治疗失败的主要原因。我们之前报道过异位MYCN表达促进神经母细胞瘤Tet21N细胞的增殖，同时使其对药物诱导的凋亡敏感。为了寻找与mycn依赖性耐药调控相关的基因，我们采用了基于功能的基因克隆方法，鉴定出了编码溶酶体天冬氨酸蛋白酶组织蛋白酶D的CTSD。通过RNA干扰或抑制组织蛋白酶D的酶活性下调组织蛋白酶D的表达会增加表达mycn的Tet21N细胞对阿霉素的敏感性。组织蛋白酶D在Tet21N细胞中的过度表达可减弱阿霉素诱导的细胞凋亡。伴有蛋白激酶B （Akt）的激活和Bcl-2的持续抗凋亡活性。在原发性神经母细胞瘤中，高CTSD信使RNA （mRNA）水平与MYCN扩增相关，MYCN是不良结局的一个强有力的预测指标。染色质免疫沉淀和荧光素酶启动子分析显示，MYCN蛋白与CTSD启动子结合并激活其转录，这表明MYCN与CTSD mRNA表达之间存在直接联系。我们进一步表明，神经母细胞瘤细胞可以分泌有丝分裂原蛋白酶D，而MYCN的表达，特别是阿霉素治疗促进了蛋白酶D的分泌。细胞外外源性组织蛋白酶D诱导敏感细胞Akt-1磷酸化和阿霉素耐药。这些结果证明了组织蛋白酶D在神经母细胞瘤细胞抗凋亡信号传导中的重要作用，并提示了神经母细胞瘤化疗耐药发展的新机制。
High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.