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首页> 外文期刊>Molecular Cancer >Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin)
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Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin)

机译：自噬通量决定了细胞对Apo2L / TRAIL（杜拉明的反应）的死亡和存活

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Background Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines. Methods Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting. Results We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation. Conclusions We show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
机译：背景技术大自噬是一种分解代谢过程，可以介导细胞死亡或存活。已知Apo2配体（Apo2L）/肿瘤坏死因子相关的凋亡诱导配体（TRAIL）处理（TR）会诱导自噬。在这里，我们研究了SQSTM1 / p62（p62）过表达是否作为自噬通量的标志物，与人类前列腺癌（PCa）的侵袭性有关，以及自噬是否调节了敏感但非耐药PCa细胞系的治疗反应。方法采用自噬标志物p62 [在PCa组织微阵列（TMA）和PCa细胞系中]和LC3（在PCa细胞系中），透射电镜和GFP-mCherry-LC3进行免疫染色和免疫印迹分析，研究自噬的诱导和通量。使用药理学（3-甲基腺嘌呤和氯喹）和遗传[（短发夹（sh）介导的ATG7和LAMP2的敲低）和小干扰（si）RNA介导的BECN1敲低]方法对TR的自噬抑制作用对TR的影响通过克隆形成存活，亚G1 DNA含量和流式细胞术对AnnexinV / PI染色评估诱导的细胞死亡。通过免疫印迹确定胱天蛋白酶8的活化。结果我们发现，p62的胞质表达增加与高级别PCa相关，这表明自噬信号可能对高级别肿瘤的存活很重要。抗TR的细胞表现出高自噬通量，在四种抗TR的PCa细胞系C4-2，LNCaP，DU145和CWRv22.1中，p62聚集体的清除效率更高。相反，TR敏感的PC3细胞中自噬通量较低，导致p62聚集体积聚。自噬的药理作用（氯喹或3-甲基腺嘌呤）和遗传作用（shATG7或shLAMP2）抑制导致TR抗性C4-2细胞死亡。表达shATG7的PC3细胞对TR诱导的细胞死亡的敏感性较低，而表达shLAMP2的PC3与表达shControl的PC3细胞一样敏感。使用氯喹抑制自噬通量阻止了p62聚集体的清除，导致caspase-8活化和C4-2细胞中的细胞死亡。在PC3细胞中，抑制自噬诱导可防止p62积累，从而阻止caspase-8激活。结论我们显示p62过度表达与晚期人PCa相关。 PCa细胞系中自噬的药理和遗传抑制作用表明，自噬通量可以通过调节caspase-8激活来确定对TR的细胞应答。因此，组合使用各种自噬抑制剂可能会对TR诱导的细胞死亡产生不同的影响。

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