Clinical Significance of Receptor-Interacting Protein 3 and Parkin,Essential Molecules for Necroptosis,in Breast Cancer

PurposeReceptor-interacting protein 3 (RIP3) is the main initiator of necroptosis. Parkin prevents the formation of the RIP1–RIP3 complex by promoting polyubiquitination of RIP3. However, the mechanism by which necroptosis affects the clinical features of breast cancer and prognosis is not known. Here, we aimed to study the effect of necroptosis on the clinical features and prognosis of breast cancer by assessing the expression of RIP3 and Parkin.MethodsTissue microarrays (TMAs) were constructed from 257 cases of breast cancer. Immunohistochemistry was performed on 4-μm tissue sections from each TMA block. The χ² test, Kaplan-Meier survival analysis with log-rank test, and Cox regression proportional hazard model were used for statistical analysis.ResultsLow RIP3 expression resulted in a large tumor size and high nuclear grade. Low RIP3 expression was correlated with human epidermal growth factor receptor 2 positivity, short overall survival (OS), and short disease-free survival (DFS). The triple negative breast cancer group with low RIP3 expression and lymph node (LN) positive group with low RIP3 expression had the shortest OS. High Parkin expression was associated with high histological grade, estrogen and/or progesterone receptor negativity, and lymphatic emboli, but was not correlated with OS and DFS. OS was correlated with LN metastasis and RIP3 loss and DFS with large tumor size, LN metastasis, and RIP3 loss.ConclusionLow RIP3 and high Parkin expression are associated with aggressive clinical features in breast cancer. RIP3, a molecular marker of necroptosis, is an independent factor associated with survival in breast cancer. Further in-depth studies are needed to investigate the role of necroptosis in breast cancer development, metastasis, and treatment in the future.     

Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines

Burkitt''s lymphoma (BL) is a highly aggressive form of B-cell non-Hodgkin''s lymphoma. The clinical outcome in children with BL has improved over the last years but the prognosis for adults is still poor, highlighting the need for novel treatment strategies. Here, we report that the combinational treatment with the Smac mimetic BV6 and TRAIL triggers necroptosis in BL when caspases are blocked by zVAD.fmk (TBZ treatment). The sensitivity of BL cells to TBZ correlates with MLKL expression. We demonstrate that necroptotic signaling critically depends on MLKL, since siRNA-induced knockdown and CRISPR/Cas9-mediated knockout of MLKL profoundly protect BL cells from TBZ-induced necroptosis. Conversely, MLKL overexpression in cell lines expressing low levels of MLKL leads to necroptosis induction, which can be rescued by pharmacological inhibitors, highlighting the important role of MLKL for necroptosis execution. Importantly, the methylation status analysis of the MLKL promoter reveals a correlation between methylation and MLKL expression. Thus, MLKL is epigenetically regulated in BL and might serve as a prognostic marker for treatment success of necroptosis-based therapies. These findings have crucial implications for the development of new treatment options for BL.     

Deoxypodophyllotoxin triggers necroptosis in human non-small cell lung cancer NCI-H460 cells

Deoxypodophyllotoxin (DPT), a naturally occurring microtubule destabilizer, inhibits tubulin polymerization and causes cell cycle arrest at G₂/M phase in tumor cells. However, the anti-tumor effect and specific mechanism of DPT in non-small cell lung cancer (NSCLC) are still poorly understood. In this study, we determined the anti-tumor effect and potential mechanism of DPT in the NSCLC cell line, NCI-H460 (H460). First, we demonstrated that DPT significantly inhibits the proliferation of H460 cells in vitro and the growth of H460 xenografts in vivo. In further studies, DPT triggered necroptosis in H460 cells with the following characteristics: (I) necrotic cell death morphology; (II) autophagy; (III) loss of plasma membrane integrity; (IV) loss of mitochondria membrane potential; (V) elevation of reactive oxygen species levels; and (VI) specific inhibition of necroptosis via a small molecule, necrostatin-1. This study also revealed that DPT has a similar effect towards the drug-sensitive cancer cell line, H460, and the drug-resistant cell line, H460/Bcl-xL. To our knowledge, this is the first report to document the induction of necroptosis by a microtubule-targeting agent to circumvent cancer drug resistance, thereby providing a new potential choice for clinical cancer therapy, especially drug-resistant cancer therapy.     

Apelin attenuates streptozotocin-induced learning and memory impairment by modulating necroptosis signaling pathway

Apelin is a neuropeptide that plays an important role in neuronal protection. In this study, we investigated the effects of apelin intracerebroventricular administration on spatial learning and memory-related behaviors, and necroptosis signaling pathways in the hippocampus of streptozotocin (STZ) -injected rats. Apelin treatment was implemented following STZ-induced dementia for 15 days. After conducting a behavioral test (Morris Water Maze), the cellular and molecular aspects were examined to detect the apelin effect on the necroptosis signaling pathway. We demonstrated that STZ administration significantly slowed down the learning capability. However apelin treatment notably reversed this neuroinflammation induced behavioral impairment. Furthermore, molecular investigations showed that apelin treatment reduced the hippocampal RIP1, RIP3, and TNF-α level. Our results suggest that apelin treatment attenuates STZ-induced dementia. This effect may be mediated by inhibition of the necroptosis signaling pathway which seems to be associated with the ability of apelin to reduce central TNF-α level. This data provides evidence of the neuroprotective effect of apelin on STZ-induced learning and memory impairment and characterize some of the underlying mechanisms.     

坏死性凋亡介导高糖引起的人脐静脉内皮细胞损伤

目的:研究坏死性凋亡是否介导高糖(HG)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤。方法:CCK-8法检测细胞存活率;Western blot法测定受体相互作用蛋白3(RIP3)、cleaved caspase-3的蛋白水平;罗丹明123染色荧光显微镜照相法检测线粒体膜电位(mitochondrial membrane potential,MMP);双氯荧光素(DCFH-DA)染色荧光显微镜照相法测定胞内活性氧簇(reactive oxygen species,ROS)的水平。结果:应用不同浓度葡萄糖(10、20和40 mmol/L)处理HUVECs 24 h,RIP3的蛋白水平随葡萄糖剂量增加而升高,40 mmol/L时达高峰;应用40 mmol/L葡萄糖处理HUVECs 3 h、6 h、9 h、12 h和24 h能上调RIP3的蛋白水平,于9 h达最高峰;应用20μmol/L凋亡蛋白酶抑制剂Z-VAD-FMK预处理HUVECs 30 min促进RIP3表达;应用100μmol/L坏死性凋亡抑制剂necrostatin-1预处理HUVECs 1 h能抑制HG诱导HUVECs的细胞存活率降低,ROS过度生成及MMP丢失,但能升高cleaved caspase-3的蛋白水平。结论:坏死性凋亡介导高糖引起的人脐静脉内皮细胞损伤,但与内皮细胞凋亡存在负相关。     

Necroptosis in inflammatory bowel disease and other intestinal diseases

For a long time, it was believed that apoptosis and necrosis were the main pathways for cell death, but a growing body of research has shown that there are other pathways. Among these, necroptosis, a regulatory caspase-independent, programmed cell death pathway, is supposed to be of importance in the pathogenesis of many diseases. The mechanism of regulating, inducing and blocking necroptosis is a complex process that involves expression and regulation of a series of molecules including receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase like protein. By blocking or downregulating expression of key molecules in the necroptotic pathway, intestinal inflammation can be affected to some extent. In this paper, we introduce the concept of necroptosis, its main pathway, and its impact on the pathogenesis of inflammatory bowel disease (IBD) and other intestinal diseases, to explore new drug targets for intestinal diseases, including IBD.     

Necrostatin-1对小鼠脑外伤脑组织损害及行为学能力的影响

目的 探讨细胞程序性坏死特异性抑制剂Necrostatin-1(Nec-1)对脑外伤导致的组织损伤和行为学能力(运动功能、学习能力)损害的影响。方法 健康成熟雄性昆明小鼠(体重20～25 g),采用改良的自由落体装置制作定量右侧脑外伤(Traumatic Brain Injury,TBI)模型,建模15 min前同侧脑室分别注射溶剂二甲基亚砜(DMSO)和DMSO配制的Nec-1溶液分别建立脑外伤DMSO组和脑外伤Nec-1组,同时设立假手术组(Sham组),利用行为学试验(Motor Test和Morris水迷宫试验)评估TBI 2周内各组小鼠的运动功能和学习能力损害,利用脑组织缺损体积(Lesion Volume,LV)评价TBI 3周后脑皮质损伤程度。结果(1)脑外伤后1周内能观察到小鼠运动能力评分降低明显(P<0.01),伤后2～7 d各组运动评分均逐渐恢复。与DMSO组比较,Nec-1预给药能明显加快运动功能的恢复过程(P<0.05);(2)所有TBI小鼠与Sham组比较,在水迷宫定位航行试验中搜找平台时需要更长的潜伏期(P<0.05); Nec-1组在伤后第9～12 d表现较短的潜伏期(P<0.05);(3)脑外伤导致各组小鼠脑组织的缺失(P<0.01),与DMSO组比较,Nec-1预处理明显减少了脑外伤后的脑组织损伤体积(P<0.05)。结论 侧脑室预注射Nec-1抑制程序性坏死可减少小鼠脑外伤后脑组织缺损,促进活动功能、学习能力的恢复。     

PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.     

肾癌中调节性细胞死亡的新动向和未来展望

肾癌是泌尿系统常见的恶性肿瘤之一。近年来,我国肾癌的发病率呈逐年上升的趋势,严重威胁着人们的健康。调节性细胞死亡是由一种细胞主动有序的死亡方式,普遍存在于生命活动过程中,在维系生命活动的平衡中发挥着至关重要的作用。近期Science杂志上报道了一种新的调节性细胞死亡方式即铜死亡,进一步强化了生命体中细胞死亡的重要性。随着对调节性细胞死亡认识的不断深入,越来越多的研究显示不同的调节性细胞死亡（如铁死亡、焦亡、自噬等）均与肾癌的发生、发展密切相关。如诱导细胞铁死亡将显著抑制肾癌的侵袭和转移、并与肾癌患者的更好预后密切相关;细胞焦亡不仅可以诱导肾癌细胞死亡还可以激活抗肾癌的免疫应答;自噬在肾癌中具有“双向”作用,增强自噬可抑制肾癌细胞生长,但也可能减弱联合用药治疗的效果;抑制细胞凋亡和坏死性凋亡可以显著促进肾癌细胞的增殖、侵袭等。本文将综述铁死亡、细胞焦亡、自噬、细胞凋亡和坏死性凋亡的分子机制和在肾癌发生、发展中作用的研究进展并进行展望,为探索肾癌的发病机制和潜在的治疗靶点提供新的视角。