Shikonin Induced Necroptosis via Reactive Oxygen Species in the T-47D Breast Cancer Cell Line

Breast cancer, the most common cancer in the women, is the leading cause of death. Necrotic signalingpathways will enable targeted therapeutic agents to eliminate apoptosis-resistant cancer cells. In the presentstudy, the effect of shikonin on the induction of cell necroptosis or apoptosis was evaluated using the T-47D breastcancer cell line. The cell death modes, caspase-3 and 8 activities and the levels of reactive oxygen species (ROS)were assessed. Cell death mainly occurred through necroptosis. In the presence of Nec-1, caspase-3 mediatedapoptosis was apparent in the shikonin treated cells. Shikonin stimulates ROS generation in the mitochondriaof T-47D cells, which causes necroptosis or apoptosis. Induction of necroptosis, as a backup-programmed celldeath pathway via ROS stimulation, offers a new strategy for the treatment of breast cancer.     

Cobalt Chloride Induces Necroptosis in Human Colon Cancer HT-29 Cells

Necroptosis, also known as “programmed necrosis”, has emerged as a critical factor in a variety of pathologicaland physiological processes and is considered a cell type-specific tightly regulated process with mechanismsthat may vary rather greatly due to the change of cell line. Here we used HT-29, a human colon cancer cellline, to establish a necroptosis model and elucidate associated mechanisms. We discovered that cobalt chloride,a reagent that could induce hypoxia-inducible factor-1α(HIF1α) expression and therefore mimic the hypoxicmicroenvironment of tumor tissue in some aspects induces necroptosis in HT-29 cells when caspase activityis compromised. On the other hand, apoptosis appears to be the predominant death form when caspases arefunctioning normally. HT-29 cells demonstrated significantly increased RIPK1, RIPK3 and MLKL expressionin response to cobalt chloride plus z-VAD treatment, which was accompanied by drastically increased IL1αand IL6 expression, substantiating the notion that necrosis can induce profound immune reactions. The RIPK1kinase inhibitor necrostatin-1 and the ROS scavenger NAC each could prevent necrosis in HT-29 cells andthe efficiency was enhanced by combined treatment. Thus by building up a necroptosis model in human coloncancer cells, we uncovered that mechanically RIP kinases collaborate with ROS during necrosis promoted bycobalt chloride plus z-VAD, which leads to inflammation. Necroptosis may present a new target for therapeuticintervention in cancer cells that are resistant to apoptotic cell death.     

Targeting of regulated necrosis in kidney disease

The term acute tubular necrosis was thought to represent a misnomer derived from morphological studies of human necropsies and necrosis was thought to represent an unregulated passive form of cell death which was not amenable to therapeutic manipulation. Recent advances have improved our understanding of cell death in acute kidney injury. First, apoptosis results in cell loss, but does not trigger an inflammatory response. However, clumsy attempts at interfering with apoptosis (e.g. certain caspase inhibitors) may trigger necrosis and, thus, inflammation-mediated kidney injury. Second, and most revolutionary, the concept of regulated necrosis emerged. Several modalities of regulated necrosis were described, such as necroptosis, ferroptosis, pyroptosis and mitochondria permeability transition regulated necrosis. Similar to apoptosis, regulated necrosis is modulated by specific molecules that behave as therapeutic targets. Contrary to apoptosis, regulated necrosis may be extremely pro-inflammatory and, importantly for kidney transplantation, immunogenic. Furthermore, regulated necrosis may trigger synchronized necrosis, in which all cells within a given tubule die in a synchronized manner. We now review the different modalities of regulated necrosis, the evidence for a role in diverse forms of kidney injury and the new opportunities for therapeutic intervention.     

RIP1/RIP3通路介导氯化血红素诱导的HT-22海马神经细胞损伤的研究

目的探索受体相互作用蛋白激酶1（RIP1）/RIP3通路是否参与氯化血红素（hemin）诱导的HT-22海马神经细胞损伤以及研究Necrostatin-1（Nec-1）在hemin诱导的HT-22细胞死亡中潜在的神经保护作用。 方法给予不同浓度的hemin（0、25、50、100 μmol/L）作用HT-22细胞24 h后测定碘化丙啶（PI）阳性细胞数和细胞存活率。用Necrostatin-1、zVAD和活性氧（ROS）清除剂叔丁基茴香醚（BHA）分别处理hemin诱导的HT-22细胞,24 h后分别测定各组PI⁺细胞数、细胞存活率,使用MitoSox Red指示ROS水平。最后研究使用siRNA敲低RIP3水平对hemin诱导HT-22细胞死亡的影响,测定各组PI⁺细胞数、细胞存活率及ROS水平。 结果Hemin可剂量依赖性的诱导HT-22神经细胞死亡;RIP1抑制剂Necrostatin-1可显著抑制hemin诱导的HT-22细胞死亡,PI⁺细胞数明显减少,细胞存活率提高,并且减少ROS积聚;BHA可显著减少hemin诱导的HT-22细胞的PI⁺细胞数。更进一步的使用siRNA敲低RIP3表达水平可以显著减少hemin诱导的HT-22细胞死亡,PI⁺细胞数明显减少,细胞存活率明显提高,ROS积聚水平显著减低。 结论RIP1/RIP3通路及ROS可能介导hemin诱导的HT-22海马神经元细胞死亡,Necrostatin-1在其中起神经保护作用。     

Naturally-occurring shikonin analogues – A class of necroptotic inducers that circumvent cancer drug resistance

We previously reported that shikonin could circumvent drug resistance mediated by P-gp, Bcl-2 and Bcl-xL, by induction of necroptosis. Here, we show that the naturally-occurring shikonin analogues (deoxyshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, α-methyl-n-butylshikonin) could bypass drug resistance mediated by not only P-gp, Bcl-2, and Bcl-xL, but also two additional important drug-resistant factors MRP1 and BCRP1, by induction of necroptosis. The results strengthen the previous findings that necroptotic induction could circumvent a broad spectrum of cancer drug resistance.     

A novel necroptosis-associated miRNA signature predicting prognosis of endometrial cancer and correlated with immune infiltration

ObjectiveNecroptosis is a form of programmed cell death identified irrelevant to caspases, which plays an important role in the tumorigenesis and development of cancer. MicroRNAs (miRNAs) are important regulators of both necroptosis and cancer.Materials and methodsExpression of sixteen necroptosis-associated miRNAs were analyzed in 546 endometrial cancer (EC) tissues and 33 paracancerous samples from the Cancer Genome Atlas (TCGA). Cox regression analysis was used to evaluate the correlations between miRNAs and overall survival. MiRNAs risk score (Mrs) and nomogram were established to assess the potential value of necroptosis-related miRNAs on prognosis. Expression of miRNA-148a-3p in endometrial cancer cells and endometrial epithelial cells was detected by quantitative real-time PCR (qRT-PCR). The targets genes of miR-148a-3p were predicted using miRDB, miRTarBase and TargetScan and the prognostic-related genes were screened. Immune infiltration analysis was conducted to explore the potential mechanism of these target genes.ResultsWe identified fourteen differentially expressed miRNAs and selected seven miRNAs (miR-15a-5p, miR148a-3p, miR-7-5p, miR-141–3p, miR-200a-5p, miR-223–3p, miR-16–5p) for prognostic-model construction. The area under the curve (AUC) of receiver operating characteristic (ROC) curve for 1-, 2- and 5-year survival were 0.678, 0.652 and 0.656 respectively. Multivariate analysis revealed that the Mrs was an independent prognostic factor considering other risk factors (HR = 1.928, 95% CI = 1.072–3.467, P = 0.028). Among these miRNAs, miRNA-148a-3p was up-regulated in cancer tissues and cells, and Kaplan–Meier analysis showed its significance in overall survival (OS). The target genes, DNAJB4 and PRNP, were associated with poor prognosis and correlated with tumor immune infiltration.ConclusionsOur study constructed a novel necroptosis-associated miRNAs model for prognosis prediction, and DNAJB4 and PRNP may be therapeutic targets for EC.     

NET formation can occur independently of RIPK3 and MLKL signaling

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor‐interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain‐like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3‐deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.     

ATP敏感性钾通道在硫化氢抑制坏死性凋亡介导的高糖致H9c2心肌细胞炎症中的作用

目的:探讨ATP敏感性钾通道(K_ATP通道)在硫化氢(H2S)抑制坏死性凋亡介导的高糖(HG)致H9c2心肌细胞炎症中的作用。方法:应用Western blot法测定受体相互作用蛋白3(RIP3)和环氧化酶-2(COX-2)的表达水平;ELISA检测细胞培养液中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平。结果:H9c2心肌细胞经HG(35 mmol/L葡萄糖)处理24 h,其RIP3的表达水平明显升高,100μmol/L K_ATP通道开放剂二氮嗪(DZ)和400μmol/L H_2S的供体硫氢化钠(Na HS)预处理心肌细胞30 min均可抑制HG对RIP3表达的上调;100μmol/L K_ATP通道阻断剂5-羟基癸酸(5-HD)预处理心肌细胞30 min可阻断Na HS对HG上调RIP3表达的抑制作用。另一方面,100μmol/L坏死性凋亡的特异性抑制剂necrostatin-1共处理或100μmol/L DZ、400μmol/L Na HS预处理心肌细胞均能抑制高糖引起的心肌细胞炎症,使COX-2表达及IL-1β和TNF-α的分泌水平均减少;而100μmol/L 5-HD能明显拮抗Na HS的上述抗炎症反应作用。结论:K_ATP通道在H_2S抑制坏死性凋亡介导的高糖致心肌细胞炎症反应中发挥重要的作用。     

抑制SHARPIN激活Rip1促进LNCaP-AI细胞坏死性凋亡

目的:探讨SHARPIN对去势抵抗性前列腺癌细胞LNCaP-AI坏死性凋亡关键因子Rip1的调控作用,以及对细胞坏死性凋亡的影响。方法:将LNCaP-AI细胞分为TNF-α+Z-VAD(caspase抑制剂)处理组与TNF-α+Z-VAD+Nec-1(Rip1抑制剂)处理组,应用MTS检测各组细胞的活力,研究坏死性凋亡机制在诱导LNCaP-AI细胞死亡中的作用。将LNCaP-AI细胞分为阴性对照组和SHARPIN干扰(si-SHARPIN)组,RT-qPCR验证抑制效率,通过免疫荧光等技术进一步探讨SHARPIN调控坏死性凋亡的具体分子机制。结果:与对照组相比,TNF-α+Z-VAD处理组的LNCaP-AI细胞活力下降28%(P0.05),而TNF-α+Z-VAD+Nec-1处理组的细胞在Rip1被抑制后,细胞活力无明显改变。在LNCaP-AI细胞中,通过siRNA抑制SHARPIN表达后,Rip1表达水平上调,同时,LNCaP-AI细胞坏死性凋亡比例升高。结论:LNCaP-AI细胞可通过坏死性凋亡机制诱导死亡,下调SHARPIN可能通过激活Rip1增强LNCaP-AI细胞坏死性凋亡。     

Necrostatin-1对创伤失血性休克大鼠肝脏高迁移率族蛋白B1表达的影响

目的 探讨程序性坏死特异性抑制剂-1(necrostatin-1,Nec-1)对创伤失血性休克大鼠肝脏HMGB-1的影响及其机制.方法 采用创伤失血性大鼠休克模型,将96只雄性SD大鼠按随机数字表法随机分为假手术组、DMSO组、Nec-1组,每组32只.假手术组仅进行麻醉、分离血管、结扎血管,并不进行创伤失血和再灌注.DMSO组建立创伤失血性休克大鼠模型,再灌注前5min前股静脉给予DMSO溶剂.Nec-1组于再灌注5 min股静脉给予Nec-1(1 mg/kg).于再灌注后分别在2、8、16、24 h各处死动物8只,取动物血清及肝脏组织.检测血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平;HE染色观察肝脏病理变化;透射电镜观察再灌注后24h后细胞器水平的细胞坏死;酶联免疫分析法(ELISA)分析血清中HMGB-1含量;蛋白质免疫印迹法(western blotting)分别检测肝脏组织中胞质和总蛋白的HMGB-1含量.结果 Nec-1组与DMSO组比较,血清ALT在8 h(P＜0.05)、16 h(P＜0.01)、24 h(P＜0.01)表达较低,Nec-1组血清AST在8 h(P＜0.01)、16h (P ＜0.01)、24h(P＜0.01)表达较DMSO组低;与DMSO组比较,Nec-1组血清HMGB-1在8 h(P＜0.05)、16 h(P＜0.01)、24h (P＜0.01)有明显下降.光镜及电镜下DMSO组及Nec-1组可见肝小叶结构破坏,淤血,炎性细胞浸润及细胞器损伤,Nec-1组肝组织损伤明显减轻;与DMSO组比较,Nec-1组肝细胞中胞质蛋白HMGB-1在8h (P＜0.01)、16h (P＜0.01)、24 h(P＜0.01)有明显下降,Nec-1组总蛋白HMGB-1在8h (P＜0.05)、24 h(P＜0.05)有明显下降.结论 Nec-1可以有效降低创伤失血性休克对肝脏的损伤,减少HMGB-1的释放,有效保护肝细胞.