Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

Aim:

To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action.

Methods:

Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting.

Results:

PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC₅₀ less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity.

Conclusion:

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.     

Systems Biology Analyses Show Hyperactivation of Transforming Growth Factor-β and JNK Signaling Pathways in Esophageal Cancer

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Inhibitors of TLR-4, NF-κB,and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells

The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90α; activities of NF-κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF-κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF-α, IL-1, and IFN-γ production. In addition, inhibitors of the NF-κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF-κB and SAPK/JNK proteins, thereby preventing the activation NF-κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90-α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF-κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors.     

SAPK/JNK信号通路在鼻咽癌放疗群集耐受中的作用研究

目的 用CNE-2Z多细胞球(multicellular spheroids,MCSs)模拟实体瘤,探讨SAPK/JNK信号通路在鼻咽癌放疗群集耐受中的作用.方法 采用liquid overlay技术进行MCSs培养,Western blot法检测X射线照射后信号通路活性变化,SAPK/JNK信号通路特异阻断剂sp-600125作用前后Caspase-3蛋白表达变化;TUNEL法检测sp-600125作用前后、X射线照射后MCSs凋亡率变化.结果 X射线照射后MCSs中SAPK/JNK信号通路表现动态磷酸化过程,其中2h磷酸化水平最高;预先阻断信号通路X射线照射后,细胞凋亡率上调(P＜0.05),caspase-3蛋白表达增高(P＜0.05).结论 SAPK/JNK信号通路短暂激活可能传递生存信号而在鼻咽癌放疗群集耐受中发挥作用,特异性阻断其信号传递上调MCSs凋亡率,这可能与促进caspase-3蛋白表达有关.     

SP600125对大鼠肝缺血再灌注损伤的保护作用

背景：SP600125作为JNK激酶抑制剂,可特异性阻断JNK细胞转导通路,对肝脏缺血再灌注又起到怎样的作用,目前尚无相关研究。 目的：应用SP600125对肝脏缺血再灌注进行干预,分析JNK信号转导通路在该病理生理过程中的作用。 方法：将30只雄性SD大鼠按随机数字表法分为3组,假手术组、缺血再灌注组及SP600125组各10只。假手术组仅行进腹手术;缺血再灌注组行进腹手术,阻断肝左中叶的血供;SP600125组于术前半小时腹腔注射SP600125 15 mg/kg,其他操作同缺血再灌注组。于复灌后2 h取材,分别测定各组血清肝功能酶活性,通过苏木精-伊红切片染色观察肝组织结构的病理变化,运用免疫组织化学法检测肝组织中p-JNK表达并进行半定量分析,以比色法检测肝脏丙二醛含量及髓过氧化物酶活性。 结果与结论：相对于缺血再灌注组,SP600125组血清肝功能酶活性明显下降,肝脏p-JNK表达较低,肝脏髓过氧化物酶活性以及丙二醛含量下降,病理损伤有所缓解。提示JNK细胞信号转导通路在肝缺血再灌注损伤过程中被广泛激活,应用JNK抑制剂SP600125对缺血再灌注损伤有保护作用。     

C-Jun氨基末端激酶信号通路在急性一氧化碳中毒迟发性脑病中的作用

目的探讨c-Jun氨基末端激酶(c-Jun N-terminal kinas,JNK)信号通路在急性一氧化碳中毒迟发性脑病中的作用。方法 120只雄性SD大鼠随机分成空白对照组(BC组)、急性一氧化碳中毒迟发性脑病组(CO组)和JNK抑制剂SP600125组(SP组)3组,每组40只,采用静态吸入式染毒法复制急性一氧化碳中毒迟发性脑病大鼠模型,选取染毒后1、3、7、14、28 d为时相点,应用Morris水迷宫试验检测平均潜伏期等学习记忆能力,免疫组化法检测海马区p-JNK表达,Tunel法检测海马区锥体细胞凋亡。结果 CO组大鼠平均潜伏期较BC组明显延长(P0.01),SP组较CO组明显缩短(P0.01),但仍比BC组延长(P0.01);p-JNK在CO组大鼠海马区表达较BC组明显增强(P0.01),SP组较CO组表达明显减弱(P0.01),较BC组明显增强(P0.01);CO组海马区锥体细胞第3天已经有凋亡增多(9.94%±1.22%),第7和14天增多最明显(39.77%±1.91%,29.72%±4.89%),第28天仍然增多(5.88%±0.55%),凋亡指数与BC组相比有明显增高(P0.01),而SP组凋亡指数与CO组相比明显降低(P0.01)。结论 JNK信号通路参与了急性一氧化碳中毒迟发性脑病的发生,应用其特异性抑制剂SP600125阻断其转导可减少海马区神经元的凋亡,减轻急性CO中毒对学习记忆能力的损害。     

SP600125抑制c-Jun的激活拮抗肾缺血/再灌注诱导的细胞凋亡

目的应用JNK抑制剂SP600125研究转录因子c-Jun的激活对肾缺血/再灌注（I/R）诱导细胞凋亡的影响。方法采用双侧夹闭大鼠肾蒂缺血45 min后再灌注3 h的方法制备肾I/R动物模型。免疫组化法分析转录因子c-Jun的激活变化情况;原位末端标记法（TUNEL）检测肾小管上皮细胞凋亡情况。结果再灌注3h,p-c-Jun的免疫活性显著增强,凋亡的细胞数明显增加。SP600125明显抑制了p-c-Jun的免疫活性且明显减少肾I/R诱导的细胞凋亡。结论肾I/R诱导的细胞凋亡与c-Jun的激活有关,SP600125能明显抑制转录因子c-Jun的激活,减轻肾小管上皮细胞的凋亡。     

SP600125对D-氨基葡萄糖衍生物诱导Eca-109细胞凋亡的影响

目的:观察特异性JNK抑制剂[specificc-junNH2terminalproteinkinase(JNK)inhibitor]SP600125对D-氨基葡萄糖衍生物2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖{[2-(3-carboxy-1-oxopropyl)a-mino-2-deoxy-D-Glucose],COPADG}诱导Eca-109细胞凋亡的影响并探讨COPADG诱导Eca-109细胞凋亡的潜在分子机制。方法:体外培养Eca-109细胞,用COPADG及SP600125对细胞进行处理。细胞间接免疫荧光染色观察P-JNK蛋白表达的改变,倒置相差显微镜观察细胞形态学变化;MTT检测不同时间点的细胞活性;流式细胞术检测细胞凋亡率。结果:经SP600125处理后,COPADG诱导的Eca-109细胞P-JNK蛋白表达明显减弱,同时,COPADG诱导的Eca-109细胞凋亡率明显减低,细胞增殖抑制率下降明显,与COPADG单独作用组之间比较差异有统计学意义。结论:SP600125对D-氨基葡萄糖衍生物CO-PADG诱导Eca-109细胞凋亡具有抑制作用,并间接证明JNK信号转导通路在COPADG诱导Eca-109细胞凋亡过程中发挥着重要作用。     

鞘内注射JNK特异性抑制剂SP600125对CCI大鼠痛行为的影响

Objective To observe the effect of intrathecal administration of SP600125 on both MWT and TWL of rats after chronic constriction injury (CCI) of the sciatic nerve. Methods 40 male SD rats were randomized to deride into 5 groups (n=8). Rats in group SP5 received SP600125 5 μg after CCI; rats in group SP25 received SP600125 25 μg after CCI; rats in group SP50 received SP600125 50 μg after CCI; rats in group DMSO received 2% DMSO 10 μl after CCI; rats in group Naive received SP600125 50 μg without sciatic nerve injury. SP600125 was dissolved in 10 μl 2%DMSO solvent. On the 7th day after CCI, MWT and TWL were determined with yon Frey filaments and thermal radiation apparatus repectively after intrathecal administration of SP600125. Results Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI rather than normal rats. Conclusion Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI.