p38 MAPK activation is required for esculetin-induced inhibition of vascular smooth muscle cells proliferation

The phenolic compound esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell-cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes. These novel results regarding the molecular mechanism of esculetin action suggest new preventive and therapeutic treatments for atherosclerosis.     

YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway

Background and purpose:

The aim of this study was to elucidate the mechanism of YC-1{3-(5′-hydroxy methyl-2′-furyl)-1-benzylindazole}-induced human renal carcinoma cells apoptosis and to evaluate the potency of YC-1 in models of tumour growth in mice.

Experimental approach:

YC-1-mediated apoptosis was assessed by analysis of MTT, SRB, DAPI staining and flow cytometry analysis. Knockdown of JNK protein was achieved by transient transfection using siRNA. The mechanisms of action of YC-1 on different signalling pathways involved were studied using western blot. Fas clustering was analysed by confocal microscopy and in vivo efficacy was examined in a A498 xenograft model.

Key results:

YC-1 displayed cytotoxicity in renal carcinoma cells at 10⁻⁷–10⁻⁸ M. Increased condensation of chromatin was observed and an increase in the cell population in subG1 phase. Moreover, YC-1 triggered mitochondria-mediated and caspase-dependent pathways. YC-1 significantly induced Fas ligand expression, but did not modify either the protein levels of death receptors or ligands. In addition, Fas clustering in cells responsive to YC-1 was observed, suggesting involvement of a Fas-mediated pathway. Furthermore, YC-1 markedly induced phosphorylation of JNK and a JNK inhibitor, SP600125, and siRNA JNK1/2 significantly reversed YC-1-induced cytotoxicity and protein expression. We suggest that YC-1 induced JNK phosphorylation, the upregulation of FasL and Fas receptor clustering to promote the activation of caspases 8 and 3, resulting in apoptosis. Finally, we demonstrated the antitumour effect of YC-1 in vivo.

Conclusions and implications:

These data suggest that YC-1 is a good candidate for development as an anticancer drug.     

Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 μmol l^? 1), it induced cell apoptosis, but caused necrosis at high dose (160 μmol l^? 1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

c-Jun N-terminal kinase 1 is required for cordycepin-mediated induction of G2/M cell-cycle arrest via p21WAF1 expression in human colon cancer cells

Cordycepin (3′-deoxyadenosine) has many anti-cancer properties. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, we investigated novel molecular mechanisms for the anti-tumor effects of cordycepin in human colon cancer HCT116 cells. After treatment of cells with cordycepin, dose-dependent cell growth inhibition was observed at an IC₅₀ value of 200 μM. Cordycepin treatment resulted in G2/M-phase cell-cycle arrest, which was associated with increased p21WAF1 levels and reduced amounts of cyclin B1, Cdc2, and Cdc25c in a p53-independent pathway. Moreover, cordycepin treatment induced activation of JNK (c-Jun N-terminal kinases). Pretreatment with SP600125, a JNK-specific inhibitor, abrogated cordycepin-mediated p21WAF1 expression, cell growth inhibition, and reduced cell-cycle proteins. Furthermore, JNK1 inhibition by small interfering RNA (siRNA) produced similar results: suppression of cordycepin-induced p21WAF1 expression, decreased cell growth, and reduced cell-cycle proteins. Together, these results suggest a critical role for JNK1 activation in cordycepin-induced inhibition of cell growth and G2/M-phase arrest in human colon cancer cells.     

Long non-coding RNA DICER1-AS1-low expression in arsenic-treated A549 cells inhibits cell proliferation by regulating the cell cycle pathway

Arsenic, an environmental pollution with diverse toxicities, incurs public health problems. Arsenic trioxide could inhibit cell proliferation in vitro experiments, but the underlying mechanisms are not fully known. LncRNAs are also involved in the arsenic-induced toxicological responses. In our study, we found that the expression of lncRNA DICER1-AS1 was significantly inhibited by sodium arsenite in a dose-dependent manner. DICER1-AS1 silencing decreased the A549 cell proliferation and inhibited cell cycle progression. Importantly, DICER1-AS1 silencing induced upregulation of p21 and downregulation of Cyclin A2, Cyclin E2, CDK1 and PCNA. In conclusion, our study provided a new lncRNA-dictated regulatory mechanism participating in arsenic-induced inhibition of cell proliferation.     

长链非编码RNA膀胱癌相关转录物1对微小RNA-503-5p的靶向关系及对结直肠癌细胞增殖和凋亡的影响

目的 研究长链非编码RNA(lncRNA)膀胱癌相关转录物1(BLACAT1)对微小RNA(miR)-503-5p的靶向关系及结直肠癌细胞增殖和凋亡的影响.方法 实时荧光定量PCR(qRT-PCR)检测结肠癌细胞株SW620,LOVO,HT29和人正常结肠黏膜上皮细胞株NCM460中BLACAT1和miR-503-5p...     

JNK信号通路与细胞凋亡关系的研究进展

c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号通路是近20年来发现的与细胞分化、细胞凋亡、应激反应以及多种人类疾病的发生和发展关系非常密切的通路之一,其生物化学功能和对细胞生理、病理情况下的调节作用一直被国内外学者所关注.近年来,研究发现JNK信号通路在调控细胞凋亡中发挥重要作用,通过调节JNK信号通路有望成为治疗细胞凋亡引起的疾病的重要靶点.本文就其生物学功能及其与细胞凋亡关系作一阐述.     

原花青素对人胆管癌细胞QBC939抑制的体内外研究

目的研究原花青素(PC)体内外对人胆囊癌细胞的抑制作用。方法常规培养细胞,24 h后随机分为阳性对照组、空白对照组和PC组。MTT法检测PC对人胆囊癌细胞增殖的抑制作用;流式细胞术检测细胞凋亡率和细胞周期;建立QBC939细胞裸鼠异种移植瘤模型。将荷瘤裸鼠随机分为5组:阴性对照组、5-Fu阳性对照组及PC 3个剂量组,每日腹腔注射给药,每隔3 d测肿瘤大小;12 d后,处死裸鼠,剥瘤称重并计算抑瘤率。结果 PC 3个剂量组显著抑制QBC939细胞增殖,且呈时间和剂量依赖性;将细胞周期阻滞在S期,并诱导QBC939细胞凋亡;PC可抑制QBC939细胞裸鼠异种移植瘤的生长。结论 PC体内外均可抑制SHG-44细胞生长,并诱导肿瘤细胞凋亡。     

Activation of JNK and PAK2 is essential for citrinin-induced apoptosis in a human osteoblast cell line

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. Previous studies by our group showed that CTN triggers apoptosis in mouse embryonic stem cells, as well as embryonic developmental injury. Here, we investigated the precise mechanisms governing this apoptotic effect in osteoblasts. CTN induced apoptotic biochemical changes in a human osteoblast cell line, including activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and caspase-3 and p21-activated protein kinase 2 (PAK2) activation. Experiments using a JNK-specific inhibitor, SP600125, and antisense oligonucleotides against JNK reduced CTN-induced activation of both JNK and caspase-3 in osteoblasts, indicating that JNK is required for caspase activation in this apoptotic pathway. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 revealed that active caspase-3 is essential for PAK2 activation. Moreover, both caspase-3 and PAK2 require activation for CTN-induced apoptosis of osteoblasts. Interestingly, CTN stimulates two-stage activation of JNK in human osteoblasts. Early-stage JNK activation is solely ROS-dependent, whereas late-stage activation is dependent on ROS-mediated caspase activity, and regulated by caspase-induced activation of PAK2. On the basis of these results, we propose a signaling cascade model for CTN-induced apoptosis in human osteoblasts involving ROS, JNK, caspases, and PAK2.     

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 microM) arsenite but inhibited at higher concentrations (5 and 10 microM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 microM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 microM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 microM SP600125 or ERK1/2 by 100 microM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.     

微小 RNA-7-5p靶向调节锌指蛋白核转录因子 4基因抑制口腔鳞状细胞癌细胞增殖和诱导凋亡的体外研究

目的 探讨微小RNA(miR)-7-5p对口腔鳞状细胞癌细胞增殖、凋亡及锌指蛋白核转录因子4(KLF4)基因表达调控的影响.方法 研究起止时间为2018年3—10月.实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-7-5p在人正常口腔黏膜成纤维细胞hOMF及口腔鳞状细胞癌细胞SCC25、Cal127和Tca8113中的表达量.将miR-7-5p模拟物(miR-7-5p mimics)及阴性对照序列(mimics Control)分别转染至SCC25细胞,分为三组:Control组(未转染的SCC25细胞)、NC组(转染mimics Con-trol的SCC25细胞)、miR-7-5p组(转染miR-7-5p mimics的SCC25细胞).以qRT-PCR检测miR-7-5p在SCC25细胞中的转染效果;MTT法检测miR-7-5p对SCC25细胞增殖的影响;流式细胞术检测miR-7-5p对SCC25细胞凋亡的影响;通过双荧光素酶报告基因实验、qRT-PCR和蛋白质印迹法(Western blotting)检测miR-7-5p对KLF4的靶向关系.结果 miR-7-5p在3种口腔鳞状细胞癌细胞SCC25、Cal127和Tca8113中的表达量[(0.21±0.02)、(0.43±0.04)、(0.48±0.05)]明显低于人正常口腔黏膜成纤维细胞hOMF(1.00±0.09)(P<0.05);转染miR-7-5p mimics能够上调SCC25细胞中miR-7-5p的表达(P<0.05);miR-7-5p过表达能够明显抑制SCC25细胞增殖能力[Control组、NC组72 h吸光度(1.43±0.13)、(1.46±0.15)比miR-7-5p组(0.81±0.09)](P<0.05),并诱导细胞凋亡[Control组、NC组凋亡率(9.48±2.65)％、(10.21±3.02)％比miR-7-5p组(24.41±8.15)％](P<0.05);双荧光素酶报告基因实验结果表明miR-7-5p过表达可抑制KLF4-Wt报告基因载体细胞相对荧光活性;qRT-PCR和Western blotting进一步证实了miR-7-5p能够靶向调节KLF4 mRNA和蛋白表达.结论 miR-7-5p直接靶向调控KLF4基因的表达来抑制口腔鳞状细胞癌SCC25细胞增殖,诱导细胞凋亡.     

去甲斑蝥素抑制人脑胶质瘤细胞增殖的体内外研究

目的研究去甲斑蝥素(Norcantharidin,NCTD)体内外对人脑胶质瘤细胞的抑制作用。方法常规培养细胞,24 h后随机分为阳性对照组、空白对照组和去甲斑蝥素6个剂量组,MTT法检测NCTD对人脑胶质瘤细胞增殖的抑制作用;流式细胞术检测细胞凋亡率;建立SHG-44细胞裸鼠异种移植瘤模型,将荷瘤裸鼠随机分为阴性对照组、顺铂阳性对照组以及NCTD 3个剂量组,腹腔注射给药12 d后,取血检测血常规及肝肾功能;处死裸鼠,剥瘤称重并计算抑瘤率。结果 NCTD 6个剂量组显著抑制SHG-44细胞的增殖,且呈时间和剂量依耐性;并提高SHG-44细胞的凋亡率;NCTD 3个剂量组可抑制SHG-44细胞裸鼠异种移植瘤的生长,对移植瘤裸鼠血常规及肝肾功能无明显影响。结论 NCTD体内外均可抑制SHG-44细胞生长,并诱导肿瘤细胞凋亡。     

JNK信号通路通过介导细胞凋亡对肺纤维化的调控作用

目的观察肺纤维化中肺组织caspase-3和磷酸化c-Jun氨基末端激酶(p-JNK)蛋白表达的变化,并探讨JNK信号转导通路对肺纤维化的调控作用。方法 72只健康雄性Wistar大鼠随机分为正常对照组(N组)、肺纤维化模型组(M组),肺纤维化+JNK抑制剂干预组(I组),每组24只,分别于第3、7、14、28天处死,每组6只。留取肺组织,对其行苏木素-伊红(HE)染色,观察其病理学改变,碱水解法测定其中羟脯氨酸的含量,免疫组织化学法测肺组织中caspase-3和p-JNK的变化。结果模型组早期肺泡炎症明显,后期肺组织实变,caspase-3和p-JNK蛋白表达含量均显著增加(P<0.05),14d时达峰值。干预组能明显阻断JNK的激活(P<0.05),干预组caspase-3表达较模型组显著减少(P<0.05),14d时减少最明显。结论细胞凋亡是肺纤维化的一个重要病理组织学特点。JNK信号转导通路在肺纤维化中被激活,发挥促细胞凋亡效应。     

JNK/SAPK is required in nitric oxide-induced apoptosis in osteoblasts

Nitric oxide(NO) induces apoptosis in human osteoblasts. Treatment with exogenous NO donors, SNAP (S-Nitroso-N-acetylpenicillamine) and SNP (sodium nitroprusside), to MG-63 osteoblasts resulted in apoptotic morphological changes, as shown by a bright blue-fluorescent condensed nuclei and chromatin fragmentation by fluorescence microscope of Hoechst 33258-staining. The activities of caspase-9 and the subsequent caspase-3-like cysteine proteases were increased during NO-induced cell death. Pretreatment with Z-VAD-FMK (a pan-caspase inhibitor) or Ac-DEVD-CHO (a specific caspase-3 inhibitor) abrogated the NO-induced cell death. The NO donor markedly activated JNK, a stress-activated protein kinase in the human osteoblasts. This study showed that the inhibition of the JNK pathway markedly reduced NO-induced cell death. But neither PD98059 (MEK inhibitor) nor SB203580 (p38 MAPK inhibitor) had any effect on NO-induced death. Taken together, these results suggest that JNK/SAPK may be related to NO-induced apoptosis in MG-63 human osteoblasts.     

慢病毒载体介导的长链非编码RNA Rpph1沉默对胆囊癌GBC-SD细胞增殖、细胞周期和凋亡的影响

目的 研究长链非编码RNA(lncRNA)Rpph1对胆囊癌细胞增殖、细胞周期和凋亡的影响和潜在机制.方法 以人胆囊上皮细胞HGBEC为对照,实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测胆囊癌细胞系GBC-SD、SGC-996和NOZ中ln?cRNA Rpph1的表达.将GBC-SD细胞分为对照组、si-con...     

Dose-dependent induction of apoptosis by R-apomorphine in CHO-K1 cell line in culture

A variety of mechanisms have been proposed as explanations for the distinctive neuropathology of Parkinson's disease, such as increased iron levels, increased oxidant stress or decreased antioxidant defences. The vulnerability of dopamine-containing neurons towards cell death has attracted much attention to the dopamine molecule itself as one of the probable neurotoxic factors leading to neurodegeneration. The similarity between apomorphine and dopamine with regards to their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study the CHO-K1 cell line was exposed to different concentrations of apomorphine, and markers of cell death and apoptosis were studied. Apomorphine reduced cell proliferation in a dose-dependent fashion after 72 h incubation. Furthermore, apomorphine induced dose-dependent cell death at concentrations of 10-50 microM. The CHO-K1 line showed specific markers of apoptosis such as the typical DNA laddering phenomenon on agarose gel, morphological changes of apoptotic nuclei as described by in situ end labelling, and annexin binding.These data strongly suggest that apomorphine, like dopamine, elicits its cytotoxic effect with an apoptotic mechanism.     

氯沙坦对心肌成纤维细胞基质金属蛋白酶-2、JNK1/2表达及增殖活性的影响

目的：研究氯沙坦通过影响高血压大鼠心肌成纤维细胞间质成分抑制心室重构的药理机制。方法：培养心肌成纤维细胞，免疫细胞化学法鉴定细胞，MTT法测氯沙坦和AngⅡ对细胞增殖活性影响的量效关系，Western Blotting测AngⅡ刺激成纤维细胞心肌JNK1／2、磷酸化JNK1／2表达的时效关系。根据实验所得AngⅡ刺激心肌成纤维细胞增殖活性作用的EC劬值、氯沙坦抑制心肌成纤维细胞活性作用的IC50值和AngⅡ刺激JNK1／2表达的最佳时间，心肌成纤维细胞分为无血清DMEM组、AngⅡ100nmol／L组、AngⅡ100nmol／L＋losartan　100nmol／L组、losartan 100nmol／L组，药物干预后分别收集各组蛋白质、培养上清液，Western blotting检测各组MMP-2、JNK1／2、磷酸化JNK1／2蛋白表达，ELISA检测分泌至培养上清液中MMP-2的量。结果：AngⅡ刺激心肌成纤维细胞增殖活性作用的EC50为53nmol／L，氯沙坦抑制心肌成纤维细胞增殖活性作用的EC50为56．3nmol／L。JNK1／2蛋白表达在AngⅡ刺激2min达高峰，随后表达逐渐降低。AngⅡ增加JNK1／2表达，氯沙坦降低AngⅡ刺激导致的JNK1／2表达。AngⅡ组MMP-2蛋白表达较无血清DMEM组明显增高，氯沙坦降低AngⅡ刺激增高的MMP-2表达。AngⅡ组MMP-2分泌较无血清DMEM组增高，氯沙坦减少AngⅡ刺激所致MMP-2分泌增高。结论：氯沙坦防治高血压引起的心室重构，可能与其对抗AngⅡ刺激心肌成纤维细胞增殖活性、MMP-2合成、分泌有关，信号通路涉及JNK1／2。     

雷帕霉素对肾小管上皮细胞增殖和凋亡的影响

目的 探讨雷帕霉素对肾小管上皮细胞增殖和凋亡的影响。方法 体外培养肾小管上皮细胞(HK-2细胞),选择100, 200, 400, 800 ng/ml雷帕霉素作用于HK-2细胞24、48、72h。利用 MTT实验分析雷帕霉素对HK-2细胞增殖的影响,并计算各浓度及不同时间的增殖抑制率优化雷帕霉素作用的浓度和时间;选择最佳的作用浓度和作用时间处理HK-2细胞,通过流式细胞分析技术,分析雷帕霉素对HK-2细胞的凋亡的影响。结果 MTT实验显示不同浓度的雷帕霉素,对增殖有抑制作用,且表现浓度依赖性和时间依赖性;RAPA可以促进HK-2细胞的凋亡。结论 通过HK-2细胞模型研究,发现雷帕霉素可抑制肾小管上皮细胞增殖、促进细胞凋亡。     

肝复康经JNK信号通路在肝星状细胞凋亡中的调节机制

目的探讨中药肝复康经JNK(c-Jun N-terminal kinase,JNK)信号通路在肝星状细胞凋亡中的调节机制。方法将SD大鼠随机分为5组:正常对照组(C),模型组(M),肝复康高(HT)、中(MT)、低(LT)剂量治疗组,每组给予相应的处理;HSC-T6细胞株随机分为3组:正常对照组、乙醛组及肝复康治疗组。HE染色法观察肝组织形态结构变化;RT-PCR法测定MKK4、MKK7、JNK1、JNK2、COX-2、Survivin、Caspase-3、α-SMA、collagenⅠ和collagenⅢ的基因表达水平;免疫组织化学法和免疫蛋白印记法观察COX-2在肝组织和肝星状细胞中的表达。结果正常组中COX-2几乎不表达。与正常组相比,模型组中COX-2表达显著增加(P<0.05)。与模型组相比,肝复康治疗组各组中COX-2表达均明显降低,其中,中剂量组最明显(P<0.05)。与正常组相比,模型组中MKK4、MKK7、JNK1、JNK2、COX-2、Survivin的基因表达水平均显著上调(P<0.05)。与模型组相比,肝复康治疗组各基因表达水平均明显减弱,中剂量组最明显(P<0.05),其中Caspase-3基因表达呈相反趋势。COX-2和Survivin的表达呈正相关,和Caspase-3的表达呈负相关。检测α-SMA、collagenⅠ与collagenⅢ基因表达,与模型组相比,中剂量治疗组表达水平明显下调(P<0.05)。结论肝复康可通过抑制JNK信号通路的传导诱导肝星状细胞凋亡,发挥抗纤维化作用。