Thymoquinone Augments Cisplatin-Induced Apoptosis on Esophageal Carcinoma Through Mitigating the Activation of JAK2/STAT3 Pathway

Background

Thymoquinone (TQ) is the major constituent of Nigella sativa seed and has shown biological activity in various human carcinomas. However, few studies have reported its effect on esophageal carcinoma (EC).

Aims

To explore the chemosensitive effect and mechanism of TQ in augmentation of cisplatin (DDP)-induced apoptosis of EC, both in vitro and in vivo.

Methods

The viability and apoptosis of esophageal carcinoma cells were detected by the Cell Counting Kit-8 assay, flow cytometry, and Hoechst 33258 staining. The expression levels of JAK2, p-JAK2, STAT3, p-STAT3, Bax, Bcl-2, Cyclin D1, Survivin, and caspase-3, 7, 9 were evaluated by western blot analysis. The histological changes were examined by TUNEL technique and immunohistochemical analysis.

Results

TQ enhanced the proapoptotic effect of DDP in human esophageal carcinoma cell line Eca-109, while blocking the activation of JAK2/STAT3 signaling pathway. The apoptosis of esophageal carcinoma cells was induced via blocking the activation of JAK2/STAT3 by using a molecular inhibitor (WP1066). Consistent with the in vivo and in vitro results, TQ increased cellular apoptosis and enriched the chemosensitivity of DDP.

Conclusions

TQ along with DDP may regulate the progression of EC and has potential to be a chemotherapeutic agent in EC.

     

七氟醚麻醉对肺癌大鼠JAK/STAT信号通路及脑神经损伤的影响

目的探讨七氟醚麻醉对肺癌大鼠JAK/STAT信号通路的影响及脑神经损伤作用。 方法24只健康雄性SD大鼠采用尾静脉注射Walker-256癌细胞悬液法建立大鼠肺癌模型,造模完成后将大鼠随机分为4组,每组6只,分别为模型组、七氟醚A组、七氟醚B组、七氟醚C组;然后七氟醚A、B、C组吸入3%七氟醚+2 ml/min氧气,吸入持续时间分别为4、6、8 h,模型组吸入空气,持续吸入时间6 h,麻醉结束24 h后,测定大鼠血清TNF-α、IL-6及IL-1β水平,利用Morris水迷宫考察大鼠学习及记忆能力,实时定量PCR检测大鼠海马组织caspase-3和caspase-12 mRNA水平,Western blot检测大鼠肺组织中JAK2、STAT3、p-JAK2及p-STAT3水平。 结果肺癌大鼠持续吸入不同时间的七氟醚进行麻醉后,与模型组相比,七氟醚各麻醉组肺癌大鼠血清TNF-α、IL-6及IL-1β水平均显著升高(P<0.05),肺癌大鼠逃避潜伏期及首次穿过平台时间均显著延长(P<0.05),90 s内穿过平台的次数显著减少(P<0.05),海马组织caspase-3、caspase-12 mRNA水平均显著升高(P<0.05),肺组织JAK2、STAT3水平均显著升高(P<0.05), JAK2及STAT3磷酸化比例显著增加(P<0.05)。 结论七氟醚麻醉能够激活肺癌大鼠JAK/STAT信号通路,并可能通过促进肺癌大鼠海马体凋亡基因的表达引起大鼠脑神经损伤,影响肺癌大鼠学习及记忆能力。     

尼古丁通过抑制JAK/STAT通路对HUVECs增殖和凋亡的影响研究

目的探究尼古丁通过抑制JAK/STAT通路促进人脐静脉血管内皮细胞(HUVECs)增殖和凋亡的作用机制。方法将HUVECs分为4组,即对照组、尼古丁1组、尼古丁2组、尼古丁3组,分别在培养基中加入终浓度为0、10-8、10-7、10-6 mol/L的尼古丁培养24 h。显微镜观察细胞形态。CCK-8和流式细胞术分别用于检测细胞活力和凋亡。Western blot和PCR分别用于检测蛋白和mRNA的表达水平。结果显微镜观察尼古丁会使HUVECs呈现萎缩状态。尼古丁可剂量依赖性的抑制HUVECs活力,并且剂量依赖性的促进HUVECs凋亡。尼古丁可显著的抑制p-JAK/t-JAK和p-STAT/t-STAT的水平。尼古丁可显著的提高Caspase-3和Bax mRNA的水平,抑制Bcl-2 mRNA的水平。结论尼古丁可能通过下调JAK/STAT通路调节凋亡相关蛋白的表达,抑制HUVECs活力并促进其凋亡。     

Janus激酶-信号转导子与转录激活子通路介导粒细胞集落刺激因子影响冠状动脉微栓塞后心肌细胞凋亡的实验研究

目的 探讨粒细胞集落刺激因子(G-CSF)对大鼠冠状动脉微栓塞(CME)后心肌细胞凋亡的影响以及Janus激酶-信号转导子与转录激活子(JAK/STAT)通路的介导作用.方法 将92只雄性成年SD大鼠,随机分成CME组(24只)、G-CSF组(24只)、JAK2特异性抑制剂(AG490)组(G-CSF+AG490,24只)和假手术组(20只).CME组、G-CSF组及AG490组升主动脉夹闭后自左室腔内注入自体微血栓,造成CME,假手术组注入等量生理盐水.G-CSF组及AG490组术后2 h起给予皮下注射重组人G-CSF(rhG-CSF)100 μg·kg-1·d-1持续5 d,AG490组同时给予AG490溶液腹腔注射5 mg·kg-1·d-1,其他组给予等量生理盐水.术后3 d、1、2及4周处死动物.各组心肌样品中以实时定量聚合酶链式反应法检测Bcl-2、Bax、Fas及FasL的mRNA表达,并计算Bcl-2/Bax比值;以Western blot法检测Caspase-3、裂解多聚二磷酸腺苷-核糖聚合酶(PARP)、总JAK2(t-JAK2)、磷酸化JAK2(p-JAK2)、t-STAT3以及p-STAT3蛋白的表达;脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法测定凋亡细胞.结果 (1)与假手术组比较,CME组术后Bcl-2、Bax、Fas及FasL的mRNA表达均升高,Bcl-2/Bax比值降低(0.28±0.04比2.98±0.49),Caspase-3(0.762±0.129比0.133±0.027)及PARP蛋白(0.992±0.146比0.386±0.074)表达增强,心肌细胞凋亡指数升高(P＜0.05或P＜0.01);t-JAK2、p-JAK2、t-STAT3以及p-STAT3蛋白表达差异无统计学意义.(2)与CME组比较,G-CSF组术后p-JAK2与p-STAT3蛋白表达明显增强,Bax、Fas及FasL的mRNA表达明显减弱,Bcl-2的mRNA表达增强,Bcl-2/Bax比值升高(2.07±0.29比0.28±0.04),Caspase-3(0.371±0.041比0.762±0.129)及PARP蛋白(0.548±0.093比0.992±0.146)表达减弱;心肌细胞凋亡指数下降(P＜0.05或P＜0.01);t-JAK2及t-STAT3蛋白表达则差异无统计学意义.(3)经AG490干预后,G-CSF引起的基因与蛋白表达的变化均有不同程度减弱(P＜0.05或P＜0.01).结论 G-CSF通过激活JAK2/STAT3细胞内信号通路减轻CME导致的心肌细胞凋亡.     

丹参酮ⅡA对心肌缺血再灌注损伤的保护作用及对JAK2/STAT3通路的影响

目的探讨丹参酮ⅡA(TA)对心肌缺血-再灌注损伤(MIRI)的影响及其对Janus激酶2(JAK2)/信号转导和转录激活因子3(STAT3)通路的影响。方法建立老年大鼠MIRI模型和心肌细胞缺氧/复氧损伤(HRI)模型,分别从体内和体外水平进行研究。采用超声心动图测定心功能,应用全自动生化检测仪检测大鼠血清肌酸激酶(CK)、乳酸脱氢酶(LDH)。四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞活力,TUNEL法检测细胞凋亡,DCFH-DA荧光探针检测活性氧(ROS)含量,全自动生化检测仪检测细胞上清液丙二醛(MDA)和超氧化物歧化酶(SOD)水平,免疫印迹法(Western Blotting)检测相关蛋白表达水平。应用JAK2/STAT3抑制剂AG490干预心肌细胞,观察各项指标变化。结果TA可改善MIRI大鼠心肌梗死面积、心脏射血分数和缩短分数、血清CK和LDH水平(P<0.05),改善HRI心肌细胞活力和凋亡水平(P<0.05),提高HRI心肌细胞抗氧化能力,改善ROS、MDA和SOD水平(P<0.05)。Western Blotting检测结果显示,TA可改善p-JAK2、p-STAT3、Bcl-2/Bax比值、cleaved Caspase-3水平(P<0.05),AG490干预可逆转上述指标变化(P<0.05)。结论TA对MIRI具有一定保护作用,可能通过激活JAK2/STAT3通路减少心肌细胞凋亡从而改善MIRI。     

汉黄芩素调节JAK/STAT信号通路并诱导肺癌A549细胞凋亡及周期阻滞的作用及机制研究

目的研究汉黄芩素对肺癌A549细胞凋亡、周期及对JAK/STAT信号通路的调节作用。方法取对数生长期的肺癌A549细胞,采用低(20μmol/L)、中(40μmol/L)、高(80μmol/L)剂量的汉黄芩素分别干预24 h、48、72 h,MTT法测定不同浓度汉黄芩素对肺癌A549细胞的增殖抑制率,实时定量PCR检测各组肺癌A549细胞中JAK2、STAT3mRNA水平,AnnexinV-FITC/PI双染法检测汉黄芩素对A549细胞凋亡的影响,流式细胞术检测汉黄芩素对A549细胞周期的影响,Western blot检测汉黄芩素对A549细胞JAK2、STAT3水平的影响。结果与对照组相比,汉黄芩素各剂量组肺癌A549细胞24 h、48、72 h增殖抑制率、p-Chk1、P53、Bax水平显著升高(P<0.05),不同浓度的汉黄芩素作用于肺癌A549细胞48 h后,CyclinB1、PCNA、Bcl-2、Survivin、JAK2及STAT3 mRNA水平显著降低(P<0.05),A549细胞凋亡率、G2/M期细胞比例显著升高(P<0.05),Western blot结果表明,汉黄芩素各剂量组A549细胞JAK2及STAT3水平显著降低(P<0.05)。结论汉黄芩素能够抑制肺癌A549细胞的增殖及迁移能力,诱导肺癌A549细胞凋亡及G2/M周期阻滞,其机制可能与调节JAK/STAT信号通路有关。     

花姜酮对胰腺癌PANC1细胞株增殖和凋亡的影响

目的 探讨花姜酮对胰腺癌PANC1细胞增殖和凋亡的影响,探讨其作用机制.方法 应用3.75、7.5、15、30、60μg/ml的花姜酮处理PANC1细胞,以未处理细胞作为对照.CCK-8法检测细胞增殖抑制率,Hoechst33342染色观察细胞形态,流式细胞术检测细胞凋亡率,Western blotting法检测细胞磷酸化STAT1(p-STAT1)、Bax和Bcl-2蛋白的表达.结果 花姜酮呈时间-剂量依赖性抑制PANC1细胞的生长,15 μg/ml花姜酮作用48 h后,细胞增殖抑制率达(72.8±2.72)%,并可观察到典型的细胞凋亡形态学改变,细胞凋亡率达14.2%;同时,PANC1细胞p-STAT1和Bax蛋白表达明显增加(0.654±0.048对0.074±0.011,0.577±0.044对0.218±0.027,P＜0.05),Bcl-2蛋白表达明显下降(0.162±0.029对0.459±0.034,P＜0.05).结论 花姜酮可能通过上调STAT1活性,升高Bax/Bcl-2比率,从而诱导PANC1细胞的凋亡和抑制细胞增殖.     

Fructose 1,6-diphosphate alleviates myocardial ischemia reperfusion injury in rats through JAK2/STAT3 pathway

Objective: To study the effect of fructose 1,6-diphosphate(FDP) on myocardial ischemia reperfusion injury in rats and its molecular mechanism.Methods: Male SPF SD rats were selected as experimental animals and randomly divided into four groups.Sham group received sham operation, I/R group were made into myocardial ischemia reperfusion injury models, FDP group were made into myocardial ischemia reperfusion injury models and then were given FDP intervention, and FDP+AG490 group were made into myocardial ischemia reperfusion injury models and then were given FDP and JAK2 inhibitor AG490 intervention.Results: CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of I/R group were significantly higher than those of Sham group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissues were significantly lower than those of Sham group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP group were significantly lower than those of I/R group whereas Bcl-2, p-JAK2 and p-STAT3 protein expression in myocardial tissue were significantly higher than those of I/R group; CK, CK-MB, c Tn I and LDH contents in serum as well as Bax and Caspase-3 protein expression in myocardial tissue of FDP+AG490 group were significantly higher than those of FDP group whereas Bcl-2 protein expression in myocardial tissue was significantly lower than that of FDP group.Conclusion: FDP could reduce the myocardial ischemia reperfusion injury in rats by activating the JAK2/STAT3 pathway.     

PIAS1基因沉默对雨蛙肽诱导胰腺腺泡细胞凋亡的影响

活化STAT蛋白抑制物(PIAS)1基因沉默可增强雨蛙肽诱导的胰腺腺泡细胞炎症反应。目的:探讨PIAS1基因沉默对雨蛙肽刺激胰腺腺泡细胞凋亡的影响。方法:AR42J胰腺腺泡细胞在Lipofectamine~(TM)2000介导下转染PIAS1-siRNA和阴性siRNA。将细胞分为PIAS1-siRNA+雨蛙肽组、阴性siRNA+雨蛙肽组、脂质体+雨蛙肽组、PBS+雨蛙肽组和对照组。以DNA Ladder、Hoechst 33258染色检测细胞凋亡情况,流式细胞术测定细胞周期和细胞凋亡率,RT-PCR和蛋白质印迹法检测1353、Bax、Bcl-2、caspase-3 mRNA和蛋白表达。结果:与其余各组相比,PIAS1-siRNA+雨蛙肽组DNA梯度裂解条带明显增加,荧光着色阳性细胞数增多;G1期细胞数增多,S期细胞数减少,细胞凋亡率增加;p53、Bax、caspase-3 mRNA和蛋白表达明显上调,Bcl-2 mRNA和蛋白表达下调(P均0.05)。结论:PIAS1基因沉默可增强雨蛙肽活化的caspase-3凋亡途径诱导的胰腺腺泡细胞凋亡,为通过调控PIAS1表达治疗急性胰腺炎提供新的理论依据。     

Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells

AIM: To investigate the effect of alternol on pancreatic cancer cells.METHODS: Pancreatic cancer cells PANC-1 and BxPC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a TdT-mediated dUTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting.RESULTS: Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and BxPC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner.CONCLUSION: Our results suggested that alternol is a candidate for treatment of pancreatic cancer.     

Icariin enhance mild hypothermia-induced neuroprotection via inhibiting the activation of NF-κB in experimental ischemic stroke

Therapeutic hypothermia (TH) is a promising neuroprotective agent for treating stroke. However, its clinical application was limited by the impractical duration. Icariin (ICA) were reported to have therapeutic effect on cerebral ischemia. In this research, our aim was to investigate whether the combination of TH and ICA had better neuroprotective effects on ischemic stroke. An ischemia-reperfusion rat model was established and treated with mild hypothermia, ICA or JSH-23 (inhibitor of NF-κB). Thermistor probe, 2′3’5′-triphenyl tetrazolium chloride (TTC), 5/12-score system, and ELISA were used to detect temperature (rectum, cortex, striatum), infarct volume, neurological deficit, and cerebral cell death of these rats. The expressions of tumor necrosis factor (TNF)-α, Interleukin- 6 (IL-6), nuclear factor-kappa B (NF-κB), nuclear factor erythroid2-related factor (Nrf2), peroxisome proliferator activated receptor gamma (PPARα), PPARγ, Janus kinase 2 (JAK2), p-JAK2, signal transducers and activators of transduction-3 (STAT3), and p-STAT3 were detected by Western blot or q-PCR. Mild hypothermia, ICA, and JSH-23 reduced the cerebral infarct volume, neurological deficit, cerebral cell death of rats, downregulated the expressions of TNF-α, IL-6, C-Caspase 3 and Bax, and the activation of PPARs/Nrf2/NF-κB and JAK2/STAT3 pathways, but elevated the expression of Bcl-2. ICA promoted the effect of mild hypothermia on infarct volume, neurological deficit, and cerebral cell death. Moreover, ICA also enhanced the regulatory effect of mild hypothermia on apoptosis/inflammation factors expressions and activation of PPARs/Nrf2/NF-κB and JAK2/STAT3 pathways. ICA could promote mild hypothermia-induced neuroprotection by inhibiting the activation of NF-κB through the PPARs/Nrf2/NF-κB and JAK2/STAT3/NF-κB pathways in experimental stroke.

     

黄芩苷诱导人肝癌HepG-2细胞凋亡作用观察

目的研究黄芩苷对人肝癌HepG-2细胞凋亡的影响,并探讨其作用机制。方法以不同浓度的黄芩苷与人肝癌HepG-2细胞共同培养,采用MTT法测定细胞增殖抑制率;采用Hoechst33258/PI染色法在荧光显微镜下观察凋亡细胞形态学变化;采用TUNEL法检测细胞凋亡率;采用Western blot法检测细胞凋亡相关蛋白Caspase-9、Caspase-3和Bcl-2表达的变化。结果在25~100μg/ml的药物浓度作用下,细胞增殖被抑制。药物浓度在50μg/ml下作用48h,细胞抑制率达50.63%,药物浓度在75μg/ml下作用48h细胞,抑制率达77.62%。抑制率呈浓度和时间依赖性;药物浓度在50μg/ml时作用48h,可见HepG-2细胞皱缩、细胞核碎裂成碎片,呈现典型的凋亡改变;随着黄芩苷作用浓度的增加,细胞凋亡率增高(P<0.05);随药物浓度的增加,Caspase-9和Caspase-3蛋白表达量呈增加趋势,而Bcl-2表达减少。结论黄芩苷能通过诱导细胞凋亡进而抑制人肝癌细胞增殖,其诱导凋亡机制可能与线粒体通路有关。     

Inactivation of PI3k/Akt signaling pathway and activation of caspase-3 are involved in tanshinone I-induced apoptosis in myeloid leukemia cells in vitro

Tanshinone I (Tan I), a diterpene quinone extracted from herbal medicine Salvia miltiorrhiza Bunge, has recently been reported to have antitumor effects. As the mechanism of its proapoptotic effects on human myeloid leukemia cells has not been extensively studied, we performed an in-depth evaluation of the effects of Tan I on apoptosis in human K562 and HL-60 cells. The results revealed that Tan I could inhibit the growth of leukemia cells and cause apoptosis in a time- and dose-dependent manner. Apoptosis was observed clearly by flow cytometry and Hoechst 33258 staining, as well as DNA fragmentation analysis. After treatment by Tan I for 48 h, the percentage of disruption of mitochondrial membrane potential (Δψm) was increased in a dose-dependent manner. Western blotting analysis demonstrated the cleavage of caspase-3 zymogen protein and a dose-dependent cleavage of poly-(ADP-ribose) polymerase. Tan I-induced apoptosis was accompanied by a significant decrease in survivin and an increase in Bax. Moreover, Tan I treatment remarkably downregulated the phosphorylation of both P85/PI3K and Akt in a time-dependent manner, and the PI3K/AKT-specific inhibitor (LY294002) mimicked the apoptosis-inducing effects of Tan I. We therefore conclude that the induction of apoptosis by Tan I in these leukemia cells is mainly related to the disruption of Δψm, the upregulation of Bax expression, and the activation of caspase-3. This process is highly correlated with the inactivation of PI3K/Akt/survivin signaling pathways. The results indicate that Tan I may serve as an effective adjunctive reagent in the treatment of leukemia.     

Involvement of VDAC1 and Bcl-2 family of proteins in VacA-induced cytochrome c release and apoptosis of gastric epithelial carcinoma cells

OBJECTIVE: It is known that the vacuolating cytotoxin (VacA) could induce apoptosis. However, the mechanism remained to be elucidated. The aim of this study is to investigate the role of Bcl family of proteins (Bcl-2 and Bax) and the mitochondrial voltage-dependent anion channel (VDAC) in VacA-induced apoptosis of AGS cells. METHODS: Plasmid pGBKT7-VacA p58 was constructed and transfected into the AGS cells. RT-PCR and Western blotting were used to determine the expressions of cytochrome c, caspase-3, Bax, Bcl-2 and VDAC1 mRNA and proteins. RESULTS: VacA p58 can induce cytochrome c release and activate caspase-3 in AGS cells. It up-regulated the expressions of Bax and VDAC1 mRNA and proteins, and decreased the expression of Bcl-2 in AGS cells. CONCLUSION: VacA p58 induces apoptosis in AGS cells. This apoptotic process is associated with the up-regulation of Bax/VDAC1 and downregulation of Bcl-2. These findings suggest that the release of cytochrome c by VacA p58 is mainly through VDAC-dependent and Bcl-2 family-dependent pathways.