Apigenin Sensitizes Huh-7 Human Hepatocellular Carcinoma Cells to TRAIL-induced Apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent for management of cancer because of its selective cytotoxicity to cancer cells. However, some cancer cells have resistance to TRAIL. Accordingly, novel treatment strategies are required to overcome TRAIL resistance. Here, we examined the synergistic apoptotic effect of apigenin in combination with TRAIL in Huh-7 cells. We found that combined treatment of TRAIL and apigenin markedly inhibited Huh-7 cell growth compared to either agent alone by inducing apoptosis. Combined treatment with apigenin and TRAIL induced chromatin condensation and the cleavage of poly (ADP-ribose) polymerase (PARP). In addition, enhanced apoptosis by TRAIL/apigenin combination was quantified by annexin V/PI flow cytometry analysis. Western blot analysis suggested that apigenin sensitizes cells to TRAIL-induced apoptosis by activating both intrinsic and extrinsic apoptotic pathway-related caspases. The augmented apoptotic effect by TRAIL/apigenin combination was accompanied by triggering mitochondria-dependent signaling pathway, as indicated by Bax/Bcl-2 ratio up-regulation. Our results demonstrate that combination of TRAIL and apigenin facilitates apoptosis in Huh-7 cells.     

Acetaminophen Induces a Caspase‐Dependent and Bcl‐xL Sensitive Apoptosis in Human Hepatoma Cells and Lymphocytes

Abstract: Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P‐450‐generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK‐Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time‐dependent manner. Staining of cells with annexin‐V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase‐3, 8, and 9, cleavage of poly(ADP‐ribose) polymerase, and degradation of lamin B₁ and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK‐Hep1 and primary lymphocytes, DNA was only degraded to 50‐kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl‐x_L prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase‐8 activation was a postmictochondrial event and occurred in a Fas‐independent manner. These results demonstrate that acetaminophen induces caspases‐dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity.     

Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells

Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.     

沉默Rock2对肝癌细胞增殖及凋亡作用的研究

目的:探讨沉默Rock2基因对人肝癌细胞Huh-7和HepG2增殖和凋亡作用的影响.方法:实验分为空白对照组、干扰无意义组、转染PBS组及干扰Rock2组.将Rock2干扰质粒shRock2转染到人肝癌细胞Huh-7和HepG2中,通过实时荧光定量PCR检测Rock2 mRNA的表达水平;Western blot检测Rock2蛋白的表达水平;MTT比色法检测沉默Rock2后对Huh-7和HepG2细胞增殖抑制的影响;流式细胞术检测沉默Rock2对Huh-7和HepG2细胞周期及早期凋亡的变化.结果:将shRock2转染肝癌细胞系Huh-7和HepG2后,Rock2 mRNA以及Rock2蛋白的表达水平均明显下降.沉默Rock2表达后,MTT结果显示Huh-7和HepG2细胞较对照组细胞增殖能力明显减弱(P＜0.01);Huh-7和HepG2细胞G0/G1期细胞比例明显升高,而S期和G2/M期细胞比例明显降低,与对照组比较差异有统计学意义(P＜0.01);肝癌细胞的早期凋亡较对照组明显增加(P＜0.01).结论:沉默Rock2能明显抑制肝癌细胞系Huh-7和HepG2的增殖,并诱导其早期凋亡,提示Rock2可作为肝癌基因治疗的一个新的分子靶点.     

丹酚酸A诱导HepG2细胞凋亡及抑制c-Met表达

目的通过研究丹酚酸A（salvianol acid A,SalA）对肝癌HepG2细胞株c-Met蛋白表达的影响,探讨SalA抑制肝癌细胞增殖,诱导细胞凋亡的可能作用机制。方法以肝癌HepG2细胞株为研究对象,采用MTT法及流式细胞术检测SalA作用后细胞存活、增殖及凋亡情况;同时运用Westernblot法及PCR法检测HepG2细胞c-Met及其下游信号通路中关键蛋白和基因表达的改变。结果肝癌HepG2细胞经SalA处理后,其细胞增殖显著抑制,细胞凋亡比例亦升高,且呈浓度依赖性;同时HepG2细胞中c-Met及其下游信号分子AKT的磷酸化水平显著下调,凋亡相关蛋白Bax、caspase-3和caspase-9的表达亦明显上调。结论SalA能有效抑制肝癌HepG2细胞的增殖并诱导细胞凋亡,其作用机制可能与其抑制HepG2细胞中c-Met蛋白及其下游信号通路中AKT蛋白的磷酸化水平有关。     

薯蓣皂苷下调ABCC1表达对人肝癌细胞HepG2/ADM耐药的影响

目的:探究薯蓣皂苷对人肝癌细胞HepG2/多柔比星(ADM)耐药性的影响及其可能的作用机制。方法:采用含0,10,20,30,40,50μmol·L^-1薯蓣皂苷的培养液培养HepG2、HepG2/ADM细胞,CCK法检测细胞增殖抑制率;MTT检测薯蓣皂苷对HepG2/ADM细胞、HepG2细胞半抑制浓度(IC50)值的影响;流式细胞术检测HepG2、HepG2/ADM细胞摄取ADM的能力。薯蓣皂苷联合ABCC1抑制剂处理HepG2/ADM细胞,MTT检测细胞IC50值变化;流式细胞术检测细胞摄取ADM的能力、细胞凋亡情况,免疫印迹法检测细胞人多药耐药相关蛋白1(ABCC1)、半胱天冬酶(caspase-3)、B淋巴细胞瘤-2(bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达情况。结果:HepG2/ADM细胞中ABCC1蛋白表达显著高于HepG2细胞(P<0.05)。薯蓣皂苷0~30μmol·L^-1对HepG2/ADM细胞、HepG2细胞毒性小。随着薯蓣皂苷处理浓度的升高,HepG2/ADM细胞IC50值均降低,摄取ADM量明显升高,具有剂量依赖性(P<0.05)。薯蓣皂苷联合ABCC1抑制剂处理HepG2/ADM细胞后能够进一步降低细胞IC50,提高细胞ADM摄取量,加速细胞凋亡,上调caspase-3、Bax蛋白表达,下调ABCC1、bcl-2蛋白表达(P<0.05)。结论:薯蓣皂苷能够逆转HepG2/ADM细胞耐药性,逆转效果与作用剂量相关,其机制可能与下调ABCC1表达,诱导细胞凋亡有关。     

索拉菲尼增强TRAIL 诱导人肝癌细胞凋亡的作用及机制探讨

目的观察索拉菲尼(Sorafenib)是否能增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导人肝癌细胞凋亡并探讨其作用机制。方法体外培养人肝癌HepG2细胞,MTT法测定细胞增殖活性,碘化丙啶(PI)染色流式细胞术(FCM)和细胞凋亡ELISA检测试剂盒检测细胞凋亡,Western blotting分析Mcl-1的表达。结果索拉菲尼具有增强TRAIL诱导人肝癌HepG2细胞凋亡的作用,并呈剂量和时间依赖的方式抑制Mcl-1的表达。Mcl-1过表达能抑制索拉菲尼增强TRAIL诱导HepG2细胞凋亡的作用。结论索拉菲尼通过抑制Mcl-1的蛋白表达增强TRAIL诱导人肝癌细胞凋亡的作用。     

Induction of Apoptosis by Lovastatin through Activation of Caspase‐3 and DNase II in Leukaemia HL‐60 Cells

Abstract: Lovastatin, an HMG‐CoA reductase inhibitor, was found to suppress growth and induce apoptosis in culture human promyelocytic leukaemic cell, HL‐60. However, the mechanisms of lovastatin‐induced apoptosis are still unclear. In this study, we attempted to elucidate the signal transduction pathway for lovastatin‐induced apoptosis in HL‐60 cells in a dose‐ and time‐dependent manner. The features of this apoptosis were attenuated by the presence of mevalonate, a metabolic intermediate of cholesterol synthesis. Treatment of lovastatin caused a rapid release of mitochondrial cytochrome c into cytosol and subsequent induction of caspase‐3, but not caspase‐1 activity. Lovastatin also stimulated proteolytic cleavage of poly‐(ADP‐ribose) polymerase (PARP), and followed by the appearance of caspase activity and DNA fragmentation. Pretreatment with caspase‐3 inhibitors, Ac‐DEVD‐CHO and Z‐VAD‐FMK, inhibited lovastatin–induced caspase‐3 activity and DNA fragmentation. Furthermore, we demonstrated that DNase II was involved in the DNA fragmentation induced by lovastatin. These results suggested that the mechanism of lovastatin induced HL‐60 cells apoptosis through activation of caspase‐3 and DNase II activities.     

尼美舒利诱导人鼻咽癌CNE2细胞凋亡作用

目的观察尼美舒利（NIM）诱导人鼻咽癌CNE2细胞凋亡作用。方法0,0.05,0.1,0.2,0.4,0.8 mmol&#8226;L－1NIM作用于体外培养的CNE2细胞株,光学显微镜观察细胞形态和数量变化,透射电子显微镜观察细胞形态和结构变化,噻唑蓝（MTT）法检测细胞悬液吸光度值,流式细胞术（FCM）检测细胞周期分布和凋亡率,蛋白免疫印迹反应（Western Blot）检测细胞环氧化酶2（COX 2）蛋白表达情况。结果不同浓度NIM作用3 d后,CNE2细胞皱缩,数量减少,与药物浓度呈正相关;透射电镜下,细胞形态结构随NIM浓度增加而出现不同程度破坏和死亡征象;相同作用时间下,NIM浓度越高,吸光度值越低;FCM结果显示,NIM各剂量组均在G0/G1峰前出现一个亚二倍体峰（凋亡峰）,随着NIM浓度增高,G0/G1期细胞所占比例逐渐下降,G2/M期细胞比例升高,凋亡率逐渐增高;Western Blot结果显示,随着NIM浓度增加,COX 2蛋白表达量逐渐减少。上述结果均与NIM浓度相关,经统计学处理,与对照组和相邻前一浓度组比较,均差异有统计学意义（P＜0.05）。结论NIM能诱导人鼻咽癌CNE2细胞凋亡,且呈剂量依赖性,该作用与抑制COX 2有关。     

羽扇豆醇抑制人肝癌Huh-7细胞增殖机制研究

摘 要 目的：研究羽扇豆醇（lupeol）对人肝癌Huh-7细胞增殖的影响。方法: 运用CCK-8法检测lupeol（10~150 μmol·L^-1）对T24细胞活力的影响;运用caspase抑制剂确证凋亡相关蛋白caspase是否与lupeol对细胞增殖抑制作用有关;运用western blot检测lupeol对c-Jun蛋白表达的影响;运用real time PCR评价lupeol对miR-21、MMP-2、MMP-9基因mRNA表达的影响;运用pGL3 Basic荧光素酶报告系统研究lupeol对MMP-9启动子活性的影响。结果: Lupeol在72 h能够抑制Huh-7细胞增殖,该抑制作用具有剂量依赖性;lupeol处理细胞72 h后半抑制浓度（IC50）为80 μmol·L^-1;与对照组相比,运用lupeol能够使Huh-7细胞中c-Jun蛋白表达下调;lupeol能够降低miR-21表达水平;与对照组相比,lupeol处理Huh-7细胞降低MMP-9的mRNA表达,并使MMP-9基因启动子活性下调40%;以上结果与对照组比较差异均有统计学意义（P<0.05）。结论: Lupeol能够抑制人肝癌Huh-7细胞增殖,该作用与抑制miR-21/c-Jun/MMP-9信号通路有关。     

Chlorpyrifos and Cypermethrin Induce Apoptosis in Human Neuroblastoma Cell Line SH‐SY5Y

Our previous in vivo studies showed that chlorpyrifos (CPF) and cypermethrin (CM) in a mixture dermally administered, strongly inhibited cholinesterase activity in plasma and the brain and were very toxic to the rat central nervous system. In this work, the mechanisms of neurotoxicity have not been elucidated. We used human undifferentiated SH‐SY5Y cells to study mechanisms of pesticide‐induced neuronal cell death. It was found that chlorpyrifos (CPF) and its mixture with cypermethrin (CPF+CM) induced cell death of SH‐SY5Y cells in a dose‐ and time‐dependent manner, as shown by MTT assays. Pesticide‐induced SH‐SY5Y cell death was characterized by concentration‐dependent down‐regulation of Bcl‐2 and Bcl‐xL as well as an increase in the caspase 3 activation. Pan‐caspase inhibitor Q‐VD‐OPh produced a slight but significant reversal effect of pesticide‐induced toxicity indicating that the major caspase pathways are not integral to CPF‐ and CPF+CM‐induced cell death. Furthermore, signal transduction inhibitors PD98059, SL‐327, SB202190, SP600125 and mecamylamine failed to attenuate pesticides effect. Atropine exhibited minimal ability to reverse toxicity. Finally, it was shown that inhibition of TNF‐α by pomalidomide attenuated CPF‐/CPF+CM‐induced apoptosis. Overall, our data suggest that FAS/TNF signalling pathways may participate in CPF and CPF+CM toxicity.     

Butein,a Plant Polyphenol,Induces Apoptosis Concomitant with Increased Caspase‐3 Activity,Decreased Bcl‐2 Expression and Increased Bax Expression in HL‐60 Cells

Abstract: In the present study we have investigated whether butein could induce apoptosis in human leukaemic HL‐60 cells. The treatment of HL‐60 cells with butein induced apoptotic cell death as determined by morphological and biochemical changes. Apoptotic DNA fragments in the butein‐treated HL‐60 cells were increased gradually as determined by flow cytometric analysis. The caspase‐3 activity was increased during butein‐induced apoptosis. However, caspase‐3 inhibitor abrogated the butein‐induced DNA fragmentation. Furthermore, the treatment of HL‐60 cells with butein decreased the expression of Bcl‐2 protein, but increased the expression of Bax protein. These results suggest that butein‐induced apoptosis is mediated through the activation of caspase‐3 and it is associated with changed expression of Bcl‐2 and Bax proteins.     

Genotoxic Effects of Cylindrospermopsin,Microcystin-LR and Their Binary Mixture in Human Hepatocellular Carcinoma (HepG2) Cell Line

Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.     

Dopamine and Iron Induce Apoptosis in PC12 Cells

Abstract: Recent studies have shown that Fe²⁺ increases the oxidation of monoamines such as serotonin, dopamine and related toxins and that the formed oxidation products can undergo co-valent binding to free sulphydryl groups of proteins such as actin and "serotonin binding proteins" which are present in soluble brain extracts. Here we have tested the ability of ferrous iron to induce [³H]dopamine association to cytoplasmic proteins and we have established that a similar oxidation mechanism evidenced in vitro studies could be applied in cell culture. When PC12 cells were incubated with ferrous iron (ferrocene), the binding of [³H]dopamine to proteins was found to be two fold increased with respect to control. The iron is likely to accelerate the oxidation of dopamine to produce quinones which covalently bind to proteins and induce high-molecular protein aggregates. We evidenced that dopamine/iron combination induced cell death in undifferentiated PC12 cells via an active cellular process evaluated in terms of morphological and biochemical changes indicative of apoptosis. We also demonstrated induction of lipid peroxidation when dopamine and ferrocene were present in high concentrations. Moreover, ascorbic acid diminished apoptosis but not the lipid peroxidation process. It might indicate that ferrocene and dopamine could produce oxidative stress of a different nature. These results show that the actions of dopamine and iron are essential in the induction of apoptosis and lipid peroxidation. However, there is no necessary causual link between lipid peroxidation and apoptosis. Our data also suggest that iron is capable of increasing the cytotoxicity of dopamine merely by increasing its rate of oxidation and without intervention of the monoamine oxidase B enzyme and, hence, both phenomenons may occur independently from each other in rat pheochromocytoma PC12. These observations may have relevance to the understanding of the mechanism by which dopaminergic neurones are destroyed in some neurodegenerative disorders.     

姜黄素对人子宫内膜癌细胞增殖和凋亡的影响

目的:探讨姜黄素对人子宫内膜癌细胞(HEC-1B)增殖和凋亡的影响。方法:应用6.67~66.67μmol/L的姜黄素分别处理HEC-1B 48h后,采用MTT法检测姜黄素对HEC-1B的增殖程度的影响;流式细胞仪进行细胞周期时相分析;荧光显微镜观察细胞核形态的变化。结果:①MTT法显示培养48h后,姜黄素组的吸光度值A490nm低于对照组(P<0.01),且在一定浓度范围内呈剂量依赖性;②流式细胞仪检测显示培养48h后,姜黄素组的G2/M比例高于对照组(P<0.05),且在一定浓度范围内呈剂量依赖性;③荧光显微镜下给药组部分细胞发生凋亡形态学改变,凋亡率为10.22%~34.72%。结论:姜黄素可抑制HEC-1B的增殖,诱导细胞凋亡,其诱导凋亡的机制可能是通过将细胞阻滞在G2/M期来实现的。     

Semi‐Synthesis and Biological Evaluation of 1,2,3‐Triazole‐Based Podophyllotoxin Congeners as Potent Antitumor Agents Inducing Apoptosis in HepG2 Cells

A series of 4β‐[(4‐substituted)‐1,2,3‐triazol‐1‐yl]podophyllotoxin congeners were synthesized by employing click chemistry and further evaluated for their antitumor activity by MTT assay. Among them, six congeners ( 10 , 11 , 12 , 13 , 22 , and 24 ) exhibited approximately 100‐fold more potent inhibitory activity against four tumor cell lines (HepG2, MKN‐45, NCI‐H1993, and B16) than etoposide as positive control. Docking studies on binding in the ATPase domain of topoisomerase II revealed perfect docking of four congeners in the active site. Furthermore, the podophyllotoxin congeners 10 , 11 , 12 , and 13 induced cell cycle arrest of HepG2 cells at the G₂/M phase in a concentration‐dependent manner, assessed by flow cytometric analysis, highlighting that they exert their antitumor activity via HepG2 cell apoptosis.