bc1-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径.方法白藜芦醇体外处理HepG2肝癌细胞24 h后,以免疫组化检测凋亡调控基因bcl-2和bax及NF-kB的表达.结果白藜芦醇处理组HepG2细胞bcl-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P＜0.01);而bax阳性积分明显高于对照组(P＜0.01).结论白藜芦醇能下调HepG2细胞bcl-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一.     

NF-kB 拮抗剂PDTC 对HepG2 细胞的抑制及对caspase-3 表达的影响

目的:研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法:以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果:不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性(P<0.05);PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论:NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

NF-kB拮抗剂PDTC对HepG2细胞的抑制及对caspase-3表达的影响

目的：研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法：以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果：不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性（P〈0.05）;PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论：NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制*

目的: 探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法: 将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化;MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果: 与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P＜0.01),形态发生改变,呈现典型的凋亡细胞形态;G1期细胞数明显增加,而G2 期细胞数量显著减少(P＜0.05);Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P＜0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P＜0.05)。结论: 四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

白藜芦醇对猪前体脂肪细胞凋亡的作用及机理

旨在研究白藜芦醇对猪前体脂肪细胞凋亡的作用,探讨其分子机制。以50 μmol/L、100 μmol/L、200 μmol/L、400 μmol/L白藜芦醇处理猪前体脂肪细胞,采用Hoechst 33258染色剂染色,光学和荧光显微镜分别观察细胞的形态学变化。semi-qRT-PCR和Western blotting方法检测凋亡相关基因sirt1、caspase-3、bcl-2、bax、p53、NF-κB的mRNA和蛋白的表达变化。结果表明,白藜芦醇处理后,前体脂肪细胞出现明显的细胞凋亡,伴随细胞体积缩小,染色质凝集,核质固缩等特征显现,与对照组相比200 μmol/L处理组细胞的凋亡率显著升高 (P＜0.05)。凋亡相关基因sirt1、caspase-3和bax的mRNA和蛋白表达水平显著上调 (P＜0.05),而bcl-2、p53、NF-κB等基因的表达水平明显下调 (P＜0.05)。进一步证实白藜芦醇特异性地增加sirt1的表达活性,而sirt1的上调影响caspase-3和bcl-2家族因子的活性,同时参与调控p53和NF-κB的转录表达。因此,推测sirt1调控凋亡相关因子表达是白藜芦醇诱导前体脂肪细胞凋亡的关键原因。     

维生素K2对肝癌HepG-2细胞增殖的抑制作用

近年研究发现维生素K2（VK2）对肝癌具有抑制作用,但目前VK2的抗肝癌的机理尚不明确。VK2对人肝癌细胞株HepG-2细胞增殖的抑制作用及其机制。具体做法如下：体外培养肝癌HepG-2细胞,分别用不同浓度的VK2（0、5、10、20和40μmol／L）处理培养1～3d,采用台盼蓝拒染法测定各时期的各组细胞的活力;收集20μmol／LVK,作用24h和48h后的HepG-2细胞和对照细胞,抽提DNA电泳检测;收集20μmol／LVK,作用48h后的HepG-2细胞和对照细胞,抽提总RNA,半定量RT—PCR检测抗凋亡基因survivin、bcl-2和促凋亡基因bax的mRNA表达水平。各VK2处理组的活细胞数明显低于对照组（P〈0．05）;经VK2处理的细胞其DNA电泳结果呈明显的梯状条带;与对照组相比,VK2处理组细胞的抗凋亡基因survivin和bcl-2的RT—PCR产物电泳的目的条带相对灰度值（目的基因灰度／GAPDH灰度）明显减少（P〈0．05）,而促凋亡基因bax则无明显变化（P〉0．05）。VK2通过下调抗凋亡基因survivin和bel－2／bax的桌浊水平诱导肝痛HenG-2细胞凋亡。     

LINC00261/miR-182-5p/PFN1对乙肝病毒相关性肝细胞癌HepG2.2.5细胞放疗抵抗的影响

[目的]探讨LINC00261调控miR-182-5p/PFN1轴对乙型肝炎病毒相关性肝细胞癌HepG2.2.15细胞放疗抵抗的作用机制。[方法]用1 Gy的X射线处理HepG2.2.15细胞得放疗抵抗细胞HepG2.2.15/R,RT-qPCR检测LINC00261和miR-182-5p表达,Western Blot检测PFN1的表达,CCK-8检测细胞增殖活力,流式细胞术检测细胞凋亡率,彗星实验检测DNA损伤情况;双荧光素酶报告基因实验验证miR-182-5p和LINC00261或PFN1的靶向关系。[结果]LINC00261在HepG2.2.15细胞中低表达(P<0.01),且在其放疗抵抗细胞HepG2.2.15/R中表达更低(P<0.01)。过表达LINC00261抑制HepG2.2.15/R细胞增殖(P<0.01),诱导细胞凋亡(P<0.05)和DNA双链断裂(P<0.01);6 Gy X射线处理可上调过表达LINC00261对HepG2.2.15/R细胞凋亡(P<0.05)和DNA损伤的促进作用(P<0.01)。双荧光素酶报告基因实验证实miR-182-5p与LINC00261或PFN1的靶向关系。过表达LINC00261或过表达PFN1可下调过表达miR-182-5p对HepG2.2.15/R细胞增殖的促进作用(P<0.05)以及对凋亡和DNA损伤的抑制作用(P<0.05)。[结论]过表达LINC00261靶向下调miR-182-5p,促进PFN1表达,促进HepG2.2.15细胞放疗抵抗。     

乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2 表达 和caspase-3 活性的影响

目的：探讨乳酸堆积和二氯乙酸钠(DCA)对肝癌细胞(HepG2)凋亡和bax、bcl-2 表达及caspase-3 活性的影响。方法：通过体 外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0 mmol/L、1.0 mmol/L、2.0 mmol/L、4.0 mmol/L、8.0 mmol/L的乳 酸培养液以及在不同浓度乳酸组中加入终浓度为10-3mmol/L DCA 培养液与HepG2共同培养,其中以0 mmol/L 乳酸组为对照 组。采用MTT法检测乳酸对HepG2 的抑制率,流式细胞仪检测乳酸和DCA 对HepG2的凋亡百分率,用Real-time PCR法测定 bax 及bcl-2 mRNA的表达,用免疫荧光法检测caspase-3 的活性。结果：乳酸对HepG2 的IC50值为13.6 mol/L,与对照组比较,随 着乳酸浓度的增加,HepG2 凋亡率增加,bax mRNA 表达升高,bcl-2 mRNA 的表达降低,caspase-3活性增加,其中1.0 mmol/L 乳 酸组与对照组比较(P>0.05),2.0 mmol/L,4.0 mmol/L 和8.0 mmol/L乳酸组与对照组比较差异有统计学意义(P<0.05)。加入DCA 后,HepG2 凋亡减少,2.0 mmol/L 乳酸+DCA 组、4.0 mmol/L乳酸+DCA 组、8.0 mmol/L乳酸+DCA 组与同浓度的乳酸组比较, bax mRNA 表达减少（P<0.05）,bcl-2 mRNA 表达增加（P<0.05）,caspase-3 活性减低（P<0.05）。结论：乳酸可诱导HepG2凋亡,且随 着乳酸浓度的增高,HepG2 的凋亡率增加,其机制可能是通过对bcl-2 及bax mRNA 表达的改变以及激活caspase-3 活性而实现, DCA可以降低HepG2 凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。     

刺五加多糖诱导人小细胞肺癌H446 细胞凋亡

研究刺五加多糖(Acanthopanax senticosus polysaccharide,ASPS)诱导H446细胞凋亡及其可能的作用机制.采用MTT法检测ASPS对小细胞肺癌H446细胞增殖的抑制作用;Hoechst 33258染色和流式细胞技术检测经ASPS处理后H446细胞凋亡的形态特征及凋亡率的变化;West ern印迹方法检测凋亡相关基因bax、bcl-2、p53 表达的变化.MTT分析表明,ASPS作用48 h后可明显抑制H446细胞的增殖,半数抑制浓度(IC50值)为476.36 mg/ml;Hoechst 染色结果: H446细胞在ASPS诱导下出现典型的凋亡形态;流式细胞术检测结果显示: 对照组及浓度为240、480、960 mg/ml 药物处理组凋亡率分别是(5.02±0.4)%、(11.12±0.8)%、(19.89±0.5)%、(22.54±0.8)%;Western印迹显示: 在ASPS的诱导下bax、p53的表达量提高,而bcl-2的表达量下降.研究表明,ASPS对H446细胞增殖有抑制作用,并能促进其凋亡;ASPS通过上调bax、 p53表达,下调bcl-2表达促进H446细胞凋亡.     

索拉菲尼联合阿霉素对人肝癌细胞株HepG2的抑制作用

目的：探讨索拉非尼（Sorafenib）和阿霉素（adriamycin）联合用药对肝癌细胞株nepG2的作用及可能的机制。方法：以不同浓度索拉非尼和不同浓度阿霉素分别组成单药组和索拉非尼＋阿霉素联合用药组作用于HepG2细胞,MTT法检测增殖抑制率、流式细胞仪分析细胞周期和凋亡率。结果：索拉非尼、阿霉素单药与联用均能抑制HepG2细胞增殖,呈剂量依赖效应,两药联用有协同效应（P〈0.01）。索拉非尼、阿霉素单药与联用均能诱导HepG2细胞凋亡,并以联合组更为明显,与对照组比较有显著的统计学意义（P〈0.01）。索拉非尼及阿霉素单药作用均可使细胞周期阻滞于G0-G1期,联合用药组G0/Gl期细胞比率低于索拉非尼及阿霉素单药组,S期细胞比率高于单药组;阿霉素能抑制HepG2细胞Survivin mRNA表达诱导细胞的凋亡。结论：索拉非尼与阿霉素联合作用于人肝癌HepG2细胞具有协同作用,其机制可能是通过多途径共同抑制HepG2细胞增殖及诱导细胞凋亡。     

A novel copper (II) complex activated both extrinsic and intrinsic apoptotic pathways in liver cancerous cells

Recent advances have put fundamental focus on the application of copper (II) (Cu [II]) complexes as agents for fighting against cancer. To determine whether [Cu(L)(2imi)] complex as a novel Cu complex can induce apoptosis in HepG2 as cancerous cells and L929 as normal cells via extrinsic or intrinsic apoptotic pathways, both cell lines were treated for 24 and 48 hours at IC₅₀ concentrations of [Cu(L)(2imi)] complex. Then, the expression of some apoptosis-related genes including p53, caspase-8, bcl-2, and bax were assayed by real-time polymerase chain reaction. The [Cu(L)(2imi)] complex seems to inhibit the expression of bcl-2 in complex-treated HepG2 cancerous cells following the 24- and 48-hour treatment. The complex upregulated the p53, bax, and caspase-8 genes, therefore treatment of HepG2 cancerous cells with [Cu(L)(2imi)] complex induces programmed cell death via the upregulation of relative bax/bcl-2 ratio. Finally, this copper complex triggered apoptosis in HepG2 cells via both intrinsic and extrinsic pathway, whereas treatment of normal L929 cells with this complex induce apoptosis only via intrinsic pathway with the upregulation of relative bax/bcl-2 ratio and does not affect the expression level of caspase-8 gene and does not trigger the extrinsic pathway. Finally, these results obtained from present study confirm the role of a novel Cu complex on the induction of apoptosis process in HepG2 and L929 cells by overexpression of bax, inhibition of bcl-2 and increase of the relative bax/bcl-2 ratio. These results support that the [Cu(L)(2imi)] complex is able to induce apoptosis in cancerous cells, therefore, it has a potential for development as a novel anticancer drug.     

Persian shallot, <Emphasis Type="Italic">Allium hirtifolium</Emphasis> Boiss,induced apoptosis in human hepatocellular carcinoma cells

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC₅₀ value (inhibiting cell growth by 50%) was 149 μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.     

白藜芦醇对人子宫内膜癌细胞系AN3CA增殖和凋亡效应及其机制探讨

目的：探讨白藜芦醇（resveratrol,Res）对人子宫内膜癌细胞AN3CA的增殖抑制和凋亡诱导效应及可能存在的机制。方法：应用噻唑蓝（MTT）法检测Res对AN3CA的增殖影响;流式细胞术检测Res对细胞周期分布和凋亡影响：荧光实时定量PCR检测Res对细胞Bcl-2、Bax和MMP-9mRNA表达水平的影响;WesternBlot方法检测Res对PCNA、Bcl-2、Bax及ERK1／2、P—ERK1／2蛋白表达水平的影响。结果：Res对子宫内膜癌细胞AN3CA具有显著的生长抑制作用（P〈0．01）,呈时间-剂量依赖性;不同浓度Res处理细胞G0／G1期比例显著增加伴随S期细胞数的减少;细胞凋亡率明显增高,200Dmol／lRes处理48h凋亡率可达30．96％±2．041％（P〈0．01）。与对照组相比,Res能抑制PCNA的蛋白表达量,增加Bax和降低Bcl-2转录和蛋白水平的表达量。Res在短时间内（0．5—1h）激活ERK1／2的磷酸化表达但随着作用时间延长（4—48h）其表现为抑制效应。结论：Res具有抑制AN3CA细胞增殖,诱导细胞G0／G1期阻滞和凋亡的效应。Res诱导凋亡可能是通过上调Bax,下调Bcl-2发挥作用,其抗癌作用机制可能与ERK1／2通路失调相关。     

Methylated chrysin induces co-ordinated attenuation of the canonical Wnt and NF-kB signaling pathway and upregulates apoptotic gene expression in the early hepatocarcinogenesis rat model

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer^32/36 in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr³⁹³ and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β^Ser9. Dietary DMF (100 mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.     

Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.