多药耐药对肝癌细胞增殖、凋亡、侵袭活性、耐药机制及丝裂原活化蛋白激酶表达的影响

目的:通过化疗药物诱导建立多株人肝癌多药耐药细胞模型,探讨多药耐药获得对细胞增殖、凋亡、侵袭能力的影响,耐药机制及与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路表达的相关性.方法:采用多柔比星( adriamycin,ADM)高浓度冲击和小剂量缓升2种方法诱导建立人肝癌细胞(HepG2、SMMC-7721和BEL-7402)耐药模型.比较耐药细胞株与亲本细胞株间的差异,主要包括采用生物发光法进行药物敏感实验并计算耐药指数;RT’-PCR法和免疫组织化学法检测相关耐药基因(p-糖蛋白、多药耐药相关蛋白1、肺癌耐药蛋白、乳腺癌耐药蛋白、蛋白激酶C、谷胱甘肽-S-转移酶-π和拓扑异构酶Ⅱ)mRNA及相应蛋白的表达.免疫组织化学法检测与细胞增殖活性相关的Ki-67和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达:FCM法检测细胞周期、细胞凋亡率的改变Transwell小室法检测细胞体外侵袭能力的改变最后,分别采用RT-PCR和蛋白质印迹法检测MAPK信号通路中ERK1、ERK2、ERK5、JNK1、JNK2和p38a基因及相应蛋白的表达.结果:共建立6株ADM耐药细胞株;与亲本细胞株相比,耐药细胞株耐药指数均＞5,并对多种化疗药耐受性增强,多药耐药相关基因及蛋白的表达均上调2～10倍,以高浓度冲击法诱导的细胞中蛋白表达上调明显.耐药细胞中Ki-67及PCNA蛋白的表达量均升高20倍以上,细胞周期被阻滞在S期,体外侵袭能力增强1.3～2.5倍;ADM干预后,细胞凋亡率减少60％以上.MAPK信号通路中不同基因和蛋白表达量均有不同比例升高,以ERK1、ERK2升高明显.结论:ADM能够诱导多株人肝癌细胞产生多药耐药,导致细胞增殖活性升高、细胞周期改变、抗凋亡能力及侵袭能力增强.MAPK信号通路中相关蛋白表达的上调与细胞多药耐药有一定关系.     

Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest

Human Hepatocellular carcinoma (HCC) cell types exhibit a major resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death, and the key determinants of mechanisms accounting for TRAIL susceptibility, still remain controversial. Our previous studies showed that overexpression of survivin reduced sensitivity of HCC cells to TRAIL. The aim of this study is to investigate how tumor cells escape TRAIL-mediated surveillance through survivin expression and how to reverse the resistance of TRAIL-inducing apoptosis. Seven tumor cell lines were treated with or without TRAIL protein and antisense oligodeoxynucleotides (ODNs) against survivin in culture. HepG(2) and SMMC7721 cells were treated with mimosine, thymidine or nocodazole to synchronize their cell cycle phases and then used to test their sensitivity to TRAIL. In vivo effects of TRAIL plasmid alone or in combination with survivin antisense ODNs on tumor growth were evaluated in a nude mouse hepatoma model of HepG(2) cell grafts. Varied levels of survivin mRNA in various cell lines were evaluated and negatively correlated to TRAIL-induced apoptosis. Hepatoma HepG(2) and SMMC7721 cells in G (1) or S phase are more sensitive to TRAIL than those in G(2) phase. Treatment with survivin antisense ODNscaused S phase arrest and significantly enhanced TRAIL-induced apoptosis. TRAIL protein caused G(2)/M arrest and resulted in an increase of survivin in HepG(2) cells. Combined TRAIL plasmid and survivin antisense ODNs significantly supressed the growth of tumor xenografts as compared to TRAIL plamid or antisense ODNs alone during four weeks of observation. The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression levels may sensitizes tumor cells to TRAIL-induced apoptosis.     

HIFU逆转人肝癌细胞HepG2/Adm多药耐药的实验研究

背景与目的：超声波能够改变细胞膜通透性,增强化疗药物对肿瘤细胞的杀伤作用。本研究评价高强度聚焦超声及其联合阿霉素(Adriamycin,ADM)对人肝癌多药耐药细胞株HepG2／Adm的效应,并探讨其作用机制。方法：取对数生长期的HepG2、HepG2／Adm细胞进行实验,分为HepG2、HepG2(超声波照射5s)、HepG2／Adm、HepG2／Adm(超声波照射5s)4组。用MTT法检测处理前后耐药细胞对抗癌药物的敏感性;用流式细胞仪检测肿瘤细胞表面mdr1基因表达产物P170的表达及细胞内阿霉素浓度;利用SP免疫组化法观察细胞膜及核膜上P-糖蛋白(P-glycoprotein,P-gp)的表达。结果：频率0.8MHz、焦域声强460W／(cm^2连续照射5s,能够部分逆转HepG2／Adm细胞的耐药性,HepG2／Adm细胞P-gp的表达活性下降83．1％,耐药细胞内ADM浓度增加88％,对ADM、cDDP、MMc、5-FU和MTX相对逆转效率分别为66．4％、63．4％、89．4％、72．4％和75．0％。结论：高强度聚焦超声可提高人肝癌细胞HepG2／Adm内药物浓度,降低细胞膜及核膜表面P-gp表达,从而部分逆转肿瘤细胞多药耐药。     

CSIG promotes hepatocellular carcinoma proliferation by activating c-MYC expression

Cellular senescence-inhibited gene (CSIG) protein significantly prolongs the progression of replicative senescence, but its role in tumorigenesis is unclear. To reveal the role of CSIG in HCC, we determined its expression in HCC tissues and surrounding tissues and its functions in tumor cell proliferation in vitro and in vivo. CSIG protein was overexpressed in 86.4% of the human HCC cancerous tissues as compared with matched surrounding tissues, and its protein expression was greater in HCC cells than the non-transformed hepatic cell line L02. Furthermore, upregulation of CSIG significantly increased the colony formation of SMMC7721 and HepG2 cells, and silencing CSIG could induce cell cycle arrest and cell apoptosis. The tumorigenic ability of CSIG was confirmed in vivo in a mouse xenograft model. Our results showed that CSIG promoted the proliferation of HepG2 and SMMC7721 cells in vivo. Finally, CSIG protein directly interacted with c-MYC protein and increased c-MYC protein levels; the ubiquitination and degradation of c-MYC protein was increased with knockdown of CSIG. CSIG could also increase the expression of c-MYC protein in SMMC7721 cells in vivo, and it was noted that the level of c-MYC protein was also elevated in most human cancerous tissues with high level of CSIG.     

POKemon和NF-κB p65在肝癌细胞中的信号调控

目的:旨在探讨原癌基因POKemon和核因子-κB(nuclear factor-kappa B,NF-κB) p65在肝癌细胞中的信号调控作用.方法:采用实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)和蛋白质印迹法分别检测POKemon、NF-κB p65 mRNA和蛋白在肝癌细胞株HepG2和SMMC7721及人胚胎肝细胞LO2中的表达情况;随后应用小干扰RNA(small interference RNA,siRNA)法依次分别抑制POKemon和NF-κB p65在肝癌细胞中的表达,观察二者在肝癌细胞中的变化,并应用FCM法检测对肝癌细胞凋亡的影响.结果:POKemon和NF-κB p65在肝癌细胞HepG2和SMMC7721中的表达量明显高于人胚胎肝细胞LO2;特异性针对POKemon基因的siRNA抑制POKemon的表达后,NF-κB p65在HepG2和SMMC7721细胞中的表达量也明显下降,转染前后分别为2.12±0.14vs 1.37±0.11和2.08±0.16vs 1.35±0.13,差异有统计学意义(P＜0.05),且肝癌细胞凋亡明显增加分别为(5.07±0.46)％vs (39.65±3.75)％和(5.71±0.83)％vs (33.21±3.66)％,差异有统计学意义(P＜0.05);特异性针对NF-κB基因的siRNA抑制NF-κB p65的表达后,POKemon在HepG2和SMMC7721细胞中的表达则无明显变化,其表达量转染前后分别为1.86±0.12vs 1.90±0.13和1.91±0.11 vs 1.85±0.11,差异无统计学意义(P＞0.05),但明显提高HepG2和SMMC7721细胞的凋亡率,差异有统计学意义(P＜0.05).结论:原癌基因POKemon可通过调控NF-κB p65的表达而阻遏肿瘤细胞凋亡,促进肝癌的发生、发展.     

反义抑制Survivin表达增强肝癌细胞系对阿霉素的敏感性

背景与目的:由于Survivin在大多数肿瘤细胞中过表达,而在正常成人终末分化组织中沉默,并参与了恶性肿瘤的发生、发展和化疗耐药,使其成为值得关注的抗癌治疗靶。反义寡核苷酸可用来封闭凋亡抑制基因Survivin,诱导肿瘤细胞凋亡或增强其化疗敏感性。本研究旨在探讨反义抑制Survivin表达对肝癌细胞系阿霉素敏感性的影响。方法:RT鄄PCR、Westernblot检测HepG2和HepG2/ADM细胞Survivin表达;MTT法评估HepG2和HepG2/ADM对反义复合物和阿霉素的敏感性;不同浓度的反义复合物分别转染HepG2和HepG2/ADM细胞,RT鄄PCR检测SurvivinmRNA表达;等辐射分析法评估不同浓度反义复合物和亚致死浓度阿霉素对HepG2和HepG2/ADM细胞的协同效应;流式细胞术检测激活型Caspase鄄3表达及凋亡率。结果:HepG2/ADM细胞SurvivinmRNA是HepG2细胞的15倍,蛋白水平是HepG2的18倍。两种细胞均显示对反义复合物敏感并呈浓度依赖关系,反义复合物对HepG2和HepG2/ADM细胞的IC50分别为317.90nmol/L和480.74nmol/L,在500nmol/L时抑制率达到71.10%和53.67%。ADM对HepG2和HepG2/ADM细胞的IC50分别为0.36μg/ml和2.12μg/ml,耐药指数为6。反义复合物剂量依赖性下调HepG2和HepG2/ADM细胞SurvivinmRNA表达,对HepG2和HepG2/ADM细胞的IC50分别为271.93nmol/和365.72nmol/L,400nmol/L时表达水平分别下调了69.12%和60.01%。反义复合物和低浓度ADM联合使HepG2细胞的敏感性增加了6倍,使HepG2/ADM细胞的敏感性增加了4倍。联合处理激活了HepG2/ADM细胞Caspase鄄3活性,诱导细胞凋亡,激活型Caspase鄄3和凋亡率随反义复合物浓度的增加而逐渐递增。结论:反义抑制Survivin表达增强肝癌细胞系对阿霉素的敏感性,反义寡核苷酸和阿霉素联合可能是耐阿霉素肝细胞癌临床治疗的一种合理策略。     

ICE基因转染联合化疗药物杀伤肝癌细胞的研究

目的：研究人ICE基因转染联合化疗药物诱导体外杀伤肝癌细胞的作用。方法：应用电穿孔法将构建成功含有目的基因的逆转录病毒载体pLXSN-hICE导入包装细胞系PA317，筛选G418抗性克隆，并将其病毒上清转入肝癌细胞株HepG2，DNA提取、电泳观察；采用^3H-TDR掺入法观察、分析化疗药物卡铂对肝癌细胞株SMMC7721及转入相应目的基因后的SMMC7721-ICE，SMMC7721-反义hICE，SMMC7721-neo细胞株体外增殖的影响。结果：ICE基因转染可诱导HepG2形成具有凋亡特征的梯状DNA；在卡铂低浓度诱导下，与对照细胞相比SMMC7721-hICE细胞株体外增殖明显受抑。结论：人ICE基因转染可直接诱导肝癌细胞株HepG2凋亡，明显提高肝癌细胞SMMC7721对化疗药物卡铂杀伤的敏感性，ICE基因转染联合化疗药物诱导极大增强了对肝癌细胞的杀伤作用，可能是治疗肝癌一个有前途的方案。     

表没食子儿茶素没食子酸酯对两株不同肝癌耐药细胞株基因表达谱的影响

背景与目的:绿茶中的儿茶索单体表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)在体内外能逆转肝癌细胞多药耐药性.鉴于多药耐药机制的复杂性,本研究拟采用高通量基因芯片技术进一步探讨EGCG逆转人肝癌细胞BEL7404/ADM、BEL7402/5-FU多药耐药的可能机制.方法:MTT法检测药物敏感性.基因芯片技术分析EGCG作用于两株不同肝癌耐药细胞的基因表达谱差异.RT-PCR和Western blot法分别检测相同处理条件下两株肝癌耐药细胞的基因MDR1、LRP和Cyclin G1蛋白表达变化以验证基因芯片技术结果.结果:EGCG对BEL7404/ADM、BELT402/5-FU细胞的IC10分别为24.76 mg/L、20.60 mg/L.20 mg/L EGCG联合0.05 mg/L ADM、100 μmol/L 5-FU对BEL7404/ADM、BEL7402/5-FU的逆转倍数分别为9.66、2.36倍.EGCG作用BEL7404/ADM双荧光通路显著差异基因210个,上调表达38个,下调表达172个.与耐药机制相关的可能基因有ABCB10(MDR/TAP)、TOP2A、TOP2B、CCNG1上调,ABCB1、MVP、ARHD、HDAC5、GSS、GSTPI、HSPA1B、HSPB7、CDKN1A、RAB11B、RAB9P40下调.作用BEL7402/5-FU双荧光通路显著差异基因179个,上调表达31个,下调表达148个.与耐药机制相关的可能基因有ABCG(BCRP)、CCNG2、GADD34、RB1、RBBP4上调,DTYMK、GPX1、USP5、BAX、BAK1、HSPA1L下调等.相同处理条件下两组肝癌耐药细胞的MDR1、LRP基因表达均减少,Cyclin G1蛋白表达均增加.结论:EGCG对肝癌多药耐药细胞BEL7404/ADM、BEL7402/5-FU具逆转作用,但作用于不同肝癌多药耐药细胞的基因表达谱变化不完全相同.     

Toll样受体4的表达及miR-TLR4干扰对HBV相关肝癌细胞生物学活性的影响

目的 观察Toll样受体4在乙肝病毒相关肝癌细胞系中的表达及对其生物学活性的影响。方法 Western blot检测Hep3B、HepG2.2.15、HepG2、SMMC7721及Huh7五种肝癌细胞中TLR4蛋白表达情况,选择TLR4表达高的HBV阳性肝癌细胞作为研究对象。构建4个miR-TLR4质粒和一个阴性对照质粒,选择干扰效果明显的质粒转染TLR4表达高的HBV阳性肝癌细胞。实验分为正常组,阴性对照组,干扰质粒3组及干扰质粒4组。通过MTT法、克隆平板形成实验、流式细胞术分别检测干扰TLR4表达对肝癌细胞增殖、克隆形成、周期及凋亡的影响。结果 TLR4基因在五种肝癌细胞株中均有表达,在Hep3B细胞中表达最高。与正常组和阴性对照组相比,干扰TLR4表达后,Hep3B细胞的增殖能力明显受到抑制（P＜0.05）,克隆形成率显著下降（P＜0.05）,周期阻滞于G₂/M期,并促进细胞凋亡（P＜0.05）。结论 TLR4表达上调促进HBV相关肝癌细胞Hep3B生长增殖、周期再分布以及抑制凋亡,在肝癌发生发展过程中起重要作用。     

AF0.3启动子调控的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异杀伤作用

背景与目的:甲胎蛋白(α-fetoprotein,AFP)启动子调控下的目的基因能在AFP阳性肝癌组织中特异性表达;嘌呤核苷磷酸化酶(Escherichiacolipurinenucleosidephosphorylase,PNP)/6-甲基嘌呤-2′-脱氧核糖核苷(6-methylpurine-2-deoxyriboside,MeP-dR)自杀基因系统具有强杀瘤效应。本研究旨在探讨AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异性杀伤作用。方法:构建甲胎蛋白启动子AF0.3调控下PNP基因表达载体pAF0.3/PNP,导入AFP阳性肝癌细胞HepG2和阴性的肝癌细胞株SMMC7721,利用G418筛选获得稳定转染PNP基因的HepG2/0.3-PNP及SMMC7721/0.3-PNP细胞。RT-PCR检测二者在细胞中的表达。台盼蓝拒染法检测细胞增殖效应,MTT法和流式细胞仪检测两株细胞对MeP-dR的敏感性及旁观者效应,高效液相色谱法(highperformanceliquidchromatography,HPLC)检测PNP基因产物活性。结果:不论有氧还是缺氧条件,HepG2/0.3-PNP对MeP-dR均较为敏感,SMMC7721/0.3-PNP则对MeP-dR完全不敏感。在任何一种条件下,HepG2/AF0.3-PNP在混合细胞中比例达25%后,就可致明显旁观者效应;而在同样条件下,SMMC7721/0.3-PNP不导致明显的旁观者效应。HPLC结果显示,pAF0.3/PNP在HepG2细胞中可以将MeP-dR转化为6-MP,但其在SMMC7721中则不具有转化活性。结论:AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性HepG2细胞有较好的杀伤作用。     

Growth arrest induced by C75, A fatty acid synthase inhibitor, was partially modulated by p38 MAPK but not by p53 in human hepatocellular carcinoma

C75, a well-known fatty acid synthase (FAS) inhibitor, has been shown to possess potent anti-cancer activity in vitro and in vivo. In this study, we reveal that C75 is a cell cycle arrest inducer and explore the potential mechanisms for this effect in hepatocellular carcinoma (HCC) cell lines with abundant FAS expression: HepG2 and SMMC7721 cells with wt-p53, and Hep3B cells with null p53. The results showed FAS protein expression and basal activity levels were higher in HepG2 cells than in the other two HCC cell lines. Treatment with C75 inhibited FAS activity within 30 min of administration and induced G(2) phase arrest accompanied by p53 overexpression in HepG2 and SMMC7721 cells. By contrast, C75 triggered G(1) phase arrest in Hep3B cells, and RNA interference targeting p53 did not attenuate C75-induced G(2) arrest in HepG2 cells. Similarly, p53 overexpression via p53 plasmid transfection did not affect C75-induced G(1) phase arrest in Hep3B cells. However, we observed a clear correlation between p38 MAPK activation triggered by C75 and the induction of cell cycle arrest in all three HCC cells. Furthermore, treatment with the p38 MAPK inhibitor SB203580 reduced p38 MAPK activity and cell cycle arrest, and also partially restored cyclin A, cyclin B1, cyclin D1 and p21 protein levels. Collectively, it was p38 MAPK but not p53 involved in C75-mediated tumor cell growth arrest in HCC cells.     

Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis

Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Africa and Asia. The mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression of HCC are not clear. We constitutively overexpressed active Notch1 in human HCC to explore the effects of Notch1 signaling on HCC cell growth and to investigate the underlying molecular mechanisms. We show here that overexpression of Notch1 was able to inhibit the growth of HCC cells in vitro and in vivo. Biochemical analysis revealed the involvement of cell cycle regulated proteins in Notch1-mediated G(0)/G(1) arrest of HCC cells. Compared with green fluorescent protein (GFP) control, transient transfection of Notch1 ICN decreased expression of cyclin A (3.5-fold), cyclin D1 (2-fold), cyclin E (4.5-fold), CDK2 (2.8-fold), and the phosphorylated form of retinoblastoma protein (3-fold). Up-regulation of p21(waf/cip1) protein expression was observed in SMMC7721-ICN cells stably expressing active Notch1 but not in SMMC7721-GFP cells, which only express GFP. Furthermore, a 12-fold increase in p53 expression and an increase (4.8-fold) in Jun-NH(2)-terminal kinase activation were induced in SMMC7721-ICN cells compared with SMMC7721-GFP cells. In contrast, expression of the antiapoptotic Bcl-2 protein could not be detected in SMMC7721-ICN cells. These findings suggest that Notch1 signaling may participate in the development of HCC cells, affecting multiple pathways that control both cell proliferation and apoptosis.     

Runx2通过促进MMP9的表达增强肝细胞肝癌迁移侵袭能力

  目的   研究Runx2对肝癌细胞中MMP9的表达以及其对肝癌细胞侵袭迁移能力的影响。  方法   通过免疫组织化学染色法对2005年12月至2015年12月天津医科大学肿瘤医院和天津医科大学总医院189例肝细胞肝癌病例标本中Runx2和MMP9的表达进行分析;将Runx2过表达质粒和干扰质粒分别转染至肝癌细胞系HepG2和SMMC7721细胞中。使用Western blot法检测转染后HepG2和SMMC7721细胞中Runx2和MMP9的表达情况;划痕、侵袭实验检测Runx2对肝癌细胞迁移和侵袭能力的影响。  结果   免疫组织化学结果显示Runx2的表达和MMP9的表达相关。在HepG2细胞中上调Runx2的表达,促进了MMP9的表达,增强了HepG2细胞迁移侵袭能力;在SMMC7721细胞中下调Runx2的表达,降低了MMP9的表达,抑制了SMMC7721细胞的迁移侵袭能力。  结论   Runx2的表达可能参与促进肝细胞肝癌细胞MMP9的表达,进而促进肝细胞肝癌的迁移侵袭能力。      

下调UHRF1的表达在抑制肝癌进展中的作用

目的 探讨UHRF1的异常表达在肝癌（hepatocellular carcinoma, HCC）进展中的作用机制。方法 采用RT-PCR和Western blot法检测UHRF1在20例HCC标本中的mRNA和蛋白表达水平, Western blot法检测不同转移潜能的肝癌细胞HepG2、SMMC7721、MHCC97L和HCCLM3中的UHRF1 的表达水平;siRNA技术干扰HCCLM3细胞中UHRF1表达后,MTT法检测细胞增殖,Western blot 法检测BAX和BCL-2表达水平的改变,流式细胞术检测HCCLM3细胞周期和细胞凋亡的变化。结 果 UHRF1在肺癌组织中的表达水平较癌旁组织显著上调（P<0.05）,随着肝癌细胞转移潜能升高而UHRF1表达水平也依次升高（P<0.01）;siRNA技术干扰HCCLM3细胞中UHRF1表达后,HCCLM3细胞生长速度明显下降（P＜0.05）,HCCLM3细胞的促凋亡蛋白BAX表达水平明显上升,而抑凋亡蛋白BCL-2表达水平明显下降;UHRF1-siRNA干扰HCCLM3细胞48 h时,细胞周期分布无明显影响（P＞0.05）;但UHRF1表达下调有促凋亡作用,与对照组相比,干扰组的细胞凋亡率显著升高,差异有统计学意义（P＜0.05）。结论 UHRF1基因在肝癌组织中表达上调,下调UHRF1的表达能够抑制肝癌进展,可能是通过诱导肿瘤细胞凋亡来实现的。     

JNK/SAPKs信号转导通路在榄香烯抗肝癌效应中的作用

目的:探讨JNK/SAPKs信号转导通路在榄香烯抗肝癌效应中的作用,为阐明榄香烯抗癌效应的分子机制提供参考.方法:气相色谱法检测榄香烯在Hca-F肝癌细胞内的分布.透射电镜观察SMMC7721凋亡细胞的超微结构变化.利用Western-blotting对榄香烯处理的HepG2肝癌细胞JNK/SAPKs进行的活性分析.利用RT-PCR检测榄香烯处理的SMMC7721肝癌细胞的DAXX基因表达.结果:榄香烯标准品在8.42 min时出现洗脱峰.榄香烯处理3 h后,SMMC7721细胞开始发生具有凋亡特征的变化.榄香烯处理组HepG2细胞的JNK/SAPKs激酶活性次于热休克处理组,高于对照组.榄香烯处理的SMMC7721细胞组未见DAXX基因的表达.结论:榄香烯能够进入细胞内.其引起的肿瘤细胞凋亡可能是通过活化JNK/SAPKs来诱导的.DAXX传人途径在榄香烯诱导的JNK/SAPKs活化过程中可能不起主要作用.     

重组人生长激素在体外对人肝癌细胞系SMMC7721的作用

[目的]研究重组人生长激素（recombinant human growth hormone，rhGH）在体外对人肝癌细胞系SMMC7721生长的影响[方法]实验分为对照组、rhGH组、rhGH＋阿霉素fAdriamycin，ADM）组及ADM组；通过体外细胞培养、四甲基俏氯唑蓝（MTT）比色技术及流式细胞术等手段．测定不同浓度的rhGH及其与ADM合用时对人肝癌细胞系SMMC7721的生长曲线、细胞抑制率、细胞周期及增殖指数（PI）的影响.[结果]与对照组相比，不同浓度的rhGH在体外对人肝癌细胞系SMMC7721的分裂增殖无明显影响（P〉0．05），生长曲线不随rhGH浓度的增加而升高；rhGH＋ADM组与ADM组比较细胞抑制率和生存率差异均无湿著性（P〉0．05），生长曲线无明显差异；与对照组及rhGH组相比较，单用ADM或rhGH＋ADM对该细胞的抑制率增加、PI明显减少（P〈0．01），而在单用ADM和rhGH＋ADM组间差异均无统计学意义（P〉0．05）：[结论]rhGH在体外对人肝癌细胞系SMMC7721的分裂增殖无明显促进作用．对ADM的抗癌作用无明湿影响。     

Reversal of Adriamycin Resistance of Hepatocellular Carcinoma by Targeting with Recombined Adenovirus Caning Antisense mdr1 RNA

OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma (HCC). However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance (MDR) is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS pCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdrl gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-Scel/l-Ceu l, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL 1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1 -mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase (P<0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM asmdr1 group compared to the ADM group at the 4th week (P<0.05). Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.     

叶绿酸铜钠对SMMC7721肝癌细胞抑制作用的体外实验研究

[目的]研究叶绿酸铜钠（CHL）体外抑制SMMC7721肝癌细胞的作用及机制。[方法]（1）MTT法测半数抑制浓度（IC50）并绘制生长曲线;（2）流式细胞仪观察CHL对SMMC7721细胞凋亡的诱导作用;（3）免疫组化方法检测增殖细胞核抗原（PCNA）、细胞周期素D1（Cyclin D1）、环氧化酶-2（COX-2）的表达。[结果]（1）CHL对SMMC7721细胞具有增殖抑制作用,且具有浓度和时间依赖效应;（2）CHL能诱导SMMC7721细胞G1期阻滞,通过抑制细胞生长周期、降低细胞增生速率抑制肿瘤细胞增殖;（3）CHL对SMMC7721细胞凋亡无明显诱导作用;（4）CHL能下调SMMC7721细胞PCNA和COX-2蛋白表达,并呈一定的浓度依赖性。对Cyclin D1的表达无明显影响。[结论]CHL能抑制SMMC7721细胞的生长,这种抑制作用同PCNA、COX-2表达下调及细胞G1期阻滞有关,但与细胞凋亡和Cyclin D1的表达无关。     

Berberine induces cell death in human hepatoma cells in vitro by downregulating CD147

The isoquinoline plant alkaloid berberine has anti-tumor effects on a variety of carcinoma cells, mainly through inhibition of cell proliferation, apoptosis induction and cell cycle arrest. However, the mechanisms underlying its role in tumor progression are unknown. In the present study, we investigated the molecular mechanisms involved in berberine-induced cell death in human hepatoma carcinoma cell (HCC) lines HepG2 and SMMC7721. Our results showed that berberine inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell death via apoptosis and autophagy. Moreover, berberine treatment significantly inhibited CD147 expression by HCC cells in a dose-dependent manner. Overexpression of CD147 protein markedly reduced berberine-induced cell death. Our data provide the first experimental evidence that berberine induces cell death in HCC cells via downregulation of CD147 and suggest a new mechanism to explain its anti-tumor effects.     

Epirubicin may enhance the inhibition of hepatocellular carcinoma induced by iodine-125 seeds through downregulating WNT pathway

Aim

To investigate the role of iodine-125 (¹²⁵I) combined with epirubicin (EPI) in inhibiting hepatocellular carcinoma (HCC) growth and promoting apoptosis.

Methods

Both in vivo and in vitro experiments were conducted. CCK-8 assay was performed to determine the cells viability after EPI treatment. HepG2 and SMMC7721 cells were treated with EPI or ¹²⁵I or in combination. Colony formation assays were performed to verify the antiproliferation effect. Annexin V–FITC/PI, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and western blotting were performed to analyze cellular apoptosis. Scratch wound healing assays and transwell assays were used to examine migration following different treatments. An isobaric tag for relative and absolute quantitation analysis was used to detect changes in protein expression after ¹²⁵I treatment, identifying the potential mediating protein cathelicidin (LL-37). LL-37 protein and WNT pathway-related proteins were detected by western blotting in SMMC7721 and HepG2 cells. Mice were treated with ¹²⁵I and EPI to evaluate whether EPI enhanced the antitumor effect of ¹²⁵I.

Results

EPI promoted ¹²⁵I-induced apoptosis and reduced the proliferation of HepG2 and SMMC7721 cells. EPI also prevented the migration of HepG2 and SMMC7721 cells. EPI combined with ¹²⁵I may interfere with the WNT signaling pathway by decreasing LL-37 to inhibit HCC development. The antitumor effects of EPI with ¹²⁵I were verified in mice.

Conclusion

EPI combined with ¹²⁵I induced apoptosis and inhibited the proliferation of HCC cells by LL-37 downregulating the WNT pathway.