姜黄素对肝癌细胞HepG2的抗癌作用及P21WAF1/CIP1表达的影响

目的：探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响。方法：体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达。结果：姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达。结论：姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达。     

姜黄素对肝癌细胞HepG2 的抗癌作用及P21WAF1/CIP1 表达的影响

目的:探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响.方法:体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达.结果:姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达.结论:姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达.     

芍药苷对结肠癌SW480细胞增殖、侵袭、迁移的影响

目的观察芍药苷对人结肠癌SW480细胞株增殖、侵袭、迁移的影响,探究其干预机制。方法含10%胎牛血清的DMEM/F12培养基常规培养人结肠癌SW480细胞株,CCK-8以及EdU-488法检测芍药苷对SW480细胞增殖的影响,Transwell小室检测芍药苷对SW480细胞侵袭、迁移的影响,Westernblot法检测beclin1、Bcl-2蛋白的表达。结果不同浓度芍药苷分别处理24h、48h、72h的结肠癌SW480细胞增殖活性受到显著抑制:相比较对照组,160μg/ml芍药苷处理48h后,SW480细胞内黄绿色荧光减弱,细胞增殖率显著下降,为(58.91±4.99)%;SW480细胞的侵袭细胞数、迁移细胞数显著下降:侵袭抑制率为26.50%,迁移抑制率为24.67%;beclin1蛋白表达高于对照组,Bcl-2蛋白表达低于对照组,beclin1与Bcl-2蛋白表达呈负相关。结论芍药苷能够抑制结肠癌SW480细胞增殖、侵袭和迁移,其机制可能通过抑制Bcl-2蛋白表达,上调beclin1蛋白的表达。     

白藜芦醇体外对肝癌HepG2细胞分化及P21WAF1/CIP1表达的影响

目的:探讨白藜芦醇(Resvratrol,Res)在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1的影响.方法:体外培养肝癌HepG2细胞.用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌.以RT-PCR方法检测HepG2细胞中P21WAF1、CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1、CIP1蛋白的表迭.结果:白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21WAF1/CIP1 mRNA和蛋白的表达.结论:白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21WAF1/CIP1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。     

脾源性酪氨酸激酶基因启动子甲基化与髓母细胞瘤细胞侵袭转移的关系

目的：探讨甲基化转移酶抑制剂5-氮杂-2-脱氧胞苷（5-aza-CdR）抑制脾源性酪氨酸激酶（Syk）基因启动子的甲基化后对髓母细胞瘤Daoy细胞侵袭转移能力的影响。方法：用甲基化转移酶抑制剂5-aza-CdR处理体外培养的髓母细胞瘤Daoy细胞,通过甲基化特异性PCR（MSP）、Real time-PCR、Western blot及Transwell实验方法分别检测不同浓度5-aza-CdR处理后髓母细胞瘤Daoy细胞中脾源性酪氨酸激酶（Syk）基因启动子区甲基化、mRNA表达、蛋白表达及细胞穿膜数的变化。结果：髓母细胞瘤Daoy细胞中Syk基因启动子存在过甲基化,与对照组比较,经不同浓度5-aza-CdR处理后,其Syk基因启动子区甲基化受到不同程度抑制,Syk mRNA的表达量最高上调（3.40±0.24）倍（P<0.01）;Syk蛋白的表达量最高上调（3.23±0.19）倍（P<0.01）;细胞侵袭及转移能力降低（P<0.05）,差异有统计学意义。结论：髓母细胞瘤Daoy细胞中Syk基因启动子甲基化导致其表达下调,可能是髓母细胞瘤发生转移的机制之一;而甲基化转移酶抑制剂5-aza-CdR可抑制其启动子区的甲基化,使Syk的表达水平上调,抑制肿瘤细胞侵袭及转移能力。     

香蜂草苷诱导人肝癌细胞株HepG2凋亡及其作用机制

为了研究香蜂草苷对肝癌细胞株HepG2凋亡的影响,并探讨其作用机制,利用MTT法检测香蜂草苷对HepG2细胞增殖的抑制作用,流式细胞术检测细胞凋亡率,试剂盒检测caspase-3和caspase-9活性,Western blot检测Bcl-2、Bax、RKIP、ERK、p-ERK蛋白表达。实验结果表明香蜂草苷可抑制HepG2细胞增殖并诱导其凋亡,其机制可能与提高caspase-3和caspase-9活性,上调Bax和下调Bcl-2表达有关。此外,我们的研究显示香蜂草苷诱导HepG2细胞凋亡可能还与其增加RKIP表达,抑制ERK/MAPK信号通路有关。     

烟酸对HepG2细胞载脂蛋白M表达的影响及其机制

目的:探讨烟酸对HepG2细胞载脂蛋白M表达的影响及其机制。方法:分别以不同浓度的烟酸(0、0.25、0.5、1.0、2.0mmol/L)干预HepG2细胞24h。提取各组细胞总RNA和蛋白质,分别采用实时R T-PCR和Western Blot检测载脂蛋白M的mRNA和蛋白的表达。采用实时R T-PCR检测肝细胞核因子-1αmRNA的表达。结果:烟酸呈剂量依赖性上调载脂蛋白M基因和蛋白、肝细胞核因子-1α基因的表达(P<0.05)。结论:烟酸可上调载脂蛋白M表达,其机制可能是通过上调肝细胞核因子-1α来实现的。     

紫草素对人肝癌HepG2细胞增殖的抑制及诱导凋亡作用

目的:观察紫草素抑制人肝癌HepG2细胞增殖及凋亡诱导的作用。方法:用不同浓度的紫草素处理HepG2细胞,MTT检测紫草素对HepG2细胞生长增殖的抑制作用;比色法测定Caspase-3酶活性;Western blot法检测磷酸化Akt蛋白(pAkt)的表达。结果:紫草素能够抑制人肝癌HepG2细胞的增殖,并呈浓度、时间依赖性,紫草素与HepG2细胞作用24小时后Caspase-3酶活性显著增强,显示紫草素诱导的调亡作用随时间的延长而增加;同时,紫草素处理HepG2细胞后,随着药物浓度的增加,磷酸化Akt蛋白表达下降。结论:紫草素可抑制人肝癌细胞HepG2的增殖,诱导HepG2细胞凋亡,凋亡机制可能与紫草素抑制PI3K/Akt信号途径有关。     

白藜芦醇体外对肝癌HepG2细胞分化及P21^WAF1/CIP1表达的影响

目的：探讨白藜芦醇（Resvratrol,Res）在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21^WAF1/CIP1的影响。方法：体外培养肝癌HepG2细胞,用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌,以RT-PCR方法检测HepG2细胞中P21^WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21^WAF1/CIP1蛋白的表达。结果：白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21^WAF1/CIP1mRNA和蛋白的表达。结论：白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21^WAF1/CIP1的表达。     

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

基于肝癌CDK2 基因敲减对CyclinE 表达及树舌多糖GF对其影响的研究

目的:探究树舌多糖GF(GAPS.GF)对rAAV-shRNA-CDK2抑制肝癌细胞Cyclin E基因表达的辅助作用。方法:将细胞培养后的人肝癌HepG2以皮下注射的方式接种于裸鼠前肢腋下,接种后的裸鼠随机分为5组:NC(非相关序列)对照组、肿瘤组、rAAV-shRNA-CDK2组、树舌多糖GF组以及树舌多糖GF+rAAV-shRNA-CDK2组,各实验组均采取尾静脉注射定量给药。采用实时定量PCR和Western blot技术研究肝癌细胞Cyclin E基因m RNA和蛋白水平的表达情况,同时观察GASP.GF对其作用的影响。结果:树舌多糖GF+rAAV-shRNA-CDK2联合应用组对肝癌HepG2细胞增殖的抑瘤率为75.6%,对Cyclin E基因mRNA表达抑制率为69%,对Cyclin E基因蛋白表达抑制率为67.5%,比rAAV-shRNA-CDK2组分别提高了3.42%、1%和2.7%。结论:树舌多糖GF与rAAV-shRNA-CDK2联合应用可以显著提高肝癌的治疗效果,说明树舌多糖GF可以辅助r AAV-shRNACDK2对肝癌细胞Cyclin E基因的表达的抑制作用。     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．     

Tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 potentates the anchorage-independent growth of human hepatoma HepG2 cells

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an adhesion molecule of the immunoglobulin superfamily, has been characterized as a putative tumor suppressor because it is frequently down-regulated in aggressive types of cancer cells. Recently, however, several studies have shown that CEACAM1 actively contributes to malignant progression or migration in some types of tumor cells, suggesting that the role of CEACAM1 might be diverse among different types of cancer cells. To investigate the functional consequences of CEACAM1 expression in hepatocellular carcinoma, we analyzed the status of CEACAM1 in hepatoma cell lines HLF, PLC/PRF/5, HepG2 and KYN-2. We found that CEACAM1 was only expressed in HepG2 cells, which show a unique property for enhanced anchorage-independent growth. When HepG2 cells were treated with small interfering RNA targeted against CEACAM1, the growth rate in monolayer culture was increased. In contrast, when HepG2 cells were cultured in suspension, inhibition of CEACAM1 expression significantly decreased the growth rate, and the speed of cell-cell attachment was repressed. Hyaluronidase treatment attenuated the growth rate of HepG2 cells in suspension culture, indicating that cell-cell attachment is a requisite for anchorage-independent growth. Our data may reveal the dual role of CEACAM1 on hepatocarcinogenesis, by showing that CEACAM1 acts as a tumor suppressor in HepG2 cells in anchorage-dependent growth conditions, while in anchorage-independent growth conditions, it augments cell proliferation by potentiating the cell-cell attachment.     

铁筷子提取物对肿瘤细胞增殖及COX-2 mRNA表达的抑制作用

研究不同铁筷子提取物对肿瘤细胞增殖及 COX-2 mRNA 表达的抑制作用。以铁筷子醇总提取物(TKZ1)、正丁醇萃取部位(TKZ2)、乙酸乙酯萃取部位(TKZ3)分别作用于 DU145、PC3、HeLa、HT-29、HepG2等肿瘤细胞,应用噻唑蓝实验(MTT 法)计算其对细胞增殖的抑制作用,应用荧光定量 PCR 技术检测TKZ1、TKZ2、TKZ3处理后的各肿瘤细胞中 COX-2 mRNA 的表达情况。结果表明：TKZ1、TKZ2、TKZ3均能显著抑制多种肿瘤细胞的增殖,与阴性对照组比较,其可以在 mRNA 水平上抑制 COX-2的表达,且呈明显的量效关系。说明铁筷子提取物对体外肿瘤细胞的增殖具有显著的抑制作用,其抗瘤机制可能与抑制肿瘤细胞中 COX-2 mRNA 的表达有关。     

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。     

GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) either with a single high dose (5-20 μM), every alternate day (0.1-10 μM) or daily (0.005-2.5 μM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 μM or greater (p<0.0001) and induction of cell death from 0.5 μM (p<0.0001). In contrast, treatment with a single high dose of 20 μM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥ 0.05 μM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥ 0.5 μM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥ 50 μM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.     

青藏高原黑果枸杞花青素对人肝癌细胞增殖和自噬的影响

探究黑果枸杞花青素在体外对人肝癌HepG2细胞增殖和自噬的影响.利用CCK-8法测定细胞活力,EdU和细胞划痕试验检测细胞增殖和迁移效果,RT-PCR和Western blot检测增殖和自噬相关基因的mRNA和蛋白表达.结果显示,黑果枸杞花青素可有效抑制人肝癌HepG2细胞的增殖和迁移;上调增殖因子(LATS1、LAT...     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.