杨梅素对人肺腺癌细胞增殖的影响及其机制

目的研究杨梅素(myricetin,Myr)对人肺腺癌(A549)细胞增殖的影响及其作用机制。方法以人肺腺癌系A549细胞为离体研究对象,通过噻唑蓝(MTT)法研究不同浓度的Myr对A549细胞生长的抑制作用并测定其半数抑制浓度(IC50);采用Westernblot法检测Myr对蛋白激酶B(Akt)磷酸化水平的影响。结果Myr对A549细胞具有明显的增殖抑制作用,随着Myr浓度的增加,细胞的存活率明显降低。Myr作用72h的IC50值为41.7μg/ml。32μg/ml杨梅素无血清作用A54912h即可显著抑制AktSer473的磷酸化水平。结论Myr能够剂量依赖性抑制A549细胞增殖,Akt的活性下调可能是Myr体外诱导人A549肺腺癌细胞增殖抑制作用的分子靶点。     

表皮生长因子受体与肝细胞生长因子受体双重抑制对肺癌H1975细胞增殖的作用

目的探讨联用不可逆性表皮生长因子受体(EGFR)抑制药BIBW2992与肝细胞生长因子受体(c-MET)抑制药SU11274对人肺腺癌H1975细胞增殖、凋亡、细胞周期及EGFR、c-MET信号通路分子表达的影响。方法分别用1μmol/LBIBW2992、2μmol/L SU11274及二者联合处理对靶向治疗获得性耐药的人肺腺癌H1975细胞48h,采用噻唑蓝(MTT)法检测细胞增殖、膜连蛋白(V)-碘化丙啶(Annexin V-PI)双染色法检测细胞凋亡、流式细胞仪检测细胞周期、免疫印记法(Western-Blot)法检测EGFR、c-MET、AKT、ERK及其磷酸化蛋白的表达。结果 BIBW2992联合SU11274可显著抑制H1975细胞的增殖,阻滞细胞周期于G0/G1期,并诱导细胞凋亡,两药联用对细胞增殖、细胞周期及凋亡的影响均强于每种药物单用。BIBW2992联合SU11274可降低H1975细胞EGFR及c-MET的磷酸化,并降低其下游分子AKT及ERK的磷酸化,而每种药物单用对AKT及ERK的磷酸化并无明显影响。结论 BIBW2992联合SU11274对H1975细胞具有显著的细胞毒性作用,其作用机制与抑制EGFR及c-MET通路下游分子的活化有一定相关性。     

曲古抑菌素A对肺癌细胞株H322抑制效应及机制的研究

目的 评价曲古抑菌素A(TSA)对肺癌细胞株H322的生长抑制效应及机制.方法 以四氮甲基唑蓝、流式细胞术观察TSA作用后,肺癌细胞株H322的生长抑制情况以及细胞周期、细胞凋亡等变化,Western blot分析细胞周期相关蛋白p21、抗凋亡蛋白survivin及细胞外信号调节激酶(ERK)的表达.结果 TSA对H322具有时间依赖性和浓度依赖性的生长抑制作用;TSA作用48h及96h后,H322细胞凋亡及G2/M期细胞明显增加(P＜0.05).p21蛋白表达水平显著提高,survivin蛋白及磷酸化ERK蛋白表达显著下降.结论 TSA对肺癌细胞株H322生长具有抑制作用,其机制可能是上调p21蛋白的表达,引起细胞周期的阻滞;同时抑制survivin的表达,阻断ERK信号通路,导致细胞凋亡.     

黄粉虫抗菌肽对K562细胞周期的影响及与羟基脲对细胞增殖抑制的比较

目的观察黄粉虫抗菌肽对K562细胞周期的影响;比较该抗菌肽与羟基脲对K562细胞的增殖抑制作用。方法用流式细胞仪检测黄粉虫抗菌肽对K562细胞周期的影响。黄粉虫抗菌肽与羟基脲分别设置5个浓度组,用MTT比色法分别求出2种药物不同浓度对K562的抑制率,根据浓度和抑制率用SPSS软件求出回归方程和IC50。结果细胞周期G0/G1期和G2/M期细胞比例增加,S期细胞比例下降。其中只有G0/G1期和S期与对照组相比,差异具有统计学意义（P〈0.01）。说明抗菌肽可使细胞周期阻滞于G0/G1期,从而抑制S期DNA的合成。抗菌肽和羟基脲的IC50分别为29.98和3644.45μg/ml。结论抗菌肽可通过影响细胞周期来发挥抗K562细胞的作用。抗菌肽明显优于羟基脲对K562细胞的增殖抑制作用,有较好的应用前景。     

DHA诱导3T3-L1前脂肪细胞G2/M期阻滞及ERK1/2通路的作用

目的探讨ERK1/2丝裂素活化蛋白激酶信号转导途径在DHA抑制3T3-L1前脂肪细胞增殖中的作用。方法四唑盐比色法(MTT法)检测DHA处理24h对体外培养的3T3-L1前脂肪细胞活力的影响,流式细胞术检测细胞周期,蛋白免疫印迹法检测ERK1/2、p-ERK1/2、p21水平。结果 MTT结果显示DHA干预24h可剂量依赖性降低3T3-L1前脂肪细胞的活力,抑制增殖,IC50为100μmol/L;流式细胞术结果表明,DHA可将3T3-L1前脂肪细胞阻滞在G2/M期;蛋白免疫印迹发现,DHA可明显升高3T3-L1前脂肪细胞ERK1/2磷酸化水平、增强p21蛋白表达。结论 DHA可能通过活化ERK1/2通路诱导G2/M期阻滞,进而抑制3T3-L1前脂肪细胞增殖。     

大蒜素影响子宫颈癌Hela细胞增殖的实验研究

目的探讨大蒜素对宫颈癌Hela细胞增殖的影响。方法不同浓度的大蒜素作用于体外培养的Hela细胞,倒置显微镜下观察Hela细胞的形态学变化,采用MTT法检测大蒜素对Hela细胞增殖抑制能力,流式细胞术分析大蒜素对Hela细胞凋亡的影响。结果 MTT显示大蒜素能抑制人宫颈癌Hela细胞的增殖,并具有时间-剂量依赖性;流式细胞术测定结果显示50μg/ml浓度大蒜素可明显诱导Hela细胞凋亡,并将细胞周期阻滞在G2/M期。结论大蒜素对人宫颈癌Hela细胞增殖有抑制作用,其抑制作用与诱导Hela细胞凋亡和阻抑细胞周期有关。     

IGFBP-rP1通过调控MEK/ERK信号通路对抑制子宫内膜癌HEC-1A细胞增殖的影响及机制探讨

目的:探讨外源性胰岛素样生长因子结合蛋白相关蛋白1 (IGFBP-rP1)联合MEK/ERK信号通路抑制剂PD98059对子宫内膜癌细胞HEC-1A增殖的影响及其机制.方法:体外培养人子宫内膜癌细胞HEC-1A细胞,向培养基中添加不同浓度人重组胰岛素生长因子结合蛋白相关蛋白1 (rhIGFBP-rP1)和PD98059,上调IGFBP-rP1的水平,采用Cell CountingKit-8(CCK-8)法检测不同浓度rhIGFBP-P1及rhIGFBP-P1联合MEK/ERK信号通路抑制剂PD98059对HEC-1A细胞增殖的影响,并用蛋白免疫印迹(western blot)法分析不同浓度rhIGFBP-rP1作用下子宫内膜癌细胞HEC-1A细胞总的ERK1/2和磷酸化ERK1/2水平变化.结果:添加rhIGFBP-rP1子宫内膜癌细胞HEC-1A增殖受到明显抑制,其抑制作用呈浓度和时间依赖性(P＜0.05);4ag/ml rIGFBP-rP1对HEC-1A增殖抑制作用随着时间延长越来越明显,12、24、48、72 h增殖抑制率比较差异有统计学意义(P＜0.05).而MEK/ERK通路抑制剂PD98059能增强其抑制作用,rIGFBP-rP1(4μg/ml)与PD98059(25 μmol/L)联用,以上各时间点细胞增殖抑制率分别增至(10.04±2.71)％、(17.02±1.58)％、(28.59±2.04)％、(35.29±1.12)％,各组比较,差异有统计学意义(P＜0.05);Western blot示添加rhIGFBP-rP1,磷酸化的ERK1/2水平显著降低,磷酸化的ERK1/2水平与rhIGFBP-rP1呈剂量依赖性.结论:IGFBP-rP1可以通过MEK/ERK信号通路抑制细胞增殖.     

榄香烯对肺癌A549细胞凋亡影响及分子机制研究

目的研究榄香烯对肺癌A549细胞的抑制作用及分子机制。方法取肺腺癌细胞株A549,分别以10μg/m L、20μg/m L、40μg/m L、80μg/m L、160μg/m L榄香烯处理,标记为观察组1~5,并设空白对照组6。于处理24 h、48 h、72 h后,以流式细胞仪检测细胞周期及细胞凋亡率,以Wsetern Bolt法检测真核起始因子4E(e IF4E)蛋白水平。结果 1处理24 h后,各组细胞周期无明显变化,G2/M期、sub-G1期细胞比例差异无统计学意义(P0.05);处理48 h、72 h后,观察组G2/M期细胞比率较对照组显著性下降,差异有统计学意义(P0.05),但观察组组内时点比较,差异无统计学意义(P0.05);榄香烯浓度越高,抑制作用越明显。2随药物浓度增长,细胞凋亡率显著性提升。3相较对照组,处理24 h后,观察组e IF4E表达呈下降趋势,且浓度越高,表达越低。结论榄香烯对肺癌A549细胞抑制作用强,其对细胞周期的影响存在剂量与时间依耐性;细胞凋亡率随药物浓度增加而增加,但过高剂量可能导致毒副作用,降低凋亡率;对A549细胞增殖抑制的生理机制可能与抑制e IF4E表达有关。     

甲基丙烯酸环氧丙酯对人支气管上皮细胞周期及凋亡影响的研究

目的探讨甲基丙烯酸环氧丙酯(GMA)对人支气管上皮细胞周期及凋亡的影响。方法人支气管上皮细胞(16HBE)经1~16μg/ml剂量的GMA染毒不同次数后,应用流式细胞仪(FCM)检测细胞周期及凋亡率的改变,同时测定细胞分裂指数(MI)。结果经1次染毒处理后,随着染毒剂量的增加,G0/G1期细胞显著减少(P<0.01),S期和G2/M期细胞显著增加,凋亡细胞数增多,细胞分裂指数下降;随着染毒次数的增加,各剂量组细胞周期均明显阻滞于G0/G1期,但高剂量组细胞仍表现出S期和G2/M期增多现象;经3次染毒后,高剂量组细胞出现凋亡率下降、分裂指数升高现象。结论经GMA染毒处理后,16HBE细胞周期由G0/G1期向S期和G2/M期移动而呈现增殖性改变。     

二十二碳六烯酸联合顺铂对人食管癌耐药细胞周期及凋亡的影响

目的探讨二十二碳六烯酸(DHA)与顺铂(DDP)联用对食管癌细胞Eca109/DDP增殖、周期和凋亡的影响。方法采用递增药物质量浓度持续作用诱导法建立耐DDP的食管癌细胞Eca109/DDP。DHA联合DDP作用于Eca109/DDP细胞,MTT法检测Eca109/DDP细胞增殖情况;流式细胞术检测细胞周期及细胞凋亡。结果递增药物质量浓度持续作用诱导6个月后,细胞可以在含1μg/ml DDP的培养液中正常生长,其对DDP的耐药指数为15.886,所得细胞命名为耐DDP的食管癌细胞Eca109/DDP。单用DHA浓度≤1.560μg/ml时,对Eca109/DDP细胞无明显抑制作用(P0.05),但与DDP 1μg/ml合用能显著促进DDP抑制Eca109/DDP细胞的增殖(P0.05),并促进DDP诱导Eca109/DDP细胞周期改变、细胞凋亡增加。细胞周期表现为G1期细胞明显增多(P0.05),S期细胞、G2期细胞明显减少(P0.05);细胞的凋亡率明显增加(P0.05)。结论 DHA促进DDP抑制Eca109/DDP细胞增殖可能与改变Eca109/DDP细胞周期、增加细胞凋亡有关。     

Potential Anticancer Activity of Myricetin in Human T24 Bladder Cancer Cells Both In Vitro and In Vivo

Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods. In this preliminary study, we evaluate the chemopreventive potential of myricetin against bladder cancer and its mechanism of action. The results of a MTT assay showed that myricetin was able to inhibit the viability and proliferation of T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G2/M in a dose-dependent manner and induced apoptosis detected by flow cytometry and DNA fragmentation analysis. Treatment with myricetin led to G2/M cell cycle arrest in T24 cells by downregulation of Cyclin B1 and cyclin-dependent kinase cdc2. Myricetin-induced apoptosis correlates with the modulation of Bcl-2 family proteins and activation of the caspase-3. Myricetin also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. Myricetin had a significantly reduced T24 cell migration that was accompanied by a decreasing MMP-9 expression in vitro. Furthermore, myricetin treatment significantly inhibited the tumor growth on T24 bladder cancer xenografts model. These findings suggest that myricetin has potential anticancer activity and could be an important chemoprevention agent for bladder cancer.     

Potential anticancer activity of myricetin in human T24 bladder cancer cells both in vitro and in vivo

Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods. In this preliminary study, we evaluate the chemopreventive potential of myricetin against bladder cancer and its mechanism of action. The results of a MTT assay showed that myricetin was able to inhibit the viability and proliferation of T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G2/M in a dose-dependent manner and induced apoptosis detected by flow cytometry and DNA fragmentation analysis. Treatment with myricetin led to G2/M cell cycle arrest in T24 cells by downregulation of Cyclin B1 and cyclin-dependent kinase cdc2. Myricetin-induced apoptosis correlates with the modulation of Bcl-2 family proteins and activation of the caspase-3. Myricetin also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. Myricetin had a significantly reduced T24 cell migration that was accompanied by a decreasing MMP-9 expression in vitro. Furthermore, myricetin treatment significantly inhibited the tumor growth on T24 bladder cancer xenografts model. These findings suggest that myricetin has potential anticancer activity and could be an important chemoprevention agent for bladder cancer.     

Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase

We examined the ability of polyphenols from tomatoes and soy (genistein, quercetin, kaempferol, biochanin A, daidzein and rutin) to modulate insulin-like growth factor-I (IGF-I)-induced in vitro proliferation and apoptotic resistance in the AT6.3 rat prostate cancer cell line. IGF-I at 50 micro g/L in serum-free medium produced maximum proliferation and minimized apoptosis. Polyphenols exhibited different abilities to modulate IGF-I-induced proliferation, cell cycle progression (flow cytometry) and apoptosis (Annexin V/propidium iodide and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling). Genistein, quercetin, kaempferol and biochanin A exhibited dose-dependent inhibition of growth with a 50% inhibitory concentration (IC(50)) between 25 and 40 micro mol/L, whereas rutin and daidzein were less potent with an IC(50) of >60 micro mol/L. Genistein and kaempferol potently induced G(2)/M cell cycle arrest. Genistein, quercetin, kaempferol and biochanin A, but not daidzein and rutin, counteracted the antiapoptotic effects of IGF-I. Human prostate epithelial cells grown in growth factor-supplemented medium were also sensitive to growth inhibition by polyphenols. Genistein, biochanin A, quercetin and kaempferol reduced the insulin receptor substrate-1 (IRS-1) content of AT6.3 cells and prevented the down-regulation of IGF-I receptor beta in response to IGF-I binding. IGF-I-stimulated proliferation was dependent on activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositide 3-kinase pathways. Western blotting demonstrated that ERK1/2 was constitutively phosphorylated in AT6.3 cells with no change in response to IGF-I, whereas IRS-1 and AKT were rapidly and sensitively phosphorylated after IGF-I stimulation. Several polyphenols suppressed phosphorylation of AKT and ERK1/2, and more potently inhibited IRS-1 tyrosyl phosphorylation after IGF-I exposure. In summary, polyphenols from soy and tomato products may counteract the ability of IGF-I to stimulate proliferation and prevent apoptosis via inhibition of multiple intracellular signaling pathways involving tyrosine kinase activity.     

Lycopene inhibits the growth of normal human prostate epithelial cells in vitro

Lycopene has repeatedly been shown to inhibit the growth of human prostate cells in vitro. However, previous studies with lycopene have focused on cancer specimens, and it is still unclear whether this carotenoid affects the growth of normal human prostate cells as well. Therefore, we investigated the effects of lycopene on normal human prostate epithelial cells (PrEC) by treating them with synthetic all-E-lycopene (up to 5 micromol/L) and assessing proliferation via [3H]thymidine incorporation. The effects of lycopene on cell cycle progression were investigated via flow cytometry. To elucidate whether lycopene modulates cyclins involved in cell cycle progression, protein expressions of cyclins D1 and E were analyzed. The results show that lycopene significantly inhibited the growth of PrEC in a dose-dependent fashion. Flow cytometry revealed a significant cell cycle arrest in the G0/G1 phase. This effect was confirmed by inhibition of cyclin D1 protein expression, whereas cyclin E levels remained unchanged. The results demonstrate that lycopene inhibits growth of nonneoplastic PrEC in vitro. We hypothesize that lycopene might likewise inhibit the growth of prostatic epithelial cells in vivo. This might have an effect on prostate development and/or on enlargement of prostate tissue as found in benign prostate hyperplasia, a potential precursor of prostate cancer.     

Src介导ERK信号通路对宫颈癌细胞增殖凋亡的作用

目的 探讨Src介导ERK信号通路对宫颈癌细胞增殖凋亡的作用。方法 采用体外细胞实验方法,以宫颈癌细胞Hela（HPV阳性）和C33A（HPV阴性）为研究对象,施加Src激酶选择性抑制剂（PP2）对Src激酶进行抑制。于抑制前后采用流式细胞术（FCM）检测各组细胞的周期及凋亡情况,分别采用Real-time PCR法和Western-blot法检测各组细胞ERK 1/2、c-Fos和c-Jun的mRNA和蛋白表达水平。应用SPSS 20.0软件进行数据录入和分析。结果 Src抑制后,Hela和C33A细胞均显示增殖指数降低,凋亡率升高,处于G0/G1期细胞比例升高,S期和G2/M期细胞比例降低,ERK 1、ERK 2、c-Fos和c-Jun的mRNA含量升高,ERK 1/2、磷酸化ERK 1/2（p-ERK 1/2）和磷酸化c-Fos（p-c-Fos）蛋白表达水平降低,c-Jun和磷酸化c-Jun（p-c-Jun）蛋白表达水平升高。Hela细胞凋亡率、p-ERK 1/2和c-Fos蛋白在Src抑制前后的变化低于C33A细胞。结论 Src活化可以上调ERK信号通路中关键因子ERK 1/2和c-Fos磷酸化蛋白的表达,促进宫颈癌细胞的生长与增殖,在宫颈癌变中发挥重要作用。HPV感染可能对Src介导的ERK信号通路调节具有调节作用。     

Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells

Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 μmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 μmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells.     

The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.     

How can food extracts consumed in the Mediterranean and East Asia suppress prostate cancer proliferation?

We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, named blueberry punch (BBP), with the ultimate aim to formulate a chemoprevention strategy to inhibit prostate cancer progression in men on active surveillance protocol. We demonstrated previously that BBP inhibited prostate cancer cell proliferation in vitro and in vivo. The purpose of this study was to determine the molecular mechanism responsible for the suppression of prostate cancer cell proliferation by BBP. Treatment of lymph node-metastasised prostate cancer cells (LNCaP) and bone-metastasised prostate cancer cells (PC-3 and MDA-PCa-2b) with BBP (up to 0·8?%) for 72?h increased the percentage of cells at the G0/G1 phase and decreased those at the S and G2/M phases. The finding was supported by the reduction in the percentage of Ki-67-positive cells and of DNA synthesis measured by the incorporation of 5-ethynyl-2'-deoxyuridine. Concomitantly, BBP treatment decreased the protein levels of phosphorylated retinoblastoma, cyclin D1 and E, cyclin-dependent kinase (CDK) 4 and 2, and pre-replication complex (CDC6 and MCM7) in LNCaP and PC-3 cells, whereas CDK inhibitor p27 was elevated in these cell lines. In conclusion, BBP exerts its anti-proliferative effect on prostate cancer cells by modulating the expression and phosphorylation of multiple regulatory proteins essential for cell proliferation.