生长抑素类似物抑制胆管癌细胞增殖的研究

目的探讨生长抑素类似物奥曲肽(OCT)对人胆管癌细胞细胞株QBC939的抑制作用及可能的作用机制。方法四氮唑蓝(MTT)法检测OCT对人胆管癌细胞株QBC939增殖的影响,流式细胞仪检测OCT对细胞周期变化的作用,免疫组织化学法研究OCT对人胆管癌细胞株QBC939P27KIP1蛋白表达变化的影响。建立胆管癌裸鼠模型,进一步探讨生长抑素的作用。结果MTT法检测不同浓度OCT(5、0.5、0.05、0.005mg/L)作用于QBC93948h后,实验组0.5、5mg/L组和对照组吸光度(A)值比较差异有统计学意义(P<0.05)。细胞生长率分别为83.80%,80.28%,77.53%,65.32%。流式细胞仪分析显示不同浓度OCT作用于QBC93948h后,G0/G1期细胞明显增多(P<0.05)。免疫组织化学P27KIP1蛋白表达随OCT浓度的增加而增强(P<0.05)。建立胆管癌裸鼠模型后,OCT干预21d后实验组瘤重明显小于对照组(P<0.05)。结论OCT可抑制人胆管癌细胞的增殖。在一定范围内呈剂量依赖性,其抗肿瘤作用机制主要是通过细胞周期阻滞来实现的,而这一作用的实现可能与P27KIP1蛋白表达的上调有关。     

miR125a对胆管癌细胞株QBC939增殖、凋亡影响及可能机制

目的探讨miR125a对胆管癌细胞株QBC939细胞增殖及凋亡的影响及可能机制。方法实时荧光定量PCR检测52例经手术和病理证实的肝外胆管癌,31例癌旁组织及10例正常胆管组织标本miR125a的表达,免疫组化检测MMP-9蛋白表达。miR125a转染胆管癌细胞株QBC939,MTT法检测细胞增殖,流式细胞分析法检测细胞周期,Caspase-3试剂盒检测凋亡,Western blot检测MMP-9蛋白表达。结果胆管癌组织miR125a表达明显下调,低于正常胆管组织(P<0.05),MMP-9明显高于正常胆管组织(P<0.05)。与对照质粒组比较,转染miR125a胆管癌细胞QBC939增殖受到抑制,流式细胞分析法表明转染miR125a的胆管癌细胞QBC939导致G1阻滞,Caspase-3试剂盒检测显示凋亡增加,Western blot分析表明转染miR125a的胆管癌细胞QBC939中MMP-9的表达出现明显的下调。结论miR125a在肝外胆管癌中低表达,它们可能与胆管癌的发生有关。胆管癌细胞株QBC939 miR125a表达上调,能够抑制细胞增殖,诱导细胞凋亡和G1阻滞,miR125a的作用可能与MMP-9的表达相关。     

黄芩苷通过调控c-Myc介导的细胞周期抑制胆管癌细胞增殖

目的　探究黄芩苷（BC）对人胆管癌（CCA）细胞QBC939和RBE增殖的影响及其潜在机制。方法　取对数生长期QBC939和RBE细胞，不同浓度的BC处理细胞24 h后，采用CCK-8法分别检测BC对CCA细胞增殖及敲低原癌基因蛋白质（c-Myc）后对BC引起细胞增殖活性改变的影响；高倍显微镜观察BC对细胞生长状态的影响；流式细胞术检测BC对CCA细胞周期的影响；蛋白免疫印迹（Western blot）实验分别检测BC及小干扰RNA（siRNA）敲低CCA细胞中c-Myc后对周期相关蛋白周期素依赖激酶抑制剂（p27）、细胞周期蛋白D1（Cyclin D1）及c-Myc表达的影响。结果　与0 μmol/L组相比，BC明显抑制QBC939和RBE细胞的增殖活性（P＜0.01），且高倍显微镜下观察到细胞生长减缓；BC可将QBC939细胞周期阻滞于S期（P＜0.05）；随着BC浓度的增加，p27的蛋白水平明显上调，而Cyclin D1则显著降低（P＜0.01）；BC可下调c-Myc的蛋白表达，且敲低c-Myc后p27和Cyclin D1蛋白表达的变化趋势与BC处理时一致（P＜0.05）；单独敲低c-Myc后可明显抑制QBC939和RBE细胞的存活率（P＜0.01），并能进一步增强BC的抑制作用。结论　BC通过抑制c-Myc信号通路阻滞细胞周期，进而抑制CCA细胞的增殖。     

阿伐他汀诱导人胆管癌QBC939细胞凋亡的研究

目的 观察阿伐他汀(ATV)对人胆管癌QBC939细胞增殖及凋亡的影响.方法 应用细胞培养技术培养QBC939细胞,经不同浓度的阿伐他汀处理后,以MTT法检测QBC939细胞的存活率,通过光镜、倒置显微镜及琼脂糖凝胶电泳检测胆管癌细胞凋亡的形态学特征、生化学特征.结果 在阿伐他汀的作用下,QBC939细胞的生长、增殖变的相对缓慢,凋亡的细胞出现膜小泡、凋亡小体等特征性改变;DNA电泳呈现典型的梯状条带.结论 阿伐他汀可抑制胆管癌细胞的生长、增殖,并可诱导细胞发生凋亡,且其作用呈剂量效应关系.     

TSA对胆道癌细胞系生长的影响

目的:研究组蛋白去乙酰化酶抑制剂在体外和体内对胆囊癌细胞系和肝外胆管癌细胞系生长的影响,并探讨其临床应用价值。方法:用组蛋白去乙酰化酶抑制剂-曲古抑菌素A(trichostatin A,TSA)作用于胆囊癌细胞系(Mz-ChA-1)和肝外胆管癌细胞系(QBC939、KMBC、OZ),用MTT法检测TSA对这些细胞系生长的影响。将Mz-ChA-1接种裸鼠皮下建立胆囊癌裸鼠种植模型,观察TSA处理前后体内种植瘤生长的变化。结果:TSA可以抑制Mz-ChA-1和QBC939、KMBC、OZ的增殖,以上作用与药物浓度在一定范围内呈正相关。成功建立胆囊癌裸鼠体内种植模型,TSA处理后裸鼠体内种植瘤生长受抑制。结论:TSA在体外能抑制胆囊癌细胞系和肝外胆管癌细胞系的增殖;TSA在体内能抑制胆囊癌细胞系的增殖。     

(-)-Syringaresinol inhibits proliferation of human promyelocytic HL-60 leukemia cells via G1 arrest and apoptosis

We examined the effect of (-)-syringaresinol, a furofuran-type lignan isolated from Daphne genkwa, on cell cycle regulation in HL-60 human promyelocytic leukemia cells in vitro. (-)-Syringaresinol decreased the viability of HL-60 cells by inducing G(1) arrest followed by apoptosis in a dose- and time-dependent manner. The G(0)/G(1) phase of the cell cycle is regulated by cyclin-dependent kinases (Cdk), cyclins and cyclin-dependent kinase inhibitors (Cdki). We show by western blot analysis, that the (-)-syringaresinol-induced G(1) arrest was mediated through the increased expression of Cdki proteins (p21(cip1/waf1) and p27(kip1)) with a simultaneous decrease in cdk2, cdk4, cdk6, cyclin D(1), cyclin D(2), and cyclin E expression. The induction of apoptosis after treatment with (-)-syringaresinol for 24 h was demonstrated by morphological changes, DNA fragmentation, altered ratio of Bax/Bcl-2, cleavage of poly(ADP-ribose) polymerase and flow cytometry analysis. (-)-Syringaresinol also induced cytochrome c release and activation of caspase-3 and caspase-9. To our knowledge, this is the first time that (-)-syringaresinol has been reported to potently inhibit the proliferation of human promyelocytic HL-60 cells through G(1) arrest and induction of apoptosis. These findings suggest that (-)-syringaresinol may be a potential chemotherapeutic agent for the treatment of cancer.     

原花青素对人胆管癌细胞QBC939抑制的体内外研究

目的研究原花青素(PC)体内外对人胆囊癌细胞的抑制作用。方法常规培养细胞,24 h后随机分为阳性对照组、空白对照组和PC组。MTT法检测PC对人胆囊癌细胞增殖的抑制作用;流式细胞术检测细胞凋亡率和细胞周期;建立QBC939细胞裸鼠异种移植瘤模型。将荷瘤裸鼠随机分为5组:阴性对照组、5-Fu阳性对照组及PC 3个剂量组,每日腹腔注射给药,每隔3 d测肿瘤大小;12 d后,处死裸鼠,剥瘤称重并计算抑瘤率。结果 PC 3个剂量组显著抑制QBC939细胞增殖,且呈时间和剂量依赖性;将细胞周期阻滞在S期,并诱导QBC939细胞凋亡;PC可抑制QBC939细胞裸鼠异种移植瘤的生长。结论 PC体内外均可抑制SHG-44细胞生长,并诱导肿瘤细胞凋亡。     

Baicalein induces a dual growth arrest by modulating multiple cell cycle regulatory molecules

Baicalein, a flavonoid present in the root of Scutellaria baicalensis Georgi, has been reported to inhibit cell proliferation in several types of cells. In this study, the effect of baicalein on cell growth and the mechanism of growth modulation were examined in primary cultured rat heart endothelial cells. Here, we report that treatment with 100-microM baicalein caused an almost complete inhibition of cell proliferation after 5 days of incubation. Baicalein mediated G1 and G2 growth arrest accompanied by the down-regulation of cyclin D2, cyclin A, cyclin-dependent kinase 1 (Cdk1) and cyclin-dependent kinase 2 (Cdk2), and up-regulation of p15(Ink4B), p21(CIP1/Waf1), p53 and cyclin E. Evaluation of the kinase activity of cyclin-Cdk complexes showed that baicalein decreased Cdk1, Cdk2, cyclin D2 and cyclin A expression in endothelial cells, leading to markedly reduced Cdk/cyclin-associated kinase activities. These results suggest that baicalein inhibits the proliferation of rat heart endothelial cells via G1 and G2 arrest in association with the down-regulation of the expression and function of Cdk1, Cdk2, cyclin D2 and cyclin A proteins, and up-regulation of cyclin E, p15(Ink4B), p53 and p21(CIP1/Waf1).     

Difference of cell cycle arrests induced by lidamycin in human breast cancer cells

Lidamycin (LDM) is a member of the enediyne antibiotic family. It is undergoing phase I clinical trials in China as a potential chemotherapeutic agent. In the present study, we investigated the mechanism by which LDM induced cell cycle arrest in human breast cancer cells. The results showed that LDM induced G1 arrest in p53 wild-type MCF-7 cells at low concentrations, and caused both G1 and G2/M arrests at higher concentrations. In contrast, LDM induced only G2/M arrest in p53-mutant MCF-7/DOX cells. Western blotting analysis indicated that LDM-induced G1 and G2/M arrests in MCF-7 cells were associated with an increase of p53 and p21, and a decrease of phosphorylated retinoblastoma tumor suppressor protein, cyclin-dependent kinase (Cdk), Cdc2 and cyclin B1 protein levels. However, LDM-induced G2/M arrest in MCF-7/DOX cells was correlated with the reduction of cyclin B1 expression. Further study indicated that the downregulation of cyclin B1 by LDM in MCF-7 cells was associated with decreasing cyclin B1 mRNA levels and promoting protein degradation, whereas it was only due to inducing cyclin B1 protein degradation in MCF-7/DOX cells. In addition, activation of checkpoint kinases Chk1 or Chk2 maybe contributed to LDM-induced cell cycle arrest. Taken together, we provide the first evidence that LDM induces different cell cycle arrests in human breast cancer cells, which are dependent on drug concentration and p53 status. These findings are helpful in understanding the molecular anti-cancer mechanisms of LDM and support its clinical trials.     

Manganese chloride-induced G0/G1 and S phase arrest in A549 cells

In the present study, we investigated the effects of manganese chloride (MnCl2) on cell cycle progression in A549 cells used as a model of Mn-induced lung toxicity. Cells were treated with various concentrations of MnCl2 (0, 0.01, 0.1, 0.5, 1.0 or 2.0 mM) for 24, 48 or 72 h. Cell proliferation was determined with MTT assay and mitotic index measurement and apoptosis was measured by flow cytometer. The results showed that MnCl2 inhibited A549 cells proliferation in a dose- and time-dependent manner, and induced apoptosis in A549 cells. When G0/G1 cells obtained by serum starvation were incubated with 0.5 mM of MnCl2 in the presence of 10% serum for several time intervals, the disruption of cell cycle progression was observed. The G0/G1 arrest was induced by MnCl2 treatment at 16 h and the arrest maintained for 8 h. Following the G0/G1 arrest, MnCl2 blocked the cells at S phase at 28 h and the S phase arrest maintained for at least 4 h. And moreover, proteasome inhibitor MG132 was able to prolong the duration of G0/G1 arrest induced by MnCl2 treatment. Results of western blotting assay revealed that cellular Cdk4, Cdk2 and phospho-Cdk2 (Thr160) levels decreased in manganese-treated cells at both 20 and 28 h. In addition, the decreasing of Cyclin A level and the increasing of p53 and WAF1/p21 were also induced by MnCl2 treatment at 20 h. The expression of Cyclin D1, Cyclin E and Cdc25A proteins was not altered in manganese-treated cells at both 20 and 28 h. Our results indicate that MnCl2 orderly induces G0/G1 and S phase arrest in A549 cells, the decreasing of Cdk4, Cdk2 and Cyclin A, and the increasing of p53 and Cdks inhibitor WAF1/p21 might be responsible for the G0/G1 arrest, and the decreasing of Cdk4 and Cdk2 levels for the S phase arrest.     

RhoC基因转染对胆管癌QBC939细胞生物学行为的影响

目的将人RhoC正义、反义基因真核表达载体转染胆管癌QBC939细胞株,研究RhoC基因对胆管癌QBC939细胞株增殖和侵袭能力的影响。方法以脂质体将包含正义、反义RhoC cDNA真核表达载体,分别转染人胆管癌QBC939细胞。应用克隆形成试验检测细胞增殖活性的变化,流式细胞仪检测转染前后细胞周期的变化,Boyden小室侵袭实验检测侵袭及运动能力的变化。结果正义RhoC基因能促进胆管癌QBC939细胞增殖,流式细胞仪分析结果显示,转染后细胞出现G1期细胞减少;侵袭实验显示,转染后细胞侵袭能力较转染前有显著加强。反义RhoC基因能抑制胆管癌QBC939细胞增殖,流式细胞仪分析结果显示,转染后细胞出现G1期细胞增加;侵袭实验显示,转染后细胞侵袭能力较转染前有显著减弱。结论RhoC基因能够促进胆管癌QBC939细胞株的体外增殖和侵袭能力,反义RhoC基因可抑制胆管癌QBC939细胞的增殖和侵袭能力。     

华蟾素对人胆管癌细胞QBC939抑制作用研究

目的观察华蟾素体内外对人胆囊癌QBC939细胞的抑制作用。方法常规培养细胞,24 h后随机分为空白对照组和华蟾素组6个剂量组,分别于3个时相采用MTT法检测华蟾素对人胆囊癌细胞增殖的抑制作用;建立QBC939细胞裸鼠异种移植瘤模型,随机分为阴性对照组、5-Fu阳性对照组及华蟾素3个剂量组,每日腹腔注射给药,观察荷瘤裸鼠的一般活动状况及进食量;12 d后处死裸鼠,剥瘤称重并计算抑瘤率;取血,ELISA法检测血清中细胞因子IL-6、TNF-α和sVCAM-1含量。结果华蟾素6个剂量组对QBC939细胞的增殖均具有抑制作用,与对照组比较差异有统计学意义,并且随着剂量的增加和时间的延长,抑制率也增加;华蟾素腹腔注射给药可改善荷瘤裸鼠的一般活动状况,增加进食量;抑制荷瘤裸鼠移植瘤的生长,与对照组比较差异有统计学意义;提高血清TNF-α水平,降低IL-6和sVCAM-1水平,差异均有统计学意义。结论华蟾素体内外均可抑制胆囊癌QBC939细胞的生长,其体内抑瘤作用机制可能与上调裸鼠血清TNF-α细胞因子水平,下调IL-6和sVCAM-1水平有关。     

Mechanism of cell cycle arrest caused by histone deacetylase inhibitors in human carcinoma cells

Inhibitors of histone deacetylase (HDAC) block cell cycle progression at G1 in many cell types. We investigated the mechanism by which trichostatin A (TSA), a specific inhibitor of HDAC, induces G1 arrest in human cervix carcinoma HeLa cells. TSA treatment induced histone hyperacetylation followed by growth arrest in G as well as hypophosphorylation of pRb. The Cdk4 kinase activity was essentially unchanged during the TSA-induced G1 arrest. On the other hand, the arrest was accompanied by down-regulation of kinase activity of Cdk2, although the total protein levels of Cdk2 and its activator Cdc25A were unaffected. Upon TSA treatment, amounts of cyclin E and the CDK inhibitor p21WAF1/Cip1 were markedly increased, while that of cyclin A was reduced. The induction of p21 and down-regulation of cyclin A correlated well with the decreased Cdk2 activity and cell cycle arrest. Furthermore, gel filtration chromatography showed the association of p21 with the cyclin E-Cdk2 complex, suggesting that the activation of Cdk2 by the enhanced expression of cyclin E is blocked by the increased p21. The elevated expression of p2 is also observed in cells treated with trapoxin and FR901228, structurally unrelated histone deacetylase inhibitors. A human colorectal carcinoma cell line lacking both alleles of the p21 gene (p21-/-) was resistant to TSA several times more than the parental line (p21+/+). These results suggest that the suppression of Cdk2 kinase activity due to p21 overexpression play a critical role in HDAC inhibitor-induced growth inhibition.     

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation

We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation.     

Cadmium induces cell cycle arrest in rat kidney epithelial cells in G2/M phase

Cadmium (Cd) has been reported to cause cell cycle arrest in various cell types by p53-dependent and -independent mechanisms. This study was designed to investigate cell cycle progression in kidney cells that are the target of chronic Cd toxicity. Rat renal proximal tubular epithelial cells, NRK-52E, were treated with up to 20 microM CdCl2 in DMEM containing 10% calf serum for up to 24 h. Flow cytometric analysis revealed time- and concentration-dependent increases in cells in G2/M phase of the cell cycle. As compared to the control cells, the cells exposed to 20 microM Cd showed a doubling of the number of cells in this phase after 24 h. The cell cycle arrest was associated with a decrease in protein levels of both cyclins A and B. Further investigation into the mechanism revealed that Cd treatment led to down-modulation of cyclin-dependent kinases, Cdk1 and Cdk2, apparently by elevating the expression of cyclin kinase inhibitors, KIP1/p27 and WAF1/p21. Furthermore, the wild-type p53 DNA-binding activity was up-regulated. Based on these observations, it appears that Cd causes G2/M phase arrest in NRK-52E cells via elevation of p53 activity, increasing the expression of cyclin kinase inhibitors p27 and p21, and decreasing the expression of cyclin-dependent kinases Cdk1 and 2, and of cyclins A and B.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDB, in chronic myeloid leukemia (HL-60) cells.

(Z)-2-(6-(Thieanisyl-2-yl)hexa-3-en-1,5-diynyl)benzenamine (THDB), an enediyne compound, was identified in our laboratory as a novel antineoplastic agent with broad spectrum of antitumor activities against many human cancer cells. THDB was found to inhibit the growth of HL-60 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in HL-60 cells following 48 h exposure to THDB. Analysis of the cell cycle regulatory proteins demonstrated that THDB did not change the steady-state levels of cyclin B1, cyclin E, Cdk1 and Cdc25C, but decreased the protein levels of Cdk2 and cyclin A. THDB also caused a marked increase in apoptosis, as characterized by DNA fragmentation (DNA ladder and sub G1 formation), and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Moreover, the THDB-induced apoptosis was significantly attenuated in the presence of specific inhibitors of caspase-3, -8 and -9. These molecular alterations provide an insight into THDB-caused growth inhibition, G2/M arrest and apoptotic death of HL-60 cells.     

姜黄素对人胆囊癌细胞株QBC939增殖和凋亡的影响

目的探讨姜黄素对人胆囊癌细胞株QBC939增殖及凋亡的影响,为胆囊癌的临床治疗提供新的思路。方法姜黄素与QBC939共同孵育后,采用MTT方法测定QBC939的增殖情况并计算抑制率,用流式细胞仪评估凋亡率,同时以蛋白免疫印迹(Western blot)方法检测姜黄素对凋亡相关蛋白Caspase 3表达水平的影响。数据比较采用t检验,P<0.05为差异有统计学意义。结果姜黄素处理组细胞生长明显受抑并存在显著凋亡,Western blot结果显示Cas-pase 3表达量远大于对照组。结论姜黄素可显著抑制胆囊癌细胞株QBC939的增殖,这种变化可能与其诱导的Cas-pase 3的高表达及促进凋亡有关。     

Induction of G(1) cell cycle arrest and p27(KIP1) increase by panaxydol isolated from Panax ginseng

Polyacetylenic compounds of Panax ginseng roots have been shown to inhibit growth of several human malignant tumor cell lines. Panaxydol is known to be one of the cytotoxic polyacetylenic compounds of P. ginseng. In this study, we first showed that panaxydol decreased markedly the proliferation, and to a lesser extent, the number of cells in a human melanoma cell line, SK-MEL-1. Next, the effect of panaxydol on cell cycle progression and its mechanism of action were investigated. Cell cycle analysis revealed that panaxydol inhibited cell cycle progression of a human malignant melanoma cell line, SK-MEL-1, at G(1)-S transition. At the same time, panaxydol increased the protein expression of p27(KIP1) as early as 1 hr after treatment. Cyclin-dependent kinase 2 (Cdk2) activity was decreased in a dose-dependent manner after 24 hr of panaxydol treatment. Protein levels of p21(WAF1), p16(INK4a), p53, pRb (retinoblastoma protein), and E2F-1 were not changed. It was also found that cycloheximide reversed the growth inhibition induced by panaxydol and partially abrogated the increase in p27(KIP1) expression. These results indicate that panaxydol induces G(1) cell cycle arrest by decreasing Cdk2 activity and up-regulating p27(KIP1) protein expression.