熊果酸诱导结肠癌HT-29细胞株凋亡的实验研究

目的探讨熊果酸诱导结肠癌HT-29细胞株凋亡的作用机制.方法用不同浓度的熊果酸处理HT-29细胞,采用四甲基偶氮唑蓝(MTT)比色检测熊果酸对HT-29细胞的增殖抑制效应,采用形态学、TUNEL法、流式细胞术检测细胞凋亡的发生,应用免疫组织化学SP法检测凋亡相关基因caspase-9, bax表达的变化.结果熊果酸在体外对HT-29细胞有中度增殖抑制效应,在熊果酸作用下,HT-29细胞出现显著的细胞凋亡征象,TUNEL显示细胞固缩,核染色质聚集或断裂,形成凋亡小体.流式细胞术检测在G1期之前出现sub-G1峰,凋亡率最高为11.63%,熊果酸的作用具有浓度和时间依赖性.在HT-29细胞凋亡过程中,凋亡相关基因caspase-9和bax的表达依次增强.结论熊果酸在体外对HT-29细胞有诱导凋亡的作用.其作用机制可能是通过促进caspase-9的活化和bax的表达上调来实现.     

全反式维甲酸诱导食管癌细胞凋亡的实验研究

目的探讨全反式维甲酸(ATRA)诱导食管癌EC9706细胞凋亡的作用及其机制。方法用不同浓度的ATRA处理EC9706细胞,采用四甲基偶氮唑盐(MTT)法检测ATRA对EC9706细胞的增殖抑制效应;采用流式细胞仪和原位末端标记(TUNEL)法检测细胞周期的变化和凋亡率;采用免疫组化S-P法,检测凋亡相关基因caspase-3和bcl-2的蛋白表达,并用病理图像分析软件进行半定量分析。结果ATRA对EC9706细胞有增殖抑制和诱导凋亡的作用;流式细胞仪检测显示,在G1期之前可出现Sub-G1峰,凋亡率最高为(32.6±0.4)%,并有浓度和时间依赖性;TUNEL法显示凋亡小体形成;EC9706细胞在凋亡过程中,凋亡相关基因caspase-3的蛋白表达增强,bcl-2的蛋白表达减弱。结论ATRA作用于食管癌EC9706细胞后将引起细胞凋亡的增加,这主要与ATRA能促进caspase-3的活化,并下调bcl-2的蛋白表达有关。     

靛玉红甲肟对HT-29细胞增殖和凋亡的影响及机制

背景与目的:近年来有报道称靛玉红甲肟(indirubin-3'-monoxime)在体内外实验中对部分实体肿瘤细胞具有明显的抑制作用,但尚未见其对人结肠癌HT-29细胞影响的研究报道,因而本文旨在研究其对HT-29细胞增殖和凋亡的影响及其机制.方法:MTT法检测不同浓度靛玉红甲肟处理HT-29细胞后细胞的增殖活性,制作生长抑制曲线.流式细胞仪检测凋亡率,RT-PCR检测细胞凋亡抑制基因survivin和bcl-2及凋亡促进基因Bax mRNA的变化.结果:靛玉红甲肟明显的抑制HT-29的增殖,其作用表现为剂量依赖性和时间依赖性(F=11.25,P<0.01).流式细胞仪检测发现:以10 μ mol/L的靛玉红甲肟处理HT-29细胞后,其凋亡率呈时间依赖性上升(F=195.25,P<0.01).RT-PCR检测发现HT-29细胞survivin(F=78.75,P<0.01)和Box(F=87.61,P<0.01)转录上升,而bcl-2转录显著下降(F=95.82,P<0.01).结论:靛玉红甲肟对人结肠癌HT-29细胞具有明显的增殖抑制和诱导凋亡作用,其作用机制与下调bcl-2/Bax比例有关.     

染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响

目的:研究染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响.方法:采用MTT比色法测定染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响,应用流式细胞术检测染料木黄酮对HT-29细胞周期及凋亡的影响.结果:染料木黄酮能够显著抑制HT-29细胞的增殖,且具有浓度依赖性,其半数抑制浓度(IC50)为23μmol/L;染料木黄酮能够引起HT-29细胞发生G0/G1期阻滞,并能够诱导HT-29细胞发生凋亡;western结果显示染料木黄酮作用后Bax表达增强,而Bcl-2表达减弱.结论:染料木黄酮能够诱导人结肠癌HT-29细胞发生周期阻滞和凋亡,从而导致HT-29细胞增殖能力下降.     

细胞周期蛋白激酶抑制剂诱导尤文肉瘤细胞凋亡作用的机制

目的探讨小分子细胞周期蛋白激酶抑制剂flavopiridol（FP）尤文肉瘤细胞株RD-ES凋亡诱导作用及其调控机制。方法FP作用RD-ES细胞后，光学显微镜观察其形态变化，MTT法测定FP对细胞增殖的抑制率；流式细胞仪检测细胞凋亡；Western blot检测bcl-2，bax，mcl-l和caspase-8的蛋白表达变化。结果MTT实验显示FP对尤文肉瘤RD-ES细胞增殖有抑制作用，其抑制效应具有时间及浓度依赖的特点，流式细胞计数分析表明FP可诱导细胞凋亡，免疫印迹检测显示FP对bcl-2及bax的表达无影响，但细胞凋亡抑制基因mcl-1活性型caspase-8的表达被上调。结论FP可诱导尤文肉瘤细胞株凋亡，其作用不依赖于bcl-2基因的变化，mcl-1基因表达的抑制及caspase-8路径的激活可能为其机制之一。     

PPARγ活化诱导细胞凋亡和周期阻滞来抑制结肠癌细胞生长

目的探讨过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)在结肠癌细胞株HT-29中的表达及其活化后对结肠癌细胞生长的影响。方法通过RT-PCR和Western blot检测HT-29中PPARγmRNA及蛋白质的表达,四甲基偶氮唑盐(MTT)微量酶反应比色法检测PPARγ配体罗格列酮(rosiglitazone,Rosi)和15d-PGJ2对HT-29细胞生长的影响,荧光显微镜、TUNEL法观察Rosi和15d-PGJ2激活PPARγ后诱导HT-29细胞凋亡形态和生化改变,流式细胞仪(FCM)以碘化丙啶(PI)单染法检测细胞周期。结果RT-PCR及Western blot检测结果表明结肠癌细胞HT-29中存在PPARγmRNA及蛋白质的表达。MTT结果显示Rosi和15d-PGJ2可抑制HT-29细胞生长,且具有时间、剂量依赖效应。荧光显微镜、TUNEL检测显示PPARγ活化后HT-29细胞出现典型的凋亡现象,10μmol/L Rosi或15d-PGJ2作用48h后的细胞凋亡率分别为(17.3±1.9)%及(20.8±2.9)%,与对照组(3.86±0.49)%相比差异均具有统计学意义(P<0.05)。FCM结果显示Rosi和15d-PGJ2激活PPARγ后诱导细胞周期阻滞于G0/G1期,与对照组相比差异有统计学意义(P<0.05)。结论结肠癌细胞HT-29表达PPARγ,其活化可通过诱导细胞凋亡及细胞周期阻滞,抑制结肠癌细胞增长。因此,PPARγ可能为结肠癌治疗的一个新靶点。     

肠积宁方促进大肠癌细胞株HT-29凋亡及干预相关基因bcl-2及bax表达的实验研究

目的：探讨肠积宁方对人大肠癌细胞株HT-29凋亡及凋亡相关基因bcl-2、bax表达的影响.方法：采用MTT法测定肠积宁方对人大肠癌细胞株HT-29的增殖抑制作用,运用流式细胞仪测定肠积宁方对大肠癌细胞株HT-29凋亡及凋亡相关基因bcl-2、bax表达的影响.结果：肠积宁方能促进人大肠癌细胞株HT-29凋亡,并抑制凋亡相关基因bc1-2、bax的表达,尤其对bcl-2的作用更显著.结论：肠积宁方具有抗肿瘤作用,其抗肿瘤作用机制可能是促进细胞凋亡并抑制凋亡相关基因的表达.     

紫杉醇对人骨肉瘤细胞凋亡及对bcl-2 、bax基因表达的影响

 目的 了解紫杉醇诱导人骨肉瘤细胞的凋亡效果,以及凋亡与bcl-2和bax表达之间的关系。方法 用不同浓度的紫杉醇作用于骨肉瘤细胞MG63。采用胎盼蓝染色法,于细胞记数板进行活细胞率检测计算。采用光镜和电子显微镜对细胞形态改变进行观测,采用原位缺口末端标记凋亡检测法（TUNEL）和流式细胞术（Annexin-V-FITC＆PI）对细胞凋亡进行检测。采用免疫组织化学法对凋亡调节基因bcl-2和bax表达进行检测。结果 骨肉瘤活细胞率随着紫杉醇作用时间延长和浓度增高而降低。紫杉醇诱导骨肉瘤细胞MG63凋亡表现为典型的凋亡特征,包括：细胞形态学改变,细胞染色质浓聚,染色质新月形变,胞核碎裂等。TUNEL法检测到了细胞DNA片段的断裂。随着细胞凋亡的发生,开始出现G2/M期阻滞。紫杉醇可以降低凋亡相关基因bcl-2的表达和增加bax基因的表达。结论 紫杉醇可以通过时间依赖性和剂量依赖性方式,促使细胞G2/M期阻滞和抑制细胞有丝分裂,从而诱导人骨肉瘤细胞凋亡。这种凋亡可能受到凋亡相关基因bcl-2低表达和bax高表达的调控。     

云南元宝枫黄酮诱导个旧肺鳞癌细胞YTMLC凋亡的实验研究

目的研究云南元宝枫黄酮对个旧肺鳞癌细胞(YTMLC)凋亡诱导作用及相关基因表达的影响,为开发应用元宝枫黄酮提供实验依据。方法采用四甲基偶氮唑蓝(MTT)比色法检测元宝枫黄酮对YTMLC细胞的生长抑制作用,流式细胞术TUNEL法检测元宝枫黄酮对YTMLC细胞凋亡的影响,RT-PCR检测元宝枫黄酮对YTMLC细胞 p53、p21、bcl-2、bax和caspase-3基因表达的影响。结果元宝枫黄酮作用YTMLC细胞48 h后,IC50为（108.53±8.22）mg/L。流式细胞术显示元宝枫黄酮既可以诱导YTMLC细胞凋亡,也可以引起YTMLC细胞坏死。元宝枫黄酮对p53、p21、bax和caspase-3的基因表达均有明显上调作用;对bcl-2基因表达有下调作用。结论云南元宝枫黄酮可以抑制个旧肺鳞癌细胞(YTMLC)体外生长,引起肿瘤细胞坏死,诱导肿瘤细胞凋亡,其诱导肿瘤细胞凋亡的可能机制是通过上调p53、p21、bax和caspase-3基因的表达,下调bcl-2基因表达。     

姜黄素诱导人结肠癌细胞株SW480凋亡及其bcl-2表达相关性研究

目的探讨姜黄素对人结肠癌细胞株SW480诱导凋亡的影响及其作用机制。方法5～50μM姜黄素分别处理SW480细胞6～48h后,MTT法检测细胞的生长活性,流式细胞仪检测细胞周期,TUNEL法检测细胞凋亡,免疫组化法检测细胞内bcl2蛋白表达水平。结果MTT显示姜黄素对SW480细胞具有细胞毒作用,呈时间浓度依赖性。FCM结果显示细胞周期中G0/G1期细胞比例增加,S期、G2/M期比例下降。TUNEL法显示凋亡细胞数增加,免疫组化结果显示bcl2基因表达明显减弱。结论姜黄素能抑制结肠癌SW480细胞的增殖并诱导细胞凋亡。其机制可能与干扰细胞周期及下调bcl2基因表达有关。     

Combined antitumor effect of ursolic acid and 5-fluorouracil on human esophageal carcinoma cell Eca-109 <Emphasis Type="Italic">in vitro</Emphasis>

Objective:To study the combined antitumor effect and possible mechanisms of ursolic acid with 5-fluorouracil (5-FU) on human esophageal carcinoma cell Eca-109 in vitro. Methods:Eca-109 cells were treated with ursolic acid (10-50 μmol/L) and/or 5-fluorouracil (48.0-768.8 μmol/L) for 48 h in vitro. And then cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). The morphological changes of apoptosis were observed by fluorescent microscopy. At last ...     

熊果酸诱导卵巢癌细胞SKOV3凋亡的研究

目的探讨三萜类化合物熊果酸（UA）诱导卵巢癌细胞SKOV3凋亡的作用。方法用不同浓度的UA处理体外培养的SKOV3细胞,用MTT法、活细胞荧光染色技术、流式细胞术（FCM）、RT—PCR、细胞免疫化学技术,检测UA对SKOV3细胞的增生抑制和诱导凋亡作用。结果UA抑制SKOV3细胞增生,30μmol／L的熊果酸在作用24h后即表现出较强的增生抑制作用,呈时间和浓度依赖性,并使细胞呈典型的凋亡形态学特征,核质浓集,出现凋亡小体,FCM检测显示40μmol／LUA作用24h后SKOV3细胞开始出现G0／G1期阻滞,72h时早期凋亡细胞达（28．7±2．4）％,并诱导野生型p53基因mRNA及蛋白表达增加。结论UA诱导SKOV3细胞凋亡,上调胞内野生型p53基因表达是所涉及的分子机制之一。     

siRNA沉默livin的表达对人结肠癌HT-29细胞增殖、凋亡及侵袭的影响

目的:探讨小干扰RNA(small interference RNA,siRNA)沉默人结肠癌HT-29细胞livin表达对HT-29细胞增殖、凋亡和侵袭的影响。方法:合成靶向livin的双链siRNA(livin-siRNA),转染HT-29细胞,RT-PCR及Western blotting检测HT-29细胞中livin mRNA及蛋白的表达,MTT实验检测HT-29细胞的增殖,流式细胞术检测HT-29细胞周期分布及凋亡,细胞侵袭实验检测HT-29细胞侵袭性的变化,caspase-3活性检测试剂盒检测caspase-3活性的变化。结果:Livin-siRNA转染后48 h,与空白组、阴性对照组及脂质体组相比,livin-siRNA转染组HT-29细胞中livin mRNA水平明显下降(0.073±0.007 vs 0.395±0.082、0.423±0.025、0.418±0.032,P<0.05),其蛋白表达也明显下调(0.106±0.003 vs 0.456±0.065、0.473±0.078、0.491±0.045,P<0.05)。转染96 h后,livin-siRNA组HT-29细胞增殖能力明显低于对照组及脂质体组(0.564±0.102 vs0.833±0.127、0.860±0.153,P<0.05),且细胞凋亡率升高[(16.5±2.8)%vs(2.4±0.5)%、(3.7±1.0)%,P<0.05]。侵袭实验显示,livin-siRNA转染后,穿过Matrigel膜的HT-29细胞数量明显少于对照组及脂质体组[(31.3±4.5)vs(101.3±8.6)、(97.4±7.8)个,P<0.05)]。livin-siRNA组HT-29细胞的caspase-3活性低于对照组(0.160±0.023 vs 0.347±0.058,P<0.05)。结论:siRNA沉默livin的表达可抑制HT-29细胞的增殖,诱导细胞凋亡,抑制细胞的侵袭。     

Gambogenic Acid Induction of Apoptosis in a Breast Cancer Cell Line

Background: Gambogenic acid is a major active compound of gamboge which exudes from the Garciniahanburyi tree. Gambogenic acid anti-cancer activity in vitro has been reported in several studies, including anA549 nude mouse model. However, the mechanisms of action remain unclear. Methods: We used nude mousemodels to detect the effect of gambogenic acid on breast tumors, analyzing expression of apoptosis-relatedproteins in vivo by Western blotting. Effects on cell proliferation, apoptosis and apoptosis-related proteins inMDA-MB-231 cells were detected by MTT, flow cytometry and Western blotting. Inhibitors of caspase-3,-8,-9were also used to detect effects on caspase family members. Results: We found that gambogenic acid suppressedbreast tumor growth in vivo, in association with increased expression of Fas and cleaved caspase-3,-8,-9 and bax,as well as decrease in the anti-apoptotic protein bcl-2. Gambogenic acid inhibited cell proliferation and inducedcell apoptosis in a concentration-dependent manner. Conclusion: Our observations suggested that Gambogenicacid suppressed breast cancer MDA-MB-231 cell growth by mediating apoptosis through death receptor andmitochondrial pathways in vivo and in vitro.     

Effects of Celecoxib on Cycle Kinetics of Gastric Cancer Cells and Protein Expression of Cytochrome C and Caspase-9

Objective: This investigation aimed to determine effects of celecoxib on the cell cycle kinetics of the gastriccancer cell line MGC803 and the mechanisms involved by assessing expression of cytochrome C and caspase-9at the protein level. Methods: Cell proliferation of MGC803 was determined by MTT assay after treatment withcelecoxib. Apoptosis was assessed using fluorescence staining and cell cycle kinetics by flow cytometry. Westernblotting was used to detect the expression of caspase-9 protein and of cytochrome C protein in cell cytosol andmitochondria. Results: Celecoxib was able to restrain proliferation and induce apoptosis in a dose- and timedependentmanner, inducing G0/G1 cell cycle arrest, release of cytochrome C into the cytosol, and cleavageof pro-caspase-9 into its active form. Conclusion: Celecoxib can induce apoptosis in MGC803 cells through amechanism involving cell cycle arrest, mitochondrial cytochrome C release and caspase activation.     

Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent on Fas/Fas ligand interaction in colon cancer HT-29 cells

Boswellic acids are the effective components of gum resin of Boswellia serrata, which has anti-inflammatory properties. Recent studies on brain tumors and leukemic cells indicate that boswellic acids may have antiproliferative and apoptotic effects with the mechanisms being not studied in detail. We studied their antiproliferative and apoptotic effects on colon cancer cells and the pathway leading to apoptosis. HT-29 cells were treated with beta-boswellic acid (BA), keto-beta-boswellic acid (K-BA) and acetyl-keto-beta-boswellic acid (AK-BA), respectively. Apoptosis was determined by flow cytometry, by cytoplasmic DNA-histone complex and the activity of caspase-3. The cleavage of poly-(ADP-ribose)-polymerase (PARP) and expression of Fas were examined by western blot. Specific caspase inhibitors, polyclonal Fas antibody, and antagonistic Fas antibody ZB4 were employed to elucidate apoptotic pathways. DNA synthesis and cell viability were examined. Both K-BA and AK-BA increased cytoplasmic DNA-histone complex dose-dependently and increased pre-G(1) peak in flow cytometer analysis, with the effects of AK-BA being stronger than K-BA. BA only increased the formation of DNA-histone complex at a high concentration. K-BA and AK-BA increased caspase-8, caspase-9 and caspase-3 activities accompanied by cleavage of PARP. The effects of AK-BA on formation of cytoplasmic DNA histone and on caspase-3 activation were 3.7- and 3.4-fold, respectively, more effective than those induced by camptothecin. The apoptosis induced by AK-BA was inhibited completely by caspase-3 or caspase-8 inhibitor and partially by caspase-9 inhibitor. ZB4 blocked exogenous Fas ligand-induced apoptosis, but had no effect on AK-BA-induced apoptosis. AK-BA had no significant effect on expression of Fas. Apart from apoptotic effect, these acids also inhibited [(3)H]thymidine incorporation and cell viability to different extent. In conclusion, boswellic acids, particularly AK-BA and K-BA have antiproliferative and apoptotic effects in human HT-29 cells. The apoptotic effect is mediated via a pathway dependent on caspase-8 activation but independent of Fas/FasL interaction.     

The combined treatment of aspirin and radiation induces apoptosis by the regulation of bcl-2 and caspase-3 in human cervical cancer cell

The antitumor mechanisms mediated by combined treatment of aspirin and radiation on human cervical cancer cells are unclear. In this paper, we studied whether aspirin and radiation induced apoptosis and whether the sensitivity to radiation was enhanced in aspirin-pretreated HeLa TG, human cervical cancer cells. We identified the regulation of apoptosis-responsive genes, bcl-2, caspase-3 and p53 after combined treatment. To investigate the growth inhibitory effect on HeLa TG cells after treatment of various nonsteroidal anti-inflammatory drugs (NSAIDs), we performed cell proliferation assay and colony-forming assay. In the presence of aspirin, sulindac and indomethacin, cell proliferation and colony formation were decreased in a time- and dose-dependent manner. According to flow cytometry analysis and Hoechst 33342 staining, we found that aspirin increased sub-G1 population and nuclear condensation of cervical cancer cells. Remarkably, the combined treatment decreased cell proliferation compared with treatment of 1 mM aspirin or 6 Gy radiation alone. Pretreatment of aspirin followed by irradiation also elevated the population of apoptotic cells. These results revealed that sensitivity to radiation was enhanced in aspirin-pretreated HeLa TG cells, and aspirin has the additive role for amplifying the radiotherapeutic effect in cervical cancer cells. Finally, combined treatment revealed bcl-2 repression and caspase-3 induction did not detect any change but p53 expression did. We have demonstrated that combined treatment of aspirin and radiation induces the antitumor effect mediated by bcl-2 and caspase-3 pathway in cervical cancer cells.