姜黄素对人结肠癌细胞SW480作用的体外实验研究

目的 研究姜黄素促进人结肠癌细胞系SW480凋亡的作用.方法 人结肠癌细胞系SW480与不同浓度的姜黄素共孵育48 h,MTT法检测细胞存活率,并用相差显微镜观察细胞形态学改变.用流式细胞术(检测细胞凋亡相关指标Annexin V/PI)和Ho-echst3258染色检测细胞凋亡.结果 姜黄素能够不同程度地抑制人结肠癌细胞系SW480的生长.姜黄素对SW480细胞的IC50(半数抑制浓度)为65 mg/mL.Hoechst3258荧光染色与流式细胞术分析SW480细胞的结果一致.结论 姜黄素可促进SW480细胞凋亡,为姜黄素治疗结肠癌提供依据.     

苯乙酸钠诱导SW480细胞凋亡及其对细胞周期各时相的影响

目的探讨苯乙酸钠(sodium phenylacetate,NaPA)诱导人结肠癌细胞株SW 480凋亡机制及对细胞周期各时相的影响。方法应用MTT比色法及流式细胞术检测NaPA对SW 480细胞的增殖抑制率及细胞周期各时相的变化,并通过电子显微镜观察SW 480细胞亚细胞结构改变。结果NaPA对SW 480细胞有明显的抑制作用,抑制率15.9%~41.7%,且呈剂量依赖性。流式细胞术结果表明,G0/G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,细胞凋亡率升高,电子显微镜下可见SW 480细胞线粒体肿胀,细胞核固缩。结论NaPA能够抑制SW 480细胞G1期向S期转化进程,诱导SW 480细胞凋亡。     

SEA对人结肠癌SW480细胞周期进程及凋亡的影响

目的 体外观察葡萄球菌肠毒素A(SEA)对人结肠癌细胞株SW480的增殖抑制作用.方法 应用MTr比色法检测不同浓度的SEA对SW480细胞的增殖抑制率,流式细胞仪检测细胞周期进程的变化及凋亡率,电子显微镜检测亚细胞水平变化.结果 不同浓度的SEA对SW480细胞增殖抑制率分别为18.5％、37.6％和41.5％,与对照组相比差异显著.流式细胞仪结果显示Go/G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,细胞凋亡率升高.电子显微镜可见,细胞线粒体肿胀、胞膜破裂、细胞核染色质趋边、凝集,可见凋亡小体.结论 SEA能显著抑制SW480细胞增殖,抑制SW480细胞周期G1期向S期转化进程,从而使G2/M期细胞相对增高,诱导SW480细胞凋亡及亚细胞结构改变.     

亚硒酸钠对胃上皮肿瘤SGC-7901细胞抑制作用的研究

应用细胞培养技术、电子显微镜技术、流式细胞仪技术及DNA末端原位标记染色法（TUNEL技术）,观察亚硒酸钠溶液对胃上皮肿瘤SGC-7901细胞的抑制作用。结果随着亚硒酸钠溶液浓度增高、作用时间延长对SGC-7901细胞的抑制率升高;可见较典型的细胞凋亡形态学变化;流式细胞仪DNA直方图上出现典型的亚二倍体的“凋亡峰”;TUNEL染色法细胞凋亡指数为8．4％～33．4％。证明亚硒酸钠溶液能抑制SGC-7901细胞生长,诱导其凋亡;抑制作用与亚硒酸钠溶液的浓度及作用时间呈正相关。     

全反式维甲酸对多种胰腺癌细胞的诱导凋亡作用

目的 观察全反式维甲酸(all-trans retinoic acid,ATRA)能否诱导人胰腺癌细胞的凋亡.方法 分别将人胰腺癌细胞SW1990、PaTu8988、BxPC3和ATRA共同孵育后,用MTT法检测细胞活性,分别用流式细胞仪检测、TUNEL和透射电镜观察细胞的凋亡.结果 MTT法显示,ATRA对3株人胰腺癌细胞株SW1990、PaTu8988和BxPC3的生长均有明显的抑制作用(P ＜ 0.05).流式细胞仪的检测、TUNEL和电镜的观察结果均证实ATRA诱导后,3株胰腺癌细胞株凋亡率均高于对照(P ＜ 0.01),并随诱导时间延长而增加.结论 ATRA在体外能显著抑制多株胰腺癌细胞株的生长,并促进其凋亡.     

全反式维甲酸对多种胰腺癌细胞的诱导凋亡作用

目的观察全反式维甲酸(all-trans retinoic acid,ATRA)能否诱导人胰腺癌细胞的凋亡。方法分别将人胰腺癌细胞SW1990、PaTu8988、BxPC3和ATRA共同孵育后,用MTT法检测细胞活性,分别用流式细胞仪检测、TUNEL和透射电镜观察细胞的凋亡。结果MTT法显示,ATRA对3株人胰腺癌细胞株SW1990、PaTu8988和BxPC3的生长均有明显的抑制作用(P<0.05)。流式细胞仪的检测、TUNEL和电镜的观察结果均证实ATRA诱导后,3株胰腺癌细胞株凋亡率均高于对照(P<0.01),并随诱导时间延长而增加。结论ATRA在体外能显著抑制多株胰腺癌细胞株的生长,并促进其凋亡。     

芥菜籽提取物对人结肠癌细胞系SW480凋亡的影响

目的观察芥菜籽提取物（MSE）对人结肠癌细胞系SW480细胞凋亡的影响并初步探讨其可能的机制。方法采用体外细胞培养技术,用CCK-8法观察MSE对人大肠癌SW480细胞生长的影响;Hoechest3325荧光染色显微镜下观察凋亡细胞形态学改变,流式细胞仪检测细胞凋亡;Caspase-3活性检测试剂盒分析其凋亡机制。结果在浓度0．2—1．0mg／ml作用24—72h范围内,MSE对SW480细胞增殖具有明显抑制作用（P〈0．01）。24h内其凋亡率随着药物浓度的增加而增加,且逐渐从早期凋亡走向晚期凋亡,呈浓度依赖性;24h内Caspase-3活性较对照组显著提高（P〈0．05）。结论MSE能有效抑制体外培养的人结肠癌细胞SW480的生长,这种抑制作用与MSE促进肿瘤细胞凋亡、激活凋亡蛋白酶Caspase-3的活性有关。     

稀释碘伏对人结肠癌细胞株SW480体外生长的影响

目的观察稀释碘伏对人结肠癌细胞株SW480体外生长的影响。方法体外培养SW480,分别用0.05%、0.025%、0.01%的碘伏处理,MTT法测算细胞生长抑制率,PI染色流式细胞术分析细胞凋亡率。结果 0.05%、0.025%、0.01%的碘伏对SW480的生长抑制率分别为84.0%、84.3%8、4.5%,三者相比,P〉0.05;0.01%的碘伏分别作用13、5、min后,SW480的生长抑制率分别为82.4%8、3.6%8、4.0%,三者相比,P〉0.05。0.01%碘伏处理SW480细胞1 min即可诱导其凋亡,凋亡率达78.3%。结论稀释碘伏在体外对人结肠癌细胞具有明显的生长抑制及诱导凋亡的作用。     

阿司匹林与二甲双胍对人结肠癌SW480细胞株的联合作用探讨

目的观察联合阿司匹林(Aspirin,ASA)与二甲双胍(Metformin,MET)对人结肠癌SW480细胞株的抑制作用,并探讨两者之间的相互作用方式。方法采用MTT法及流式细胞术检测空白对照组、ASA组、MET组及ASA与MET共同作用组对人结肠癌SW480细胞增殖及细胞凋亡的影响。结果单独使用ASA、MET对结肠癌SW480抑制率及其凋亡率均高于空白对照组,且72 h时抑制率及其凋亡率均高于24 h者;在干预24 h、72 h时,ASA与MET共同作用组的抑制率和凋亡率均明显高于单独使用MET组或ASA组,Q值分别为1.19和1.23。结论 ASA和MET单独作用均对结肠癌SW480细胞有一定的抑制作用,且随着时间延长,抑制作用明显加强;联合使用MET和ASA对人结肠癌SW480细胞具有协同抑制作用。     

奥沙利铂对人结肠癌细胞凋亡及FADD的影响

以不同浓度（0、25、50和100μg/ml）的奥沙利铂干预体外培养的人结肠癌细胞株（SW480）,分别于12、24和48 h后,应用四甲基偶氮唑盐比色法检测细胞的增殖活性,流式细胞仪检测细胞周期和细胞凋亡率,半定量RT-PCR检测Fas相关死亡结构域蛋白（FADD）mRNA水平,Western blotting检测FADD蛋白水平。结果奥沙利铂对SW480生长增殖具有明显抑制作用,其效应呈浓度和时间依赖性,SW480细胞呈G2/M期阻滞;SW480细胞的凋亡率呈浓度依赖性的增加（P〈0.05）。不同浓度的奥沙利铂处理SW480细胞株24 h后,FADD mRNA和蛋白水平呈浓度依赖性增加。提示奥沙利铂能诱导SW480细胞凋亡,其作用机制可能与其激活凋亡死亡受体途径使FADD表达上调有关。     

Apoptosis induction with polo-like kinase-1 antisense phosphorothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM:To investigate the effects of polo-like kinase-1 (PLKl) antisense phosphorothioate oligodeoxynudeotide (ASODN) on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS:After SW480 colon cancer cells were transfected with PLKl ASODN, Northern and Western blot analyses were used to examine PLKl gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynudeotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS:The levels of PLKl mRNA and protein were greatly inhibited by PLKl ASODN in SW480 cancer cells transfected with PLKl ASODN. Apoptosis index (AI) induced PLKl ASODN in a time-and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G_2/M compared with control groups. CONCLUSION:PLKl ASODN can induce apoptosis of human colon cancer cell line SW480.     

New anti-proliferative agent, MK615, from Japanese apricot ＂Prunus mume＂ induces striking autophagy in colon cancer cells in vitro

AIM: To investigate the anti-neoplastic effects of MK615, an extract from the Japanese apricot (Prunus mume), against colon cancer cells. METHODS: Three colon cancer cell lines, SW480, COLO, and WiDr, were cultured with MK615. Growth inhibition was evaluated by cell proliferation assay and killing activity was determined by lactate dehydrogenase assay. Induction of apoptosis was evaluated by annexin Ⅴ flow cytometry. Morphological changes were studied by light and electron microscopy, and immunofluorescence staining with Atg8. RESULTS: MK615 inhibited growth and lysed SW480, COLO and WiDr cells in a dose-dependent manner. Annexin Ⅴ flow cytometry showed that MK615 induced apoptosis after 6 h incubation, at which point the occurrence of apoptotic cells was 68.0%, 65.7% and 64.7% for SW480, COLO, and WiDr cells, respectively. Light and electron microscopy, and immunofluorescence staining with Atg8 revealed that MK615 induced massive cytoplasmic vacuoles (autophagosomes) in all three cell lines. CONCLUSION: MK615 has an anti-neoplastic effect against colon cancer cells. The effect may be exerted by induction of apoptosis and autophagy.     

Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50&gt;l mg&#183;L^1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25&#177;3.48μmol&#183;L^-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg&#183;L^-1),combined with subtoxic doxorubicin (0.86 μmol&#183;L^-1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12&#177;2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82&#177;1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (p&gt;0.05).CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentra~on of doxorubidn to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubidn against colon cancers.     

雌激素受体β过表达对大肠癌细胞增殖凋亡的影响

目的观察雌激素受体β（ERβ）过表达对大肠癌细胞增殖和凋亡的影响,并探讨其机制。方法以脂质体介导的ERβ基因转染SW480细胞,G418筛选阳性克隆（SW480-C1-ERβ）。RT—PCR及WesternBlot鉴定ERβ的过表达。以正常SW480细胞和转染了空质粒的SW480细胞（SW480-pEGFP—C1）作为对照,在无雌激素或有雌激素作用的条件下,采用MTT法检测细胞增殖,流式细胞术检测细胞凋亡率,实时荧光定量RT—PCR方法检测凋亡相关基因survivin和Bax表达。结果SW480和SW480-pEGFP—C1细胞中ERβ mRNA和蛋白表达较低,而稳定转染的SW480-C1-ERβ细胞中ERβmRNA和蛋白表达明显增加。在有或无雌激素作用的条件下均可发现,与SW480细胞或SW480-pEGFP—C1细胞比较,SW480-C1-ERβ细胞增殖速度减慢,凋亡率明显增高,survivin表达减低,Bax表达增高;在SW480-C1-ERβ细胞中,有雌激素作用者较无雌激素作用者以上变化更明显。结论ERβ过表达可以同时以配体非依赖性和配体依赖性的方式抑制细胞增殖和增加细胞凋亡,可能与调控凋亡相关基因survivin和Bax表达有关。     

Synergistic antitumor effect of TRATL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosisinducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms. METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT)assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detectapoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50＞1 mg@L1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25+3.48μmol@L-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg.L-1),combined with subtoxic doxorubicin (0.86 μmol@L1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+2.67%, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P＞0.05). CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein i snot involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.     

Mechanism of counterattack of colorectal cancer cell by Fas/Fas ligand system

AIM: To determine the role of Fas/Fas ligand (FasL) in the immune escape of colon cancer cells. METHODS: Immunohistochemistry was used to observe the expression of Fas and FasL in the tissues of colon cancer patients. In situ hybridization was used to detect the localization of FasL mRNA expression in cancer tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and CD45 staining were performed to detect the apoptosis of tumor-infiltrating lymphocytes (TILs). Co-culture assays of colon cancer cells (SW480) and Jurkat cells (Fas-sensitive cells) were performed to observe the counterattack of colon cancer cells to lymphocytes. RESULTS: Of 53 cases of colon carcinomas, 23 cases (43.4%) expressed Fas which was significantly lower as compared to the normal colonic mucosa (73.3%, P<0.01), and 45 cases (84.9%) of colon carcinomas expressed FasL, whereas only two cases (3.75%) in normal mucosa expressed FasL. FasL expression in the colon cancer cells was found to be associated with increased cell death of TILs. The apoptotic rate of TIL in the FasL-positive staining regions of tumor cells was significantly higher than that in the FasL-negative staining region (54.84±2.79% vs 25.73±1.98%, P<0.01). The co-culture of SW480 cells and Jurkat cells confirmed the function of FasL on the SW480 cells. The apoptotic rates of Jurkat cells were found to be related with the amount of SW480 cells. CONCLUSION: Colon cancer cells can escape the immune surveillance and killing via decreasing Fas expression, and can counterattack the immune system via increasing FasL expression. Fas/FasL can serve as potential targets for effective antitumor therapy.     

Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer

AIM:To explore the effect of antisense oligocleoxynucletide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.RESULTS:The telomerase activity in SW480 cells transfected with 1.0μmol/L of As-ODN for 2-5days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis.The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes.CONCLUSION:As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.     

黑米花青素对结肠癌细胞增殖、细胞周期及凋亡的影响

目的探讨黑米花青素对结肠癌细胞增殖、细胞周期及凋亡的影响。 方法利用MTT法和流式细胞分析技术分别观察不同浓度同一时间和相同浓度不同时间,黑米花青素对结肠癌细胞(SW480)在细胞增殖、细胞周期分布和细胞凋亡的影响。 结果黑米花青素对结肠癌细胞的增殖、细胞周期分布和凋亡产生了影响,而且这种作用有浓度依赖性和时间依赖性。 结论黑米花青素可以抑制结肠细胞的增殖、阻滞细胞周期的进展、促进肿瘤细胞的凋亡。     

姜黄素对结肠癌细胞SW480 FasL基因表达和侵袭能力的影响

目的研究姜黄素对人结肠癌SW480细胞FasL mRNA表达及对其侵袭能力的影响,为中药抗肿瘤提供实验依据。方法根据MTT法得到姜黄素对SW480细胞的半数有效抑制浓度（IC50）,确定药物作用浓度。SW480细胞分别经姜黄素不同浓度（0．5 IC50、IC50）作用后,应用逆转录-聚合酶链反应（RT-PCR）法检测姜黄素作用前后人结肠癌SW480细胞FasL mRNA的变化;应用Transwell细胞侵袭试验检测姜黄素对SW480细胞侵袭能力的影响。结果姜黄素处理后SW480细胞FasL mRNA表达水平均明显高于对照组（P〈0．01）;而且FasL mRNA表达水平随姜黄素作用浓度增加显著上调,姜黄素不同浓度组比较均有显著性差异（P〈0．01）;随姜黄素作用浓度升高,SW480细胞侵袭能力明显增强,不同浓度组比较均有显著性差异（P〈0．01）。结论姜黄素在一定时间内均可上调人结肠癌SW480细胞FasL mRNA的表达,而且这种上调作用在一定范围内呈剂量依赖性,可使结肠癌细胞的侵袭能力增强。