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。     

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。     

氧化苦参碱诱导人结肠癌SW480细胞凋亡的机制

目的探讨氧化苦参碱诱导人结肠癌SW480细胞凋亡的作用及机制。方法以不同剂量的氧化苦参碱作用于人结肠癌细胞株SW480,四甲基偶氮唑蓝法(MTT)检测细胞的增殖能力,Hoechst 33258染色法观测细胞凋亡。免疫印迹法测定B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达,荧光法测定半胱氨酸蛋白酶(caspase)-3的活性。结果氧化苦参碱可剂量依赖性地抑制细胞的增殖,促进细胞凋亡。Western印迹方法显示氧化苦参碱可抑制抗凋亡蛋白Bcl-2的表达,提高促凋亡蛋白Bax的表达,同时激活凋亡效应分子caspase-3。结论氧化苦参碱能够抑制结肠癌SW480细胞的增殖,通过提高Bax的表达、抑制Bcl-2的表达、激活caspase-3发挥其诱导细胞凋亡的作用。     

姜黄素对人结肠癌SW620细胞凋亡机制的影响

目的 探讨体外姜黄素对人结肠癌SW620细胞生长及凋亡的影响.方法 体外培养SW620细胞,用不同浓度的姜黄素作用为实验组,同时设对照组,以CCK8法测细胞增殖抑制作用、流式细胞仪检测细胞凋亡及p53和Bcl-2蛋白表达情况.结果 不同浓度姜黄素作用SW620细胞24 h增殖抑制显著(P<0.05),其抑制效应具有剂量依赖性,同时诱导SW620细胞凋亡、上调p53基因表达、下调Bcl-2基因表达(P<0.05).结论 姜黄素可抑制SW620细胞的生长且具有剂量依赖性,并可诱导肿瘤细胞凋亡,其抗肿瘤效应可能与细胞凋亡相关基因表达的调控有关.     

雷替曲塞对人结肠癌细胞SW480增殖、凋亡的影响及其机制

目的观察雷替曲塞对人结肠癌细胞(SW480)细胞增殖及细胞凋亡的影响,并探讨其机制。方法将SW480随机分为A组、B组、C组与对照组,A组加入雷替曲塞0. 1μg/mL,B组加入雷替曲塞0. 5μg/mL,C组加入雷替曲塞2. 5μg/mL,对照组加入等量培养液。培养24、48、72 h后采用CCK-8试剂盒检测各组SW480增殖活性;培养10 d后,计算各组SW480克隆形成率;培养72 h后,采用实时荧光定量PCR检测Bax、Bcl-2及Caspase-3 mRNA,采用Western Blotting法检测Bax、Bcl-2及Caspase-3蛋白。结果随着雷替曲塞浓度的增加以及干预时间的延长,SW480的增殖速度减慢(P均<0. 05),且在浓度达到一定程度后,该现象不再明显;孵育10 d后,各组SW480克隆形成率组间两两比较,P均<0. 05;随着雷替曲塞浓度的增加,SW480 Bax及Caspase-3蛋白及mRNA表达升高,Bcl-2蛋白及RNA表达降低(P均<0. 05)。结论雷替曲塞可通过调节SW480 Bax、Caspase-3和Bcl-2蛋白及...     

姜黄素对人结肠癌细胞HT-29中β-catenin、血管内皮生长因子的影响

目的研究姜黄素对体外培养人结肠癌HT-29细胞增殖及对细胞株中β-catenin、血管内皮生长因子(VEGF)表达的影响。方法采用不同浓度的姜黄素作用于HT-29细胞,MTT法检测细胞增殖情况;Western印迹检测药物处理后β-catenin及VEGF蛋白的表达。结果姜黄素对HT-29细胞的增殖具有抑制作用,且呈剂量-时间依赖性(P<0.05),测得IC50为19.27μmol/L;在蛋白水平,姜黄素处理后,β-catenin、VEGF的蛋白表达水平降低。结论姜黄素能够抑制HT-29细胞的增殖,并下调结肠癌细胞HT-29中β-catenin、VEGF的表达。     

携带凋亡素VP3基因重组腺病毒的制备及其在人结肠癌SW480细胞中的表达

目的利用细菌内同源重组法构建携带凋亡素VP3基因的重组腺病毒并观察其在人结肠癌SW480细胞中的表达。方法扩增目的基因VP3 cDNA,与EcoRⅤ酶切线性化的pShuttle-IRES-hrGFP穿梭质粒连接构建重组穿梭质粒pShuttle-VP3-hrGFP,PmeⅠ酶切线性化pShuttleVP3-hrGFP并转化含pAdeasy-1的超感受态BJ5183大肠杆菌,细菌内同源重组法构建重组腺病毒质粒pAd-VP3-hrGFP,并经PCR、EcoRⅤ、PacⅠ酶切电泳及测序鉴定,线性化的重组腺病毒质粒经脂质体包裹转染AD293细胞进行重组腺病毒的包装和扩增,CsCI密度梯度离心法行病毒浓缩和纯化并将其感染人结肠癌SW480细胞中进行表达,观察其感染效率及VP3蛋白表达水平。结果 pShuttle-VP3-hrGFP重组穿梭质粒构建鉴定成功,PCR反应扩增出402 bp的片段;pAd-VP3-hrGFP重组腺病毒质粒经酶切获得大于23 kb的大片段和4.5 kb的片段,重组腺病毒质粒经测序证实VP3-hrGFP编码区成功克隆入腺病毒p Ad中,且其序列与Gen Bank中VP3 CDNA序列完全一致。成功包装出携带VP3基因的重组腺病毒,滴度为1.738×10~(12)opu/ml,重组腺病毒可在SW480细胞内得到有效表达。结论用细菌内同源重组法可快速、高效地制备携带凋亡素VP3基因的重组腺病毒,并可在人结肠癌细胞中高效表达。     

蛹虫草提取物对人结肠癌细胞SW111C生长的抑制作用

目的探讨蛹虫草提取物（RW）对结肠癌细胞株SW111C的抑制作用及可能机制。方法将不同浓度的RW作用于体外培养的SW111C细胞。采用四氮唑盐（MTT）比色法检测不同作用时间SW111C增殖抑制率;姬姆萨染色法观察细胞形态学变化;流式细胞术（FCM）PI染色法检测细胞凋亡率和细胞周期的分布;端粒重复序列扩增（TRAP）-银染法检测端粒酶活性变化。结果 RW对SW111C细胞有明显抑制作用,且呈现浓度和时间依赖性;经RW作用72 h后,细胞发生凋亡形态学变化,凋亡率随浓度的增高而增高,细胞周期阻滞于G1期,S期细胞减少;随着药物浓度升高,端粒酶活性逐渐减弱。结论 RW对体外培养的SW111C细胞有增殖抑制作用,并呈现浓度和时间依赖性,其机制可能与诱导SW111C细胞凋亡、改变其细胞周期分布、降低端粒酶活性有关。     

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。     

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.     

Anti-proliferation effect of zoledronic acid on human colon cancer line SW480

Objective: To investigate the anti-proliferation effect and mechanism of zoledronic acid(ZOL) on human colon cancer line SW480. Methods: SW480 cells were treated with 0, 12.5, 25, 50, 100 and 200 μmo L/L of ZOL for 48 h, and CCK-8 assay was employed to obtain the survival rate of SW480 cells. SW480 cells were treated with 25 μmo L/L of ZOL for 0, 12, 24, 48 and 72 h, and then the survival rate was obtained. SW480 cells of the ZOL group were treated with 25 μmo L/L of ZOL for 48 h, while cells of the Cs A+ZOL group were pretreated with 10 μmo L/L of Cs A for 0.5 h and then treated with 25 μmo L/L of ZOL for 48 h. Then the survival rates of SW480 cells of the control group, ZOL group and Cs A+ZOL group were determined. Flow cytometry was employed to detect the apoptosis rate and the mitochondrial transmembrane potential(△ψm) of the three groups and Western blot was used to detect the expressions of cyt C in the cytosol of the three groups. Results: ZOL inhibited the proliferation of SW480 cells, and the inhibition rate positively correlated with the concentration of ZOL and the action time(P 0.01). The cell survival rate and the △ψm of the ZOL group were greatly lower than those of the control group, while the apoptosis rate and the expression of cyt C in the cytosol were obviously higher than those of the control group. All the differences showed distinctly statistical significances(P 0.01). The cell survival rate and the △ψm of the Cs A+ZOL group were all lower than those of the control group, but substantially higher than those of the ZOL group; while the apoptosis rate and the expression of cyt C in the cytosol were higher than those of the control group, but distinctly lower than those of the ZOL group. All the differences were statistically significant(P 0.01). Conclusions: ZOL can induce the apoptosis in human colon cancer line SW480 and then inhibit the proliferation of SW480 cells directly by opening the mitochondrial permeability transition pore abnormally, decreasing △ψm, and releasing the cyt C into the cytosol. And the effect enhances with the increases of the concentration of ZOL and the action time.     

片仔癀对人结肠癌SW480细胞株的抑制作用研究

目的探究片仔癀对人结肠癌SW480细胞株的抑制效果。 方法通过对人结肠癌SW480细胞株常规体外培养,随机设定空白组、5-氟尿嘧啶（5-FU）组和不同浓度片仔癀组,分别采用对应药物干预24 h、48 h、72 h,并通过细胞增殖活力检测法（MTT法）和荧光显微镜观察其抑制效果。采用人结肠癌SW480细胞株建立结肠癌小鼠模型,把造模成功的150只小鼠随机分为模型组,5-FU组和片仔癀低、中、高剂量组,每组30只;分别以不同剂量药物外敷3 d,通过抑瘤率和原位凋亡检测（TUNEL法）解释抑制效果。 结果1 mg/mL、2 mg/mL、3 mg/mL浓度片仔癀对人结肠癌SW480细胞体外抑制率最高,为70.05%、71.39%、70.12%,与5-FU组比较差异具有统计学意义（χ²=4.49,4.97,4.52;均P<0.05）。4 mg/mL片仔癀抑制率为67.39%,与5-FU组比较差异无统计学意义（χ²=3.57,P>0.05）;荧光显微镜显示5-FU组与片仔癀各组人结肠癌SW480细胞株数量均显示不同程度的减少、体积变小、折光性差、细胞悬浮及脱落死亡。片仔癀高、中、低剂量组对人结肠癌SW480细胞的体内抑瘤率分别为51%、39%、23%,其中高、中剂量组与5-FU组比较差异无统计学意义（χ²=0.05,0.49;均P>0.05）,低剂量组与之比较差异有统计学意义（χ²=4.09,P<0.05）;荧光显微镜显示片仔癀各剂量组和5-FU组对人结肠癌SW480细胞均有不同程度的凋亡反应。 结论片仔癀外用对人结肠癌SW480细胞生长有明显的抑制作用,高、中剂量体内抑制作用与5-Fu相近,优于低剂量,临床可考虑采用高剂量可能取得更佳效果,体外抑制作用上显示则优于5-Fu,但剂量差异影响不大。     

PRL-3基因对结直肠癌细胞增殖能力的影响

目的:探讨PRL-3的过表达或敲低对结直肠癌细胞增殖能力的影响.方法:利用MTT法、平板克隆形成实验检测PRL-3对细胞体外增殖的影响;应用流式细胞术检测PRL-3对细胞周期的影响.结果:应用MTT法,检测PRL-3对SW480/EGFP、SW480-EGFP-PRL-3、SW480/EGFP/Mock及SW480-PRL-3-KD1细胞体外增殖能力的影响,经析因方差分析,4组差异具有显著性(F=23.463,P=0.000);不同时间点对细胞体外增殖的影响差异具有显著性(F=71.515,P=0.000);各组细胞与各时间组两因素交互效应显著(F=2.128,P=0.008);除第1天外,其他各时间点细胞组间的细胞增殖差异具有显著性.经LSD法多重比较,结果表明,与SW480/EGFP/Mock和SW480/EGFP细胞相比,SW480-EGFP-PRL-3细胞的增殖速度加快,而SW480-PRL-3-KD1细胞的增殖速度减慢.平板克隆形成实验显示SW480-EGFP-PRL-3细胞克隆形成能力明显增强,而SW480-PRL-3-KD1细胞克隆形成能力显著下降,差异具有显著的统计学意义(F=44.411,P=0.000).结论:PRL-3基因可促进结直肠癌细胞的增殖.     

Chitooligosaccharides promote radiosensitivity in colon cancer line SW480

AIM: To investigate the anti-proliferation and radiosensitization effect of chitooligosaccharides(COS) on human colon cancer cell line SW480.METHODS: SW480 cells were treated with 0, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/m L of COS for 48 h. CCK-8 assay was employed to obtain the cell survival ratio of SW480 cells, and the anti-proliferation curve was observed with the inhibition ratio of COS on SW480 cells. The RAY + COS group was treated with 1.0 mg/m L of COS for 48 h, while both the RAY and RAY+COS groups were exposed to X-ray at 0, 1, 2, 4, 6 and 8 Gy, respectively. Clonogenic assay was used to analyze cell viability in the two groups at 10 d after treatment, and a cell survival curve was used to analyze the sensitization ratio of COS. The RAY group was exposed to X-ray at 6 Gy, while the RAY+COS group was treated with 1.0 mg/m L of COS for 48 h in advance and exposed to X-ray at 6 Gy. Flow cytometry was employed to detect cell cycle and apoptosis rate in the non-treatment group, as well as in the RAY and RAY + COS groups after 24 h of treatment.RESULTS: COS inhibited the proliferation of SW480 cells, and the inhibition rate positively correlated with the concentration of COS(P 0.01). Cell viability decreased as radiation dose increased in the RAY and RAY+COS groups(P 0.01). Cell viabilities in the RAY+COS group were lower than in the RAY group at all doses of X-ray exposure(P 0.01), and the sensitization ratio of COS on SW480 cells was 1.39. Compared with the non-treatment group, there was a significant increase in apoptosis rate in both the RAYand RAY + COS groups; while the apoptosis rate in the RAY+COS group was significantly higher than in the RAY group(P 0.01). In comparing these three groups, the percentage of G2/M phase in both the RAY and RAY + COS groups significantly increased, and the percentage of the S phase and G0/G1 phase was downregulated. Furthermore, the percentage in the G2/M phase was higher, and the percentage in the S phase and G0/G1 phase was lower in the RAY + COS group vs RAY group(P 0.01). CONCLUSION: COS can inhibit the proliferation of SW480 cells and enhance the radiosensitization of SW480 cells, inducing apoptosis and G2/M phase arrest.     

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.     

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.