TSA对甲状腺鳞癌细胞株Akt、p21、mTOR基因表达的影响

目的观察曲古抑菌素A（Trichostatin A,TSA）对甲状腺鳞癌SW579细胞株Akt、mTOR和p21基因表达的影响,探讨TSA抗甲状腺癌的作用机制。方法体外培养SW579细胞,用CCK-8法检测TSA对SW579的生长抑制作用,计算半数抑制浓度。实验分为对照组、TSA组、Wortmannin组、Wortmannin＋TSA组、Rapamycin组,用RT-PCR方法检测SW579细胞Akt、p21及mTOR的mRNA表达,分析各处理组Akt、p21及mTOR基因表达变化。结果 CCK-8结果显示,TSA可明显抑制SW579的增殖,并呈剂量依赖性,半数抑制浓度约为147nmol/L。RT-PCR检测结果表明,与对照组相比,TSA组、Wortmannin组和Wortmannin＋TSA组Akt、mTOR mRNA表达水平明显降低,TSA组p21表达显著上调,Wortmannin组p21表达明显降低,各组mTOR表达均明显降低。结论 TSA在一定浓度范围内对甲状腺鳞癌细胞株SW579有剂量依赖性的增殖抑制作用,其机制可能与TSA降低SW579细胞Akt、mTOR基因的表达,进而再上调p21的表达有关。     

曲古抑菌素A抑制乳腺癌细胞MCF-7生长增殖及促进凋亡的机制

目的 探讨曲古抑菌素A(TSA)对乳腺癌细胞系MCF-7细胞生长增殖、凋亡及p21、p53表达的影响.方法 分别以不同浓度的TSA处理MCF-7细胞,采用MTT比色法测定细胞增殖活性;用流式细胞仪检测细胞凋亡率;用RT-PCR方法检测细胞p21、p53mRNA的表达水平;用Western印迹法检测MCF-7细胞的p21、p53蛋白表达.结果 TSA作用后,各组细胞均出现显著的生长抑制作用,存活率明显降低,并呈剂量依赖性.流式细胞仪分析,在100 ～ 400 nmol/L范围内凋亡率随TSA浓度的升高而升高(P＜0.01).经不同浓度TSA处理的细胞p21mRNA和蛋白表达水平明显上调(P＜0.01);、而p53mRNA和蛋白表达水平则没有明显变化.结论 TSA显著抑制MCF-7细胞生长,促进凋亡可能与TSA维持p53的稳定表达,从而促进其下游因子p21的表达有关.     

维甲酸抑制甲状腺鳞癌细胞株生长增殖作用机制的探讨

目的观察9-顺维甲酸（9-cis-RA）对甲状腺鳞癌SW579细胞株生长增殖及维甲酸受体B（RARβ）和p21表达的影响，进而探讨维甲酸的抗癌作用机制。方法将甲状腺鳞癌细胞株SW579分为对照组和实验组。实验组加入不同浓度的9-cis-RA，使其终浓度分别为1×10^-7mol／L、5×10^-7mol／L、1×10^-6mol／L、5×10^-6mol／L、1×10^-5mol／L，各组细胞均在培养24h后加药，继续培养48h。在倒置显微镜下观察细胞生长情况及形态变化；采用MTT比色法测定细胞增殖活性；流式细胞仪检测细胞周期；用RT-PCR方法检测SW579细胞RARβ及p21mRNA的表达。结果低浓度9-cis-RA组（1×10^-7mol／L、5×10^-7mol／L、1×10^-6mol／L）肿瘤细胞有向良性细胞形态转化的趋势；高浓度组（5×10^-6mol／L、1×10^-5mol／L）细胞生长密度较对照组明显降低，与低浓度组相比，向成熟分化的趋势不明显。MTT结果显示，实验组细胞均出现显著的生长抑制作用，且呈剂量依赖性。流式细胞仪分析，实验组随着9-cis-RA的升高，S期细胞在细胞周期中所占比例逐渐减少，G1期细胞则明显增加（P〈0．01），细胞滞留在G1期。RT-PCR检测结果表明，经不同浓度维甲酸处理的各组细胞RARβ和p21mRNA表达水平均显著上调。结论9-顺维甲酸在一定浓度范围内对甲状腺鳞癌细胞株SW579有剂量依赖性的增殖抑制作用，其机制可能与提高SW579细胞RARβ基因的表达，进而再上调p21的表达有关。     

野生型p53基因对肠癌细胞增殖及凋亡的影响

目的研究人野生型p53基因对肠癌细胞系SW480增殖及凋亡的影响。方法阳离子脂质体介导转染人野生型p53基因至SW480细胞系,利用MTT检测该基因对SW480细胞增殖的影响;利用Western blot检测CyclinD1及Bcl-2、Bax蛋白表达水平。结果与对照组比较,转染人野生型p53基因后的SW480细胞的增殖能力受到抑制;Cy-clinD1蛋白水平下调,Bcl-2、Bax表达呈负相关趋势。结论野生型p53基因在一定程度上可诱导肿瘤细胞凋亡。     

四溴苯三唑对甲状腺鳞癌SW579细胞凋亡的影响

目的观察四溴苯三唑(TBB)对甲状腺鳞癌SW579细胞株生长增殖和凋亡的影响。方法将甲状腺鳞癌细胞株SW579分为对照组和实验组。实验组细胞均在培养24 h后加药,加入不同浓度的TBB,使其终浓度分别为12.5、25、50、75、100μmol/L。培养到一定时间后,在倒置显微镜下观察细胞生长情况及形态变化;四甲基偶氮唑蓝(MTT)法检测培养细胞的增殖情况;流式细胞术检测细胞凋亡率;RT-PCR方法检测细胞凋亡相关基因Caspase-3 mRNA表达。结果低浓度TBB组(12.5、25、50μmol/L)肿瘤细胞有向良性细胞形态转化的趋势;高浓度组(75、100μmol/L)细胞生长密度较对照组明显降低,与低浓度组相比,向成熟分化的趋势不明显。MTT结果显示,实验组细胞均出现显著的生长抑制作用,且呈剂量依赖性。实验组随着TBB药物浓度的升高,S期细胞在细胞周期中所占比例逐渐减少,G1期细胞则明显增加,细胞滞留在G1期。RT-PCR方法检测发现实验组细胞凋亡相关基因Caspase-3 mRNA表达水平上调。结论 TBB可抑制甲状腺鳞癌SW579细胞增殖,促进细胞凋亡,且呈剂量依赖性;其机制可能与上调Caspase-3基因表达,激活Caspase途径有关。     

ATRA对甲状腺鳞癌细胞株SW579细胞CDK4、CyclinD1、Rb表达的影响

目的观察全反式维甲酸（ATRA）对甲状腺鳞癌细胞株SW579细胞CDK4、CyclinD1、Rb表达的影响,以探讨其抑癌机制。方法分别以终浓度为10-7、10-6、5×10-6、10-5mol/L的ATRA作用SW579细胞株,对照组加入5μl无水乙醇,各组细胞均在培养24 h后加药。继续培养48 h,RT-PCR、Western blot检测SW579细胞CDK4、CyclinD1、Rb mRNA及其蛋白的表达。结果 ATRA作用后,RT-PCR检测结果表明,经不同浓度ATRA处理的细胞CDK4、Rb mRNA表达水平没有明显变化;CyclinD1的表达明显下调;Western blot检测结果表明经不同浓度ATRA处理的细胞CDK4蛋白表达水平没有明显变化,pRb蛋白的磷酸化水平明显下降,CyclinD1蛋白表达水平明显下降。结论 ATRA在一定浓度范围内可能通过下调甲状腺鳞癌细胞株SW579细胞CyclinD1的表达及Rb蛋白的磷酸化水平抑制细胞增殖。     

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.     

抑制干扰素诱导蛋白p204表达促进大鼠血管外膜成纤维细胞生长与迁移

目的观察干扰素诱导蛋白p204表达抑制后对大鼠血管外膜成纤维细胞(VAF)凋亡、增殖与迁移的影响及其部分机制。方法应用Ifi204小干扰RNA(si RNA)转染VAF使Ifi204基因沉默(Ifi204-si RNA组),以非特异性si RNA转染作为其转染阴性对照组(Con-si RNA组)和未经处理的VAF作为未干预阴性对照组(Neg组)。用MTT法检测VAF细胞增殖,流式细胞仪检测细胞凋亡,细胞划痕法和Transwell法测定细胞迁移情况,应用实时荧光q RT-PCR和Western blot分别检测p204、p53及p21的m RNA和蛋白表达。结果与Con-si RNA组和(或)Neg组相比,Ifi204-si RNA组的p204、p53及p21的m RNA和蛋白表达减少(P0.05),细胞凋亡率下降(P0.05),细胞增殖及迁移速度提高(P0.05)。结论抑制p204表达可减少大鼠VAF细胞凋亡,促进其增殖与迁移,其机制可能部分与抑制p53及p21表达有关。     

miR-485-5p通过靶基因FLOT2调控甲状腺癌细胞增殖、凋亡的分子机制

目的探讨微小RNA-485-5p(miR-485-5p)靶向脂筏标记蛋白(FLOT)2对甲状腺癌细胞增殖、凋亡的影响及其作用机制。方法采用qRT-PCR与Western印迹分别检测不同甲状腺癌细胞及正常甲状腺上皮细胞中miR-485-5p与FLOT2 mRNA及蛋白表达。四甲基偶氮唑蓝(MTT)法检测甲状腺癌SW579细胞转染miR-485-5p mimic、miR-NC、si-FLOT2、si-NC后的细胞增殖活性,并采用流式细胞术检测细胞凋亡率。采用双荧光素酶活性检测鉴定miR-485-5p与FLOT2的靶向关系。Western印迹检测SW579细胞中细胞周期蛋白(Cyclin)D1、p21、p27、B淋巴细胞瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶(cleaved-caspase)-3蛋白表达。结果与TEC细胞相比,甲状腺癌细胞SW579、FTC-133、KAT-18中miR-485-5p的相对表达量显著降低(P0.05),而FLOT2 mRNA及蛋白表达显著升高(P0.05);miR-485-5p过表达或抑制FLOT2表达后SW579细胞OD值、CyclinD1及Bcl-2蛋白表达均显著降低(P0.05),细胞凋亡率、p21、p27及Bax、cleaved-caspase-3蛋白表达均显著升高(P0.05);双荧光素酶活性检测鉴定FLOT2是miR-485-5p的靶基因,miR-485-5p可负向调控FLOT2表达;FLOT2过表达可逆转miR-485-5p过表达对甲状腺癌SW579细胞增殖及凋亡的作用。结论 miR-485-5p通过负向调控靶基因FLOT2抑制甲状腺癌细胞增殖并诱导细胞凋亡。     

他汀药物对p53突变肿瘤细胞的作用及机制

目的研究他汀药物对p53突变的肿瘤细胞的作用及分子机制。方法采用具有突变p53背景的人结肠癌HT29细胞,通过MTT实验检测他汀药物对细胞增殖的影响,用Western印迹检测他汀药物浓度梯度和时间梯度处理后细胞内突变p53,分子伴侣热休克蛋白(HSP)90、p53信号通路下游靶蛋白PUMA、p21、凋亡相关蛋白PARP的表达情况。结果 MTT实验结果显示,他汀药物会显著抑制HT29细胞的增殖,美伐他汀IC50为(39.95±3.81)μmol/L,辛伐他汀IC50为(24.99±0.70)μmol/L。Western印迹结果显示,随着浓度和时间梯度增加,突变p53和HSP90表达水平没有改变,但是,p53下游靶蛋白PUMA、p21的表达水平升高,PARP蛋白切割增强。结论他汀药物能够抑制p53突变的肿瘤细胞的增殖,其机制可能是将突变型p53恢复为野生型p53从而发挥其转录激活下游靶基因的功能。     

Effect of indomethacin on cell cycle proteins in colon cancer cell lines

AIM: To study the effect of indomethacin (IN) on human colon cancer cell line SW480 with p53 mutant and SW480 transfected wild-type p53 (wtp53/SW480) in vitro and investigate molecular mechanism of anti-tumor effect of IN on colon cancer. METHODS: SW480 cells and wtp53/SW480 cells were treated with different concentrations of IN respectively, the expressions of CDK2, CDK4 and p21WAF1/CIP1 protein were detected by Western blotting. RESULTS: IN gradually down-regulated the expression of CDK2, CDK4 protein of wtp53/SW480 cells in a dose-dependent manner, and inhibitory effect reached the maximum level at 600 μmol/L; IN up-regulated the expression of p21WAF1/CIP1 protein in a dose-dependent manner at a certain concentration range, and the expression reached the maximum level at 400 μmol/L, and returned to the base level at 600 μmol/L. The expression of CDK2, CDK4 and P21WAF1/CIP1 protein of SW480 cells did not change. CONCLUSION: IN exerts antitumor effect partly through down regulation of the expression of CDK2, CDK4, protein and up regulation of the expression of p21WAF1/PIC1.     

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

ObjectiveTo investigate the effects of miR-25-3p on the occurrence, development and proliferation of tongue squamous cell carcinoma cells.MethodsTo establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant retroviral vector-mediated gene transfer method. The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assays. cyclinD1, p21^cip1 and p27^kip1 mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR. cyclinD1, p21, p27^kip1, AKT, p-AKT, FOXO1 and p-FOXO1 expressions in the transfected Tca8113 were detected by western blot analysis. In addition, miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.ResultsQuantitative PCR showed that miR-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue. MTT and cell colony formation assays showed that after miR-25-3p overexpression, the proliferation of transfected Tca8113 was obviously attenuated. Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression, p21^cip1 and p27^kip1 expressions were upregulated, while cyclinD1, AKT, FOXO1 expressions were downregulated, and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.ConclusionsMiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression, playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.     

Expressions of p53, VEGF C,p21: could they be used in preoperative evaluation of lymph node metastasis of esophageal squamous cell carcinoma?

The prognosis of esophageal squamous cell carcinoma is primarily determined by staging. Although radiological methods have revealed lymph node metastasis preoperatively, these radiological findings cannot be correlated with pathological staging. The aim of this study was to compare the expressions of p53, vascular endothelial growth factor C (VEGF C) and p21 with lymph node metastasis in preoperative endoscopic biopsy and postoperative resection material. Tissue samples were taken from 40 patients who had undergone endoscopic biopsies and radical esophagectomies. The expressions of p53, VEGF C and p21 proteins in these sections were immunohistochemically examined. The expression of each antibody was characterized as a negative or positive reaction according to the pattern and intensity of semiquantitative immunostaining. The staining pattern of antibodies was divided into three groups: < 10% cancer cells were accepted to be (-), 10-50% were (+), heterogenous and > 50% were (+ +), homogenous. For each antibody, statistical correlation with conventional prognostic parameters such as localization, microscopic grade, stage, pathological lymph node metastasis and survival, were investigated. p53 expression was observed in 65.5% (19/29) of lymph node positive cases, whereas p53 was in 50% (20/40) of cases. VEGF C was in 65% (26/40) and p21 was in 15% (6/40) of cases. p53 has the specificity of 90.9% and sensitivity rate of 65.5% in detecting lymph node metastasis and positive predictive value was 95%. Expression of p53 was significantly correlated with stage and lymph node metastasis (P = 0.02 and P = 0.03, respectively). Prediction of lymph node metastasis by p53 was correlated independently and in coexpression with VEGF C (P < 0.01). There was no relation detected between p21 and other parameters. In esophageal squamous cell carcinoma (SCC), p53 and VEGF C expressions were correlated with pathologically positive lymph nodes. When preoperative staging has been insufficient in esophageal carcinoma cases, immunohistochemical analysis of p53 and VEGF C staining in tissues could be an aid to clinicians regarding lymph node metastasis.     

The clinicopathological significance of p21 and p53 expression in esophageal squamous cell carcinoma: an analysis of 153 patients

OBJECTIVE: The p21 gene is thought to play a central role in tumor suppression. The aim of this study was to examine the clinicopathological role of p21 and p53 in esophageal squamous cell carcinomas. METHODS: The expression of p21 and p53 proteins in 153 Chinese patients (131 men, 22 women) with resected esophageal squamous cell carcinomas was investigated by the immunohistochemical method. Correlation between p21 and p53 expression and clinicopathological features was examined. RESULTS: The expression of p21 and p53 was detected in 70% and 64% of the tumors, respectively. The staining of p21 and p53 was also found in squamous carcinoma in situ, dysplasia, and nontumor epithelium. p21 expression was often weak in the suprabasal cells and found in better differentiated tumors. There was no significant correlation between the expression of p21 and the abnormal accumulation of p53. The prognosis of the patients depended on the size, stage, and p21 expression of the lesion. In stage III lesions with tumor diameter < or = 7.5 cm (n = 93), patients with loss of p21 expression had better survival. The survival rates of patients were worse if they had expression of both p21 and p53. CONCLUSIONS: Thus, p21 and p53 had prognostic value for esophageal squamous cell carcinomas. Loss of p21 expression was shown without p53 alternations, indicating that other mechanisms are also involved in turning off the gene. The pattern of p21 and p53 expression predicts an aggressive clinical course of esophageal squamous cell carcinomas.     

Changes in p53 and Waf1p21 expression and cell proliferation in esophageal carcinogenesis

AIM: To study the correlation between changes in p53 and Waf1p21 expression and cell proliferation, determined by proliferating cell nuclear antigen (PCNA), at different stages of human esophageal carcinogenesis.METHODS: Biopsied and resected esophageal tissues from a high risk population of esophageal cancer in northern China were used in this study. All specimens were fixed in 85% alcohol and processed for routine histology. The avidin biotin peroxidase complex (ABC) method was used to detect p53, Waf1p21 and PCNA.RESULTS: Strong nuclear staining of p53, Waf1p21 and PCNA was observed in normal esophageal epithelium and epithelia with different lesion severities. As the lesions progressed to dysplasia (DYS) and to esophageal squamous cell carcinoma (SCC), the Waf1p21 immunoreactivity percentage decreased. The number of Waf1p21-positive cells slightly increased from normal to basal cell hyperplasia (BCH), but did not further increase in DYS and SCC. The total number of Waf1p21-positive cells was lower than the number of p53-positive cells in normal and BCH esophageal epithelia and much lower in DYS and SCC. Waf1p21-positive cells were located in the third and fourth cell layers in half of the samples examined, which was 2-4 cell layers higher than the cells expressing PCNA and p53 in the same histological categories of normal, BCH and DYS.CONCLUSION: Low Waf1p21 levels at the DYS stage may be related to a functional loss of p53. Other mechanisms may also be responsible for the decreased Waf1p21 expression in DYS and SCC.     

曲古菌素A对人胃癌细胞增殖和细胞周期的作用

目的 观察曲古菌素A(TSA)对人胃癌细胞株SGC-7901细胞增殖及细胞周期的影响,探讨其可能的机制.方法 TSA干预人胃癌SGC-7901细胞24 h后.采用四甲基偶氮唑盐法检测其对细胞增殖的影响,流式细胞技术检测细胞周期,实时PCR检测细胞周期素D1和p21 mRNA的表达情况.结果 经TSA干预24 h后,人胃癌SGC-7901细胞增殖受抑制,TSA 0.1、0.5和2.0μmol/L组抑制率分别为3.52%±6.11%、13.29%±4.13%和14.24%±2.80%;同时TSA 0.5μmol/L组(71.26%±0.51%)和TSA 2.0μmol/L组(71.03%±0.12%)的G0/Gl期细胞比例明显高于对照组(51.12%±1.17%);TSA 0.5μmol/L组(13.55%±0.44%)和TSA 2.0 μmol/L组(10.63%±0.63%)的S期细胞比例明显低于对照组(34.60%±0.60%).出现G0/G1细胞周期阻滞.TSA干预后细胞周期相关基因细胞周期素D1 mRNA表达下调和p21 mRNA表达上调.结论 TSA通过调控细胞周期相关基因细胞周期素D1和p21的表达,抑制人胃癌SGC-7901细胞的增殖,引起G0/G1期细胞周期阻滞,最终影响肿瘤细胞的生长.