反义p73基因诱导人肝癌细胞HepG2的凋亡

目的：构建表达重组反义p73基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并进一步探讨其作用机制。方法：克隆p73基因的反义片段,重组法构成逆转录病毒载体pBabe-p73（pBP73）,以脂质体Lipofectamine2000将其转染293A细胞进行病毒包装;将逆转录病毒感染人肝癌细胞HepG2,用MTT法检测细胞生长抑制情况,Western blotting检测p73的表达;再分别用琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;最后检测p53,caspase-3和bcl-2蛋白的表达变化。结果  重组质粒pBP73经鉴定连接正确,其转染293A细胞后上清液中可得到病毒,滴度达5×107pfu;MTT检测见pBP73病毒组48和72h细胞抑制率高于对照组（45.1% vs. 5.3%,69.5% vs.17.3%,均p<0.05）。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达20.47%;p73蛋白高表达组p53和caspase-3蛋白的表达亦有显著升高（p<0.05）,但bcl-2蛋白未见表达差异。结论：成功构建了p73逆转录病毒,反义p73基因在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能机制是通过激活caspase-3而发生作用。     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

RTN4对于结肠癌细胞增殖的调控作用

2018年全球癌症统计调查显示,结直肠癌约占患癌新病例的12.1%。因此,寻找新的结肠癌发生有关的基因,发现新的治疗靶点显得尤为迫切。通过数据库分析发现,RTN4基因的表达水平与结肠癌患者生存率的相关性具有统计学意义。针对RTN4基因构建其干扰质粒,将慢病毒作为载体转染结肠癌HCT116细胞中构建敲低RTN4的结肠癌细胞系,最后检测了低表达后RTN4基因的细胞增殖。结果发现,敲低RTN4基因后显著促进了结肠癌细胞HCT116的增殖,研究通过Western blot观察敲低RTN4后HCT116细胞自噬通路相关蛋白p62和LC3的表达情况,发现与对照组相比较,敲低RTN4组LC3转化量（LC3-II/LC3-I）增多,而p62蛋白减少。研究分析了RTN4的潜在抑癌作用,发现敲低RTN4基因会显著增强结肠癌细胞的增殖能力,并且诱导自噬,说明RTN4可能与激活LC3/p62自噬途径有关。     

Effect of Ducrosia flabellifolia and Savignya parviflora Extracts on Inhibition of Human Colon and Prostate Cancer Cell Lines

The goal of this study was to investigate whether Ducrosia flabellifolia and Savignya parviflora methanol extract the have effect on colon and prostate cancer cell lines. Analysis of total content of phenolics and flavonoids of each plant extract was carried out. Cytotoxic effect, cell cycle analysis, induction of apoptosis and gene expression of Bcl-2 and Bax genes were studied. Obtained results indicated that, the plant extracts exhibit growth inhibition of used cancer cell lines and induced apoptosis as well as arresting of cell cycle. At the molecular level, changes in gene expression were detected via qPCR and confirmed by western blotting. The exhibited anticancer potentialities of plant extracts against utilized cancer cell lines are due to its containing bioactive compounds. Further detailed isolation, fractionation and characterization of bioactive compounds are needed.     

结肠炎相关结肠癌分泌蛋白质体外富集方法的建立

目的:建立小鼠结肠炎相关结肠癌分泌蛋白质体外富集的方法;方法:利用结肠炎相关结肠癌小鼠模型,取不同阶段实验小鼠的结肠体外培养48h,收集分泌蛋白质。使用SDS-PAGE分离分析分泌蛋白质,并通过Western blot检测分泌蛋白质是否存在胞浆蛋白或者核蛋白的污染。结果:在实验条件下收集得到的分泌蛋白重现性好,分泌蛋白中核蛋白和胞浆蛋白的污染较少,且能够明显分辨与病程相关差异表达蛋白质条带。结论:成功建立了结肠炎相关结肠癌分泌蛋白质的体外富集方法。     

Increased Expression and Aberrant Localization of Mucin 13 in Metastatic Colon Cancer

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.     

抑瘤基因NGX6对人结肠癌细胞凋亡的影响

NGX6基因是中南大学肿瘤研究所分子遗传室在对鼻咽癌研究中筛选克隆出的一个新基因．以稳定转染并表达NGX6基因的HT-29细胞为实验组,转染空白质粒的HT－29细胞以及HT－29细胞为对照组,利用PI／Annexin—V双染流式细胞仪检测结肠癌细胞的凋亡情况,通过EMSA分析体外培养的结肠癌细胞及种植瘤细胞内核转录因子－κB（NF－κB）的激活情况．研究结果表明：在裸鼠结肠癌种植瘤中,转染了NGX6基因的与未转染及空白载体组比较,结肠癌细胞凋亡率明显增高;EMSA（electrophoretic mobility shif tassay）分析体外培养的结肠癌细胞及种植瘤细胞内核转录因子－κB（NF—κB）的激活情况,发现转染了NGX6基因的细胞NF—κB的激活均明显受到抑制．此研究说明,NGX6基因只有诱导结肠癌细胞凋亡的能力,其可能的机制是抑制了结肠癌细胞NF—κB的激活．     

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids

1. Download : Download high-res image (191KB)
2. Download : Download full-size image

     

抑制Hedgehog信号通路改变结肠癌细胞基因表达谱

Hedgehog（Hh）信号通路在机体发育和肿瘤发生中发挥着重要作用。在该研究中,Western blot检测三株结肠癌细胞Hedgehog信号通路组分的表达,结果表明三株结肠癌细胞中HT-29细胞Hedgehog信号通路组分较完整。采用MTT和BrdU法检测Hedgehog信号通路膜受体Smo特异性抑制剂环杷明和末端转录因子Gli1/2的特异性抑制剂GANT61对HT-29细胞的影响,提示这两种抑制剂均显著抑制HT-29细胞生存率和细胞增殖率,且GANT61比环杷明更敏感。表达谱芯片检测阻断Hedgehog信号通路后HT-29细胞基因谱的变化,结合生物信息学分析,揭示HT-29细胞经环杷明和GANT61处理后基因表达呈现抑制特征,其差异基因表达主要以下调为主,其中环杷明主要影响细胞内源刺激等,而GANT61主要影响代谢和类固醇合成,并与MAPK信号通路有关联,两者均能影响细胞免疫及凋亡相关通路。这些结果提示,Hh信号通路有可能作为结肠癌的治疗靶点。     

Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In Vitro Metastasis

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells'' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.     

Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

胰岛素样生长因子1（IGF-I）通过PI-3K/Akt途径对结肠癌细胞凋亡的影响

目的：探讨胰岛素样生长因子-1（IGF-I）通过磷酯酰肌醇3-激酶/蛋白激酶B（PI-3K/Akt）信号通路对结肠癌细胞株SW480凋亡率的影响及其凋亡抑制蛋白survivin表达水平的变化。方法：培养结肠癌SW480细胞株,实验分成三组：未加IGF-I空白组、IGF-I刺激组、IGF-I＋LY294002阻断组,检测阻断剂LY294002是否阻断PI-3K/Akt通路（Western Blot检测三组P-Akt表达情况）;Western Blot及免疫荧光观察三组survivin蛋白表达变化;MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果：Western blot结果显示LY294002可抑制IGF-I诱导的p-Akt的表达（P〈0.05）;阻断IGF-I诱导的PI-3K/Akt通路后MTT显示结肠癌细胞SW480增殖抑制率升高（P〈0.05）,流式细胞术分析显示凋亡率明显上升（P〈0.05）;Western blot及免疫荧光结果显示LY294002可抑制IGF-I诱导的survivin的表达（P〈0.05）。结论：IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

Antiproliferative Activity of Kinetin Riboside on HCT-15 Colon Cancer Cell Line

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity both in vitro and in vivo. N⁶-furfuryladenosine (kinetin riboside, KR) displays antiproliferative and apoptogenic activity against various human cancer cell lines and has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, we demonstrate that KR is able to inhibit the proliferation in HCT-15 human colon cancer cells in a dose-dependent manner with a concentration of 2.5 μM, which causes 50% inhibition of cell viability. The cell cycle analysis by flow cytometry showed that KR arrested cell cycle progression in the S Phase by blocking through G₂/M and G₀/G₁ phase in HCT-15 colon cells. Moreover, suppression of clonogenic activity occurs after exposure to KR at a concentration of 2.5 μM for HCT-15.     

RNA干扰敲减PIWIL1表达对人结肠癌细胞SW620增殖，粘附及侵袭能力影响

PIWIL1为AGO蛋白(Argonaute proteins )PIWI亚家族的成员之一,在睾丸中特异表达. 其在干细胞自我更新、RNA干扰和翻译调节中起着重要的作用.本文采用实时PCR方法检测PIWI基因家族中PIWIL1、PIWIL2、PIWIL3、PIWIL4在人结肠癌SW620细胞中mRNA表达水平,首次证实了在SW620细胞中,PIWIL1相对PIWIL2、PIWIL3、PIWIL4,其表达水平最高（P﹤0.05）.构建表达针对PIWIL1的小发卡结构干扰RNA的重组质粒,并用Western 印迹验证其达到较高的干扰效率.用脂质体将重组质粒转染入SW620细胞中,通过MTT实验、集落形成实验、细胞聚集实验及细胞侵袭转移实验,分别观察到其可抑制SW620细胞的生长、增殖、侵袭转移能力,增强了SW620细胞的粘附能力.提示PIWIL1可能在结肠癌发生、发展过程中发挥作用.     

左右半结肠癌多组学分子特征差异的研究进展

结肠癌(colon cancer,CC)是一种常见的恶性肿瘤,其发病率和死亡率均占癌症前列.根据解剖学位置,CC可分为左半结肠癌(left-sided colon cancer,LCC)和右半结肠癌(right-sided colon cancer,RCC),两者在临床特征上表现出较大的差异.近些年来,随着生物学技术和...     

Irradiation Decreases the Neuroendocrine Biomarker Pro-Opiomelanocortin in Small Cell Lung Cancer Cells In Vitro and In Vivo

Background

Small cell lung cancer (SCLC) is an extremely aggressive disease, commonly displaying therapy-resistant relapse. We have previously identified neuroendocrine and epithelial phenotypes in SCLC tumours and the neuroendocrine marker, pro-opiomelanocortin (POMC), correlated with worse overall survival in patients. However, the effect of treatment on these phenotypes is not understood. The current study aimed to determine the effect of repeated irradiation treatment on SCLC cell phenotype, focussing on the neuroendocrine marker, POMC.

Results

Human SCLC cells (DMS 79) were established as subcutaneous xenograft tumours in CBA nude mice and then exposed to repeated 2Gy irradiation. In untreated animals, POMC in the blood closely mirrored tumour growth; an ideal characteristic for a circulating biomarker. Following repeated localised irradiation in vivo, circulating POMC decreased (p< 0.01), in parallel with a decrease in tumour size, but remained low even when the tumours re-established. The excised tumours displayed reduced and distinctly heterogeneous expression of POMC compared to untreated tumours. There was no difference in the epithelial marker, cytokeratin. However, there were significantly more N-cadherin positive cells in the irradiated tumours. To investigate the tumour response to irradiation, DMS79 cells were repeatedly irradiated in vitro and the surviving cells selected. POMC expression was reduced, while mesenchymal markers N-cadherin, β1-integrin, fibroblast-specific protein 1, β-catenin and Zeb1 expression were amplified in the more irradiation-primed cells. There were no consistent changes in epithelial marker expression. Cell morphology changed dramatically with repeatedly irradiated cells displaying a more elongated shape, suggesting a switch to a more mesenchymal phenotype.

Conclusions

In summary, POMC biomarker expression and secretion were reduced in SCLC tumours which regrew after irradiation and in repeatedly irradiation (irradiation-primed) cells. Therefore, POMC was no longer predictive of tumour burden. This highlights the importance of fully evaluating biomarkers during and after therapy to assess clinical utility. Furthermore, the gain in mesenchymal characteristics in irradiated cells could be indicative of a more invasive phenotype.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

P-糖蛋白对结肠癌多药耐药的影响

结肠癌是常见的消化道恶性肿瘤。对术后患者以及无法采用手术治疗的患者,临床多采用化疗、放疗等综合性治疗方法。随着大量化疗药物在临床的广泛使用,结肠癌多药耐药性成为化疗失败的最主要原因。研究表明,P-糖蛋白(P-glycoprotein, P-gp)作为ATP结合盒(ABC)转运蛋白超家族成员之一,与多种肿瘤的多药耐药相关,其介导的多药耐药已经成为目前研究的热点。本文旨在通过对P-糖蛋白的结构、耐药机制以及逆转P-糖蛋白介导的结肠癌多药耐药新发现进行阐述,引导读者对P-糖蛋白在结肠癌多药耐药中的作用有更深入的了解。     

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.