Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombined and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

CEA启动子控制HSV—TK基因表达对人结直肠癌细胞的杀伤作用

An expression plasmid pCEA-TK, in which HSV-TK gene was under the control of CEA promoter, was constructed. The human colorectal carcinoma cell line LoVo or the human uterine cervical cancer cell line HeLa was co-transfected with pSV2-neo and pCEATK, respectively. After G418 selection, both transgenic cell clones (LoVo/CEATK and HeLa/CEATK) were obtained. LoVo/CEATK cells were 1300 times more sensitive to the cytotoxicity of ganciclovir than LoVo cells. However, the elevation of GCV sensitivity induced by pCEATK gene in HeLa line was only 8 times. Injection of GCV resulted in significant regression of HSV-TK transfected LoVo tumor in nude mice. These data suggested that the expression of TK gene driven by CEA promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electromicroscopy and DNA fragmentation assay demonstrated that GCV could induce apoptosis in LoVo/CEATK cells. The possibility of the CEATK/GCV system in the treatment of human colorectal carcinoma was discussed.     

DLC-1基因表达对结肠癌细胞侵袭转移能力的影响

目的:研究DLC-1基因对结肠癌细胞侵袭迁移能力的影响.方法:将DLC-1 shRNA(短发夹状RNA,short hairpin RNA)序列克隆到质粒pGCsi-U6/Neo载体,采用脂质体介导的转染方法将构建的DLC-1 shRNA表达质粒转入结肠癌细胞系LoVo细胞.采用RT-PCR技术和Western Blot技术分别检测LoVo细胞中DLC-1mRNA和蛋白表达水平的变化.Transwell小室人工重组基底膜侵袭转移实验观察LoVo细胞侵袭迁移能力的改变.结果:结肠癌细胞系LoVo细胞表达DLC-1分子.所构建质粒表达载体能有效地干扰LoVo细胞DLC-1 mRNA和蛋白质表达水平;Transwell小室人工重组基底膜侵袭转移实验结果显示,转染后LoVo细胞侵袭转移能力明显增强(p＜0.05).结论:结肠癌细胞系LoVo细胞表达DLC-1基因,应用RNAi技术可特异性降低其表达.DLC-1的表达水平与结肠癌细胞侵袭转移相关.     

Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner

Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estradiol (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. These data strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms remain unexplored. LoVo cells were transient transfected to overexpress ER-beta, DNA fragmentation and caspase activity assay were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the TNF-alpha promoter activity. Our data clearly demonstrated that E2 and ER-beta alone could upregulate p21 and p27 proteins, which further activate caspase-8 and caspase-9 to induce apoptosis in LoVo cell, and the ER-beta. effects were enhanced by E2. However, overexpressed ER-beta did not influence the expression and promoter activity of TNF-alpha. In addition, E2 and overexpressed ER-beta downregulated the beta-catenin proteins which cause the downregulation of its target genes, cyclin D1 and Rb, to inhibit the cell cycle and cell proliferation. The results indicate that overexpressed ER-beta may induce LoVo cell apoptosis and anti-proliferation by increasing p53 signaling in a ligand-dependent manner, and without hTNF-alpha involvement. Efforts aiming at enhancing ER-beta expression and/or activity may prove to be an attractive alternative therapy against colorectal cancer.     

Faecalibacterium prausnitzii对大肠癌细胞增殖和凋亡的影响

目的:通过观察Faecalibacterium prausnitzii(F.prausnitzii)及其上清体外对大肠癌细胞增殖和凋亡的影响,探讨F.prausnitzii的抗肿瘤作用。方法:将大肠癌Lo Vo细胞培养于96孔板,待细胞贴壁后加入不同浓度的F.prausnitzii(l×107/m L、1×108/m L、1×109/m L)及其上清(1/2、1/5浓度),共同培养24 h或48 h后,以MTT法检测F.prausnitzii及其上清对Lo Vo细胞的增殖抑制作用;将大肠癌Lo Vo细胞培养于6孔板,待细胞贴壁后加入不同浓度的F.prausnitzii及其上清(同前),共同培养48 h后用Annexin V-FITC/PI双染色法对细胞染色后在流式细胞仪上检测F.prausnitzii及其上清对Lo Vo细胞凋亡的影响。结果:MTT结果显示,与对照组相比,不同浓度的F.prausnitzii及其上清对大肠癌Lo Vo细胞具有显著的增殖抑制作用(P0.05),且F.prausnitzii及其上清浓度越大、作用时间越长,其抑制作用越明显(P0.05),具有浓度和时间依赖性。流式细胞结果显示,与对照组相比,不同浓度的F.prausnitzii及其上清均具有诱导大肠癌Lo Vo细胞凋亡的作用,且浓度越大,诱导作用越明显(P0.05),具有浓度依赖性。结论:F.prausnitzii及其上清在体外具有抗肿瘤作用,其机制可能是通过诱导肿瘤细胞凋亡。     

紫草素促进caspase-9活化诱导大肠癌细胞凋亡的分子机制研究

目的探讨紫草素对大肠癌（CRC）细胞LoVo的抗肿瘤作用及其机制。 方法以不同紫草素浓度梯度（0、2、4、6 μmol/L）处理CRC细胞LoVo 24 h，以4 μmol/L紫草素处理不同时间梯度（0、12、24、48 h）的CRC细胞LoVo。流式细胞技术结合Annexin V-FITC/PI双染色测定细胞凋亡率，Western blot检测细胞中caspase-9蛋白的表达及切割情况。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果与0 μmol/L处理CRC细胞LoVo 24 h比较，2、4、6 μmol/L的细胞凋亡率[（6.94±1.02）﹪比（10.61±1.12）﹪、（15.55± 1.35）﹪、（36.51±1.46）﹪]均升高；与4 μmol/L紫草素处理0 h的CRC细胞LoVo比较，12、24、48 h的细胞凋亡率[（1.33±0.59）﹪比（19.23±1.24）﹪、（22.24±1.41）﹪、（28.41±1.52）﹪]均升高，差异具有统计学意义（P均< 0.001）。当紫草素剂量≥2 μmol/L，处理时间≥12 h时，caspase-9蛋白表达上调并被诱导活化，而caspase-9抑制剂（Z-LEHD-FMK）预处理后，LoVo细胞凋亡率下降38.7﹪，差异有统计学意义（P < 0.05）。 结论紫草素可以通过caspase-9蛋白的表达及其切割活性诱导CRC细胞凋亡。     

Peroxiredoxin 2 is upregulated in colorectal cancer and contributes to colorectal cancer cells’ survival by protecting cells from oxidative stress

Peroxiredoxin 2 (Prdx2) is a member of the peroxiredoxin family, which is responsible for neutralizing reactive oxygen species. Prdx2 has been found to be elevated in several human cancer cells and tissues, including colorectal cancer (CRC), and it influences diverse cellular processes involving cells’ survival, proliferation, and apoptosis, which suggests a possible role for Prdx2 in the maintenance of cancer cell. However, the mechanism by which Prdx2 modulates CRC cells’ survival is unknown. The current study aimed to determine the effect of elevated Prdx2 on CRC cells and to further understand the underlying mechanisms. The results of this study showed that Prdx2 was upregulated in CRC tissues compared with the matched noncancer colorectal mucosa tissues and that Prdx2 expression was positively associated with tumor metastasis and the TNM stage. In the LoVo CRC cell line, Prdx2 was upregulated at both the RNA and protein levels compared with the normal FHC colorectal mucosa cell line. In addition, the LoVo CRC cell line was significantly more resistant to hydrogen peroxide (H₂O₂)-induced apoptosis because of a failure to activate pro-apoptotic pathways in contrast to Prdx2 knockdown cells. Suppression of Prdx2 using a lentiviral vector-mediated Prdx2-specific shRNA in the LoVo cell line restored H₂O₂ sensitivity. Our results suggested that Prdx2 has an essential role in regulating oxidation-induced apoptosis in CRC cells. Prdx2 may have potential as a therapeutic target in CRC.     

组织专一性表达自杀基因治疗人结肠癌的研究

构建了以癌胚抗原(CEA)基因启动子控制的HSV-TK和ECCD的表达质粒PCEA-TK和pCEA-CD. 将它们分别与pSV2-neo共转染人结肠癌细胞株LoVo和人宫颈癌细胞株HeLa. G418筛选得到细胞克隆LoVo/CEA-TK、toVo/CEA-CD、HeLa/CEA-TK和HeLa/CEA-CD. 与野生型LoVo细胞相比, LoVo/CEA-TK和LoVo/CEA-CD形态无明显改变, 生长曲线也相似, 但对GCV或5-FC的细胞毒的敏感性分别提高了2000倍或700倍.而HeLa/CEA-TK(或HeLa/CEA-CD)仍对低浓度GCV(或5-FC)不敏感. 以上结果显示了应用组织专一性表达的自杀基因治疗人结肠癌的可能性.     

siRNA沉默VEGF基因对大肠癌细胞生物学特性的影响

目的研究VEGF和受体FLT-1、FLK-1在大肠癌组织中的表达及与临床病理因素之间的关系;观察SiRNA干扰VEGF基因对人大肠癌细胞系Caco-2生物学特性的影响。方法应用免疫组织化学S?P法,检测82例大肠癌及14例大肠正常黏膜组织中VEGF和FLT-1、FLK-1的表达;以脂质体lip-2000为载体,用针对VEGF特异靶点的siRNA转染入人大肠癌细胞系Caco-2后,免疫细胞化学S-P法、Western Blot检测VEGF蛋白表达变化,MTT法检测对细胞增殖的影响;流式细胞技术检测细胞凋亡。结果 (1)大肠癌组织VEGF和受体FLT-1、FLK-1阳性表达率显著高于正常大肠组织(P0.05)。VEGF的表达在有无淋巴结转移组和不同的Duke’s分期中亦有显著差异(P0.05)。(2)VEGF-siRNA转染Caco-2细胞后,免疫细胞化学结果显示转染VEGF-siRNA组阳性细胞呈弱阳性表达,明显减弱;Westernblot结果显示VEGF-siRNA组细胞的蛋白条带亮度明显降低;MTT检测VEGF-siRNA组细胞生长出现明显的抑制(P0.05),不同时段(24h、48h、72h)肿瘤细胞增殖抑制率之间有显著差异(P0.05);流式细胞术检测VEGF-siRNA组出现明显亚二倍体峰,凋亡率显著高于正常对照组(P0.01)。结论 VEGF在大肠癌的进展过程中起重要作用。siRNA干扰VEGF基因能有效抑制人大肠癌细胞系VEGF蛋白表达,细胞增殖能力减弱,促进细胞凋亡。以VEGF基因为靶点,利用siRNA技术进行基因治疗有可能成为一种新的有效手段。     

糖皮质激素抑制结肠癌细胞增殖和侵袭的作用机制

本研究目的是为了证实地塞米松对结肠癌LoVo细胞增殖的抑制作用,并阐明其中的分子机制。LoVo细胞经不同浓度梯度地塞米松干预,再加入TGF-β1受体抑制剂SB431542阻断TGF-β1信号传导途径,通过MTS分析各组细胞增殖情况,借助Hoechst 33342和Annexin V/PI染色法检测细胞凋亡率;结合Western blotting对TGF-β1、Smad2和caspase-3蛋白表达情况的检测结果,分析地塞米松诱导结肠癌LoVo细胞凋亡的作用机理。LoVo细胞在1.0 mmol/L和10.0 mmol/L地塞米松干预48 h后,细胞增殖率与对照组相比分别降低32%(p<0.01)和47%(p<0.001),2组细胞凋亡率分别为28%和36%(p<0.001)。Western blotting结果显示,与对照组相比,地塞米松以浓度依赖性方式显著上调LoVo细胞TGF-β1、Smad2和Cleavedcaspase-3蛋白水平(p<0.01),而TGF-β1受体抑制剂SB431542明显下调TGF-β1、Smad2和Cleaved-capase-3蛋白表达(p<0.05)。流式细胞术检测结果表明,SB431542+地塞米松干预组与地塞米松处理组LoVo细胞凋亡率分别为8%和23%(p<0.001)。地塞米松可显著诱导LoVo细胞凋亡,而SB431542能够挽救这一过程,这表明,地塞米松通过TGF-β1/Smad2通路诱导LoVo细胞凋亡。     

siRNA抑制Survivin基因对大肠癌细胞HCT116增殖及凋亡的的影响

目的探讨si RNA沉默Survivin基因后对人大肠癌细胞系HCT116的Survivin蛋白表达、细胞增殖及凋亡的影响。方法以脂质体lip-2000为载体,用针对Survivin特异靶点的si RNA转染人大肠癌细胞系HCT116后,应用免疫细胞化学S-P法、Western Blot检测Survivin蛋白表达变化;MTT法检测细胞增殖;流式细胞技术检测细胞凋亡。结果免疫细胞化学结果显示:HCT116的正常对照组、脂质体对照组及阴性错配对照组细胞浆均呈强阳性表达,Survivin-si R-NA组细胞浆Survivin呈弱阳性表达;Western blot结果显示:HCT116细胞系Survivin-si RNA组细胞的蛋白条带亮度均明显低于正常对照组、脂质体对照组和阴性错配对照组;MTT检测结果:与阴性错配对照组相比,Survivin-si RNA组细胞生长出现明显的抑制(P0.05),不同时段(24h、48h、72h)肿瘤细胞增殖抑制率之间有显著差异(P0.05)。流式细胞术检测Survivin-si RNA组细胞凋亡比例为9.72%,明显高于空白对照组及阴性错配对照组(P0.01)。结论si RNA抑制Survivin基因可以抑制大肠癌细胞增殖,促进凋亡;Survivin有望成为大肠癌基因治疗的新靶点。     

苦瓜MAP30蛋白的原核表达及其生物活性研究

以天然苦瓜基因组为模板PCR扩增去前导肽后成熟的MAP30蛋白基因,克隆至可诱导表达载体pET28a中。将含MAP30基因的表达载体pET28a-MAP30转化至E. coli Rostta（DE3）中并通过IPTG诱导表达。经聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白杂交（Western blot）以及液相色谱-质谱（LC-MS）对表达的重组MAP30蛋白进行鉴定,并通过镍柱亲和层析纯化。将pUC19质粒与不同浓度的纯化后的重组MAP30蛋白孵育,分析其切割DNA的活性。同时将纯化后的重组MAP30蛋白体外作用于人乳腺癌细胞（MCF-7）,采用MTT、AO/PI双染等方法进行抗肿瘤活性分析。实验结果表明纯化后的蛋白经质谱鉴定和Western blot分析,目的蛋白成功地与His-tag融合表达。首次发现大肠杆菌异源表达的重组MAP30蛋白同天然蛋白一样可以切割超螺旋DNA活性。MTT、AO/PI双染结果证实重组MAP30体外可诱导MCF-7细胞发生凋亡。通过基因工程技术大量制备MAP30蛋白,进一步研究其体外生物学活性,为以后的临床应用奠定基础。     

RNAi 介导的组织因子基因沉默抑制裸鼠移植性结肠癌的生长

组织因子(tissue factor,TF) 是机体外源性凝血途径的启动因子,发挥生理性止血的重要作用.近来研究表明,TF 除凝血功能外尚与多种恶性肿瘤的血管生成,侵袭转移及预后密切相关.为探讨 TF 对人结肠癌细胞（LoVo 细胞）生长能力的影响,构建带有 TFsiRNA 的重组腺病毒Ad pTF 和不带有 TFsiRNA 的对照病毒 Ad pDC,分别感染 LoVo 细胞,采用RT PCR 和 Western 免疫印迹法检测被感染 LoVo 细胞的 TF mRNA 和蛋白表达水平,并通过体内成瘤实验,进一步分析对 LoVo 细胞生长能力的影响.结果显示,与感染 Ad pDC 的 LoVo 细胞相比,感染 Ad pTF 的 LoVo 细胞的 TF mRNA 和蛋白表达水平更低,皮下种植瘤的体积更小,局部侵袭范围也更局限.研究表明,腺病毒介导的 RNAi 能特异而有效地沉默 LoVo 细胞中 TF 基因的表达,进而抑制 LoVo 细胞体内种植瘤的生长侵袭能力.     

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies.In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer.