DAL-1基因与肿瘤

人类DAL-1基因编码4.1B蛋白,是膜/细胞骨架相关蛋白4.1超家族成员之一,参与维持细胞正常形态和生理功能。在人类大多数肿瘤中DAL-1基因表达下调,研究表明其功能失活与该基因启动子区CpG岛甲基化密切相关。本综述将侧重DAL-1基因在肿瘤发生发展中的研究进展。     

非小细胞肺癌DAL-1基因SNP+1810T/C及其蛋白表达临床意义分析

目的:探讨非小细胞肺癌(NSCLC)中肺腺癌差异表达基因(DAL-1)单核苷酸多态性(SNP)+1810T/C(rs8082898)与蛋白表达的关系及其对肿瘤发生发展的影响。方法:收集204例NSCLC石蜡组织及其配对正常淋巴结组织,以及112例NSCLC新鲜血液标本,采用PCR-RFLP检测DAL-1基因+1810T/C位点基因型;其中167例NSCLC及其配对癌旁组织SP法检测DAL-1蛋白表达;分析+1810T/C基因型与DAL-1蛋白表达及临床病理特征的关系。结果:在NSCLC中,TT组+1810T/C等位基因频率为92.72%(293/316),CT组为6.33%(20/316),CC组为0.95%(3/316),多态CT+CC组为7.28%(23/316);DAL-1蛋白表达阳性率TT组(59.74%,92/154)与CT+CC组(38.46%,5/13)差异无统计学意义,χ2=0.000,P>0.05。+1810T/C多态在Ⅲ、Ⅳ期高于Ⅰ、Ⅱ期,χ2=11.003,P<0.05;淋巴结转移组高于无转移组,χ2=4.548,P<0.05。NSCLC组DAL-1蛋白表达阳性率(58.08%,97/167)低于对照组(90.42%,151/167),χ2=45.665,P<0.05;高中分化组(63.44%,59/93)高于低分化组(47.30%,35/74),χ2=4.365,P<0.05;Ⅰ期(72.31%,47/65)、Ⅱ期(54.29%,19/35)高于Ⅲ、Ⅳ期合组(46.27%,31/67),χ2=9.450,P<0.05;淋巴结转移组(37.08%,33/89)低于无转移组(82.05%,64/78),χ2=34.532,P<0.05。结论:DAL-1基因+1810T/C位点多态和蛋白表达降低可能在NSCLC的生长及淋巴结转移中发挥作用。     

DAL-1表达下调与大肠腺癌发生发展的关系

目的探讨DAL-1表达下调与大肠腺癌发生发展的关系。方法RT-PCR检测6株大癌细胞系和51例组织标本DAL-1 mRNA表达;BSP和MSP法分别检测大肠癌细胞系和组织标本DAL-1基因启动子甲基化状态;5-aza-dC作用实验验证大 肠癌细胞系中DAL-1启动子甲基化情况。 结果6株大肠癌细胞系DAL-1均有表达,各细胞系间表达无明显差异;部分大肠腺癌组织中DAL-1表达下调,且在淋巴结转移患者和Dukes C、D期患者更常见（P＜0.05）。DAL-1基因启动子高甲基化是少见现象。结论DAL-1表达下调在大肠腺癌发生发展中发挥重要作用,特别是在进展期大肠腺癌;但该基因启动子高甲基化不是下调其表达的主要机制。     

DAL-1对非小细胞肺癌细胞侵袭和迁移能力影响的初步研究

鉴定和测序鉴定序列及插入方向正确,其中pGPU6/GFP/Neo-DAL-1-T4表达载体在mRNA和蛋白质水平能有效抑制DAL-1的表达,抑制率分别为(87 41±1 994)%和(82 73±2 147)%.与对照组细胞比较,DAL-1稳定抑制的实验组细胞迁移和侵袭能力(P=0 000)和增殖能力显著增强(P=0 000).结论: 成功设计并构建了靶向DAL-1的shRNA表达载体,建立了DAL-1稳定抑制的NSCLC细胞株,为进一步研究DAL-1在NSCLC细胞中的作用提供了实验模型.     

RNAi沉默结肠癌细胞LOVO中livin基因的表达

目的：运用RNA干扰（RNA interference,RNAi）技术阻断结肠癌细胞系LOVO中livin基因的表达,并研究livin基因沉默后对LOVO细胞增殖和克隆形成产生的影响。方法：用真核转录载体pSilencerTM4.1-CMV neo构建针对livin基因的重组RNAi真核转录载体pSilencer4.1-L1和pSilencer4.1-L2,脂质体法转染结肠癌细胞系LOVO,通过RT-PCR、免疫印迹实验检测livin的表达变化,并用克隆形成实验、MTT法检测转染后LOVO细胞在细胞增殖、克隆形成等方面的变化。结果：重组载体pSilencer4.1-L1有效地阻断了LOVO细胞中livin基因在mRNA和蛋白水平上的表达（P〈0.01）。pSilencer4.1-L1转染LOVO细胞后,与对照组相比细胞生长速度明显变慢,其细胞数在72h时与对照组相比减少约30%;克隆形成率仅为15%,与对照组相比下降了约70%。结论：成功构建了可有效沉默livin基因的RNAi干涉载体,初步证明livin基因在结肠癌细胞的分化增殖等方面所起的重要作用,为进一步阐明livin基因与结肠癌的关系以及以livin基因为靶点的结肠癌基因治疗研究奠定了基础。     

RNAi沉默结肠癌细胞LOVO中livin基因的表达

目的:运用RNA干扰(RNA interference,RNAi)技术阻断结肠癌细胞系LOVO中livin基因的表达,并研究livin基因沉默后对LOVO细胞增殖和克隆形成产生的影响。方法:用真核转录载体pSilencerTM4.1-CMV neo构建针对livin基因的重组RNAi真核转录载体pSilencer4.1-L1和pSilencer4.1-L2,脂质体法转染结肠癌细胞系LOVO,通过RT-PCR、免疫印迹实验检测livin的表达变化,并用克隆形成实验、MTT法检测转染后LOVO细胞在细胞增殖、克隆形成等方面的变化。结果:重组载体pSilencer4.1-L1有效地阻断了LOVO细胞中livin基因在mRNA和蛋白水平上的表达(P<0.01)。pSilencer4.1-L1转染LOVO细胞后,与对照组相比细胞生长速度明显变慢,其细胞数在72h时与对照组相比减少约30%;克隆形成率仅为15%,与对照组相比下降了约70%。结论:成功构建了可有效沉默livin基因的RNAi干涉载体,初步证明livin基因在结肠癌细胞的分化增殖等方面所起的重要作用,为进一步阐明livin基因与结肠癌的关系以及以livin基因为靶点的结肠癌基因治疗研究奠定了基础。     

P53基因与结直肠癌

1979年,Lane和Crawford在猿猴病毒(SV_(40))感染的小鼠细胞中首先鉴定出能与SV_(40)T抗原相互作用的P53蛋白。编码该蛋白的基因称之为P53基因。研究表明,该基因结构的改变和表达异常与结直肠癌等51种人类肿瘤有关。 1 野生型P53基因和P53蛋白 P53基因定位于人类染色体17P13.1,     

蛋白4.1家族在不同乳腺癌细胞株中的表达与定位

背景与目的:目前,已在多种人类肿瘤中发现蛋白4.1家族成员表达异常.本研究旨在通过检测蛋白4.1家族成员(4.1R/B/G/N)在3株转移能力不同的人乳腺癌细胞株MCF-7、T-47D和MDA-MB-231中的表达和定位,探讨蛋白4.1家族成员与乳腺癌细胞转移能力之间的关系.方法:采用Western blot检测蛋白4.1家族成员在MCF-7、T-47D和MDA-MB-231细胞中的表达;免疫荧光标记对蛋白4.1家族成员在3株细胞中的表达进行定位.结果:4.1R/B/G在3种细胞系中均有表达,在T-47D细胞中的表达量均明显高于在MCF-7细胞中的表达量(P<0.05):但在高转移性的MDA-MB-231细胞中它们的表达水平转而下降.4.1N在T-47D细胞中的表达量明显低于在MCF-7细胞中的表达量(P<0.01),而在MDA-MB-231细胞中的表达则几乎检测不到,提示4.1N表达下调或缺失与乳腺癌细胞转移能力密切相关.4.1R/B/G/N在MCF-7和T-47D细胞中均主要定位于细胞膜及胞间连接处,同时4.1B在细胞核中也有表达;而在高转移的MDA-MB-231细胞中蛋白4.1家族成员均定位于细胞质中,提示蛋白4.1家族成员亚细胞定位的改变可能是乳腺癌细胞转移过程中的重要事件.结论:4.1N表达缺失和蛋白4.1家族成员亚细胞定位的改变与乳腺癌细胞MDA-MB-231的高转移性密切相关.     

4．1B蛋白在食管鳞状细胞癌中的异常表达及其分子机制

目的: 研究4.1B蛋白在食管鳞状细胞癌组织中的表达及异常表达的分子机制.方法: 采用免疫组织化学法检测110例石蜡包埋食管鳞状细胞癌及癌旁组织中4.1B蛋白的表达水平.随机选取其中29例,应用微卫星PCR技术检测4.1B等位基因的杂合子丢失情况.应用甲基化特异PCR技术检测33例新鲜食管鳞状细胞癌手术标本的4.1B基因启动子区域的甲基化状态.结果: 食管鳞状细胞癌组织中4.1B蛋白的阳性表达率为60.9%(67/110),癌旁正常组织的阳性表达率为94.5%(104/110),2组之间差异有统计学意义(X2=35.945,P<0.01).食管鳞状细胞癌高、中、低分化组的阳性表达率分别为74.4%(29/39)、61.8%(21/34)和45.9%(17/37),3组之间差异有统计学意义(X2=6.453,P<0.05).在20.7%(6/29)的食管鳞状细胞癌组织中,分别于D18S481、D18S62和D18S391这3个微卫星位点检测到4.1B等位基因的杂合子缺失.在33例新鲜手术标本中,有69.7%(23/33)的食管鳞状细胞癌组织检测出4.1B基因启动子区域的甲基化.结论: 4.1B蛋白在食管鳞癌组织中的阳性表达率明显低于癌旁正常组织,食管鳞状细胞癌的分化程度与其表达量呈正相关;启动子区域的异常甲基化可能是4.1B蛋白阴性表达的重要原因.     

NET-1 shRNA抑制肺癌细胞A549中NET-1表达及对癌细胞增殖的影响

目的:探讨转染PSilencer4.1 - NET -1 shRNA进入肺癌细胞株A549后对细胞中NET -1基因的表达及细胞增殖的影响.方法:转染PSilencer4.1 - NET -1 shRNA进入肺癌细胞株A549,同时设立空转染对照组与空白组,Western blot检测细胞NET -1蛋白,逆转录聚合酶链反应(RT - PCR)检测癌细胞中NET -1mRNA的含量,WST -8法检测细胞的增殖,流式细胞仪检测细胞周期的相关变化.结果:PSilencer4.1 - NET-1 shRNA转染组中NET -1 mRNA的含量及细胞NET -1蛋白与对照组比较明显降低,实验组细胞的增殖明显慢于对照组与空白组,癌细胞阻滞在G1期占63.24％,S期细胞比例明显下降,与对照组比较均有统计学意义(P＜0.05).结论:肺癌细胞中NET -1持续低表达可导致癌细胞增殖抑制、生长减慢,在肺癌患者的预后判断中有一定的参考价值.     

Hypoxia-inducible factor-1alpha expression and angiogenesis in gastrointestinal stromal tumor of the stomach

Hypoxia inducible factor (HIF)-1 is reported to transactivate expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. The aim of this study was to elucidate the clinical significance of HIF-1alpha expression in gastrointestinal stromal tumors (GIST). Specimens obtained from 53 patients who underwent surgical resection for GIST of the stomach were used in this study. Specimens were examined immunohistochemically for HIF-1alpha, VEGF, and Ki-67 expression. Tumor microvessel density (MVD) was determined immunohistochemically with anti-CD31 antibody and was estimated by averaging the counts from three high-power fields in the area showing the greatest neovascularization. HIF-1alpha expression was detected in 17 (32.1%) of 53 lesions and was correlated significantly with tumor size, liver metastasis, VEGF expression, and MVD. Prognosis was significantly poorer in patients with tumors expressing HIF-1alpha than in patients with tumors lacking HIF-1alpha expression. HIF-1alpha may play a role in angiogenesis and tumor progression of GIST through regulation of VEGF.     

Expression of vascular endothelial growth factor and angiogenesis in gastrointestinal stromal tumor of the stomach

Vascular endothelial growth factor (VEGF) is associated with the malignant potential of several types of carcinoma. The aim of this study was to elucidate the clinical significance of VEGF expression in gastrointestinal stromal tumor (GIST). METHODS: Specimens obtained from 53 patients who had underwent surgical resection for GIST of the stomach were used in this study. Specimens were examined immunohistochemically for VEGF expression and Ki-67 expression. Tumor microvessel density (MVD) was determined immunohistochemically with anti-CD31 antibody, and was estimated by averaging the counts from three high-power fields in the area showing the greatest neovascularization. RESULTS: VEGF expression was detected in 14 (26.4%) of the 53 lesions and correlated significantly with tumor size, liver metastasis, Ki-67 labeling index, and MVD. Prognosis was significantly poorer than in patients with tumors expressing VEGF than in patients with tumors lacking VEGF expression. Multiple logistic regression analysis for 10-year survival showed VEGF expression and high mitotic rate to be independent predictor of a poor outcome. CONCLUSIONS: Angiogenesis associated with VEGF may play an important role in the progression of GIST. VEGF expression may serve as an indicator of a poor prognosis.     

The mobilization, recruitment and contribution of bone marrow-derived endothelial progenitor cells to the tumor neovascularization occur at an early stage and throughout the entire process of hepatocellular carcinoma growth

Obvious neovascularization is a key feature of hepatocellular carcinoma (HCC) and the status of neovascularization in HCC is closely correlated with the tumor growth and patient prognosis. The actual effect of current antivascular treatment including embolization to HCC is not satisfactory. Compensatory angiogenesis is one of the primary causes responsible for failure of antiangiogenic therapy. Bone marrow-derived endothelial progenitor cells (BM-EPCs) are considered as important building blocks for adult neovascularization. However, the role of mobilized BM-EPCs in HCC remains unknown. In this study, GFP+-BM orthotropic HCC mice were established to investigate whether BM-EPCs are involved in HCC-induced neovascularization. We found that a large number of BM-EPCs were mobilized into the circulation with the development of HCC, recruited into the HCC region and incorporated into the vascular endothelium directly by differentiation into vascular endothelial cells, including sinus, capillary vessels and great vessels. Dynamic observation revealed that the mobilization and the incorporation of BM-EPCs into different types of vessels were present in early phases and throughout the whole process of HCC growth. The proportion of BM-EPCs in vessels increased gradually, from 17 to 21% with tumor growth. Moreover, injected GFP+-EPCs also specifically homed to tumor tissue and incorporated into tumor vessels directly. In this initial study, we demonstrated that BM-EPCs play a prominent role in HCC neovascularization. Blockade of BM-EPC-mediated vasculogenesis may improve the efficacy of current anti-vascularization therapy for patients with HCC.     

Myeloid cell trafficking and tumor angiogenesis

Tumor growth and metastasis depend on neovascularization, the growth of new blood vessels. Recent findings have revealed that tumor neovascularization is regulated in part by monocytes, which are myeloid lineage cells from the bone marrow. Tumors exhibit significant monocyte infiltrates, which are actively recruited to the tumor microenvironment. Upon tumor infiltration, monocytes can participate in tumor neovascularization. Monocytes can either differentiate into macrophages, which express proangiogenic growth factors, or into endothelial-like cells, which may directly participate in neovascularization. Preliminary studies in animals suggest that modulation of bone marrow-derived cell trafficking into tumors will provide a useful new approach in cancer therapy.     

Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis

There is a constant requirement for vascular supply in solid tumors. Tumor-associated neovascularization allows the tumor cells to express their critical growth advantage. Axillary lymph node status is the most important prognostic factor in operable breast cancer, and experimental and clinical evidence suggests that the process of metastasis is also angiogenesis-dependent. Various angiogenic growth factors and cytokines induce neovascularization in tumors, namely members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) gene families. A strong correlation has been found between VEGF expression and increased tumor microvasculature, malignancy, and metastasis in breast cancer. Anti-angiogenic therapy approaches offer a new promising anti-cancer strategy and a remarkably diverse group of over 20 such drugs is currently undergoing evaluation in clinical trials.     

Role of bone marrow-derived cells in tumor angiogenesis and treatment

The role of bone marrow-derived cells in tumor neovascularization is currently the subject of intense research and debate. Two recent studies offer novel yet somewhat conflicting evidence for the role of these cells in tumor growth and neovascularization. These results have significant implications for tumor biology and treatment. At the same time, they raise many questions, which must be addressed for translating these important findings into new, improved treatment strategies.     

Prolonged tumor dormancy by prevention of neovascularization in the vitreous.

Tumors release a diffusible substance that stimulates neovascularization. To study the neovascularization that occurs in diabetic retinopathy, we implanted V2 carcinomas and mouse ependymoblastomas into the vitreous of experimental animals. In the vitreous, unlike previous sites, the tumors failed to stimulate neovascularization. They grew for weeks as small, unvascularized, three-dimensional aggregates of cells. Explosive growth into a large, vascularized mass occurred when the avascular tumors reached the retinal surface. The vitreous proved to be a valuable model for observing the in vivo growth of small, solid tumors. Xenografts survived for months without evidence of immune rejection. The consequence of the prolonged avascular state is the restriction of tumor size. The normal vitreous may act to inhibit capillary proliferation. An understanding of the mechanism for maintaining the avascular state may lead to therapeutic blockade of neovascularization. This would be important in the management of diabetic retinopathy and neoplasia.     

Potent antitumor effects of the conditioned medium of bone marrow-derived mesenchymal stem cells via IGFBP-4

Cell transfer therapy using mesenchymal stem cells (MSCs) has pronounced therapeutic potential, but concerns remain about immune rejection, emboli formation, and promotion of tumor progression. Because the mode of action of MSCs highly relies on their paracrine effects through secretion of bioactive molecules, cell-free therapy using the conditioned medium (CM) of MSCs is an attractive option. However, the effects of MSC-CM on tumor progression have not been fully elucidated. Herein, we addressed this issue and investigated the possible underlying molecular mechanisms. The CM of MSCs derived from human bone marrow greatly inhibited the in vitro growth of several human tumor cell lines and the in vivo growth of the SCCVII murine squamous cell carcinoma cell line with reduced neovascularization. Exosomes in the MSC-CM were only partially involved in the inhibitory effects. The CM contained a variety of cytokines including insulin-like growth factor binding proteins (IGFBPs). Among them, IGFBP-4 greatly inhibited the in vitro growth of these tumors and angiogenesis, and immunodepletion of IGFBP-4 from the CM significantly reversed these effects. Of note, the CM greatly reduced the phosphorylation of AKT, ERK, IGF-1 receptor beta, and p38 MAPK in a partly IGFBP4-dependent manner, possibly through its binding to IGF-1/2 and blocking the signaling. The CM depleted of IGFBP-4 also reversed the inhibitory effects on in vivo tumor growth and neovascularization. Thus, MSC-CM has potent inhibitory effects on tumor growth and neovascularization in an IGFBP4-dependent manner, suggesting that cell-free therapy using MSC-CM could be a safer promising alternative for even cancer patients.     

抗肿瘤血管生成靶向治疗进展

血管生成是肿瘤生长和转移的关键。抗血管生成是一种癌策略,其目标是抗为活跃增殖的肿瘤细胞提供氧气和营养的新生血管。通过阻断新血管的生成抑制癌症的生长和转移。目前,最确定的抑制肿瘤血管生成的方法是阻断血管内皮生长因子（vascular endothelial growth factor,VEGF）通路。但最近,一些非VEGF因子如血小板衍生因子（platelet-derived growth factor,PDGF）、成纤维细胞生长因子（fibroblast growth factor,FGF）、肝细胞生长因子（hepatocyte growth factor,HGF）及血管生成素（angiogenin,Ang）等也参与肿瘤血管生成,强调有必要发展药物针对多种促血管生成途径。     

作用于血管生成的抗肿瘤药物进展

肿瘤生长转移具有血管依赖性，肿瘤血管生成抑制药物能破坏或抑制血管生成，有效阻止肿瘤的生长、转移和复发，是近年来肿瘤研究的新热点之一，也是肿瘤防治的一条新途径。本文对肿瘤血管生成抑制药物的种类，临床研究的新进展作一综述。