EGFR单克隆抗体抗结肠癌作用的实验研究

目的:观察表皮生长因子受体单克隆抗体(EGFRMcAb)对结肠癌的作用。方法:用不同剂量的EGFRMcAb处理LST174结肠癌细胞系, 采用细胞计数、生长曲线测定及MTT法测定体外培养细胞的生长和增殖抑制率。结果:可见一定程度的增殖抑制并呈剂量依赖性。抗-EGFR抗体组细胞数明显低于对照组(P<0.01).不同浓度之间比较:当EGFRMcAb为0.625mL/L时细胞计数相对高, 是对照组的61.3%;当EGFRMcAb为2.5mL/L时细胞计数最低, 是对照组的33.8%.EGFRMcAb组细胞生长受到抑制, 细胞生长曲线较对照组速度减慢。MTT值显示实验组细胞增殖能力低于对照组, 抑制率为42.3%(P<0.01).结论:EGFRMcAb可抑制人结肠癌LST174细胞生长, 具有一定的抗结肠癌作用。     

Mutation in the platelet-derived growth factor receptor alpha inhibits adeno-associated virus type 5 transduction

Due to its non-pathogenic lifecycle, little is known about the cellular determinants of infection by adeno-associated virus (AAV). To identify these critical cellular factors, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient cells followed by selection with furin-dependent toxins. A population of cells specifically resistant to AAV5 transduction was identified and sequence analysis suggested all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRα) gene. Characterization of this mutation suggested it inhibited PDGFRα trafficking resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRα trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRα.     

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P < 0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.     

Activation of human eosinophils by platelet-derived growth factor.

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.     

Extrachromosomal amplification of the epidermal growth factor receptor gene in a human colon carcinoma cell line

In situ hybridization, using a biotinylated cDNA probe for the epidermal growth factor receptor (EGFR) gene, indicates that the amplified EGFR genes in the colon tumor cell line, DiFi, are localized in many small double minute chromosomes (dmin) of varying size and visibility. Analysis of the electrophoretic mobility of gamma-irradiated DNA from DiFi by pulsed-field gel electrophoresis and Southern blot hybridization using EGFR probe, indicates that the amplified EGFR in DiFi exists in extrachromosomal, covalently closed circular episomes, presumably equivalent to dmin. Two major and one minor species were observed which had estimated sizes of 650, 1,300, and 2,000 kb, respectively. The DiFi cell line appears to represent a unique case of extrachromosomal EGFR gene amplification in human cells.     

Epidermal growth factor receptor expression in human pancreatic cancer: Significance for liver metastasis

Pancreatic cancer is a malignant tumor with an extremely poor prognosis. The mechanisms of the aggressive growth and metastasis are not yet extensively understood. Over-expression of epidermal growth factor receptor (EGFR) was suggested to be associated with malignant transformation of pancreatic cancer. We examined EGFR expression in 77 cases of invasive ductal adenocarcinoma of the pancreas, and analyzed the relation between the EGFR expression pattern and clinicopathological factors. EGFR immunoreactivity was detected in 41.6% (32/77) of human pancreatic cancers; i.e. diffuse expression in 32.5% (25/77) and focal expression in 9.1% (7/77). The EGFR expression was associated with gender (p<0.05), histological differentiation (p<0.05) and metastatic status of TNM classification (p<0.01). The observations suggested that EGFR expression plays important roles in metastasis, especially liver metastasis and recurrence of human pancreatic cancer.     

Modification of the primary tumor microenvironment by transforming growth factor alpha-epidermal growth factor receptor signaling promotes metastasis in an orthotopic colon cancer model

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.     

Microvessel morphology and vascular endothelial growth factor expression in human colonic carcinoma with or without metastasis

We quantified microvessel morphology and vascular endothelial growth factor (VEGF) expression in human colonic carcinoma with or without metastasis. The cancerous growth and the noncancerous section of surgical specimens from 36 patients with colorectal carcinoma (14 without metastasis and 22 with metastasis) were studied. Tissue slices immunostained with CD34 were processed for microvessel counts (per mm(2)), the mean diameter of microvessels (microm), and the mean spatial direction of microvessels (degree), defined by the angle between the longitudinal axis of microvessels and the direction perpendicular to the surface of the mucosa. Tissue slices immunostained with anti-VEGF antibody were processed for total epithelial cell counts (per mm(2)), VEGF-positive cell counts (per mm(2)), and VEGF-positive ratio (%). Carcinoma without metastasis had significantly larger microvessel counts (213 +/- 77, p < 0.01), larger microvessel diameter (7.99 +/- 1.77, p < 0.05), and larger spatial direction (47.2 +/- 8.3, p < 0.01) than normal tissue (144 +/- 49 for microvessel counts; 7.03 +/- 0.90 for microvessel diameter; 39.5 +/- 6.6 for spatial direction). Compared with carcinoma without metastasis, carcinoma with metastasis had a significantly larger microvessel diameter (9.75 +/- 2.65, p < 0.03) and lower microvessel counts (180 +/- 92, p = 0.51). Carcinoma without metastasis had a significantly larger VEGF-positive cell count (1276 +/- 805, p < 0.05) and larger VEGF-positive ratio (53.6 +/- 39.3, p < 0.05) than normal tissue (571 +/- 553 for VEGF-positive cell counts; 24.6 +/- 23.2 for VEGF-positive ratio). Carcinoma with metastasis had a significantly lower total cell count (1443 +/- 237, p < 0.001) and lower VEGF-positive cell count (716 +/- 463, p < 0.05) than carcinoma without metastasis. With tumor progression, microvessel diameter significantly increased and microvessel counts decreased, which can be in part explained by VEGF expression. The microvessel diameter seems to be the dominant parameter responsible for cancer cell intravasation as the first step of metastasis.     

Peroxiredoxin I, platelet-derived growth factor A, and platelet-derived growth factor receptor alpha are overexpressed in carcinoma ex pleomorphic adenoma: association with malignant transformation

Carcinoma ex pleomorphic adenoma is a rare salivary gland malignancy. It constitutes an important model for the study of carcinogenesis, as it can display the tumor in different stages of progression, from benign pleomorphic adenoma to frankly invasive carcinoma. Growth signaling pathways undergo continuous activation in human tumors, commonly as a consequence of the overexpression of ligands and receptors such as platelet-derived growth factor and platelet-derived growth factor receptor. Hydrogen peroxide is produced after platelet-derived growth factor receptor activation, and it is essential for the sequential phosphorylation cascade that drives cell proliferation and migration. By their ability to degrade hydrogen peroxide, peroxiredoxins are involved in growth factor signaling regulation and in the oxidative stress response. To verify the potential association of peroxiredoxin I, platelet-derived growth factor-A, and platelet-derived growth factor receptor-alpha with carcinoma ex pleomorphic adenoma progression, we investigated the expression of these molecules in carcinoma ex pleomorphic adenoma showing different degrees of invasion. The peroxiredoxin I, platelet-derived growth factor-A, and platelet-derived growth factor receptor-alpha proteins were present in remnant pleomorphic adenoma to only a small extent, but, collectively, they were highly expressed as soon as the malignant phenotype was achieved and remained at elevated concentrations during progression to the advanced stages of carcinoma ex pleomorphic adenoma. In addition, their locations overlapped significantly, strengthening their connection to this growth-signaling pathway. Our results indicate that carcinoma ex pleomorphic adenoma cells acquire at least 2 significant advantages relative to their normal counterparts: resistance to oxidative stress-induced apoptosis, conferred by high peroxiredoxin I concentrations, and sustained growth, reflecting platelet-derived growth factor-A and platelet-derived growth factor receptor-alpha overexpression.     

SDF-1和VEGF-C在结肠癌中的表达及与淋巴结转移的关系

目的探讨结肠癌组织中基质细胞衍生因子SDF-1、血管内皮生长因子-C（VEGF-C）表达的相互关系及其在淋巴结转移过程中的作用和可能机制。方法应用免疫组化法检测70例结肠癌组织和12例癌旁组织中SDF-1及VEGF-C的表达水平。结果结肠癌组织中SDF-1、VEGF-C阳性表达率分别为71．4％、64．3％,显著高于癌旁非癌组织的16．7％和8．3％（P〈0．01）。SDF-1、VEGF-C的表达与结肠癌的浸润深度、淋巴结转移及TNM分期有关（P〈0、01或P〈0．05）,与结肠癌的分化程度、远处转移无关（P〉0．05）。SDF-1、VEGF-C之间表达呈正相关（r=0．608,P〈0．05）。结论结肠癌组织中SDF-1、VEGF-C表达均显著增高,两者在结肠癌淋巴结转移中可能具有协同效应,共同促进结肠癌的淋巴结转移。     

Epidermal growth factor receptor expression in anal canal carcinoma

Most anal canal carcinomas (ACCs) are squamous cell carcinomas (SCCs). SCCs in other tumor sites strongly express epidermal growth factor receptors (EGFRs), the inhibition of which might result in favorable changes in tumor growth. A review of the published scientific literature reveals no information regarding the expression of EGFR in ACCs. Therefore, we obtained archived pathology samples from ACC biopsies and examined the frequency and level of expression of EGFR and other cell surface and cell cycle markers. The 21 samples studied universally and strongly expressed EGFR and were negative for HER-2. Clinical studies of EGFR inhibitors in advanced ACC are warranted.     

Preeclamptic sera stimulate increased platelet-derived growth factor mRNA and protein expression by cultured human endothelial cells.

Monolayer cultures of human endothelial cells were incubated with pre- and postdelivery sera from five women with preeclampsia and four matched, normal pregnancies. Conditioned media collected from endothelial cells pretreated in vitro with prepartum sera from preeclamptic women contained greater mitogenic activity and elevated levels of platelet-derived growth factor (PDGF)-like peptides than cells exposed to normal pregnancy sera or postpartum preeclamptic sera. Under the same experimental conditions, predelivery preeclamptic sera stimulated greater expression of endothelial cell PDGF-B-chain mRNA than that accumulated in the presence of matched postdelivery sera. By contrast, no differences in endothelial cell PDGF-B mRNA levels were noted when pre- and postdelivery sera from normal parturients were tested. The results suggest that a factor(s) in the blood of preeclamptic women can stimulate the synthesis and release of a potent growth factor and vasoconstrictor from human endothelial cells.     

EGFR、COX-2及P63蛋白表达与甲状腺乳头状癌侵袭转移的关系

目的　探讨甲状腺乳头状癌 (PTC)中表皮生长因子受体 (EGFR)、环氧化酶 2 (COX 2 )和 p6 3蛋白的表达与肿瘤侵袭转移的关系。方法　采用免疫组化S P法检测 6 7例PTC(其中腺内侵袭 4 1例、腺外侵袭 2 6例 ;有淋巴结转移 2 9例、无淋巴结转移 38例 )、3例滤泡癌和 15例甲状腺腺瘤中EGFR、COX 2及 p6 3蛋白的表达。结果　EGFR、COX 2和 p6 3蛋白在PTC组织中的阳性表达率分别为 88 1%、82 1%和 70 2 % ,均显著高于甲状腺腺瘤 (P <0 0 5 ) ;EGFR与COX 2、COX 2与p6 3蛋白在PTC组织中的的表达呈明显正相关 (P <0 0 5 ) ;EGFR和COX 2在PTC腺外侵袭组和有淋巴结转移组阳性表达率均明显高于腺内侵袭组和无淋巴结转移组 (P <0 0 5 ) ,p6 3蛋白阳性表达率与淋巴结转移有关 (P <0 0 5 )、与浸润程度无关。结论　EGFR与COX 2在PTC组织中的高表达可能促进肿瘤的侵袭和转移 ,p6 3蛋白在PTC的高表达提示其与PTC的细胞增殖相关     

miR-496过表达抑制结肠癌细胞生长和转移

目的:研究miR-496过表达对结肠癌细胞生长和转移的影响及其分子机制。方法:运用生物信息学软件筛选miR-496靶向相互作用蛋白;real-time PCR和Western blot法测定结肠癌细胞系HT29、HCT116、SW480以及正常结肠上皮细胞NCM460中miR-496、CTNNB1 mRNA和β-catenin蛋白的表达;运用Lipofectamine 2000将miR-496 mimics转染HT29、HCT116和SW480细胞,分别命名为HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞,转染scramble为阴性对照;运用MTT法、乳酸脱氢酶(LDH)试剂盒法、克隆形成实验和Transwell分别测定细胞活力、LDH漏出率、克隆形成能力和转移能力;萤光素酶报告基因实验测定miR-496启动子活性;Western blot法测定β-catenin、真核细胞翻译起始因子4E结合蛋白1(4E-BP1)、p-4E-BP1、低密度脂蛋白受体相关蛋白6(LRP6)、p-LRP6、MMP-7、MMP-9、MMP-13以及TIMP-2的蛋白水平。结果:miR-496与β-catenin内源性相互作用;miR-496在HT29、HCT116和SW480细胞中低表达,而在NCM460高表达;β-catenin在HT29、HCT116和SW480细胞中高表达,而在NCM460低表达;培养24 h、48 h、72 h、96 h的HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞活力、LDH漏出率、克隆形成率和转移的细胞数均显著低于对照组(P0.05);萤光素酶报告基因实验结果显示转染miR-496 mimics细胞中的miR-496启动子活性明显增加(P0.05),分别是对照组的1.75倍、2.04倍和1.61倍。Western blot实验结果显示miR-496过表达抑制β-catenin蛋白表达,p-4E-BP1和p-LRP6的蛋白水平降低;siRNA或miR-496过表达介导的β-catenin表达下调能显著抑制MMP-7和MMP-9的表达,促进TIMP-2的表达。结论:miR-496在结肠癌细胞中低表达,在正常结肠上皮细胞中高表达;miR-496过表达抑制结肠癌细胞的生长和转移,其机制是通过抑制Wnt/β-catenin通路进一步抑制MMP-7和MMP-9表达,促进TIMP-2表达,从而抑制结肠癌细胞的恶性表型。     

Oestrogen receptor and epidermal growth factor receptor expression in male breast carcinoma.

Male breast carcinomas are probably hormone-dependent, but receptor studies are few because this is a relatively rare tumour. We have studied 21 cases of male breast carcinoma immunohistochemically for oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) expression employing the antibodies ER-ICA and 12E on formalin-fixed, paraffin-embedded material. In our series, 86 per cent of male breast cancers were ER-positive and 76 per cent were EGFR-positive. Male breast carcinomas do not exhibit the inverse correlation between ER and EGFR expression that characterizes female breast carcinomas. Owing to the limitations of a small series, we were unable to comment on the relationship between ER and EGFR expression and patient survival. However, the relatively high incidence of ER expression may provide a growth advantage for this tumour in a male environment characterized by low levels of oestrogen. In addition, high EGFR expression may also contribute to a poor prognosis independent of ER status.     

Relative platelet-derived growth factor receptor subunit expression determines cell migration to different dimeric forms of PDGF

Platelet-derived growth factor (PDGF) receptor transfectants of a fibroblastoid cell line (BHK) have been used to investigate the ability of the three dimeric forms of PDGF to elicit a chemotactic response. Cells transfected with the beta receptor subunit were only responsive to PDGF-BB, whereas cells expressing the alpha-receptor subunit were equally responsive to all three dimeric forms, PDGF-AA, PDGF-AB, and PDGF-BB. A positive chemotactic response correlated with rearrangement of actin organization. In a study of human arterial smooth muscle cells that express both PDGF receptor subunits endogenously, we again found that recombinant PDGF-AA could elicit a chemotactic response. However, the two smooth muscle cell isolates we examined differed in their chemotactic response to PDGF-AA. This difference correlated closely with their ability to respond mitogenically to this PDGF dimeric form, and the magnitude of both chemotactic and mitogenic responses was related to the proportion of the two receptor subunit species at the cell surface.