Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro (K(d)=1 x 10(-9) M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8- to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(-) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(-) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.     

Cyclooxygenase-2 gene expression in human breast cancer

Expression of the inducible isoform of the cyclooxygenase gene (PGHS-2, COX-2) which codes for the enzyme that catalyzes formation of prostaglandins, was detected in 13/13 human breast tumors of high grade but not in samples of normal breast tissue. There was a statistically significant linear association between COX-2 gene expression and high (>50%) tumor cell density (p<0.01), with COX-2 protein localized to tumor cells. These results indicate that COX-2 gene expression may be useful as a molecular biomarker for human breast tumors and may also predict sensitivity to treatment with nonsteroidal anti-inflammatory drugs.     

白藜芦醇对不同乳腺癌细胞增殖的抑制作用

Objective To investigate the effects of resveratrol (Res) on the proliferation, cell cycle and apoptosis of three breast cell lines DU4475, MDA-MB-231 and MCF-7 with different estrogen receptor (ER) expressions. Methods Res was added to the drug treatment group at 6.25, 12.5, 25, 50, 100 and 200 μmol/L, respectively. Three observation periods at 24, 48 and 72 hours were set up respectively. MTT assay was used to detect the effects of Res on proliferation of breast cells. Cell cycle and apoptosis were analyzed by flow cytometry (FCM). The concentrations of drugs were 0, 12.5, 25, 50 μmol/L. The results were analysed by the statistical software SPSS17.0. Results After Res intervention, the proliferations of three cell lines were inhibited to different extent. After 48 and 72 hours of Res, inhibitions of Res with different concentration were significant different (F=15.26, P﹤0.05). Inhibition rates of DU4475 and MDA-MB-231 with ER-negative were higer than that of MCF-7 with ER-positive. FCM results prompted that these three kinds of cells were blocked in S phase after 48 hours treatment of 12.5-50 μmol/L Res and the block percentages had significant difference (F=34.81, P﹤0.05). The percentages of S phase for DU4475 and MDA-MB-231 arresting were higher than that of MCF-7. For DU4475, the apoptotic and necrosis percentage were higher than that of MCF-7 at 100 μmol/L (t=16.082, P<0.05). Conclusion Res can inhibit proliferation, induce cell cycle changing and apoptosis of DU4475, MDA-MB-231 and MCF-7 cells. The inhibitions of Res on DU4475 and MDA-MB-231 are better than that of MCF-7 with ER-positive.     

Metabolism of resveratrol in breast cancer cell lines: impact of sulfotransferase 1A1 expression on cell growth inhibition

Resveratrol is a polyphenolic compound present in grapes and wine with anticancer activities that undergoes pronounced metabolism in humans. In order to determine whether metabolism of resveratrol also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MB-MDA-231 human breast cancer cells. Along with resveratrol, it was possible to identify one metabolite, namely, resveratrol-3-O-sulfate in both cell lines. Its concentration in the cytoplasm and culture medium was 5.4- to 9-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 3.1-fold higher IC(50) value in the ZR-75-1 cell line (74 microM compared to 38 microM). By using RT-PCR, expression of sulfotransferase (SULT)1A1 mRNA, but not of other SULTs investigated, showed a close correlation with resveratrol 3-O-sulfate formation which was particularly high in ZR-75-1 and very low in MDA-MD-231 cells. In conclusion, we demonstrate that SULT1A1-based biotransformation reduces the anticancer activity of resveratrol in breast cancer cells, which must be considered in humans following oral uptake of dietary resveratrol as a chemopreventive agent.     

白藜芦醇对人胃癌SGC7901细胞的抑制及其可能的机制

目的:探讨白藜芦醇(resveratrol,Res)对人胃癌SGC7901细胞的抑制及其可能的作用机制.方法:以Res(10、20、40 μg/ml)作用人胃癌SGC7901细胞,同时设溶剂二甲亚砜(DMSO)和培养液作用为对照组;采用MTT法检测Res对人胃癌SGC7901细胞生长的抑制情况,相差显微镜观察细胞形态变化,比色法检测Res对SGC7901细胞Caspase-3活性的影响,流式细胞术检测Res对SGC7901细胞周期的影响.结果:Res对SGC7901细胞生长有明显抑制作用,且有剂量和时间依赖性,最大抑制率达(53.39±5.32)%;Res作用于SGC7901细胞后可见悬浮细胞增多,细胞胞体缩小、变圆、碎裂,细胞内出现颗粒样物质,在40 μg/ml作用48 h变化最明显;Res能明显上调SGC7901细胞Caspase-3活性,这种作用呈时间与浓度依赖性;流式细胞术发现,Res通过阻滞SGC7901于S期而抑制细胞分裂.结论:Res明显抑制人胃癌SGC7901细胞的生长,其机制可能是与激活Caspase-3从而诱导细胞凋亡、以及影响肿瘤细胞周期有关.     

HMGA1 protein overexpression in human breast carcinomas: correlation with ErbB2 expression.

We measured, by immunohistochemistry, HMGA1 protein expression in 212 breast tissue specimens: 6 normal samples, 28 hyperplastic lesions (13 with cellular atypia), 11 fibroadenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, and 13 lobular carcinomas. HMGA1 was not expressed in normal breast tissue; HMGA1 staining was intense in 40% of hyperplastic lesions with cellular atypia and in 60% of ductal carcinomas and weak in fibroadenomas and in hyperplastic lesions without cellular atypia. Because HMGA1 expression was similar among ductal breast carcinomas with different histologic grading, we evaluated the association between HMGA1 expression and that of other markers of breast carcinoma invasion (estrogen and progesterone receptors, Ki-67 antigen, and ErbB2) in 21 cases of grade 3 breast ductal carcinomas and 7 cases of breast lobular carcinomas. We found that HMGA1 expression tended to be associated only with c-erbB-2 expression (Spearman rho: 0.36; P=0.065). Taken together, these results suggest that HMGA1 expression might be a novel indicator for the diagnosis and prognosis of human breast cancer.     

Anticancer effect and enhanced chemotherapy potential of resveratrol in human pancreatic cancer cell lines

Objective Gemcitabine, the only approved drug for the treatment of pancreatic cancer, is not very effective. Novel and effective cancer chemopreventive agents are urgently needed. Recently, emerging studies determined resveratrol possessed anticancer effects on various cancer cells. We explored the anticancer effect of resveratrol in pancreatic cancer cells and investigated the involved moleculars of action. We also examined whether resveratrol enhanced antitumor activity of gemcitabine in vitro.Methods Proliferation inhibition was assessed by cell count kit-8 assay. Cell cycle phase distribution and apoptotic cells were measured by flow cytometric analysis. We determined the expression of bcl-2, cyclinD1, and activation of caspases-3 and poly(ADP-ribose) polymerase1 proteins used Western blot analysis.Results Resveratrol inhibited the proliferation of three pancreatic cancer cell lines in a dose dependent fashion, and induced accumulation of cells at the G1 phase as well as apoptosis. Our data also demonstrated that resveratrol enhanced gemcitabine-induced apoptosis in pancreatic cancer cells. In addition, resveratrol inhibited the expression of cyclinD1, bcl-2, and induced activation of caspase-3 and poly(ADPribose) polymerase1. Conclusion Our results suggested that resveratrol might be not only a potential regimen, but also an effective chemosensitizer for the chemotherapy of pancreatic cancer.     

胰岛素对人乳腺癌细胞影响的体外实验研究

目的:探讨胰岛素能否促进人乳腺癌细胞增殖。方法:将胰岛素直接作用于体外培养的人乳腺癌细胞株(MDA-MB-543),运用四氮唑盐,直接细胞计数及流式细胞仪测定等方法观察胰岛素对MDA-MB-543细胞株活力,细胞总数,细胞周期等的影响。结果:胰岛素(0.5~16mu/ml,8~18小时)使MDA-MB-543细胞株活力,细胞总数增加,呈明显的量效关系,流式检测证实胰岛素(7.5mu/ml)使S期细胞增多。结论:在体外,胰岛素可诱导人乳腺癌细胞株MDA-MB-543增殖。     

Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer

The ErbB2 (Neu) receptor tyrosine kinase is frequently overexpressed in human breast cancers, and this phenotype correlates with a poor clinical prognosis. We examined the effects of the mammalian target of rapamycin inhibitor, rapamycin, on mammary tumorigenesis in transgenic mice bearing an activated ErbB2 (NeuYD) transgene in the absence or presence of a second transgene encoding vascular endothelial growth factor (VEGF). Treatment of NeuYD or NeuYD x VEGF mice with rapamycin dramatically inhibited tumor growth accompanied by a marked decrease in tumor vascularization. Two key events that may underlie the antitumor activity of rapamycin were decreased expression of ErbB3 and inhibition of hypoxia-inducible factor-1-dependent responses to hypoxic stress. Rapamycin exposure caused only a modest inhibition of the proliferation of tumor-derived cell lines in standard monolayer cultures, but dramatically inhibited the growth of the same cells in three-dimensional cultures, due in part to the induction of apoptotic cell death. These studies underscore the therapeutic potential of mammalian target of rapamycin inhibitors in ErbB2-positive breast cancers and indicate that, relative to monolayer cultures, three-dimensional cell cultures are more predictive in vitro models for studies of the antitumor mechanisms of rapamycin and related compounds.     

维生素D_3对人乳腺癌细胞株表皮生长因子受体mRNA表达的影响

目的　探讨维生素D3 对人乳腺癌细胞株细胞增殖等生物学行为的影响。方法　选用两种人类乳腺癌细胞株MCF 7及ZR 75 1,应用细胞培养及核酸分子杂交方法 ,研究使用维生素D3 以及并用三苯氧胺后 ,上述细胞株生长及表皮生长因子受体 (EGFR)mRNA表达的改变。结果(10 -10 ～ 10 -7)mol/L维生素D3 对MCF 7及ZR 75 1细胞呈现明显的抑制生长作用 ,并能抑制 (10 -9～10 -7)mol/L三苯氧胺对MCF 7细胞的刺激生长作用。低浓度 (10 -10 ～ 10 -9)mol/L维生素D3 与半数抑制浓度三苯氧胺合并使用 ,可使抑制作用进一步加强 ,并使MCF 7及ZR 75 1两细胞株的EGFRmRNA降低至对照组以下。结论　维生素D类化合物有可能成为乳腺癌药物治疗的发展方向     

Challenges in the treatment of ErbB2 (HER2)-positive breast cancer

This article reviews the challenges and future options in the treatment of ErbB2- positive metastatic breast cancer. It summarizes a presentation from a symposium that was held at the ECCO 14 congress in 2007.Despite great progress in the management of ErbB2-positive metastatic breast cancer, significant challenges remain in these patients. Many patients do not respond to trastuzumab-containing regimens or develop progressive disease within 1 year. Continuing trastuzumab in these patients has yet to show proven clinical benefit in prospective studies. Several targeted agents have been developed to overcome the limitations of current targeted therapy for ErbB2-positive breast cancer. One such agent, lapatinib, was recently approved in Europe in combination with capecitabine in patients previously treated with trastuzumab in the metastatic setting. The intracellular site of action of lapatinib may underlie substantial clinical benefit.Based on available data to date, novel ErbB2-targeted therapies may hold significant promise in the management of ErbB2-positive metastatic breast cancer.     

Molecular mechanisms underlying ErbB2/HER2 action in breast cancer

Overexpression of ErbB2, a receptor-like tyrosine kinase, is shared by several types of human carcinomas. In breast tumors the extent of overexpression has a prognostic value, thus identifying the oncoprotein as a target for therapeutic strategies. Already, antibodies to ErbB2 are used in combination with chemotherapy in the treatment of metastasizing breast cancer. The mechanisms underlying the oncogenic action of ErbB2 involve a complex network in which ErbB2 acts as a ligand-less signaling subunit of three other receptors that directly bind a large repertoire of stroma-derived growth factors. The major partners of ErbB2 in carcinomas are ErbB1 (also called EGFR) and ErbB3, a kinase-defective receptor whose potent mitogenic action is activated in the context of heterodimeric complexes. Why ErbB2-containing heterodimers are relatively oncopotent is a function of a number of processes. Apparently, these heterodimers evade normal inactivation processes, by decreasing the rate of ligand dissociation, internalizing relatively slowly and avoiding the degradative pathway by returning to the cell surface. On the other hand, the heterodimers strongly recruit survival and mitogenic pathways such as the mitogen-activated protein kinases and the phosphatidylinositol 3-kinase. Hyper-activated signaling through the ErbB-signaling network results in dysregulation of the cell cycle homeostatic machinery, with upregulation of active cyclin-D/CDK complexes. Recent data indicate that cell cycle regulators are also linked to chemoresistance in ErbB2-dependent breast carcinoma. Together with D-type cyclins, it seems that the CDK inhibitor p21waf1 plays an important role in evasion from apoptosis. These recent findings herald a preliminary understanding of the output layer which connects elevated ErbB-signaling to oncogenesis and chemoresistance.     

p57kip2蛋白在乳腺癌组织和细胞系中的表达和定位

目的：研究p57kip2在人乳腺癌组织和细胞系中的表达和定位.方法：免疫印迹法检测人乳腺肿瘤细胞系中p57kip2的表达.人乳腺肿瘤组织进行免疫组化染色,检测p57kip2在组织中的表达.激光共聚焦扫描显微镜检测内源p57kip2在MCF-7细胞中的定位.结果：p57kip2在乳腺癌细胞系内表达,免疫组化染色分析和激光共聚焦扫描显微镜证实p57kip2主要定位于细胞核内.结论：p57kip2在乳腺癌细胞内表达,p57kip2主要定位于乳腺癌组织细胞核内,可能是参与肿瘤细胞基因转录调控和蛋白表达的重要蛋白之一.     

Loss of Nrdp1 enhances ErbB2/ErbB3-dependent breast tumor cell growth

Dysregulation of ErbB receptor tyrosine kinases is thought to promote mammary tumor progression by stimulating tumor cell growth and invasion. Overexpression and aberrant activation of ErbB2/HER2 confer aggressive and malignant characteristics to breast cancer cells, and patients displaying ErbB2-amplified breast cancer face a worsened prognosis. Recent studies have established that ErbB2 and ErbB3 are commonly co-overexpressed in breast tumor cell lines and in patient samples. ErbB2 heterodimerizes with and activates the ErbB3 receptor, and the two receptors synergize in promoting growth factor-induced cell proliferation, transformation, and invasiveness. Our previous studies have shown that the neuregulin receptor degradation protein-1 (Nrdp1) E3 ubiquitin ligase specifically suppresses cellular ErbB3 levels by marking the receptor for proteolytic degradation. Here, we show that overexpression of Nrdp1 in human breast cancer cells results in the suppression of ErbB3 levels, accompanied by the inhibition of cell growth and motility and the attenuation of signal transduction pathways. In contrast, either Nrdp1 knockdown or the overexpression of a dominant-negative form enhances ErbB3 levels and cellular proliferation. Additionally, Nrdp1 expression levels inversely correlate with ErbB3 levels in primary human breast cancer tissue and in a mouse model of ErbB2 mammary tumorigenesis. Our observations suggest that Nrdp1-mediated ErbB3 degradation suppresses cellular growth and motility, and that Nrdp1 loss in breast tumors may promote tumor progression by augmenting ErbB2/ErbB3 signaling.     

雌激素对人乳腺癌细胞系基质细胞衍化因子-1影响的观察

目的:探讨雌激素对基质细胞衍化因子-1(SDF-1)的影响.方法:选取乳腺癌细胞系MCF-7和MDA-MB-231为研究对象,分成对照组、雌激素组和雌激素+雌激素受体阻断组,分别加入不同生理浓度的17-β雌二醇作用一定时间以及同一浓度17-β雌二醇作用不同时间点,用酶联免疫吸附实验(ELISA)方法测定培养液中SDF-1的浓度,半定量逆转录聚合酶链反应(RT-PCR)法检测细胞SDF-1 mRNA的表达.结果:MDA-MB-231细胞系加与未加雌激素,均未检测到SDF-1的分泌.而MCF-7细胞基础培养液中可检测到SDF-1分泌.不同生理浓度的17-β雌二醇均可增加MCF-7细胞SDF-1的分泌水平.当加入1×10-7 mol/L的生理高浓度17-β雌二醇时,细胞分泌SDF-1水平在2 h达到高峰,是对照组的6倍[(1 823.17±325.18)ρg/mL vs (307.23±5.42)ρg/mL,F=201.02,P<0.01],该作用可被雌激素受体拮抗剂(ICI182,780)消除.此外,SDF-1mRNA的表达水平与测得的SDF-1蛋白水平相一致.结论:在某些乳腺癌细胞系,生理浓度的雌激素可促进SDF-1的分泌,而这种作用主要是通过雌激素受体来实现.雌激素可通过SDF-1来影响乳腺癌的生物学特性.     

A peptide conjugate of vitamin E succinate targets breast cancer cells with high ErbB2 expression

Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic alpha-tocopheryl succinate (alpha-TOS). Treating erbB2-low or erbB2-high cells with alpha-TOS induced similar levels of apoptosis, whereas alpha-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. alpha-TOS rapidly accumulated in erbB2-high cells exposed to alpha-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by alpha-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. alpha-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting alpha-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors.