大豆异黄酮激活PPARγ抑制人乳腺癌MCF-7细胞增殖

目的：探讨两种大豆异黄酮主要成分染料木黄酮（genistein,GEN）和大豆苷元（daidzein,DAI）抑制人乳腺癌MCF-7细胞增殖的作用与过氧化物酶体增殖激活物受体γ（peroxisome proliferators-activated receptorγ,PPARγ）信号途径的关系。方法：采用免疫细胞化学染色方法观察MCF-7细胞的PPARγ表达情况,PPARγ介导的荧光素酶报告基因检测大豆异黄酮和PPARγ配体罗格列酮（rosiglitazone,ROS）对MCF-7细胞PPARγ的激活作用,MCF-7细胞分别经8×10-5mol/L GEN、DAI和1×10-5mol/L的ROS单独或联合1×10-5mol/L的PPARγ特异性抑制剂GW9662联合处理24、48和72 h后,用CCK-8法检测细胞增殖。结果：MCF-7细胞存在有PPARγ表达,GEN、DAI呈剂量依赖性增强报告基因荧光素酶活性,且这种作用可被GW9662明显阻断;GEN、DAI和ROS呈时间依赖性明显抑制MCF-7细胞增殖（P〈0.05）,而GW9662可以显著削弱GEN、DAI和ROS对MCF-7细胞的增殖抑制作用（P〈0.05）。结论：大豆异黄酮可通过激活乳腺癌MCF-7细胞的PPARγ信号途径抑制其增殖。     

Herpes simplex virus-enhanced cationic lipid/DNA-mediated transfection

Liposome plasmid DNA complexes (lipoplexes) are often inefficient in mediating gene transfer and expression because of DNA degradation in lysosomal vesicles. Because herpes simplex virus (HSV) enters cells by fusion of the virus envelope with the plasma membranes, thereby overriding the endosomal pathway, HSV/lipoplex mixtures could be useful for improving gene transfer particularly when the mixture uses highly defective HSV particles that fail to express cytotoxic viral gene products. To evaluate this possibility, lipoplexes composed of cationic liposomes and lacZ reporter plasmids were compared for their ability to transduce cells in culture in the presence and absence of infectious HSV particles. The results showed that HSV increased the efficiency of cell transduction by approximately 4-100-fold compared with lipoplex vector alone, depending on the cell type targeted for gene delivery. The increased efficiency of transduction was virus dose dependent and required virus entry.     

14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation

We investigated the role of 14-3-3sigma protein in insulin-like growth factor-I (IGF-I) receptor signaling. It has been previously shown that 14-3-3sigma negatively regulates cell cycle especially in response to p53-sensitive DNA damage. In this study we demonstrated that 14-3-3sigma is a positive mediator of IGF-I receptor-induced cell proliferation. Treatment with IGF-I increased 14-3-3sigma mRNA and protein levels about 4-fold, in a time-dependent manner in MCF-7 breast cancer cells. Preincubation with the phosphoinositide 3'-kinase inhibitor LY294002 significantly reduced the effects of IGF-I on 14-3-3sigma gene expression in these cells, suggesting that this effect of IGF-I occurs via the phosphoinositide 3'-kinase pathway. 14-3-3sigma is induced by IGF-I in MCF-7 cells, which express wild-type p53, as well as in MCF-7 cells transfected with a small interference RNA targeting duplex that reduced p53 expression levels. These results suggest that IGF-I induces 14-3-3sigma expression in a manner that is independent of p53. Using the small interference RNA strategy, we demonstrated that a 70-75% reduction of 14-3-3sigma mRNA levels resulted in a similar decrease in the effects of IGF-I on cell cycle progression and proliferation in MCF-7 cells. This effect was also associated with a reduction in IGF-I-induced cyclin D1 expression. Taken together, these results suggest that 14-3-3sigma positively mediates IGF-I-induced cell cycle progression.     

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.     

Differences in the starvation-induced autophagy response in MDA-MB-231 and MCF-7 breast cancer cells

Breast cancer is a heterogeneous disease with distinct subtypes that have made targeted therapy of breast cancer challenging. Previous studies have demonstrated that an altered autophagy capacity can influence the development of breast cancer. However, the molecular differences in starvation-induced autophagic responses in MDA-MB-231 and MCF-7 cells have not been fully elucidated. In this study, we found that an increase of LC3B-II protein expression level and a decrease of the p62 protein expression level in both cells treated by Earle’s balanced salt solution. Meanwhile, we observed an increase of autophagosome using transmission electron microscopy and an enhancement in the green fluorescence intensity of LC3B protein by confocal microscopy. Furthermore, we detected the expression of 13 autophagy-related (ATG) genes and 11 autophagy signaling pathway-related genes using qPCR. Among 13 ATG genes, we found that 6 genes were up-regulated in treated MDA-MB-231 cells, while 4 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. In addition, among 11 autophagy signaling pathway-related genes, 7 genes were up-regulated in treated MDA-MB-231 cells, while 5 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. These findings suggest that the autophagic response to starvation was different in the two treated cell lines, which will contribute to further study on the molecular mechanism of starvation-induced autophagy and improve the targeted therapy of breast cancer.     

Investigation of the efficacy of paclitaxel on some miRNAs profiles in breast cancer stem cells

Understanding of the functions of microRNAs in breast cancer and breast cancer stem cells have been a hope for the development of new molecular targeted therapies. Here, it is aimed to investigate the differences in the expression levels of let-7a, miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-200c, miR-221, miR-222 and miR-335, which associated with gene and proteins in MCF-7 (parental) and MCF-7s (Mammosphere/stem cell-enriched population/CD44+/CD24-cells) cells treated with paclitaxel. MCF-7s were obtained from parental MCF-7 cells. Cytotoxic activity of paclitaxel was determined by ATP assay. Total RNA isolation and cDNA conversion were performed from the samples. Changes in expression levels of miRNAs were examined by RT-qPCR. Identified target genes and proteins of miRNAs were analyzed with RT-qPCR and western blot analysis, respectively. miR-125b was significantly expressed (2.0946-fold; p = 0.021) in MCF-7s cells compared to control after treatment with paclitaxel. Downregulation of SMO, STAT3, NANOG, OCT4, SOX2, ERBB2 and ERBB3 and upregulation of TP53 genes were significant after 48 h treatment in MCF-7s cells. Protein expressions of SOX2, OCT4, SMAD4, SOX2 and OCT4 also decreased. Paclitaxel induces miR-125b expression in MCF-7s cells. Upregulation of miR-125b may be used as a biomarker for the prediction of response to paclitaxel treatment in breast cancer.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

On the Mechanism of Cationic Amphiphile-mediated Transfection. To Fuse or not to Fuse: Is that the Question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.     

miR-200c-3p靶向FOSL1增加乳腺癌细胞化疗敏感性

化疗耐受是乳腺癌复发转移率居高不下、综合治疗效果难以提高的主要瓶颈。前期研究证实,miR-200c-3p在乳腺癌敏感细胞MCF-7中的表达量显著高于耐药细胞MCF-7/5Fu,提示miR-200c-3p可能参与乳腺癌化疗增敏,但是具体机制不详。生物信息学预测联合双荧光素酶报告基因实验证实,miR-200c-3p靶向调控FOSL1,且在多种肿瘤中miR-200c-3p与FOSL1表达负相关。实时荧光定量PCR技术和Western印迹技术证实,FOSL1在耐药细胞MCF-7/5Fu中的表达量显著高于亲本细胞MCF-7。在MCF-7细胞中,过表达FOSL1能够显著提高该细胞对5-Fu的化疗耐受;在MCF-7/5Fu中,使用siRNA技术沉默FOSL1,将提高该细胞对5-Fu的化疗敏感性。此外,MTT实验还发现,miR-200c-3p抑制剂能够显著上调MCF-7细胞对5-Fu的耐受,但是在此细胞中干扰FOSL1的表达,又可以增加其对5-Fu的化疗敏感性;miR-200c-3p mimics显著增加MCF-7/5Fu细胞的化疗敏感性,上调FOSL1表达后又可逆转miR-200c-3p mimics的化疗增敏作用。总之,miR-200-3p能够通过靶向FOSL1增加乳腺癌细胞对5-fluorouridine化疗敏感性。     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptor TRAIL-R3 is up-regulated by p53 in breast tumor cells through a mechanism involving an intronic p53-binding site

Tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (TRAIL-R3) is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors. We studied the regulation of TRAIL-R3 gene expression in breast tumor cells treated with the genotoxic drug doxorubicin (DXR). The breast tumor cell line MCF-7 (p53 wild type) responded to DXR with a marked elevation of TRAIL-R3 expression at the mRNA, total protein, and cell surface levels. In contrast, in EVSA-T cells (p53 mutant) DXR did not induce increased expression of TRAIL-R3. In MCF-7 cells overexpressing the human papillomavirus protein E6, which causes p53 degradation, DXR-induced TRAIL-R3 expression was notably reduced. Furthermore, in MCF-7 cells overexpressing a temperature-sensitive p53 mutant (Val135), shifting the cultures to the permissive temperature was sufficient to induce the expression of TRAIL-R3. We also cloned and characterized a p53 consensus element located within the first intron of the human TRAIL-R3 gene. This element binds p53 and confers responsiveness to genotoxic damage to constructs of the TRAIL-R3 promoter in transient transfection experiments. Our results indicate that genotoxic treatments such as DXR, frequently used in cancer therapy, may also induce genes such as TRAIL-R3 that potentially have antiapoptotic actions and thus interfere with the TRAIL signaling system. This is particularly important in view of the proposed use of TRAIL in antitumor therapy.     

Intracellular delivery of VIP-grafted sterically stabilized phospholipid mixed nanomicelles in human breast cancer cells

The purpose of this study was to determine whether biocompatible and biodegradable vasoactive intestinal peptide-grafted sterically stabilized phospholipid mixed nanomicelles (VIP-SSMM; size, approximately 15 nm), a novel nanosized actively targeted drug delivery platform for breast cancer, accumulate in human MCF-7 breast cancer cells. Using hydrophobic CdSe/ZnS quantum dots (QD), we found that QD-loaded VIP-SSMM accumulated significantly faster and in greater quantity in MCF-7 cells than did QD-loaded SSMM alone (p<0.05). This process was mediated, in part, by VIP receptors because excess human VIP, but not PACAP(6-38) or galanin, significantly attenuated this response (p<0.05). Taken together, these data indicate that VIP-SSMM are actively targeted to human breast cancer cells through VIP receptors. We suggest that VIP-SSMM could be used as an actively targeted nanosized drug delivery platform for breast cancer cells over-expressing VIP receptors.     

Cellular uptake of cationic lipid/DNA complexes by cultured myoblasts and myotubes

Several cationic lipids which are highly efficient for delivering genes in vitro do not increase gene delivery in vivo after an intramuscular injection. In order to elucidate the origin of this phenomenon, we have studied the cellular uptake and intracellular fate of cationic lipid/DNA complexes in vitro on myogenic mouse cells (myoblasts and myotubes) of the C2 cell line and of primary cultures. We used a cationic lipid with a spermine head group and its fluorescent analog, and a fluorescent plasmid obtained by nick-translation. In myoblasts, transgene expression was obtained and lipoplexes were internalized in cytoplasmic vesicles. In myotubes, no transgene expression could be detected and we observed an absence of lipoplex internalization. The in vitro uptake of cationic lipid was inversely correlated with the degree of fusion of C2 cell myotubes cultures.     

Regulation of sodium iodide symporter gene expression by Rac1/p38β mitogen-activated protein kinase signaling pathway in MCF-7 breast cancer cells

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.     

Enhanced gene delivery to HER-2-overexpressing breast cancer cells by modified immunolipoplexes conjugated with the anti-HER-2 antibody

Cationic liposome-mediated gene delivery to tumors has met with only limited success due to the low transfection efficiency and lack of target specificity. We developed a gene delivery system for HER-2-overexpressing cells by adding modified anti-HER-2 Fab' fragments to liposome/DNA complexes (lipoplexes). The modified anti-HER-2-Fab' was conjugated to liposomes containing cationic lipids such as 1,2-dioleoyl-3-(trimethylammonium) propane and cholesterol (1:1 w/w) using a maleimido-polyethyleneglycol-3400-1,2-dioleoyl-3-sn-phosphatidylethanolamine linker. The specific modification constricted the sizes of these immunolipoplexes to a range of 0.3- 0.7 microm, and they remained stable for a longer duration of time compared to the lipoplex controls (0.8-3.2 microm at 4 h). In addition, a 10-fold increase in luciferase activity was achieved after transfecting human breast cancer SK-BR3 cells with immunolipoplexes as compared to the control lipoplexes. Flow cytometry analysis demonstrated that 80% of SK-BR3 cells expressed the green fluorescent protein (GFP) 48 h after being transfected with immunolipoplexes, while only 40% of those with control lipoplexes and 3% of those with naked DNA alone expressed GFP. Furthermore, the anti-HER-2 immunolipoplexes showed specific enhancement of transfection efficiency in HER-2-overexpressing SK-BR3 cells (a 6-fold increase in luciferase activity) but not in HER-2-negative MCF-7 breast cancer cells. The enhancement of gene delivery by anti-HER-2 immunoliposomes was not affected by the presence of serum. These results demonstrate the feasibility of improving target-specific gene delivery to HER-2-overexpressing cells by insertion of lipid-modified anti-HER-2-Fab' into the preformed liposomes.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.