Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells

To better understand the barriers associated with cationic lipid-mediated gene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filter supports at an air-liquid interface were used to study the binding and uptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of binding and uptake of cationic lipid:pDNA complexes by these cell systems were monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiated from internalized probe by adding trypan blue, which quenched the fluorescence of bound but not internalized probes. For proliferating cells, binding and internalization of the cationic lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the complexes was observed using polarized epithelial cells. However, after aspirating a small area of cells from the filter support, virtually all of the cells adjoining this newly formed edge bound and internalized the cationic lipid:pDNA complexes. To determine if their uptake in edge cells was related to the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies known to disrupt tight junctions. Cells treated in this manner bound and internalized cationic lipid:pDNA complexes efficiently and also expressed significant levels of transgene product. Control cells with intact tight junctions neither bound complexes nor expressed significant transgene product. These data confirm and extend earlier observations that the polarized apical membranes of airway epithelial cells are resistant to transfection by lipid:pDNA complexes. Further, in contrast to previous studies that have shown the entry step of complexes is not an important barrier for COS and HeLa cells, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tight junctions of polarized epithelial cells may improve gene delivery to these cells for diseases such as cystic fibrosis (CF).     

Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts

Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies.     

环指蛋白157通过下调小凹蛋白-1表达参与ERK通路激活促进黑素瘤细胞增殖

目的 探讨环指蛋白157(RING finger protein 157, RNF157)在黑素瘤细胞增殖中的作用及相关机制。方法采用慢病毒转染技术构建RNF157过表达黑素瘤细胞株，小干扰RNA(siRNA)技术构建敲减RNF157的黑素瘤细胞株，采用实时荧光定量聚合酶链反应（qRT-PCR）和Western印迹实验验证转染效果。采用MTT实验、克隆形成实验检测RNF157过表达黑素瘤细胞和敲减RNF157黑素瘤细胞的细胞增殖能力。采用裸鼠皮下成瘤实验及免疫组化染色检测RNF157过表达黑素瘤细胞体外增殖能力。采用免疫共沉淀（Co-IP）结合质谱分析、蛋白质组学和泛素化组学，探究RNF157在ERK通路中的潜在作用机制。结果 免疫组化染色结果显示，RNF157在黑素瘤组织中的表达高于瘤旁组织（P<0.01）。qRT-PCR和Western印迹实验结果显示，过表达组的RNF157表达水平显著高于对照组（P<0.000 1）;si3转染效率最高，si3组的RNF157表达水平显著低于对照组（P<0.01）。MTT实验、克隆形成实验结果显示，RNF157过表达可促进黑素瘤...     

Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice

A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-gamma, TNF-alpha, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-alpha were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.     

Differential growth suppression of human melanoma cells by tea (Camellia sinensis) epicatechins (ECG, EGC and EGCG)

We previously reported that catechins of green tea have different antiproliferative effects on cell lines derived from gender-dependent cancers; epicatechin 3-gallate (ECG) had the strongest inhibitory effect. In the present study, we examined the effects of epigallocatechin (EGC), epicatechin-gallate (ECG) and EGC 3-gallate (EGCG) on the viability, density, doubling time and cycle number of cell lines derived from melanoma metastasized to lymph nodes (MB-1133 and SE-0154) or distant organs (CH-0356, JK-0346, SA-1171, GE-0208, NS-1176 and LF-0023). These catechins have been documented to have no growth suppressive or apoptotic effects on normal melanocytes (Nihal et al., Int J Cancer 2005;114:513-21). EGCG (50 muM) showed greater inhibitory potency than EGC (50 muM) in SE-0154, NS-1176, GE-0208 and LF-0023 cell lines but the two catechins produced similar inhibitory effects in CH-0356, JK-0346 and SA-1171 cell lines. The IC(50) (50% inhibitory concentration) was lower for EGC than EGCG in MB-1133 and CH-0356 cells, higher for EGC than EGCG in GE-0208 cells and comparable (11-12 muM) for both the catechins in LF-0023 cells. When compared with EGC, the cytotoxic effect (% dead cell counts) and the suppression of the growth (change in cell number) of all melanoma cell lines tested were pronounced with EGCG. This investigation validates the hypothesis that anticancer action of the various catechins may vary with the type of malignancy and provides a model for tumor cell heterogeneity based on susceptibility and resistance of tumor cells to different green tea catechins. Therefore, this information is critical for undertaking chemopreventive or chemotherapeutic trials against melanoma and gender-based cancers.     

Potentiation of lipid peroxidation in B16F10 and B16F1 melanoma cells by caffeine,a methylxanthine derivative: relationship to intracellular glutathione

BACKGROUND: Caffeine has shown an inhibitory role in invasion and proliferation in melanoma pulmonary metastasis as well as in high-grade tissue sarcoma. However, little is known about its mechanism and possible role in metastatic cell lines. MATERIALS AND METHODS: B16F10 and B16F1 cell lines of high and low metastatic potential were treated with caffeine at different time intervals with different doses. Reduced glutathione, glutathione S-transferase and lipid peroxides were estimated to evaluate the effect of caffeine. RESULTS: Caffeine treatment showed glutathione depletion and increased lipid peroxidation with higher glutathione S-transferase activity in both B16F10 and B16F1 cell lines. However the effect of caffeine was dependent on the time factor as well as on the dose. CONCLUSIONS: Caffeine was an effective inhibitor of metastatic activity. Glutathione depletion in conjunction with increased lipid peroxidation was a potent indicator in the regulation of metastatic behavior of B16F10 and B16F1 melanoma cell lines.     

Copper complexes as prospective anticancer agents: in vitro and in vivo evaluation,selective targeting of cancer cells by DNA damage and S phase arrest

A series of six new bis(thiosemicarbazone)copper(i) complexes of the type [Cu(L^1–6)₂Cl] (1–6) have been synthesized and characterized. The molecular structure of the ligand L⁴ was determined by the single crystal XRD method. All the complexes adopted trigonal planar (Y-shaped) geometry. All the complexes strongly bind with CT-DNA via intercalative mode, which was further supported by molecular docking studies. Further, the complexes were effectively bind with BSA as observed by UV-Vis and fluorescence spectra. All the complexes effectively cleave pBR322 DNA through hydrolytic pathway as evidenced from T4 ligase experiments. All the complexes interact with the anticancer receptor focal adhesion kinase (FAK) via electrostatic, van der Waals, hydrogen bonding, σ–π and π–π interactions. In vitro cytotoxicity of the complexes were assessed by MTT assay against four cancer cell lines such as human breast adenocarcinoma (MCF-7), cervical (HeLa), epithelioma (Hep-2) and Ehrlich ascites carcinoma (EAC), and two normal cell lines namely normal human dermal fibroblasts (NHDF) and L6 myotubes with respect to the commercially used anticancer drug cisplatin. All the complexes induce apoptosis in EAC cells, which was confirmed by AO/EB, Hoechst 33258 and PI staining methods. The complexes block cell cycle progression of EAC cells in S phase (DNA synthesis). The cellular uptake studies confirmed the ability of the complexes to go into the cytoplasm and accumulation in the cell nuclei. In the in vivo anticancer studies, the complexes significantly reduce the tumour volume in female Swiss albino mice. Overall, our results ensure the role of thiosemicarbazone-based copper(i) complexes as prospective anticancer agents, induction of apoptosis and S phase arrest with the mitochondrial controlled pathway.

The thiosemicarbazone-based copper(i) complexes causing S phase arrest and apoptosis involving the mitochondrial controlled pathway has been investigated.     

原发性巨球蛋白血症颅内浸润一例报告附文献复习

目的 总结原发性巨球蛋白血症颅内浸润的诊疗体会.方法 对1例原发性巨球蛋白血症颅内浸润(Bing-Neel综合征)患者的临床资料进行分析,并进行相关文献复习,为其诊断和治疗提供思路.结果 患者1年前诊断为原发性巨球蛋白血症,治疗中患者出现持续性头痛.颅脑MRI平扫+增强:右侧额颞叶可见一团块状异常占位病变,T2呈高信号,增强后病灶强化明显;手术切除肿瘤组织行病理检查示额叶小细胞恶性肿瘤浸润;免疫组化染色检查示淋巴-浆细胞瘤样细胞颅内侵犯.结论 原发性巨球蛋白血症颅内浸润罕见,化疗疗效有限,理想的治疗应该是放疗联合化疗.     

Multimodal imaging of micron‐sized iron oxide particles following in vitro and in vivo uptake by stem cells: down to the nanometer scale

In this study, the interaction between cells and micron‐sized paramagnetic iron oxide (MPIO) particles was investigated by characterizing MPIO in their original state, and after cellular uptake in vitro as well as in vivo. Moreover, MPIO in the olfactory bulb were studied 9 months after injection. Using various imaging techniques, cell–MPIO interactions were investigated with increasing spatial resolution. Live cell confocal microscopy demonstrated that MPIO co‐localize with lysosomes after in vitro cellular uptake. In more detail, a membrane surrounding the MPIO was observed by high‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM). Following MPIO uptake in vivo, the same cell–MPIO interaction was observed by HAADF‐STEM in the subventricular zone at 1 week and in the olfactory bulb at 9 months after MPIO injection. These findings provide proof for the current hypothesis that MPIO are internalized by the cell through endocytosis. The results also show MPIO are not biodegradable, even after 9 months in the brain. Moreover, they show the possibility of HAADF‐STEM generating information on the labeled cell as well as on the MPIO. In summary, the methodology presented here provides a systematic route to investigate the interaction between cells and nanoparticles from the micrometer level down to the nanometer level and beyond. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.