Effects of anticancer agents and scavengers on CMV‐promoter‐driven exogenous gene expression in genetically modified cells

Objectives This study aimed to investigate whether the levels of rsGFP mRNA and the fluorescence levels of cytomegalovirus (CMV)‐promoter‐driven rsGFP (red‐shifted green fluorescent protein) could be changed by using anticancer agents and also to examine the effects of co‐treatment with anticancer agents and scavengers. Methods The pQBI25 vector, which encodes the CMV promoter and the cDNA for rsGFP, was transfected into FR cells (rat skin fibroblast cell line). FR‐pQBI25 cells were then exposed to doxorubicin, 5‐fluorouracil, methotrexate or paraquat with or without scavengers such as N‐acetyl cysteine (NAC) and edaravone for 48 h. Key findings The levels of rsGFP mRNA were found to be significantly higher following doxorubicin, 5‐fluorouracil and paraquat treatment but were not changed by methotrexate. These levels of rsGFP mRNA were found to be significantly lower after paraquat/edaravone co‐treatment compared with paraquat alone. The fluorescence levels of rsGFP were found to be significantly higher following doxorubicin and paraquat treatment but were not changed by 5‐fluorouracil and methotrexate. The levels were also found to be significantly lower after paraquat/edaravone co‐treatment compared with paraquat alone and also after doxorubicin/NAC co‐treatment compared with doxorubicin alone. Conclusions These findings suggest that CMV‐promoter‐driven exogenous gene expression may be partly regulated by reactive oxygen species.     

Dual ligands modified double targeted nano-system for liver targeted gene delivery

Abstract

Context: It is now well established that the surface of nanocarriers with specific ligands defines a new biological identity, which assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs.

Objective: The aim of this study is to develop systemically administrable dual ligands modified nano-system which could both target cancer cells and macrophages in the liver.

Methods: Transferrin (Tf) and mannan (M) were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) and PE separately to get transferrin-PEG-PE (T-PEG-PE) and mannan-PE (M-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors were evaluated in tumor bearing animal models.

Results: D-modified solid lipid nanoparticles/enhanced green fluorescence protein plasmid (D-SLN/pEGFP) has a particle size of 198?nm and a gene loading quantity of 89%. D-SLN/pEGFP displayed over 25% higher transfection efficiency than M-PE modified SLN/pEGFP (M-SLN/pEGFP) in HepG2 cells and T-PEG-PE modified SLN/pEGFP (T-SLN/pEGFP) in Kupffer cells (KCs) isolated from mice.

Conclusion: It could be concluded that T-PEG-PE and M-PE could function as excellent active targeting ligands to improve the cell targeting ability of the carriers and the dual ligands modified vectors could be applied as a promising active targeting gene delivery system.     

NO-1886 ameliorates glycogen metabolism in insulin-resistant HepG2 cells by GSK-3β signalling

Objectives The aim of the study was to elucidate the possible role and mechanism of NO‐1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. Methods HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. Key findings We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin‐induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO‐1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin‐resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO‐1886 for 24 h. Compared with the dimethyl sulfoxide‐treated group, 2.5 µm NO‐1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO‐1886 increased the protein content of P‐GSK‐3βser⁹ and decreased the protein level of GSK‐3β in insulin‐resistant HepG2 cells, but NO‐1886 didn't change the protein levels of PI3‐Kp85 and Akt2. Conclusion Lipoprotein lipase activator NO‐1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK‐3 signalling.     

antagomiR-221抑制肝癌细胞增殖的初步研究

目的 研究小分子RNA抑制剂antagomiR-221对肝癌细胞增殖的影响.方法 原位杂交法检测肝癌组织及癌旁组织中miR-221表达情况:实验分3组重组质粒pEGFPantagomiR-221转染HepG2细胞为实验组,空质粒pEGFP-N2转染HepG2细胞为阴性对照组,HepG2细胞为空白对照组.采用细胞计数法和...     

Heat shock enhances CMV-IE promoter-driven metabotropic glutamate receptor expression and toxicity in transfected cells

In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca2+ and cAMP. Hyperthermia increased average amplitudes of Ca2+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.     

灯盏花素对高糖环境肾系膜细胞c-fos、c-jun蛋白表达的影响

目的　观察高葡萄糖环境中 ,蛋白激酶C(PKC)抑制剂灯盏花素对肾小球系膜细胞 (GMC)c fos、c jun蛋白表达和Ⅳ型胶原 (C Ⅳ )合成的影响 ,探索糖尿病肾病防治的新途径。方法　原代培养大鼠GMC ,分别置于正常葡萄糖 (对照组 )、高葡萄糖 (高糖组 )和高葡萄糖加灯盏花素 (高糖加灯盏花素组 )环境中 ,观察干预 2 4h、4 8h和 1wk后GMCc fos、c jun蛋白表达、C Ⅳ合成和PKC活性的变化。结果　与对照组比较 ,高糖组干预 2 4h后c fos、c jun蛋白表达同时明显增高 ,4 8h后c fos开始下降 ,而c jun 1wk后仍保持高水平 ,高糖组C Ⅳ合成 1wk后增加 ,各观察时点PKC活性均较对照组明显增高 ;而高糖加灯盏花素组各时点c fos、c jun蛋白表达、C Ⅳ合成和PKC活性均低于高糖组。结论　高葡萄糖可促使GMC中c fos、c jun蛋白表达和C Ⅳ合成增加 ,此可能为PKC活化所介导 ,灯盏花素可通过抑制PKC活化而有效阻止高葡萄糖引起的上述变化。     

聚乙二醇-壳聚糖共聚物作为基因传递载体的体外研究

本文通过将单甲氧基聚乙二醇(mPEG)的端羟基氧化为醛基，进而与壳聚糖(CS)链节上的氨基反应，合成了聚乙二醇-壳聚糖(mPEG-CS)共聚物。用MTT法检验不同浓度共聚物对HeLa细胞和A549细胞的毒性，结果显示5~100 μg·mL^-1聚合物的细胞毒性较低。通过考察不同PEG取代度的共聚物与质粒DNA所形成复合物的粒径、zeta电位及凝胶阻滞分析，筛选出最佳共聚物为取代度3.55%的mPEG(3.55)-CS。将mPEG(3.55)-CS作为基因传递载体，介导绿色荧光蛋白基因(pEGFP-C1)转染HeLa细胞和A549细胞，荧光显微镜下观察到荧光蛋白的表达，流式细胞仪测定HeLa细胞与A549细胞的最高转染率分别为8.1%和4.8%，证实了mPEG-CS共聚物是一种有效的非病毒类基因传递载体。     

Sulfur dioxide and benzo(a)pyrene modulates CYP1A and tumor‐related gene expression in rat liver

Sulfur dioxide (SO₂) and benzo(a)pyrene (B(a)P) are common industrial and environmental contaminants. However, few data are available on the effects of SO₂ on proto‐oncogenes and tumor suppressor genes, as well as the interactions between SO₂ and other xenobiotics regulating proto‐oncogenes or tumor suppressor genes expression. To investigate the interactions between SO₂ and B(a)P, male Wistar rats were exposed to intratracheally instilled with B(a)P or SO₂ inhalation alone or together. We detected mRNA expression of CYP1A1 and 1A2, 7‐ethoxyresorufin O‐deethylase (EROD), and methoxyresorufin O‐demethylase (MROD) activities in livers. The mRNA and protein levels of several cancer‐related genes were analyzed in livers by real‐time RT‐PCR and Western blot, respectively. The EROD/MROD activities and CYP1A1/2 expression were down‐regulated by SO₂ but up‐regulated by B(a)P alone. Exposure of SO₂ alone induced c‐fos, c‐jun, c‐myc, H‐ras, and p53 expression, and depressed p16 and Rb expression in livers. The effects of B(a)P on the above gene were similar to SO₂ except c‐fos expression. Furthermore, SO₂ + B(a)P exposure increased the expression of c‐fos, c‐jun, c‐myc, and p53, and decreased p16 and Rb expression in livers compared with exposed to SO₂ or B(a)P alone. However, no synergistic effects were observed on H‐ras and CYP1A1/2 after SO₂ + B(a)P exposure. Our findings indicate that multiple cell cycle regulatory proteins play key roles in the toxicity of SO₂ and B(a)P in livers. It might involve the activation of c‐fos, c‐jun, c‐myc, and p53. And p16‐Rb pathway might also participate in the progress. Although the gene products we studied are classed as oncogenes and tumor suppressor genes, their functions actually relate to more general processes of control of cell proliferation, survival, and/or apoptosis. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Proteomic analysis of liver cancer cells treated with 5‐Aza‐2′‐deoxycytidine (AZA)

5‐Aza‐2′‐deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti‐tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti‐tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre‐ and post‐treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up‐regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up‐regulated proteins: glutathione S‐transferase P, protein DJ‐1, peroxiredoxin‐2, UMP‐CMP kinase, cytochrome c‐type heme lyase, enhancer of rudimentary homolog, profilin‐1, and one down‐regulated protein, heat‐shock protein β?1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up‐regulated proteins, glutathione S‐transferase P and peroxiredoxin‐2, using RT‐PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation‐specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley‐Liss, Inc.     

Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus

Purpose. The objective of this study was to examine glucose-modulated reporter gene expression via recombinant adeno-associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10^-7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV-transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV-treated diabetic C57BL/6J mice.     

绞股蓝皂苷对小鼠白血病P388细胞内原癌基因表达的影响

目的　研究绞股蓝皂苷 (gypenosides ,GP)体外给药对小鼠白血病P 388细胞内原癌基因c fos和c myc基因表达的影响。方法　采用Northern杂交和狭线杂交的方法检测c fos和c myc基因表达的水平。 结果　GP在给药剂量0 0 15和 0 0 30 g·L-1的条件下 ,体外给药 2 4h ,c fos和c myc的表达水平分别为 0 5 2 2 ,0 947和 0 16 5 ,0 12 0 ,而对照组则为 0 36 0和 0 30 8。结果表明 ,GP作用 2 4h使P 388细胞内的c fos基因表达水平明显升高 ,c myc基因的表达水平明显降低。结论　GP对原癌基因c fos,c myc表达水平的影响是其抑制P 388细胞生长的机制之一。     

Modulation of aryl hydrocarbon receptor‐regulated genes by acute administration of ammonium metavanadate in kidney,lung and heart of C57BL/6 mice

We recently reported that vanadium (V⁵⁺) was able to decrease the 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD)‐mediated induction of Cyp1a1 and Nqo1 at mRNA, protein and catalytic activity levels in mouse hepatoma Hepa 1c1c7 and human hepatoma HepG2 cells. However, little is known regarding the in vivo effects. Thus, the objective of this study was to investigate whether similar effects would occur at the in vivo level. Therefore, we examined the effect of exposure to V⁵⁺ (5 mg kg^?1) with or without TCDD (15 µg kg^?1) on the AhR‐regulated genes in kidney, lung and heart of C57BL/6 J mice. Our results demonstrated that V⁵⁺ alone significantly decreased Cyp1b1 protein and catalytic activity levels in kidney at 24 h. Moreover, it significantly potentiated Nqo1 and Gsta1 gene expression in the heart, and only Gsta1 gene expression in the lung. Upon co‐exposure, we found that V⁵⁺significantly inhibited the TCDD‐mediated induction of Cyp1a1, Cyp1a2 and Cyp1b1 mRNA, protein and catalytic activity levels in the kidney at 24 h. On the other hand, V⁵⁺ significantly potentiated the TCDD‐mediated induction of Nqo1 and Gsta1 protein and activity levels in the kidney. Cyp1a1, Cyp1b1, Nqo1 mRNA, protein and catalytic activity levels in the lung were significantly potentiated at 6 h. Interestingly, all tested genes in the heart were significantly decreased at 6 h with the exception of Gsta1 mRNA. The present study demonstrates that V⁵⁺ modulates TCDD‐induced AhR‐regulated genes. Furthermore, the effect on one of these enzymes could not be generalized to other enzymes even if it was in the same organ. Copyright © 2012 John Wiley & Sons, Ltd.     

Bile Acid‐Induced Toxicity in HepaRG Cells Recapitulates the Response in Primary Human Hepatocytes

Cholestatic liver injury is a pathological component of numerous disease states. Much of the current literature on cholestatic liver injury is derived from in vitro studies using rodent hepatocytes or cell lines transfected with bile acid (BA) uptake transporters. While these studies demonstrate BA‐driven apoptosis, it is debatable whether these models reflect the human pathophysiology, as primary human hepatocytes undergo primarily necrosis. HepaRG cells are a bipotential, human hepatoma line that express apical and basolateral BA transporters. Thus, we sought to determine whether HepaRG cells could replicate the response of primary human hepatocytes to BA exposure in vitro. HepG2 cells, primary murine hepatocytes (PMH) or HepaRG cells, were exposed to taurocholic acid (TCA), or glycochenodeoxycholate (GCDC) and lactate dehydrogenase release were measured to determine cell death. Cell death occurred dose‐responsively in HepaRG cells when exposed to GCDC; however, HepG2 cells died acutely only at very high concentrations of GCDC. In HepaRG cells, pre‐treatment with the caspase inhibitor z‐VD‐FMK had no effect on cell death, indicating a lack of apoptotic cell death, and while c‐jun N‐terminal kinase (JNK) protein was activated by GCDC treatment in HepaRG cells, the inhibition of JNK did not protect. Although previous data indicate that TCA stimulates pro‐inflammatory gene induction in PMH, there was no change in gene expression after TCA stimulation in HepaRG cells, which mimicked previous data found in primary human hepatocytes. These data provide evidence for HepaRG cells as a new model for the study of the effect of BA on human hepatocytes.     

In vitro toxicity of aflatoxin B1 and its photodegradation products in HepG2 cells

The effects of aflatoxin B₁ (AFB₁) were studied using the HepG2 cell line. Cytotoxicity, apoptosis and p53 expression were assessed after exposure to different concentrations of AFB₁ (0–100 μm ) and its two types of degradation products, namely the mixtures of photodegradation products in water (Pw) and the mixtures of photodegradation products in peanut oil (Po) for different time periods (0, 24, and 48 h). After exposure of the HepG2 cells to these compounds for different times and concentrations, the cytotoxicity of Pw and Po decreased approximately 40 and 100% compared with AFB₁, respectively. The expression of p53 protein decreased significantly in AFB₁‐exposed cells, decreased slightly in Pw‐treated cells and did not decrease compared to the untreated cells. The results of the in vitro cytotoxicity assay indicate that Pw is less toxic than AFB₁, and Po has almost no toxicity, which can be explained by the differences in the chemical nature of the various kinds of the test compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Nanostructured lipid carriers as novel drug delivery system for lung cancer gene therapy

Context: Nanostructured lipid carriers (NLC) are potentially good colloidal drug carriers for gene delivery. They are advised to be the second lifetime of lipid nanocarriers.

Objective: The aim of this study is to develop novel modified NLC as nanomedicine for delivery of plasmid-containing enhanced green fluorescence protein (pEGFP). This system could target the lung cancer cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials.

Methods: In the present study, pEGFP-loaded NLC (NLC/pEGFP) were prepared. Transferrin (Tf) containing ligands were used for the surface coating of the vectors. In vitro transfection efficiency of the modified vectors was evaluated in human alveolar adenocarcinoma cell line (A549 cells) and in vivo transfection efficiency of the modified vectors was evaluated on mice bearing A549 cells model.

Results: Tf-modified NLC/pEGFP (Tf-NLC/pEGFP) has a particle size of 157?nm, and ～82% of gene loading quantity. Tf-NLC/pEGFP displayed remarkably higher transfection efficiency than non-modified NLC/pEGFP both in vitro and in vivo.

Conclusion: The results demonstrate that the novel NLC gene delivery system offers an effective strategy for lung cancer gene therapy.