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Current strategies of gene transfection are not efficient at achieving a notable therapeutic effect. The aim of the present study was to combine ultrasound‑targeted microbubble destruction (UTMD) with a polyethylenimine/pEGFP‑N3 plasmid/nuclear localization sequence (PEI/DNA/NLS) complex gene delivery system, and evaluate the transfection efficiency of enhanced green fluorescent protein (EGFP) gene delivery to 293T cells using this system. The formation of PEI/DNA/NLS complexes and the protective effects of PEI/NLS were verified by gel electrophoresis. Solutions consisting of the plasmid alone, PEI/DNA complexes, PEI/DNA/NLS complexes, UTMD+DNA, UTMD+PEI/DNA complexes, and UTMD+PEI/DNA/NLS complexes were transduced into 293T cells via ultrasound irradiation. The expression of GFP was observed using an inverted microscope and transfection efficiency was detected by flow cytometry following 24 h incubation in vitro. Cell activity was detected using a Cell Counting kit (CCK)‑8 assay. Gel electrophoresis confirmed the formation of PEI/DNA/NLS complexes and demonstrated that PEI/NLS exhibited protective effects on plasmid integrity for a limited time. Inverted microscope observations revealed that a greater GFP signal was observed with the combined action of PEI/DNA/NLS complexes with UTMD, and flow cytometry analysis demonstrated the highest level of transfection efficiency in this group. In addition, the viability of the cells detected by CCK‑8 and treated with PEI/DNA/NLS complexes with UTMD was >80%. In conclusion, the combination of UTMD and PEI/DNA/NLS complexes was highly effective for the efficient transfection of 293T cells without causing excessive cell damage. This method may provide a novel and effective gene transduction system to be applied in clinical treatments. [ABSTRACT FROM AUTHOR]

Ultrasound-targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post-transfection, the rates of Cy3-labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD-mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post-transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein from the plasmid were significantly increased in the cNLS group compared with the control group. The mNLS group displayed no significant difference compared with the control group, while the WGA group exhibited significant inhibition in most indicators of transfection efficiency compared to the cNLS group. These results suggest that combining a cNLS peptide with pDNA during UTMD-mediated transfection significantly improved transfection efficiency. Thus, a cNLS peptide may be an important mediator and a new strategy in enhancing the efficiency of UTMD-mediated gene transfection. [ABSTRACT FROM AUTHOR]

Epigallocatechin-3-gallate (EGCG) is an active and major constituent of green tea. As a non-nucleoside inhibitor of DNA methylation, EGCG is able to inhibit the hypermethylation of newly synthesised DNA, resulting in the reversal of hypermethylation and recovery in expression of the silenced genes. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a novel tumour suppressor gene, which negatively regulates matrix metalloproteinases, and inhibits tumour invasion, angiogenesis and metastasis. The present study aimed to examine the effects of EGCG on the methylation status of the RECK gene and tumour invasion in a salivary adenoid cystic carcinoma (SACC) cell line in vitro. Marked levels of methylated and weak levels of unmethylated RECK promoter were detected in the SACC83 cells, which was determined using methylation-specific polymerase chain reaction (PCR). In addition, the treatment of SACC83 cells with EGCG partially reversed the hypermethylation status of the RECK gene. Western blot analysis and reverse transcription-PCR demonstrated that EGCG significantly enhanced the protein and mRNA expression levels of RECK, and significantly reduced the invasive ability of the SACC83 cells, as determined using a Transwell assay. These results suggested that EGCG possesses novel anti-metastatic therapeutic potential for the treatment of SACC. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction and apoptosis have key roles in the initiation and progression of atherosclerosis (AS). AS has been demonstrated to be associated with a high‑fat diet, which may increase endothelial permeability and apoptosis; however, the exact mechanisms underlying the development of AS remain poorly understood. MicroRNAs (miRNAs) are vital for the regulation of cardiovascular disease, and dysregulated miRNAs have been implicated in AS. The present study investigated whether miRNA (miR)‑126 regulates high‑fat diet‑induced endothelial permeability and apoptosis by targeting transforming growth factor β (TGFβ), a secreted protein that controls cellular proliferation and apoptosis. In the present study, apolipoprotein E (apoE)‑/‑ mice were fed a high‑fat diet in order to establish a model of AS. Mice were subcutaneously injected with a miR‑126 mimic, a miR‑126 antagomir or control miRNA. Reverse transcription‑quantitative polymerase chain reaction was used to assess miR‑126 expression, and a fluorometric assay was used to evaluate caspase‑3 activity. The effects of miR‑126 on the endothelial permeability of the aortic intima were also explored. Western blotting and immunohistochemical analysis were used to investigate the effects of miR‑126 on B‑cell lymphoma‑2 (Bcl‑2) and transforming growth factor (TGF) β protein expression levels. Furthermore, a luciferase assay was performed to verify whether TGFβ may be a direct target gene of miR‑126. In apolipoprotein E‑knockout mice, a high‑fat diet reduced miR‑126 expression and induced apoptosis as determined by the upregulation of caspase‑3 activity. A miR‑126 antagomir increased endothelial permeability and apoptosis in mice fed a high‑fat diet. By contrast, an miR‑126 mimic attenuated endothelial permeability and apoptosis. The reduction in miR‑126 was associated with a reduction in protein expression levels of Bcl‑2 and an increase of TGFβ in mice fed a high‑fat diet. In addition, the present study demonstrated that miR‑126 reduced TGFβ expression following binding to the 3'‑untranslated region of TGFβ mRNA. The current study demonstrated a role for miR‑126 in AS and identified TGFβ as a direct target of miR‑126. Furthermore, the present study demonstrated that miR‑126 contributed to endothelial permeability and apoptosis, and suggested that the downregulation of TGFβ may be involved in the molecular mechanisms underlying the actions of miR‑126. miR‑126 may therefore have potential as a novel therapeutic target for the treatment of AS. [ABSTRACT FROM AUTHOR]

Inefficient gene delivery poses a challenge for non-viral gene therapy. Cytoplasmic and nuclear membrane barriers are responsible for the inefficiency as they restrict the import of exogenous genes. The present study aimed to improve the transfection efficiency using a novel gene delivery system, which consisted of two components: ultrasound microbubbles and the nuclear factor κB (NFκB) binding motif. Ultrasound-targeted microbubble destruction (UTMD) was used to enhance the cytoplasmic import of plasmids and the NFκB binding motif was added to promote the nuclear intake of the plasmid from the cytoplasm. In the present study, human umbilical vein endothelial cells were transfected using UTMD with two different Cy3-labeled plasmids, phSDF-1α and phSDF-1α-NFκB. phSDF-1α-NFκB was constructed by inserting a specific DNA targeting sequence (five optimal repeats of the binding motif for the inducible transcription factor NFκB) into phSDF-1α. The nuclear import and gene expression efficiency of phSDF-1α-NFκB were compared with those of phSDF-1α to investigate the effect of the NFκB binding motif on transfection. The results showed that UTMD significantly increased the cytoplasmic intake of pDNA and maintained high cell viability. The nuclear import and gene expression of phSDF-1α-NFκB-transfected cells were significantly higher than those transfected with phSDF-1α. Compared with the NFκB-free plasmids, the quantity of NFκB plasmids in the nucleus increased 6.5-fold and the expression of SDF-1α was 4.4-fold greater. These results suggest that UTMD combined with NFκB binding motif significantly improve transfection efficiency by enhancing the cytoplasmic and nuclear import of exogenous plasmid DNA. [ABSTRACT FROM AUTHOR]

Melatonin is predominately produced and secreted by the pineal gland, and inhibits cell growth in various cancer cell lines such as colorectal cancer. However, the precise mechanisms involved have not been fully elucidated. In the present study, the potential molecular mechanism underlying the efficacy of melatonin on migration in RKO colon cancer cells was investigated. The effects of melatonin and H-1152, a selective inhibitor of Rho-associated protein kinase (ROCK), on the migration of RKO cells were analyzed by an in vitro wound healing assay. The localization of zonula occludens-1 (ZO-1) and occludin were observed by immunofluorescence. Reverse transcription-quantitative polymerase chain reaction (qPCR) was performed to analyze the relative mRNA levels of ROCK, ZO-1 and occludin. In addition, western blot analysis was implemented to examine the expression of ROCK, phospho (p)-myosin phosphatase targeting subunit 1 (MYPT1), p-myosin light chains (MLC) and p-p38. The results revealed that the expression levels of ROCK2, p-MYPT1 and p-MLC in RKO cells were decreased, and the membrane protein expression of ZO-1 and occludin increased when the cells were treated with melatonin. qPCR demonstrated that melatonin downregulated ROCK2 gene expression, and upregulated the expression of the ZO-1 and occludin genes. The levels of ZO-1 and occludin localized in the tight junctions were markedly increased in the immunofluorescence assay. In addition, the phosphorylation levels of p38 were reduced when the cells were treated with melatonin, and treatment with H-1152 downregulated p38 phosphorylation. The results indicated that melatonin may inhibit the migration of RKO colon cancer cells by downregulating ROCK expression via the p38/mitogen-activated protein kinase signaling pathway. [ABSTRACT FROM AUTHOR]

The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non-viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound-targeted microbubbles and peptide nucleic acid (PNA)-binding nuclear localization signals (NLS). Ultrasound-targeted microbubble destruction (UTMD) and PNA-binding NLS were used to improve the cytoplasmic and nuclear importation of the plasmid, respectively. Experiments were performed using antibody-targeted microbubbles (AT-MCB) that specifically recognize the SV40T antigen receptor expressed on the membranes of 293T cells, resulting in the localization of ultrasound microbubbles to 293T cell membranes. Furthermore, PNA containing NLS was inserted into the enhanced green fluorescent protein (EGFP)-N3 plasmid DNA (NLS-PNA-DNA), which increased nuclear localization. The nuclear import and gene expression efficiency of the AT-MCB with PNA-binding NLS were compared with AT-MCB alone or a PNA-binding NLS. The effect of the AT-MCB containing PNA-binding NLS on transfection was investigated. The ultrasound and AT-MCB delivery significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. The nuclear import and gene expression of combined microbubble- and PNA-transfected cells were significantly greater compared with cells that were transfected with AT-MCB or DNA with only PNA-binding NLS. The quantity of EGFP-N3 plasmids in the nuclei was increased by >5.0-fold compared with control microbubbles (CMCB) and NLS-free plasmids. The gene expression was ~1.7-fold greater compared with NLS-free plasmids and 1.3-fold greater compared with control microbubbles. In conclusion, UTMD combined with AT-MCB and a PNA-binding NLS plasmid significantly improved transfection efficiency by increasing cytoplasmic and nuclear DNA import. This method is a promising strategy for the noninvasive and effective delivery of target genes or drugs for the treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of tauroursodeoxycholic acid (TUDCA) on the lipolytic action of tumor necrosis factor (TNF)-α in 3T3-L1 adipocytes. Following treatment with TNF-α, cell viability was determined by MTT assay to select the optimum concentration and duration of TNF-α treatment in 3T3-L1 adipocytes. Intracellular lipid droplet dispersion and glycerin content in culture media were determined to evaluate the effect of TUDCA on TNF-α-induced lipolysis in 3T3-L1 adipocytes. Western blotting was performed to detect protein expression levels of perilipin-A and protein markers of endoplasmic reticulum stress: Immunoglobulin-binding protein (BiP), inositol-requiring enzyme (IRE), c-Jun N-terminal kinase (JNK), phosphorylated (p)-IRE and p-JNK. Following treatment with 50 ng/ml TNF-α for 24 h, glycerin content increased significantly and lipid droplets were dispersed. Glycerin content was reduced significantly and dispersal of lipid droplets reduced following pretreatment of 3T3-L1 adipocytes with 1 mmol/l TUDCA. TNF-α additionally activated the expression of BiP, p-IRE and p-JNK in a time-dependent manner; following pretreatment of 3T3-L1 adipocytes with 1 mmol/l TUDCA, the expression levels of these three proteins decreased. Therefore, TUDCA may inhibit TNF-α-induced lipolysis in 3T3-L1 adipocytes and reduce production of free fatty acids. Its underlying molecular mechanisms are potentially associated with the inhibition of activation of the IRE-JNK signaling pathway, which influences perilipin-A expression levels. [ABSTRACT FROM AUTHOR]

Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet-induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)-1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high-fat diet-fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high-fat diet (AS group) or a high-fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium-dependent relaxation and endothelium-independent relaxation were measured using high-frequency ultrasound. Administration of a high-fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high-fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high-fat diet. A similar change was observed for the TJ proteins ZO-1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ, is associated with remodeling from cell membrane to cytoplasm. The present study was the first, to the best of our knowledge, to indicate that ML7 may ameliorate VED and AS by regulating the TJ proteins ZO-1 and occludin via mechanisms involving MLCK and MLC phosphorylation. [ABSTRACT FROM AUTHOR]

Melatonin (MLT) is an endogenous indole compound with numerous biological activities that has been associated with atherosclerosis (AS). In the present study, rabbits were used as an AS model in order to investigate whether MLT affects endothelial cell permeability, myosin light chain kinase (MLCK) activity and MLCK expression via the mitogen-activated protein kinase (MAPK) pathway. Expression and activity of MLCK were measured using western blot analysis, quantitative polymerase chain reaction, immunohistochemistry and γ-32P-adenosine triphosphate incorporation. Endothelial permeability was detected using rhodamine phalloidin fluorescence staining. The phosphorylation of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in endothelial cells were also analyzed using western blot analysis. Atheromatous plaques were formed in rabbits with a high cholesterol diet; however, following treatment with MLT, the number and areas of atheromatous plaques were significantly reduced. In addition, MLT treatment reversed the increase of MLCK activity and expression that occurred in rabbits with high cholesterol intake. Furthermore, levels of phosphorylated ERK, JNK and p38 decreased following MLT treatment. In conclusion, the results of the present study indicated that AS may be associated with increased MLCK expression and activity, which was reduced following treatment with MLT. The mechanism of action of MLT was thought to proceed via modulating MAPK pathway signal transduction; however, further studies are required in order to fully elucidate the exact regulatory mechanisms involved. [ABSTRACT FROM AUTHOR]