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High-fat diet (HFD) induced obesity is associated with low-grade inflammation, insulin resistance (IR), and glucose intolerance. The objective of this study is to assess the effect of interleukin 10 (IL10), an anti-inflammatory cytokine, on blocking HFD-induced obesity and obesity-associated metabolic disorders by hydrodynamic delivery of IL10-containing plasmid. Animals fed a regular chow or HFD received two injections (one on day 1 and the other on day 31) of plasmids containing green fluorescence protein (GFP) or mouse IL10 (mIL10) gene. Blood concentration of mIL10 reached ~200 ng/ml on day 7 in animals receiving mIL10 plasmid DNA. The transfection efficiency of liver cells was the same in animals fed a regular chow or HFD. No difference was seen in animals on regular chow when injected with plasmids containing either gfp or mIL10 gene. Overexpression of mIL10 prevented weight gain of animals on HFD. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) showed that mIL10 maintained insulin sensitivity and prevented glucose intolerance. The mechanistic study reveals that mIL10 suppressed macrophage infiltration and reduced the development of crown-like structures in adipose tissue (AT). Collectively, these results suggest that maintaining a higher level of IL10 through gene transfer could be an effective strategy in preventing diet-induced obesity.

Glioblastomas are the most aggressive forms of primary brain tumors due to their tendency to invade surrounding healthy brain tissues, rendering them largely incurable. The water channel protein, Aquaporin-4 (AQP4) is a key molecule for maintaining water and ion homeostasis in the central nervous system and has recently been reported with cell survival except for its well-known function in brain edema. An increased AQP4 expression has been demonstrated in glioblastoma multiforme (GBM), suggesting it is also involved in malignant brain tumors. In this study, we show that siRNA-mediated down regulation of AQP4 induced glioblastoma cell apoptosis in vitro and in vivo. We further show that several apoptotic key proteins, Cytochrome C, Bcl-2 and Bad are involved in AQP4 signaling pathways. Our results indicate that AQP4 may serve as an anti-apoptosis target for therapy of glioblastoma.

Liver X receptor (LXR) activation improves glucose homeostasis in obesity. This improvement, however, is associated with several side effects including hyperlipidemia and hepatic steatosis. Activation of peroxisome proliferator-activated receptor alpha (PPARα), on the other hand, increases fatty acid oxidation, leading to a reduction of hyperlipidemia. The objective of this study was to investigate whether concurrent activation of LXR/PPARα can produce synergistic benefits in treating obesity-associated metabolic disorders. Treatment of high fat diet-induced obese mice with T0901317, an LXR activator, or fenofibrate, the PPARα agonist, or in combination alleviated insulin resistance and improved glucose tolerance. The combined treatment dramatically exacerbated hepatic steatosis. Gene expression analysis in the liver showed that combined treatment increased the expression of genes involved in lipogenesis and fatty acid transport, including srebp-1c, chrebp, acc1, fas, scd1 and cd36. Histochemistry and ex vivo glycerol releasing assay showed that combined treatment accelerated lipid mobilization in adipose tissue. Combined treatment also increased the transcription of glut4, hsl, atgl and adiponectin, and decreased that of plin1, cd11c, ifnγ and leptin. Combined treatment markedly elevated the transcription of fgf21 in liver but not in adipose tissue. These results suggest that concurrent activation of LXR and PPARα as a strategy to control glucose and lipid metabolism in obesity is beneficial but could lead to elevation of lipid accumulation in the liver.

The strategy of encapsulating small interference RNA (siRNA) into the mesopores enables mesoporous silica nanoparticles (MSNs) to be an attractive material for siRNA delivery. Nevertheless, the gene silencing efficiency mediated by this sort of vehicles has not been systematically investigated yet. In this work, through quantifying gene silencing efficiency by flow cytometry (FCM) and performing intracellular tracking by confocal laser scanning microscopy (CLSM), we found that the RNA interference (RNAi) efficiency mediated by MSNs-based delivery vehicles was strongly dependent on their endosomal escape capability. Thereafter, we investigated the correlation between the gene knockdown efficiency and the endosomal escape kinetics of such carriers. The results indicated that highly efficient MSNs-based siRNA delivery vectors must possess the capability of initiating effectively endosomal escape before the degradation of their packaged siRNA in endolysosomes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: To examine the effect of farnesoid X receptor (FXR) activation by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) on diet-induced obesity and hepatic steatosis., Methods: Fifteen week-old C57BL/6 mice fed with high-fat diet (HFD) or high-fat, high-cholesterol diet were treated by twice weekly injection of GW4064 (50 mg/kg) intraperitoneally or DMSO (carrier solution) for 6 weeks. Body weight, body composition and food intake were monitored weekly. Serum glucose and insulin levels and lipid content in the liver were measured at the end of study. Additionally, genes involved in lipogenesis, gluconeogenesis and inflammation were analyzed by real time PCR. CD36 protein level was detected by western blot., Results: Activation of FXR by GW4064 suppressed weight gain in C57BL/6 mice fed with either HFD or high-fat and high-cholesterol diet. GW4064 treatment of mice significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter Cd36 gene expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis., Conclusions: The results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders.

RNA interference (RNAi) is widely regarded as a promising technology for disease treatment, yet one major obstacle for its clinical application is the lack of efficient siRNA delivery vehicles. In this study, we described a magnetic mesoporous silica nanoparticles (M-MSNs)-based, polyelectrolyte (polyethylenimine, PEI) and fusogenic peptide (KALA)-functionalized siRNA delivery system (denoted as M-MSN_siRNA@PEI-KALA), which was highly effective for initiating target gene silencing both in vitro and in vivo. The construction of this delivery system began with the encapsulation of siRNA within the mesopores of M-MSNs, followed by the coating of PEI on the external surface of siRNA-loaded M-MSNs and the chemical conjugation of KALA peptides. The as-prepared delivery vehicles, with notable siRNA protective effect and negligible cytotoxicity, could be easily internalized into cells, readily escape from the endolysosomes and release the loaded siRNA into the cytoplasm. As a result, the knockdown of enhanced green fluorescent protein (EGFP) and vascular endothelial growth factor (VEGF) in tumor cells were observed, both with excellent RNAi efficiencies. In the following in vivo experiments, the intratumoral injection of M-MSN_VEGF siRNA@PEI-KALA significantly inhibited the tumor growth, possibly by the suppression of neovascularization in tumors. To sum up, we have established a highly effective MSNs-based delivery system, which has great potential to serve as therapeutic siRNA formulation for cancer treatment., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Human arylamine N-acetyltransferase 1, (HUMAN)NAT1, is a phase II xenobiotic-metabolizing enzyme that plays an important role in drug and carcinogen biotransformation and cancer development. Its gene expression has been shown to be regulated by environmental factors. The purpose of the current study is to determine the involvement of nuclear receptors in transcriptional regulation of (HUMAN)NAT1 gene. We show that among the nuclear receptors examined, including the glucocorticoid receptor, retinoid acid receptor-related orphan receptor alpha, constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and retinoic acid receptor, the glucocorticoid receptor plays a dominant role in regulating (HUMAN)NAT1 gene expression through distal promoter (P3). The involvement of the glucocorticoid receptor in transcription regulation of (HUMAN)NAT1 gene expression was demonstrated by dexamethasone treatment, reporter assay using plasmid-containing 3 kbp of 5'-end region of promoter 3, and treatment of anti-glucocorticoid RU486 in primary culture of human hepatocytes and transfected HepG2 cells. In addition, translation inhibition did not affect dexamethasone-induced gene expression through P3, suggesting that dexamethasone effect is directly mediated by glucocorticoid receptor activation. Furthermore, deletion analysis revealed the presence of multiple responsive elements within the 3 kbp fragment of P3. Transfection assays in mice using hydrodynamics-based procedure and reporter gene assay in a mouse cell line revealed that glucocorticoid-induced NAT gene expression is species dependent. Dexamethasone treatment of transfected mice and mouse cell line decreased (MOUSE)Nat2 gene expression, (HUMAN)NAT1 homologue. These results suggest that glucocorticoids serve as a modulator for (HUMAN)NAT1 gene expression via the P3-containing 5'-flanking region.

Long circulation time is critical for blood pool contrast agents used in high-resolution magnetic resonance angiography. For iron oxide particle contrast agents, size and surface properties significantly influence their in vivo performance. We developed a novel long-circulating blood pool contrast agent by introducing zwitterionic structure onto the particle surface. Zwitterionic structure was fabricated by 3-(diethylamino)propylamine (DEAPA) grafted onto the surface of ployacrylic acid coated magnetite nanoparticles via EDC/NHS [N-(3-dimethylaminopropyl)-N'-ethylcarbo-diimide hydrochloride/N-hydroxysuccinimide] coupling chemistry. Zwitterionic particles demonstrated five times lower macrophage cell uptake than the original particles and low cell toxicity. Magnetic resonance angiography indicated that zwitterionic nanoparticles had much longer in vivo circulation time than the original particles and were an ideal candidate for blood pool contrast agent. We suppose that zwitterionic modification by DEAPA and EDC/NHS can be used generally for coating nanoparticles with carboxyl surface and to prolong their circulating time., (Copyright © 2012 John Wiley & Sons, Ltd.)

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ∼1 μM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1β. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.

Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.

The Notch pathway displays several functions related to tumor progression. Breast carcinomas commonly express Notch1, Notch2, Notch3 and Notch4 at variable levels and these are mainly involved in differentiation, proliferation and survival. Notch1 can also induce the invasion of breast cancer cells. However, the precise role and mechanism of Notch1 in tumor invasion remains unclear. In this report, we used small interference RNA technology to knock down the expression of Notch1, resulting in reduced migration and invasion of breast cancer cells. Meanwhile, F-actin polymerization, which is essential for cellular generation of the forces needed for motility, was also impaired in Notch1 knockdown cells. We further investigated the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteases-2 (MMP-2) and MMP-9, and found that the expression of functional EMMPRIN and MMP-2 was significantly decreased in Notch1 knockdown cells, while the expression of MMP-9 was constant. Additionally, the silencing of Notch1 expression likewise impaired cell-to-matrix and cell-to-cell adhesion. Western blotting results showed that reduction of Notch1 levels impacted the phosphorylation of PAK, phosphorylation of Akt, phosphorylation of FAK, the phosphorylation of integrin β1, ICAM-1 and β-catenin. Collectively, these findings suggest that targeting Notch1 has important therapeutic value in breast cancer.

Magnetofection is an efficient new physical gene transfection technology. Despite its effective gene delivery capability, till now relatively little work has been conducted on the mechanism of magnetofection, especially the intracellular fates of the components of magnetofectins and their effects on magnetofection. In this study, we investigated the mechanism of magnetofection using magnetofectins that were prepared via electrostatic self-assembly of the three components: polyethyleneimine (PEI)-coated magnetic nanoparticles (MNPs-PEI), plasmid DNA (pDNA) and PEI in the free form (free PEI). TEM observation and agarose gel electrophoresis assays have indicated MNPs play the role of driving magnetofectins to the cell surface without entering into the nucleus. Confocal microscopic tracking of fluorescence-labeled PEI has shown that the free PEI (green) can be found in the nucleus but almost all of the MNPs-PEI (red) are confined in the cytoplasm in COS-7 cells 30 min post-transfection or in SPC-A1 cells 90 min post-transfection, implying that the pDNA/PEI complex must separate from MNPs-PEI before entering into the nucleus. In addition, reporter gene assays showed the magnetofectins, in which the free PEI was absent, failed to transfect SPC-A1 or COS-7 cell lines; and there was an optimal ratio of the constituents of magnectofectins to achieve optimal transfection efficiency by balancing stable complex formation and facile release of PEI/pDNA from the complex. In summary, our findings further the knowledge of magnetofection and can be helpful for the design and preparation of gene delivery vehicles for effective magnetofection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s inhibited migration and invasion of glioma cells. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in migration and invasion. Several key proteins, including cofilin, LIMK, PAK, FAK, integrin β1, and MMP-9, which are critical for cells migration and invasion, were probably involved in ITSN1-s signaling pathways. These results suggest that ITSN1-s contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP-9. Our results indicate that ITSN1-s is a critical factor in gliomas invasion and identify that ITSN1-s is a new potentially antiinvasion target for therapeutic intervention in gliomas., (Copyright © 2011 Wiley-Liss, Inc.)

Glioblastoma is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering tumors of this type largely incurable. Aquaporin-4 (AQP4) is a key molecule involved in maintaining water and ion homeostasis in the central nervous system and has been recently reported to play a role in cell migration in addition to its well-known function in brain edema. Increased AQP4 expression has been demonstrated in glioblastoma multiforme (GBM), suggesting that it is also involved in malignant brain tumors. Here, we identify a novel role for aquaporin-4 in glioblastoma cell migration and invasion. In the present study, we used small-interference RNA technology and a pharmacological inhibitor to knock down the expression of AQP4, which resulted in specific and massive impairment of glioblastoma cell migration and invasion in vitro and in vivo. In addition, we demonstrated the possible mechanisms by which AQP4 functions in the process of glioblastoma cell invasion. The downregulation of matrix metalloprotease-2 (MMP-2) expression in LN229 cells with AQP4 reduction coincided with decreased cell invasive ability. Furthermore, our study showed that AQP4 may also be involved in the regulation of glioblastoma cell adhesion. The expression of β-catenin and connexin 43 were increased in AQP4-downregulated LN229 cells consistent with their enhanced cell-cell adhesion ability. In summary, our results indicate that AQP4 is involved in the control of glioblastoma cell migration and invasion and may be a potential therapeutic target for glioblastoma cell infiltration.

[Gd@C82(OH)22]n, a fullerene-based nanoparticle, exhibits potent anti-tumor effects in mouse tumor-bearing models without detectable toxicity and the pathological studies revealed a massive infiltration of leukocytes in the residual tumors of [Gd@C82(OH)22]n-treated mice. We report here that [Gd@C82(OH)22]n promotes macrophages secreting pro-inflammatory cytokines IL-6 and TNF-alpha, enhances the expression of MHC-II and costimulatory molecules such as CD40 and CD54, increases endocytosis and cell adhesion. Furthermore, [Gd@C82(OH)22]n-treated macrophages became functionally activated as illustrated by their capacity to activate allogeneic T cells. Taken together, our results indicate that [Gd@C82(OH)22]n nanoparticle is a potent activator of macrophages, which may in part account for its potent anti-tumor effect.

Malignant gliomas have a high proliferation ability and high tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (also called ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s induced glioma cells apoptosis. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in glioma cells apoptosis. Our data demonstrate that several key proteins, including FAK, Akt, Bcl-2, BAD which are critical for cells apoptosis were probably involved in ITSN1-s signaling pathways. Our results indicate that ITSN1-s is an effecter in regulation of gliomas cells apoptosis, and identify that ITSN1-s may be a new potentially anti-apoptosis target for therapeutic of gliomas., (2010 Elsevier B.V. All rights reserved.)

The collagen gel droplet embedded culture-drug sensitivity test (CD-DST) is an anticancer drug sensitivity test developed about 10 years ago. This study reports the application of this test in the choice of neoadjuvant chemotherapy for the treatment of one patient with a large synovial sarcoma in the right shank. A 28-year old man presented at our hospital with a large mass in his right shank which had a hard texture and an obscure boundary. The histopathological diagnosis of excisional biopsy specimens was synovial sarcoma with low differentiation. Theprubicin/Cisplatin (THP/CDDP) neoadjuvant chemotherapy was selected based on the results obtained from the CD-DST. After three courses, a computed tomography (CT) scan was performed which indicated that the volume of the tumor had decreased significantly. Additionally, tumor necrosis, as well as the clinical response, showed complete response. The histopathological diagnosis of wide excision specimens indicated a grade III chemotherapy response. The patient was alive and without recurrence after a follow-up of 16 months. Our results indicated that the CD-DST is a useful tool for selecting neoadjuvant chemotherapeutic drugs for patients with synovial sarcoma.

In our previous study, [Gd@C(82)(OH)(22)](n), a fullerene-based nanoparticle, exhibited potent anti-tumor effects in mouse tumor-bearing models without detectable toxicity. The mechanism involved in the anti-tumor effect exerted by [Gd@C(82)(OH)(22)](n) remains to be elucidated. This study found that glioblastoma cells treated with [Gd@C(82)(OH)(22)](n) nanoparticles showed a significant impairment in migration and adhesion by cell chemotaxis, scratch and adhesion assays in vitro. Furthermore, our data showed that the key proteins, CD40 and ICAM-1, were involved in the inhibition of adhesion in the [Gd@C(82)(OH)(22)](n) nanoparticle-treated glioblastoma cells. Thus, our study suggests that the [Gd@C(82)(OH)(22)](n) nanoparticle is a new potential anti-tumor effector and a therapeutic component for malignant glioblastoma infiltration.

Cisplatin is the most important chemotherapeutic agent involved in treatment of head and neck squamous cell carcinoma (HNSCC), but cisplatin resistance in HNSCC is still a serious problem in clinic. The reasons why patients fail chemotherapy are unclear. We examined 25 HNSCC patients who were all tested for cisplatin sensitivity by CD-DST (collagen gel droplet embedded culture-drug sensitivity) method and expression of Stat3 and Notch1. We found that high expression levels of Stat3 and Notch1 were closely associated with cisplatin resistance respectively (P=0.014, P=0.000). In addition, cisplatin resistance of HNSCC was decreased after inhibition of Stat3 or Notch signaling in vitro. Our results provide first evidence that both high Stat3 and Notch1 expression are associated with cisplatin resistance in HNSCC patients, supporting the hypothesis that co-activation of Stat3 and Notch1 by their crosstalk induces the reprogrammed survival pathways in HNSCC responding to chemotherapy.

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. The serine/threonine kinase Akt2 is well known as an important regulator of cell survival and growth. In this study, we show that siRNA-mediated depletion of Akt2 inhibited migration and invasion of glioma cells. In addition, we demonstrate the mechanisms by which Akt2 functions to promote cell migration and invasion. Phosphorylation of cofilin, a critical step of actin polymerization, and phosphorylation of Girdin, essential for the integrity of the actin cytoskeleton and cell migration, were impaired. Furthermore, epidermal growth factor-induced ACAP1 phosphorylation and integrin beta1 phosphorylation were also blocked, consistent with defects in adhesion. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during migration. Decreased MMP-9 expression in Akt2 knocked-down glioma cells was subsequently confirmed by Western blotting, consistent with the decreased invasion in vitro and in vivo. These results suggest that Akt2 contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP-9. Our immunohistochemistry results by using human gliomas tissue sections also indicated that Akt2 expression was closely related with the malignancy of gliomas. This is coincident with our in vivo and in vitro results from cell lines. All of these results indicate that Akt2 is a critical factor in gliomas invasion. This study identifies that Akt2 is a potentially antiinvasion target for therapeutic intervention in gliomas.

The crosstalk, mediated by chemoattractants, between cancer cells and tumor-associated macrophages, plays an important role in tumor invasion and metastasis. Our previous study reported that atypical protein kinase C zeta (PKCzeta) regulates epidermal growth factor-induced chemotaxis of human breast cancer cells. In this study, we investigated the role of PKCzeta in CSF-1-induced chemotaxis of macrophages. Knockdown of PKCzeta by small interference RNA impaired CSF-1-induced chemotaxis of human acute monocytic leukemia cell line THP-1, which was probably a result of a decrease in CSF-1-induced phosphorylation of LIN-11, Is11, and MEC-3 protein domain kinase (LIMK)/cofilin and actin polymerization. Furthermore, silencing PKCzeta expression also impaired migration of mouse peritoneal macrophages. Scratch analysis indicated that PKCzeta was required for macrophage migration. Therefore, PKCzeta is required for CSF-1-induced chemotaxis of macrophages. Blocking activation of PKCzeta will be a novel strategy to inhibit cancer metastasis by blocking migration of cancer cells and macrophages.

Glioblastomas are the most aggressive forms of primary brain tumors with their tendency to invade surrounding healthy brain tissues, rendering them largely incurable. In this report, we used small-interference RNA technology to knock down the expression of protein kinase C (PKC) zeta, which resulted in specific and massive impairment of glioblastoma cell migration and invasion. We also explained the fundamental molecular processes of glioblastoma migration and invasion in which PKCzeta is a participant. The silence of PKCzeta expression likewise impaired the phosphorylation of LIN-11, Isl1 and MEC-3 protein domain kinase (LIMK) and cofilin, which is a critical step in cofilin recycling and actin polymerization. Consistent with the defects in cell adhesion, phosphorylation of integrin beta1 was also dampened. Therefore, PKCzeta regulated both cytoskeleton rearrangement and cell adhesion, which contributed to cell migration. Additionally, there was down-regulation of matrix metalloprotease-9 expression in siPKCzeta/LN-229 cells, which coincided with decreased invasion both in vitro and in vivo. These results indicate that PKCzeta is involved in the control of glioblastoma cell migration and invasion by regulating the cytoskeleton rearrangement, cell adhesion, and matrix metalloprotease-9 expression. Collectively, these findings suggest that PKCzeta is a potential therapeutic target for glioblastoma infiltration.

Tumor-associated macrophages play an important role in tumorigenesis and metastasis. Trafficking of macrophages to the proximity of tumors is mediated by CSF-1, a growth factor. In this study, we investigated the role of PKB/Akt in CSF-1-induced macrophage migration. Disruption of Akt2 expression by small interference RNA impaired chemotaxis of both THP-1 cells and mouse peritoneal macrophages. Phosphorylation of PKCzeta, an essential component in chemotaxis signaling pathway, was reduced. LIMK/Cofilin, downstream of PKCzeta, regulated cytoskeleton rearrangement during cell migration. Disruption of Akt2 expression inhibited CSF-1-induced LIMK/Cofilin phosphorylation, which contributed to defects in actin polymerization and chemotaxis. Furthermore, MCP-1, a chemokine, -induced macrophage chemotaxis was also impaired. Taken together, our results demonstrated that Akt2 plays an essential role in both CSF-1- and chemokine-induced chemotaxis of macrophages.

The oxysterol receptor LXR is a key transcriptional regulator of lipid metabolism. LXR increases expression of SREBP-1, which in turn regulates at least 32 genes involved in lipid synthesis and transport. We recently identified 25-hydroxycholesterol-3-sulfate (25HC3S) as an important regulatory molecule in the liver. We have now studied the effects of 25HC3S and its precursor, 25-hydroxycholesterol (25HC), on lipid metabolism as mediated by the LXR/SREBP-1 signaling in macrophages. Addition of 25HC3S to human THP-1-derived macrophages markedly decreased nuclear LXR protein levels. 25HC3S administration was followed by dose- and time-dependent decreases in SREBP-1 mature protein and mRNA levels. 25HC3S decreased the expression of SREBP-1-responsive genes, acetyl-CoA carboxylase-1, and fatty acid synthase (FAS) as well as HMGR and LDLR, which are key proteins involved in lipid metabolism. Subsequently, 25HC3S decreased intracellular lipids and increased cell proliferation. In contrast to 25HC3S, 25HC acted as an LXR ligand, increasing ABCA1, ABCG1, SREBP-1, and FAS mRNA levels. In the presence of 25HC3S, 25HC, and LXR agonist T0901317, stimulation of LXR targeting gene expression was repressed. We conclude that 25HC3S acts in macrophages as a cholesterol satiety signal, downregulating cholesterol and fatty acid synthetic pathways via inhibition of LXR/SREBP signaling. A possible role of oxysterol sulfation is proposed.

This is a comparative study on the efficacy of differential cell apoptosis induced by three methods (intracellular hyperthermia, with water bath hyperthermia and extracellular hyperthermia) in human lung adenocarcinoma SPC-A1 cells in vitro. The effects of hyperthermia on cell apoptosis were determined by Transmission electron microscopy(TEM), agarose gel electrophoresis and flow cytometry methods, respectively. The intracellular effect of particle heating was compared with that of water bath hyperthermia and extracellular hyperthermia; significant differences between these heating methods were detected, the rate of apoptosis being 36.59%, 5.66%, 7.78% respectively. When treated with intracellular hyperthermia, the SPC-A1 cells manifested typical morphological characters of apoptosis by TEM observation, and the SPC-A1 cell DNA was degraded into large fragments by agarose gel electrophoresis assay. Our results showed that amino-silane Fe3O4 induced intracellular hyperthermia was superior to water bath hyperthermia and extracellular hyperthermia. It is mainly the interaction between intracellular nanoparticles and cell that induced apoptosis. Therefore, the aminosilane-coated Fe3O4 may be used in hyperthermia or chemotherapeutics on cancer cells for further clinical application.

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives (oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a sulfated oxysterol, 5-cholesten-3beta,25-diol 3-sulfate (25HC3S), that is localized to liver nuclei. The present study reports a biosynthetic pathway for 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and sterol 27-hydroxylase (Cyp27A1) gene knockout mice indicated that 25-hydroxycholesterol (25HC) is synthesized by CYP27A1. Incubation of cholesterol or 25HC with mitochondrial and cytosolic fractions in the presence of 3'-phosphoadenosyl 5'-phosphosulfate resulted in the synthesis of 25HC3S. Real-time RT-PCR and Western blot analysis showed the presence of insulin-regulated hydroxycholesterol sulfotransferase 2B1b (SULT2B1b) in hepatocytes. 25HC3S, but not 25HC, decreased SULT2B1b mRNA and protein levels. Specific small interfering RNA decreased SULT2B1b mRNA, protein, and activity levels. These findings demonstrate that mitochondria synthesize 25HC, which is subsequently 3beta-sulfated to form 25HC3S.

Based on the study of extraction of DNA using carboxyl-coated Fe3O4, here we provide a rapid, high efficient, safe and economical method. Using fluorescent dye-Sybr Green I, the quantification of double-strand DNA exhibits the advantages of high sensitivity,good repeatability and convenience.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system. In this experiment, we first demonstrated that StAR expressed in endothelial cells as well. Immunochemistry showed positive staining of StAR in endothelial cells. To investigate whether steroids and oxysterols regulate StAR expression in endothelial cells, mouse brain microvascular endothelial cell line (bEnd.3) was treated with various steroids and oxysterols, including free cholesterol (CHO), low density lipoprotein (LDL), and 25-hydroxycholesterol (25-OH). All these three compounds increased StAR mRNA and protein expression in a time- and dose-dependent manner. When treated with CHO and LDL, the StAR mRNA change was prior to the protein change, suggesting that transcription may be one of the mechanisms of CHO and LDL regulation. In contrast to CHO and LDL, 25-OH increased StAR protein levels independently of mRNA amount. It suggested that 25-OH might regulate StAR activity at post-transcriptional level.

Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-kappaB first increased at 1 h after tryptase-treatment (1 ng/ml) and reached the plateau after 4 h. The NF-kappaB mRNA increased by 3-fold (n = 3, P < 0.05), AP-1 by 2-fold (n = 3, P < 0.05), and PKB by 10-fold (n = 3, P < 0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 microM or antisense PKB cDNA transfection. The up-regulation of NF-kappaB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.