Bromosulfophthalein suppresses inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

OBJECTIVE: It has been reported that glutathione (GSH), the most abundant cellular antioxidant, can inhibit production of pro-inflammatory cytokines by activated macrophages. Bromosulfophthalein (BSP) has been recognized as an inhibitor of the efflux of reduced GSH from cells, leading to an increase in the intracellular GSH level. In this study, we evaluated, for the first time, whether BSP possessed anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages. MATERIALS AND METHODS: RAW 264.7 cells were treated with BSP and the levels of proinflammatory cytokines, GSH, and nitrite were assessed. Gene expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) was analyzed via quantitative RT-PCR. We also examined various inflammatory signaling pathways including Akt/forkhead box protein O1 (FoxO1)/toll-like receptor 4 (TLR4), mitogen-activated protein kinases (MAPKs), and Fas protein by Western blot and flow cytometry analysis. RESULTS: Our study demonstrated that BSP induced an increase in intracellular GSH level in LPS-stimulated macrophages. BSP inhibited production of nitric oxide and proinflammatory cytokines. BSP increased phosphorylation of Akt and nuclear exclusion of FoxO1 and suppressed TLR4 expression. Additionally, BSP decreased MAPKs activation and Fas expression. DISCUSSION AND CONCLUSION: Taken together, these data suggest that BSP can attenuate inflammation through multiple signaling pathways. These findings highlight the potential of BSP as a new anti-inflammatory agent.

Detection of AD-BMP-2-IRES-HIF-1α MU on local promoting angiogenic and osteogenic capacity of necrosis area.

The induced EPCs were transfected by Ad-BMP-2-IRES-HIF-1αmu, and then transplanted into femoral head necrotic zone, the effect on osteogenesis and agiogenesis of necrosis zone was detected. The Ad-BMP-2-IRES-HIF-1α was transfected into induced EPCs and then transplanted into avascular necrotic parts of the femoral head (ANFH).Afterwards, the promotion effect on angiogenic and osteogenic capabilities of the necrosis parts from Ad-BMP-2-IRES-HIF-1α was detected. Rabbit bone marrow MNCs were obtained by density gradient centrifugation method, and were induced into EPCs by M199 medium; EPCs were identified in accordance with the cell morphology, specific surface markers and uptake abilities. The Ad-BMP-2-IRES-HIF-1α was transfected to EPCs and then transplanted into parts of ANFH. The models were euthanized 2 and 4 weeks after operation and then the angiogenic and osteogenic indexes of necrotic parts were detected. The results showed that more blood vessels generated in group A than that in group B and C (P<0.05), and the statistical differences were found between group B and C (P<0.05). The detection of histology and BMP-2 immunohistochemistry showed that there were statistically significant differences between group A and B, group A and C (P<0.05). There was no significant difference between group B and C (P<0.05). To sum up, this experiment shows that the EPCs transfected by Ad-BMP-2-IRES-HIF-1α have stronger angiogenic and osteogenic capabilities.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and ß-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells.

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.

Targeted antitumor effect induced by hTERT promoter mediated ODC antisense adenovirus.

The expression of Ornithine decarboxylase (ODC) which is the first key enzyme of polyamine biosynthesis is increased in cancer cells. We had blocked the polyamine synthesis pathway using the adenoviral-mediated antisense ODC in some cancer cells such as prostate cancers and colorectal cancers. These researches demonstrated that ODC antisense expression could inhibit tumor cell growth. In order to reach the goal of applying the targeting gene therapy in clinical practice, we cloned the antisense ODC RNA which was driven by cancer specific promoter (hTERT promoter; telomerase reverse transcriptase promoter) into the adenovirus vector (rAd-CMV-GFP-hTERTp-ODC). Human cancer cell lines (HepG2, Bel-7402, A549) and normal cell lines (HELF, LO2) were infected separately with rAd-CMV-GFP-hTERTp-ODC as well as with control vector (rAd-CMV-GFP). Luciferase activity assay was performed to determine hTERT promoter activity. Cell growth curves analysis, western blot analysis, flow cytometry analysis and Matrigel invasion assays were performed to assess properties of cell growth and invasiveness. The results showed that there was significant inhibition of ODC expression and cell proliferation in cancer cells treated with rAd-CMV-GFP-hTERTp-ODC compared with cells treated with PBS or rAd-CMV-GFP, and no significant inhibition was detected in normal cells. Our research offers a powerful and safe new therapeutic strategy for cancer targeted treatment.

Antioxidative nanofullerol inhibits macrophage activation and development of osteoarthritis in rats.

Background: There is emerging evidence which suggests that cellular ROS including nitric oxide (NO) are important mediators for inflammation and osteoarthritis (OA). Water-soluble polyhydroxylated fullerene C60 (fullerol) nanoparticle has been demonstrated to have an outstanding ability to scavenge ROS. Purpose: The objective of this study is to assess the effects of fullerol on inflammation and OA by in vitro and in vivo studies. Methods: For in vitro experiments, primary mouse peritoneal macrophages and a macrophage cell line RAW264.7 were stimulated to inflammatory phenotypes by lipopolysaccharide (LPS) in the presence of fullerol. For the animal study, OA model was created by intra-articular injection of monoiodoacetate into the knee joints of rats and fullerol was intravenously injected immediately after OA induction. Results: NO production and pro-inflammatory gene expression induced by LPS was significantly diminished by fullerol in both macrophage cell types. Meanwhile, fullerol could remarkably reduce phosphorylation of p38 mitogen-activated protein kinase, and protein level of transcription factors nuclear factor-kappaB and forkhead box transcription factor 1 within the nucleus. The animal study delineated that systematic administration of fullerol prevented OA, inhibiting inflammation of synovial membranes and the damage toward the cartilage chondrocytes in the OA joints. Conclusion: Antioxidative fullerol may have a potential therapeutic application for OA.

Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription.

BACKGROUND: NKX3.1 and PCAN1 are both prostate-specific genes related to prostate development and prostate cancer. So far, little is known about the regulatory mechanisms of the expression of these two genes. In the present study, we found that NKX3.1 upregulated PCAN1 gene transcription in LNCaP prostate cancer cells. To understand the regulatory mechanisms, our work focused on identifying the functional NKX3.1 binding sites upstream of the PCAN1 gene, which might be involved in the positive regulation of PCAN1 expression by NKX3.1. RESULTS: We cloned and characterized a 2.6 kb fragment upstream of the PCAN1 gene. Analysis of the 2.6 kb sequence with MatInspector 2.2 revealed five potential binding sites of NKX3.1 transcription factor. Luciferase reporter assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and RNA interference were performed to study the effects of NKX3.1 on PCAN1 gene expression in prostate cancer cells. Our results showed that PCAN1 promoter activity and mRNA expression were increased by transfection with the NKX3.1 containing plasmid (pcDNA3.1-NKX3.1) and that PCAN1 mRNA expression was decreased by RNA interference targeting human NKX3.1 in LNCaP prostate cancer cells. The results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that NKX3.1 bound to NBS1 (-1848 to -1836) and NBS3 (-803 to -791) upstream of the PCAN1 gene. The luciferase reporter assays showed that NBS1 and NBS3 enhanced the promoter activity in pGL3-promoter vector with cotransfection of the NKX3.1 containing plasmid. Furthermore, the deletion of NBS1 or both NBS1 and NBS3 reduced PCAN1 promoter activity and abolished the positive regulation of PCAN1 expression by NKX3.1. CONCLUSION: Our results suggested that two functional NKX3.1 binding sites located at -1848 to -1836 and -803 to -791 upstream of the PCAN1 gene were involved in the positive regulation of PCAN1 gene transcription by NKX3.1.

Arrestin-biased AT1R agonism induces acute catecholamine secretion through TRPC3 coupling.

Acute hormone secretion triggered by G protein-coupled receptor (GPCR) activation underlies many fundamental physiological processes. GPCR signalling is negatively regulated by ß-arrestins, adaptor molecules that also activate different intracellular signalling pathways. Here we reveal that TRV120027, a ß-arrestin-1-biased agonist of the angiotensin II receptor type 1 (AT1R), stimulates acute catecholamine secretion through coupling with the transient receptor potential cation channel subfamily C 3 (TRPC3). We show that TRV120027 promotes the recruitment of TRPC3 or phosphoinositide-specific phospholipase C (PLCγ) to the AT1R-ß-arrestin-1 signalling complex. Replacing the C-terminal region of ß-arrestin-1 with its counterpart on ß-arrestin-2 or using a specific TAT-P1 peptide to block the interaction between ß-arrestin-1 and PLCγ abolishes TRV120027-induced TRPC3 activation. Taken together, our results show that the GPCR-arrestin complex initiates non-desensitized signalling at the plasma membrane by coupling with ion channels. This fast communication pathway might be a common mechanism of several cellular processes.

Long non-coding RNA LOC283070 mediates the transition of LNCaP cells into androgen-independent cells possibly via CAMK1D.

AIMS: The present study is to investigate the role of long non-coding RNAs (lncRNAs) in the development of androgen independence in prostate cancer and its underlying mechanism. METHODS: We established an androgen-independent prostate carcinoma (AIPC) cell line LNCaP-AI from androgen-dependent prostate carcinoma (ADPC) cell line LNCaP. Different expression profiles of lncRNAs and mRNAs between LNCaP and LNCaP-AI cells were investigated using microarray analysis. The expression of RNAs was determined using quantitative real-time polymerase chain reaction. Protein levels were measured using Western blotting. MTT assay was used to test cell viability. Tumor formation assay was performed in nude mice to detect tumor growth in vivo. Flow cytometry was performed to detect cell cycles. Transwell assay was employed to test cell migration and invasion. RESULTS: According to bioinformatics prediction, lncRNA LOC283070 could possibly play an important role in the transition of LNCaP cells into LNCaP-AI cells. LOC283070 was up-regulated in LNCaP-AI cells and frequently up-regulated in AIPC cell lines. Overexpression of LOC283070 in LNCaP cells accelerated cell proliferation and migration, even under androgen-independent circumstances. Knockdown of LOC283070 inhibited LNCaP-AI cell proliferation and migration. Moreover, overexpression of LOC283070 promoted tumor growth in vivo in both normal mice and castrated mice. CAMK1D overexpression had similar effect with LOC283070, and CAMK1D knockdown fully abrogated the effect of LOC283070 overexpression on the transition of LNCaP cells into androgen-independent cells. CONCLUSIONS: The present study shows that overexpression of LOC283070 mediates the transition of LNCaP cells into androgen-independent LNCaP-AI cells possibly via CAMK1D.

Crystal Structure and Substrate Specificity of PTPN12.

PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.

[Inhibition of the expression of prostate specific antigen by curcumin].

AIM: To study the effect of curcumin on the expression of prostate specific antigen (PSA). METHODS: AXSYM system-chemical luciferase method was used to examine the content of PSA in prostate cancer cell lines, LNCap after treated with different doses of curcumin. pGL3-PSA luciferase expression vector, containing 640 bp DNA of PSA gene 5' promoter region was constructed and transfected into LNCap cell with lipofectin. Through detecting the activity of luciferase, the effect of curcumin on the promoter of PSA was studied. Western blotting was used to detect expression of androgen receptor (AR) in LNCap cell with different concentrations of curcumin. RESULTS: The expression of PSA was inhibited and activity of luciferase was reduced by curcumin. There was also significant difference in AR expression as shown by Western blotting experiment after treatment of different doses of curcumin. CONCLUSION: Through inhibiting AR expression, curcumin reduced the function of PSA promoter and inhibited PSA protein expression.

The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop.

Protein tyrosine phosphatases have diverse substrate specificities and intrinsic activities that lay the foundations for the fine-tuning of a phosphorylation network to precisely regulate cellular signal transduction. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs. Here, we find that a conserved second-sphere residue, Thr263, located in the surface Q-loop is important for both the function and activity of PTPs. Using PTP1B as a study model, we found that mutations of Thr263 impaired the negative regulation role of PTP1B in insulin signaling. A detailed mechanistic study utilizing steady-state kinetics, Brønsted analysis and pH dependence in the presence of pNPP or phosphopeptide substrates revealed that Thr263 is required for the stabilization of the leaving group during catalysis. Further crystallographic studies and structural comparison revealed that Thr263 regulates the general acid function through modulation of the WPD-loop by the T263:F182/Y/H interaction pair, which is conserved in 26 out of 32 classical PTPs. In addition, the hydrophobic interaction between Thr263 and Arg1159 of the insulin receptor contributes to the substrate specificity of PTP1B. Taken together, our findings demonstrate the general role of the second-sphere residue Thr263 in PTP catalysis. Our findings suggest that the second sphere residues of PTP active site may play important roles in PTP-mediated function in both normal and diseased states.

Adenoviral Delivery of the VEGF and BMP-6 Genes to Rat Mesenchymal Stem Cells Potentiates Osteogenesis.

The combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP) to sites of bone injury results in enhanced repair compared to the administration of a single factor or a combination of two factors. Based on these findings, we hypothesized that coexpression of VEGF and BMP-6 genes would enhance the osteoblastic differentiation of rat bone-marrow-derived stem cells (rMSCs) and osteogenesis by comparison to rMSCs that do not express VEGF and BMP-6. We prepared a GFP tagged adenovirus vector (Ad-VEGF+BMP-6) that contained DNA encoding the hVEGF and hBMP-6 genes. rMSCs were transduced with the virus, and the successful transduction was confirmed by green fluorescence and by production of VEGF and BMP-6 proteins. The cells were cultured to assess osteoblastic differentiation or administered in the Fischer 344 rats to assess bone formation. Mineralization of rMSCs transduced with Ad-VEGF+BMP-6 was significantly enhanced over the nontransduced rMSCs. Only transduced rMSCs could induce osteogenesis in vivo, whereas Ad-VEGF+BMP-6 or nontransduced rMSCs alone did not induce osteogenesis. The data suggests that the combined delivery of MSCs, VEGF, and BMP-6 is an attractive option for bone repair therapy.

Regulation of zinc transporters by dietary zinc supplement in breast cancer.

Zinc is essential for cell growth and is a co-factor for more than 300 enzymes, representing over 50 different enzyme classes. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. However, the mechanisms involved and the relations to zinc transporters are still unknown. A series of zinc transporters are characterized in this article and several of that are emphasized in view of their unique tissue-specific expressions. Established human breast cancer in a nude mice model is used. With a dietary zinc supplement treatment, ZnT-1 mRNA expression in established human breast cancer is raised by 24%, and is nearly 2 times of that in basal diet. ZIP1, ZIP2 and LIV-1 mRNA are the same between two treatment groups. Moreover, no significant changes of these zinc transporters expressions are found between differential breast cancer cell lines in the nude mice model. This is the first report, which detects the zinc transporters expressions in established human breast cancer in nude mice model. These results lead to the constitutive expression and response to zinc in different tissues. In addition to that, ZnT-1 seems to have played an important role in zinc homeostasis, even in breast cancer.