Xun Qu

Fucoidan stimulation induces a functional maturation of human monocyte-derived dendritic cells.

Fucoidan is a complex sulfated polysaccharide with a wide variety of biological activities for modulating immune cell functions. However, the effects of fucoidan on maturation process and activation of human monocyte-derived dendritic cells (DCs) remain to be elucidated. The present study demonstrated that the level of special marks and polarization phenotype of DCs was altered by fucoidan. Human monocytes were cultured with GM-CSF and IL-4 for 5 days followed by another 2 days in the presence of fucoidan or LPS. Then DCs were harvested on day 7 and were examined using functional assays. We demonstrated that fucoidan up-regulated the expression of HLA-DR and co-stimulatory molecules of DCs. However the endocytic activity was impaired markedly. Fucoidan induces their Th1-promoting tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) secretion, and enhances their allostimulatory capacity. In an allogeneic MLR assay, DCs treated with fucoidan were potent in the secretion of IL-12p70, TNF-alpha and IFN-gamma. Naive T cells stimulated by fucoidan-treated DCs differentiated towards a helper T cell type 1 (Th1) response depending on IL-12 secretion. These results suggest that fucoidan may induce immature DCs maturation and drive their differentiation towards a Th1-polarizing phenotype. Moreover, our data suggest that DCs appear to be a potential target for the immunomodulatory capacity of fucoidan and fucoidan may be used on DC-based vaccines for cancer immunotherapy.

Hypoxia skews dendritic cells to a T helper type 2-stimulating phenotype and promotes tumour cell migration by dendritic cell-derived osteopontin

It is well recognized that tissue microenvironments are involved in regulating the development and function of dendritic cells (DC). Oxygen supply, which varies in different tissues, has been accepted as an important microenvironmental factor in regulating the biological functions of several immune cells and as being involved in tumour progression and metastasis. However, little is known about the effect of hypoxia on the biological functions of DC and the effect of these hypoxia‐conditioned DC on tumour metastasis. In this study, we analysed the transcriptional profiles of human monocyte‐derived immature DC (imDC) and mature DC (mDC) cultured under normoxia and hypoxia by microarray, and found a body of potential targets regulating the functions of DC during hypoxia. In addition, the phagocytic ability of hypoxic imDC markedly decreased compared with that of normoxic imDC. Importantly, hypoxic DC poorly induced the proliferation of allogeneic T cells, but polarized allogeneic CD4+ naive T cells into a T helper type 2 (Th2) response. Moreover, hypoxic DC secreted large amounts of osteopontin, which were responsible for the enhanced migration of tumour cells. Therefore, our study provides new insights into the biological functions of DC under hypoxic conditions and one of mechanisms underlying tumour immune escape during hypoxia.

BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1

Inadequate vascularization limits the repair of bone defects. In order to improve angiogenesis and accelerate osteogenesis, the synergism of co-cultured cells with genetic modification in bone regeneration was investigated in this study. Endothelial progenitor cells (EPCs) and bone marrow stem cells (BMSCs) were transfected with the genes of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) by adenovirus, respectively. The co-cultured cells, designated as four groups including BMSC + EPC, Ad-BMP2-BMSC + EPC, BMSC + Ad-VEGF-EPC, and Ad-BMP2-BMSC + Ad-VEGF-EPC groups, were seeded on an alginate gel and then implanted into rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis. Both VEGF and BMP2 could induce the overexpression of inhibitor of DNA-binding 1(Id1) gene which significantly promoted tube formation in vitro and increase the amount of blood vessels in the Ad-BMP2-BMSC + Ad-VEGF-EPC group after implantation. Nevertheless, overexpression of Id1 retarded the terminal differentiation of osteoblasts and the bone formation. Later, osteogenic gene expression at transcriptional level, calcium nodules, and alkaline phosphatase (ALP) activity showed a gradual decrease and the amount of newly formed osteogenesis area exhibited a small increase in the Ad-BMP2-BMSC + Ad-VEGF-EPC group. This finding suggests that a balanced regulation of Id1 expression in VEGF-EPCs and BMP2-BMSCs may be critical to cell-based and gene-based approaches for bone regeneration.

Hypoxia suppresses the production of MMP-9 by human monocyte-derived dendritic cells and requires activation of adenosine receptor A2b via cAMP/PKA signaling pathway.

The migration of dendritic cells (DCs) from the site of antigen-encounter to regional lymphoid organs is crucial for DCs to function as potent antigen-presenting cells. Matrix metalloproteinase-9 (MMP-9) is critically for DCs migration across extracellular matrix (ECM). We verified in previous studies that hypoxia diminished the production of MMP-9 in human monocyte-derived DCs via an unknown mechanism. In this study, we found, for the first time to our knowledge, that hypoxia altered the expression of adenosine receptors on matured DCs (mDCs) toward the predominant expression of adenosine receptor A(2b). MRS1754 (an A(2b)-receptor specific antagonist) was able to counteract the inhibition of hypoxia on MMP-9 by mDCs. We also found that forskolin (a direct adenylate cyclase activator) can mimic the action of hypoxia on the production of MMP-9 by DCs, whereas the adenylate cyclase inhibitor SQ22536 and the PKA inhibitor H89 can abrogate the inhibition of MMP-9 produce by mDCs under hypoxia. The results herein provide initial evidence that the inhibitory effect of hypoxia on MMP-9 by mDCs requires the activation of A(2b) in a cAMP/PKA-dependent pathway. These data offer new insights into our understanding of the molecular mechanisms underlying the migratory function of DCs in local-tissue hypoxic microenvironments.

RhoBTB2 (DBC2) functions as tumor suppressor via inhibiting proliferation, preventing colony formation and inducing apoptosis in breast cancer cells

RhoBTB2 was isolated recently as a tumor suppressor gene from sporadic breast cancer. Although RhoBTB2 was found to be frequently lost in breast cancer and a variety of cancers, its antitumor effect, however, remains unclear. In this study, we constructed a recombinant expression vector pEGFP-N1-RhoBTB2 and transfected it into RhoBTB2-negative breast tumor cell line T-47D. Stable transformanted cells were identified by fluorescence microscope, RT-PCR and Western blot. Cell viability was measured by MTT assay. Colony forming efficiency of breast tumor cells was detected by colony formation assay. Morphological change of apoptotic cells was observed by hematoxylin-eosin staining. Apoptotic ratio was determined by flow cytometry. Cell invasion and migration ability assay were performed using transwell system. Overexpression of RhoBTB2 in breast tumor cells significantly inhibited the proliferation and colony formation of tumor cells. In addition, RhoBTB2 also elevated the apoptotic ratio and caused typical changes of apoptotic morphology in breast tumor cells of RhoBTB2 overexpression. But RhoBTB2 did not influence the invasion and migration ability of breast tumor cells. Therefore, RhoBTB2 is an important tumor suppressor gene related with breast cancer and may play antitumor roles by inhibiting proliferation, preventing colony formation and promoting the apoptosis of tumor cells. However, the precise mechanism behind the antitumor effects of RhoBTB2 needs to be investigated further.

Comparison of the effects of marchantin C and fucoidan on sFlt-1 and angiogenesis in glioma microenvironment.

Objectivesu2002 This study aimed to examine the effects of marchantin C and fucoidan on angiogenesis induced by glioma cells and monocytes, and to elucidate the role of sFlt‐1 in this process.

OPN promotes survival of activated T cells by up-regulating CD44 in patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa, which represents cell-mediated autoimmune diseases. Pathological study demonstrated that abundant T lymphocytes infiltrated the oral mucosa, in which the activated T cells that trigger apoptosis of oral epithelial cells is an important mechanism for OLP. However, to date the molecular mechanisms underlying the T lymphocytes infiltration and accumulation in OLP remain unclear. In this paper, we found that the levels of plasma OPN were elevated and were associated with the up-regulated expressions of CD44 in OLP patients. In vitro, the addition of exogenous OPN can suppress the apoptosis of activated CD8(+) T cells via CD44, and this T cell resistance to apoptosis may be attributed to the reduction of endogenous mature granzyme B. Our results suggested that the abnormally elevated levels of OPN may contribute to the abnormal infiltration and accumulation of the activated T cells by up-regulating CD44 in OLP.

Expression of RGC32 in human normal and preeclamptic placentas and its role in trophoblast cell invasion and migration

INTRODUCTIONnPreeclampsia (PE) is a pregnancy-specific complication and it is related to insufficient extravillous trophoblast invasion. To date, the pathophysiology of PE has not yet been fully elucidated. Response gene to complement 32 (RGC32) is a novel cellular protein, and it plays important roles in the regulation of cell differentiation, angiogenesis, migration, and invasion. This study aimed to determine the RGC32 expression and function in human placentas and to explore the underlying mechanisms.nnnMETHODSnRGC32 expression in term placentas collected after cesarean section from pregnant women with PE and normal pregnant women was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The effects of RGC32 expression on trophoblast invasion, migration, and the underlying mechanisms were studied in HTR8/SVneo cells.nnnRESULTSnThe messenger RNA (mRNA) and protein levels of RGC32 were significantly downregulated in preeclamptic placentas compared with normal controls (P < 0.05). RGC32 silencing significantly inhibited HTR8/SVneo cell migration and invasion (P < 0.001, respectively). These effects were associated with decreased activities and expression of matrix metalloproteinase (MMP)-2/9, and with the reduced phosphorylation level of Akt.nnnDISCUSSIONnRGC32 may play important roles in the pathophysiology of PE by directly affecting the invasion/migration of trophoblast.

Stable gene-silence of Kif2a synergistic with 5-fluorouracil suppresses oral tongue squamous cell carcinoma growth in vitro and in vivo.

OBJECTIVEnSquamous cell carcinoma of the oral tongue (SCCOT) is one of the most common malignant carcinomas in the head and neck. Recurrence and/or metastasis often results in failure of treatment and decreases the survival of the patients. The purpose of this study is to investigate the effect of gene-silence of Kif2a on SCCOT in viro and in vivo.nnnSTUDY DESIGNnPlasmid-mediated expression of Kif2a-siRNA (pGPU6/GFP/Kif2a) was employed to silence the expression of Kif2a in Tca8113 cells at both mRNA and protein levels. Tca8113 cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and growth of Tca8113 tumors was determined by intra-tumor injection of pGPU6/GFP/Kif2a in nude mice.nnnRESULTSnGene-silence of Kif2a suppressed Tca8113 cell proliferation. pGPU6/GFP/Kif2a synergized the tumor suppression effect of 5-Fluorouracil (5-Fu) on Tca8113 cells.nnnCONCLUSIONSnOur data support that Kif2a is a potential molecular target for the therapeutics of recurrent and metastatic SCCOT.

Single-molecule-counting protein microarray assay with nanoliter samples and its application in the dynamic protein expression of living cells

A novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of proteins was provided using total internal reflection fluorescence microscopy coupled with quantum dot (QD)-labeling. In the SMCMA, the microarray consisting of ∼ 300 μm diameter microspots with the spot-to-spot pitch distance of 500 μm was fabricated by spotting 1.8 nL of solutions containing the target protein onto the substrate which was modified with primary antibody of the protein and blocked with ethanolamine and BSA using a pin-tool type microarraying robot. Then, biotinylated secondary antibody of the protein was bound to the protein to form sandwich immunocomplexes. After labeling with streptavidin-coated QDs, the whole image of the microarray was acquired using a homemade single-molecule microarray reader. The target protein was quantified based on the number of bright dots from the QDs corresponding to single target protein molecules on the microarray. Using the SMCMA, an amount as low as 1.5 × 10(-21) mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells.