Yucun Liu

Protective Effect of 1,25-Dihydroxyvitamin D3 on Lipopolysaccharide-Induced Intestinal Epithelial Tight Junction Injury in Caco-2 Cell Monolayers

Lipopolysaccharide was found to be elevated in the plasma of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD) patients and may play an important role in the pathogenesis and propagation of these intestinal diseases. To illustrate the destructive effect of lipopolysaccharide (LPS) and to test the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on LPS-induced barrier injury, an in vitro intestinal epithelia barrier model was established with Caco-2 monolayers and treated with clinically relevant concentrations (1–10 ng/ml) of LPS with or without 1,25(OH)2D3. Transepithelial electrical resistance (TEER) and FITC-Dextran 40kda (FD-40) flux were measured to reflect monolayer permeability. We found that LPS at clinically relevant concentrations increased intestinal permeability by downregulating and redistributing tight junction (TJ) proteins. 1,25(OH)2D3 added at baseline or at day 4 abrogated the destructive effect of LPS on monolayer permeability by restoring the expression and localization of TJ proteins. LPS, at clinically relevant concentrations, also downregulated the expression of vitamin D receptor (VDR); 1,25 (OH)2D3, however, could restore the expression of VDR. Our findings illustrate the mechanism underlying the destructive effect of clinically relevant concentrations of LPS on intestinal TJ barrier and provide evidence for the clinical application of vitamin D in LPS-related intestinal barrier dysfunction.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Suppresses Angiogenesis by Down-Regulating the Expression of VEGF and MMP-7 in Gastric Cancer

Background Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that functions to inhibit angiogenesis, proliferation, and invasion in different types of cancer. The ability of SPARC to modulate neovascularisation is believed to be mediated in part by its ability to modulate the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we aimed to determine the effect of SPARC expression in gastric cancer cells on proliferation and angiogenesis in vitro and in vivo. Method We evaluated expression of SPARC in seven human gastric cancer cell lines. Then we established a stably transfected SPARC overexpressed cell line (BGC-SP) and a stably transfected SPARC knock-down cell line (HGC-sh). The effect of SPARC overexpression and SPARC silencing was studied by examining capillary formation of HUVECs in vitro and a dorsal skin-fold chamber model in vivo. Quantitative real-time PCR and western blotting were performed to detect if the expressions of VEGF and MMP-7 were modulated by SPARC expression. To further determine the effect of SPARC expression on angiogenesis in vivo, xenograft models were established and microvessel density (MVD) of different clones were detected by immunohistochemistry. Results Endogenous SPARC overexpression inhibited the expression of VEGF and MMP-7, as well as the angiogenesis induced by BGC-SP cells. Correspondingly, SPARC silencing increased the expression of VEGF and MMP-7, as well as the angiogenesis induced by HGC-sh cells. Elevated angiogenesis induced by SPARC silencing in HGC-sh cells was decreased when VEGF was neutralised by antibodies, and MMP-7 was knocked down in vitro. Conclusion SPARC suppresses angiogenesis of gastric cancer by down-regulating the expression of VEGF and MMP-7.

Downregulation of SPARC expression decreases gastric cancer cellular invasion and survival

BackgroundSecreted protein acidic and rich in cysteine (SPARC) plays a key role in the development of many tissues and organ types. Aberrant SPARC expression was found in a wide variety of human cancers, contributes to tumor development. Because SPARC was found to be overexpressed in human gastric cancer tissue, we therefore to explore the expression of SPARC in gastric cancer lines and the carcinogenic mechanisms.MethodsSPARC expression was evaluated in a panel of human gastric cancer cell lines. MGC803 and HGC 27 gastric cancer cell lines expressing high level of SPARC were transiently transfected with SPARC-specific small interfering RNAs and subsequently evaluated for effects on invasion and proliferation.ResultsSmall interfering RNA-mediated knockdown of SPARC in MGC803 and HGC 27 gastric cancer cells dramatically decreased their invasion. Knockdown of SPARC was also observed to significantly increase the apoptosis of MGC803 and HGC 27 gastric cancer cells compared with control transfected group.ConclusionsOur data showed that downregulating of SPARC inhibits invasion and growth of human gastric cancer cells. Thus, targeting of SPARC could be an effective therapeutic approach against gastric cancer.

1,25(OH)2D3 attenuates TGF-β1/β2-induced increased migration and invasion via inhibiting epithelial-mesenchymal transition in colon cancer cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been reported to inhibit proliferation and migration of multiple types of cancer cells. However, the mechanism underlying its anti-metastasis effect is not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TGF-β1/β2-induced epithelial-mesenchymal transition (EMT) is tested in colon cancer cells. The results suggest that 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased invasion and migration of in SW-480 and HT-29 cells. 1,25(OH)2D3 also inhibited the cadherin switch in SW-480 and HT-29 cells. TGF-β1/β2-induced increased expression of EMT-related transcription factors was also inhibited by 1,25(OH)2D3. 1,25(OH)2D3 also inhibited the secretion of MMP-2 and MMP-9 and increased expression of F-actin induced by TGF-β1/β2 in SW-480 cells. Taken together, this study suggests that the suppression of EMT might be one of the mechanisms underlying the anti-metastasis effect of 1,25(OH)2D3 in colon cancer cells.

1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway.

Substantial studies have demonstrated the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on intestinal barrier function, but the mechanisms are not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TNF-α induced barrier dysfunction was further investigated in Caco-2 cell monolayers. The barrier function of Caco-2 monolayers was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. ZO-1 and Occludin were chosen as markers of the localization of tight junction (TJ) proteins for immunofluorescence. The expression of MLCK and phosphorylation level of myosin light chain (MLC) were measured by immunoblotting. The activation of NF-kB p65 was analyzed by EMSA and immunofluorescence. The results suggest that 1,25(OH)2D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated activation of MLCK-P-MLC signaling pathway.

Tissue factor/activated factor VIIa induces matrix metalloproteinase-7 expression through activation of c-Fos via ERK1/2 and p38 MAPK signaling pathways in human colon cancer cell

PurposeIncreased expression of tissue factor (TF) is associated with tumor invasion and metastasis in human colorectal cancer. We have previously observed that TF/FVIIa upregulates matrix metalloproteinase-7 (MMP-7) expression at the transcriptional level in colon cancer cells. MMP-7 overexpression is believed to play an important role in tumor invasion and metastasis. The aim of this study is to elucidate the molecular mechanisms by which TF/FVIIa induced MMP-7 expression and cell invasion in vitro.MethodsReverse transcription polymerase chain reaction, Western blot, luciferase assay, and chromatin immunoprecipitation (ChIP) were used to determine the potential mechanism and signaling pathways by which TF/FVIIa induced MMP-7 expression and cell invasion in LoVo cells. Small interfering RNA (siRNA) and cell invasion assay was used to examine whether blocking c-Fos expression could abolish FVIIa-mediated upregulation of MMP-7 and cell invasion in vitro.ResultsThe results showed that FVIIa induced the upregulation of MMP-7 both at the mRNA and protein levels in a time- and dose-dependent manner and increased the invasive behavior of LoVo cells. FVIIa enhanced the promoter activity of MMP-7, and the activator protein-1 (AP-1) binding site was responsible for the activation. Site mutation of the AP-1 binding site in the promoter almost completely abolished FVIIa-mediated response. Furthermore, ChIP assay confirmed that FVIIa promoted the direct binding of c-Fos with the MMP-7 promoter in vivo. FVIIa also induced the expression and nuclear accumulation of the AP-1 subunit c-Fos. siRNA-mediated knockdown of c-Fos eliminated FVIIa-stimulated MMP-7 expression and cell migration in vitro. In addition, selective mitogen-activated protein kinase (MAPK) kinase (MEK1/2) inhibitor (PD98059) and p38 MAPK inhibitor SB203580 suppressed MMP-7 upregulation induced by FVIIa.ConclusionsOur data suggest that a novel TF/FVIIa/MAPK/c-Fos/MMP-7 axis plays an important role in modulating the invasion of colon cancer cells and blockage of this pathway holds promise to treat colon cancer metastasis.

SPARC expression is negatively correlated with clinicopathological factors of gastric cancer and inhibits malignancy of gastric cancer cells

Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein which plays multiple roles in different types of cancer. Our previous study showed that SPARC overexpression inhibited the growth and angiogenesis of tumors, and reduced expression of vascular endothelial growth factor (VEGF). However, the relationship between SPARC expression and clinicopathological factors of gastric cancer (GC) is controversial, and the role of SPARC in GC remains unclear. We evaluated expression of SPARC in 65 human GC tissues using immunohistochemistry (IHC). The results indicated that SPARC expression was negatively correlated with clinicopathological factors of GC. In vitro assay showed that SPARC overexpression decreased proliferation and clonogenicity by suppressing CD44 expression. In addition, SPARC overexpression inhibited VEGF induced proliferation and arrested cell cycle of GC cells by reducing the activation of VEGFR2, ERK1/2 and AKT signaling pathways. SPARC suppressed the invasion and migration of GC by reducing MMP-7, MMP-9, N-cadherin, Sp1 and p-ERK1/2 expression. In the in vivo assay, cancer metastasis mouse models were established by tail vein injection. The results revealed that the lung metastases of SPARC-overexpressing GC cells in the mice were much fewer than those of control cells.

Aberrant methylation of the SPARC gene promoter and its clinical implication in gastric cancer

Secreted protein acidic and rich in cysteine (SPARC) gene has been shown to be epigenetically silenced in several cancers. We investigated the loss of expression and promoter methylation of this tumor suppressor gene in gastric cancers and correlated the data with clinicopathological features. We observed the loss of SPARC mRNA and SPARC protein expression in 7 of 10 (70%) gastric cancer cell lines. Upon treatment of expression-negative cell lines with a demethylating agent, expression of mRNA and protein was restored in all cells. Methylation rate of SPARC gene was 80% in ten gastric cancer cell lines and 74% (163 of 220) in primary tumors, while it was 5% in normal gastric mucosa (n = 40). In intestinal gastric cancer, SPARC methylation correlated with a negative prognosis (P < 0.001; relative risk 2.754, 95% confidence interval 1.780–4.261). Immunostaining revealed that SPARC protein was overexpressed in stromal fibroblasts adjacent to neoplastic epithelium but rarely expressed in the primary gastric cancer cells. These results implicate SPARC promoter methylation as an important factor in the tumorigenesis of gastric carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and the potential clinical importance in human gastric cancers.

H19 Overexpression Induces Resistance to 1,25(OH)2D3 by Targeting VDR Through miR-675-5p in Colon Cancer Cells

The long noncoding (lnc) RNA H19 was involved in the tumorigenesis of many types of cancer. However, the role of H19 in the tumorigenesis of colon cancer has not been fully illustrated. Recent studies suggested a potential relationship between H19 and vitamin D receptor (VDR) signaling. Considering the pivotal role of VDR signaling in the colon epithelium both physiologically and pathologically, the correlation between H19 and VDR signaling may have an important role in the development of colon cancer. In this study, the correlation between H19 and vitamin D receptor (VDR) signaling and the underlying mechanisms in colon cancer were investigated both in vitro and in vivo. The results suggested that VDR signaling was able to inhibit the expression of H19 through regulating C-Myc/Mad-1 network. H19, on the other hand, was able to inhibit the expression of VDR through micro RNA 675-5p (miR-675-5p). Furthermore, H19 overexpression induced resistance to the treatment with 1,25(OH)2D3 both in vitro and in vivo. Together, these results suggested that H19 overexpression might be one of the mechanisms underlying the development of resistance to the treatment with 1,25(OH)2D3 in the advanced stage of colon cancer.

The FVIIa-tissue factor complex induces the expression of MMP7 in LOVO cells in vitro

Background and aimsThe extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on the cell surface trigger intracellular signaling events involved in multiple physiological processes. TF expression is related to the metastatic potential of tumor cells and is a significant risk factor in the development of hepatic metastases in patients with colorectal cancer. At present, it is unclear how the interaction between TF and FVIIa influences the development of metastasis in colon cancer.Materials and methodsWe used a stable LOVO cell line derived from colorectal adenocarcinoma for our model Western blot analysis, Northern blot analysis, polymerase chain reaction, and RNA inference (RNAi), and the Dual-Luciferase Reporter Assay System technology were utilized to determine if MMP7 can be up-regulated by the VIIa/TF complex.ResultsNorthern blot analysis confirmed that the plasma clotting factor FVIIa/TF complex resulted in a marked increase in MMP7 expression in a time- and dose-dependent manner via the p38 pathway in vitro. The proximal promoter of the human MMP7 gene was cloned into a luciferase reporter construction (MMP7.luc1592). Upon treatment with FVIIa, reporter activity in LOVO cells was increased by 2.5-fold. TF RNAi almost completely abolished FVIIa-mediated MMP7.luc induction. Deletion constructs from MMP7.luc1592 further defined an active promoter region.InterpretationTaken together, these data provide evidence that expression of MMP7 in colon cancer may be regulated by FVIIa and TF at the transcriptional level. MMP7 may act as a downstream mediator of FVIIa/TF signal transduction to facilitate the development of metastasis in colon cancer.