Implication of EMT Induced by TGF-β1 in Pancreatic Cancer

This study examined the implication of EMT induced by TGF-β1 in pancreatic cancer invasion. TGF-β1 expression was determined in 29 cases of human pancreatic carcinoma （PC） by immunohistochemistry and the results were compared with those of pathological examination. Moreover, the effects of TGF-β1 on the phenotype and invasion of pancreatic cancer cell line Panc-1 were also investigated. TGF-β1 was detected in 12 cases （41.4 %） of PC. Significant correlation was found between the expression of TGF-β1 and lymph node involvement （P=0.047） and the depth of invasion （P=0.035）. TGF-β1 obviously promoted EMT of Panc-1 cell lines and their invasion ability was substantially enhanced. TGF-β1 may promote the malignancy of pancreatic cancer by triggering EMT.     

Involvement of TLR2-My D88 in Abnormal Expression of mi R-146a in Peripheral Blood Monocytes of Patients with Chronic Hepatitis C

miR-146 a is an immunoregulatory micro RNA closely associated with viral infection. This study investigated the expression changes of mi R-146 a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, mi R-146 a expression was upregulated. After treated by interferon/ribavirin, mi R-146 a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce mi R-146 a. Silencing TLR2 and My D88 could significantly inhibit the expression of mi R-146 a. It was concluded that the expression of mi R-146 a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-My D88 signaling pathway may take part in the overexpression of mi R-146 a in monocytes stimulated with HCV core protein.     

Erbin interacts with Sema4C and inhibits Sema4C-induced epithelial-mesenchymal transition in HK2 cells

Erbin, a member of Leucine-rich repeat and PDZ-containing protein family, was found to inhibit TGF-β-induced epithelial-mesenchymal transition （EMT） in our previous study. However, the mechanism of Erbin in regulating EMT is unclear. Semaphorin protein Sema4C, with PDZ binding site at C-terminal has been recognized as a positive regulator of EMT. Here, we aimed to examine the inter- action between Erbin and Sema4C. HK2 cells were treated with TGF-β1, or transfected with Erbin and （or） Sema4C. Interaction of Erbin and Sema4C was identified by immunoprecipitation. RT-PCR was used to detect the expression of Erbin and Sema4C at mRNA level after transfection. The expression levels of Erbin, Sema4C, and markers of EMT were measured by using Western blotting or ELISA. Af- ter HK2 cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the protein expression levels of Erbin and Sema4C were both up-regulated, and immunoprecipitation results showed Erbin interacted with Sema4C in HK2 cells both at endogenous and exogenous levels. Furthermore, overexpression of Sema4C suppressed E-cadherin, induced vimentin and promoted fibronectin secretion, indicating Sema4C promotes the process of EMT. However, HK2 cells overexpressing Erbin were resistant to Sema4C-induced EMT. In contrast, Erbin specific siRNA promoted EMT induced by Sema4C. Taken together, these results suggest that Erbin can interact with Sema4C, and co-expression of Erbin blocks the process of Sema4C-induced EMT.     

Pien Tze Huang(片仔癀) Overcomes Doxorubicin Resistance and Inhibits Epithelial-Mesenchymal Transition in MCF-7/ADR Cells

Objective: To evaluate the effect of Pien Tze Huang(片仔癀, PZH) on breast cancer chemoresistance and related epithelial-mesenchymal transition(EMT) and investigate the underlying mechanisms. Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay was used to determine the cell viability. Adriamycin(ADR) staining observed by fluorescence microscope was performed to detect the accumulation of ADR. Transwell assay was used to analyze the cell migration and invasion. Western-blot was performed to detect the protein expression of related genes. Results: MCF-7/ADR cells were resistant to ADR treatment, and PZH treatment inhibited the viability of MCF-7/ADR cells in a dose-dependent manner. PZH treatment also increased the intercellular accumulation of ADR and down-regulated the expression of ABCG2 and ABCB1 in MCF-7/ADR cells(P0.05). In addition, PZH treatment inhibited EMT, migration and invasion of MCF-7/ADR cells(P0.05). Moreover, PZH suppressed activation of transforming growth factor β1(TGF-β) signaling in MCF-7/ADR cells(P0.05). Conclusion: PZH treatment can effectively overcome chemoresistance via down-regulating ABCG2, ABCB1 and inhibit EMT in ADR resistant human breast cancer cells via suppression of the TGF-β1 pathway.     

MicroRNA-378a-3p Downregulation as a Novel Biomarker with Poor Clinical Outcomes in Cervical Cancer

Objective Cervical cancer(CC)is one of the most common malignant tumors in gynecology.This study aimed to investigate the prognostic significance of serum micro RNA(mi R)-378 a-3 p in CC and the effect of mi R-378 a-3 p on tumor growth.Methods Real-time quantitative polymerase chain reaction analysis was used to measure the expression of mi R-378 a-3 p in serum from patients with CC and healthy control subjects as well as from CC tissues and adjacent normal tissues.The association between serum mi R-378 a-3 p levels and clinicopathological factors was analyzed.The correlation between mi R-378 a-3 p levels and overall survival(OS)of CC patients was determined by Kaplan-Meier analysis.The CC cell proliferation and migration abilities after transfection of mi R-378 a-3 p mimics were detected by Cell Counting Kit-8 and scratch wound healing assays,respectively.Tumor volume and weight in mice treated with mi R-378 a-3 p were measured using a caliper and an electronic balance.Results Mi R-378 a-3 p expression was downregulated in the serum and tissues of CC patients compared to that in healthy control subjects and normal tissues,respectively.Low expression of mi R-378 a-3 p was positively correlated with large tumor size,advanced tumor stage,and lymph node metastasis.The OS of patients with low expression of mi R-378 a-3 p was significantly lower than that of patients with high expression.Overexpression of mi R-378 a-3 p suppressed the proliferation and migration of CC cells.In vivo studies indicated that overexpression of mi R-378 a-3 p was associated with decreased tumor volume and weight in mice.Conclusion Mi R-378 a-3 p downregulation is associated with the development and prognosis of CC,suggesting that it may be a potential biomarker for CC.     

Upregulated DJ-1 promotes renal tubular EMT by suppressing cytoplasmic PTEN expression and Akt activation

Recently,phosphatase and tensin homolog deleted on chromosome 10(PTEN) is suggested as a new agent in the fighting against fibrogenesis.In tumor,DJ-1 is identified as a negative regulator of PTEN.But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear.Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model.Human proximal tubular epithelial cells(HKC) were treated with transforming growth factor-beta 1(TGF-β1),or transfected with DJ-1 or PTEN.Confocal microscope was used to investigate the localization of DJ-1 and PTEN.The selective phosphoinositide-3 kinase(PI3K) inhibitor,LY294002,was administered to inhibit PI3K pathway.The DJ-1 and PTEN expression,markers of epithelial-mesenchymal transition(EMT) and Akt phosphorylation were measured by RT-PCR,Western blotting or immunocytochemistry.In vitro,after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h,the expression of DJ-1 was increased,and that of PTEN was decreased.In vivo,the same results were identified in 5/6-nephrectomized rats.In normal HKC cells,most of DJ-1 protein localized in cytoplasm,and little in nucleus.TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei.In contrary,TGF-β1 emptied cytoplasmic PTEN protein into nucleus.Overexpression of DJ-1 decreased the expression of PTEN,promoted the activation of Akt and the expression of vimentin,and also led to the loss of cytoplasmic PTEN.Contrarily,overexpression of PTEN protected HKC cells from TGF-β1-induced EMT.In conclusion,DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.     

The expression of matrix metalloproteinases-9, transforming growth factor-β1 and transforming growth factor-β receptorⅠ in human atherosclerotic plaque and their relationship with plaque stability

Background Transforming growth factor-beta (TGF-β) and matrix metalloproteinases-9 (MMP-9) have been implicated in the pathogenesis of human atherosclerosis but their relationship during lesion progression are poorly understood. The objective of this study was to investigate the expression of MMP-9, TGF-β1 and TGF-β receptor Ⅰ (TβR-Ⅰ) in human atherosclerotic plaque and their relationship and plaque stability.Methods Specimens of human coronary artery atherosclerotic plaques were obtained from 41 patients undergoing coronary endarterectomy, and were paraffin embedded, sectioned at 4 μm intervals then stained with haematoxylin and eosin. They were divided into stable (with no or only little lipid core) and unstable plaque groups (with lipid core size＞40%): the immunohistochemical staining were performed for MMP-9,TGF-β1 and TβR-Ⅰ.Results The expression of MMP-9 in the unstable plaques was much higher than in the stable ones, but the expression of TGF-β1 was higher in the stable plaques. There was no similar significant difference for TβR-Ⅰ. Correlation analysis showed that there was a negative correlation between the expression of MMP-9 and TGF-β1 (r=-0.332, P=0.034 for average areal density; r=-0.373, P= 0.016 for average optical density).Conclusions There were close relationships between MMP-9, TGF-β1 and plaque stability. Enhanced production of MMP-9 may participate in the formation of unstable plaque, while TGF-β1 maybe an important stabilizing factor in preventing transition into an unstable plaque phenotype.     

Effect of Rapamycin on TGF-β1- induced epithelial-mesenchymal transition in LoVo colonic adenocarcinoma cells

Objective:To investigate the effect of Rapamycin on epithelial-mesenchyrnal transition(EMT) of LoVo colonic adenocarcinoma cells in vitro.Methods:Cultured LoVo colonic adenocarcinoma cells were divided into three groups: negative control group,EMT-inducing group(TGF-β1) and EMT-interfering group(TGF-β1 plus Rapamycin).E-cadherin expression in LoVo cells was detected by Western Blot,while the expression of vimentin was evaluated through immunocytochemistry.The Snail mRNA in LoVo cells was examined by RT-PCR.Results:TGF-β1 induced LoVo cell switching from polygonal to spindle-shaped.TGF-β1 enhanced the expression of vimentin,but lowered the level of E-cadherin.In contrast,Rapamycin impaired the transition induced by TGF-β1.Rapamycin dramatically abrogated TGF-β1-induced vimentin expression and restored E-cadherin expression in LoVo cells.Rapamycin significantly repressed the up-regulation of Snail mRNA expression induced by TGF-β1.Conclusion:Rapamycin dramatically abrogated TGF-β1 induced Snail mRNA expression in LoVo cells,hence inhibiting EMT of these cells in vitro.     

Effects of emodin on the proliferation of the glomerular mesangial cell and correlative cytokines in rats

Objective：To investigate the effects of emodin（EMD） on cell proliferation and correlative cytokines secretion of glomerular mesangial in rats. Methods：The effects of EMD on cell proliferation and IL-6, TGF-β1 secretion of glomerular mesangial in rats were observed. Cell proliferation was measured by MTT method. IL-6 and TGF-β1 secretion was detected with ELISA. Results：EMD was able to inhibit the cell proliferation and down-regulate the IL-6 and TGF-β 1 secretion of glomerular mesangial, as compared to the model group in rats （P 〈 0.05）. Conclusion：EMD could significantly inhibit the cell proliferation, and reduce the creation of extracellular matrix（ECM）, this indicated that it could play an important role in alleviation and prevention of glomerular sclerosis. The mechanism may be that EMD can reduce the IL-6 and TGF-β1 secretion ofglomerular mesangial cell in rats.     

Influence of High Level TGF-β1 on Scleral Thickness

In order to explore the role of TGF-β1 in scleral remodeling and the possible mechanism,the influence of high level TGF-β1 on scleral thickness and the expression of MMP-2 and TIMP-2 was investigated in a TGF-β1 transgenic mouse model.Alb/TGF-β1(Cys223,225Ser) TGF-β1 transgenic mice were used as experimental subjects and non-transgenic littermates as controls.Plasma levels of TGF-β1 were determined by ELISA.TGF-β1,MMP-2 and TIMP-2 levels in sclera were detected by using Western blot.The thickness of posterior sclera was measured by computerized image analysis of a midsagittal section.Mean difference was analyzed with independent t-test.The results showed plasma levels of TGF-β1 in transgenic mice were 1.68 times as much as that in the controls(P<0.01).TGF-β1 levels in the sclera of transgenic mice were 2.68 times of the controls(P<0.01).Posterior scleral thickness in transgenic mice were significantly thicker than in the controls.There was no significant difference in the MMP-2 levels between transgenic mice and controls,but the TIMP-2 levels were increased significantly in transsgenic mice as compared with those in the controls.It was suggested that high levels of TGF-β1 in transgenic mice could result in the increased scleral thickness by inducing the expression of TIMP-2 to suppress the activity of MMP-2,finally inhibiting the degradation of collagen.     

Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

Background Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-β(1) (TGF-β(1)) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-β(1) delivery and the effect of released TGF-β(1) on the chondrogenic potential of chondrocytes.Methods Chitosan scaffolds and chitosan microspheres loaded with TGF-β(1) were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-β(1) microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-β(1) on cell adhesivity, proliferation and synthesis of the extracellular matrix.Results TGF-β(1) was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-β(1) was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-β(1) could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-β(1) was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7%±10.4%) was significantly lower than that in the controlled TGF-β(1) release group (92.4%±4.8%, P&lt;0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-β(1) microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-β(1) was retained during microsphere fabrication and after growth factor release.Conclusion Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-β(1), and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-β(1) possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.     

The Expression of the Plasmid DNA Encoding TGF—β1 in Endothelium after Injection into the Anterior Chamber

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected.Specific TGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior chamber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foundation for further study on TGF-β1 participating in local induction of corneal immune tolerance.     

The Chondrogenic Potential of Rabbit Bone—mar—orw—derived Mesenchymal Stem Cells（MSCs）As—sayed under the Influence of Growth Factors

Objective To establish a method of inducing rabbit bone marrow mesenchymal stem cells(MSCs)to express chondrocytic phenotype.Methods MSCs were seperated from bone marrow extracted from the iliac of New Zealand rabbit,TGF-β1,IGF-Ⅰand Vitamin C were applied into culture medium to induce proliferation and transformation of MSCs,Colony forming efficiency(CFE)was measured to reflect cell proliferation rate;procollagenαl（Ⅱ）mRNA in cells was detected by RT-PCR;ultrastructure of cells was observed under scanning electronic microscope(SEM);alkline phosphatase(ALP)in culture medium was also detected.Results Under the influence of TGF-β1,IGF-Ⅰand VitamineC,CFEof MSCs rose from control level of 3/106to21/106,expression of articular cartilage specific procollagenαl（Ⅱ）mRNA by cells was promoted;At the same time,floccu-lent matrixes synthesized were observed between connecting polygonal MSCs,ALP in culture medium ed-clined to control level with culture time,Conclusion Expression of chondrocytic phenotype by MSCs could be induced by the symergistic action of TGF-β0281and Vitamine C.Growth factor-induced MSCs could be a good cell source for tissue-engineered cartilage.     

Combined Use of RGD-peptide Modified PLGA and TGF-β1 Gene Transfected MSCs to Improve Cell Biobehaviors in vitro

In order to improve the surface properties of PLGA polymer for a better material/cell interface to modulate the cells behaviors, we prepared a novel three-block copolymer, PLGA-[ASP-PEG], and immobilized an RGD-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys （GRGDSPC） on the surface of it. Transforming growth factor-β1 （TGF-β1） was transfected into bone marrow stromal cells （MSCs） employed as seeded cells. Cell adhesion, spreading, proliferation and differentiation on this material were investigated. The results showed that the cell adhesive ratio on RGD-modified materials was higher than on un-modified materials （P〈0.05）. The extent of cell spreading was also wider on RGD-modified materials than on un-modified materials. Cell proliferation indices of transfected MSCs were increased as compared with the un-transfected MSCs （P〈0.05）. The ALP activities in the MSCs cultured with RGD-modified materials were higher than on un-modified materials after 14 days （P〈0.05）, and those in transfected MSCs were higher than in un-transfected MSCs （P〈0.05）. It was suggested that the combined use of RGD-modification and TGF-β gene transfection could improve the interaction of biomaterial and cells.     

COA: Bone Morphogenetic Protein 2 Promoted Transforming Growth Factor β3 Induced Chondrogenesis of Human Osteoarthritic Synovium-Derived Stem Cells

Background Synovium-derived stem cells (SDSCs) with greater chondrogenic potential are attracting more considerable attention as a cell source for cartilage regeneration. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2) on transforming growth factor-beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods Nucleated cells isolated from human osteoarthritic synovium were plated at an optimal cell density to allow the selective proliferation of SDSCs. The clonogenicity, stem cell marker expression and multi-differentiation potential were determined by CFU assay, flow cytometry assay and specific staining including alizarin red S staining, Oil red staining and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium without or with TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II (COL2A1), aggrecan (ACAN), SOX9, link-protein (HAPLN1), collagen type X (COL10A1) and BMP receptor II (BMPR-II). Results Cells isolated under the optimized culturing density (104/60cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99% positive) for CD44, CD90, CD105 and negative (<10% positive) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Metachromatic staining of the extracellular matrix with Safarnin O was positive and the expression of collagen type II was detected. The combination of TGF-β3 and BMP-2 produced cell pellets with larger diameter and weight, produced more sGAGs, expression higher levels of collagen type II and chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation of BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.     

The effect of endogenous transforming growth factor β_1 on the growth of bladder cancer cells in vitro

Objective To investigate the influence of endogenous transforming growth factor β1(TGFβ1) on the cell cycle regulation and proliferation of bladder cancer. Methods A constructed replication defective retroviral vector pRevTβ-AS, which carried antisense RNA of TGFβ1.was transfected to a bladder cancer cell line EJ. The proliferation and clone-formation of transferred cells were observed in vitro,and the alteration of cell cycle was also detected by flow cytometric analysis. Results TGFβ1 antisense RNA was transferred into EJ cell and expressed efficiently. After the inhibition of target gene expression in EJ cells, the reduced growth and clone-formation rates were demonstrated, and the proliferative indexes were decreased by 12 % . The ratios of GO and G1 stage cells to June 2003 Vol12 No2 the antisense RNA-transfected EJ cells were increased, simultaneously,the ratio of S stage cells to the antisense RNA-transfected EJ cells ratios were decreased, compared with the control group. Conclusion The pro     

Caveolin-1 is involved in radiation-induced ERBB2 nuclear transport in breast cancer cells

This study examined the radiation-induced ERBB2 nuclear transport in the BT474 breast cancer cell line and the relationship between caveolin-1 and radiation-induced ERBB2 nuclear transport. The BT474 cells were treated with herceptin (200 nmol/L), PP2 (a caveolin-1 inhibitor, 100 nmol/L) and irradiation combined or alone. Confocal microscopy was used to observe the nuclear import of ERBB2 and caveolin-1 after irradiation. Western blotting was employed to detect the expression of ERBB2, caveolin-1 and DNA-PKcs after irradiation, and immunoprecipitation to identify the ERBB2 and caveolin-1 complex before perinuclear ERBB2 localization. Confocal microscopy showed the transport of ERBB2 and caveolin-1 from the cell membrane to the nucleus 15 min after irradiation and the proteins accumulated at the perinuclear region within 45 min. Western blotting revealed that the expression levels of ERBB2, caveolin-1 and DNA-PKcs were increased after irradiation and reached a peak 45 min later. Both herceptin and PP2 treatments were found to decrease ERBB2 expression. An immune complex composed of ERBB2 and caveolin-1 was found in the herceptin group after irradiation. It was concluded that after irradiation, ERBB2 may be transported from the cell membrane to the nucleus and activate DNA-PKcs to trigger DNA double-strand break (DSB) repair; caveolin-1 may participate in this process. Treatments involving the downregulation of caveolin-1 may increase the radio-sensitization of breast cancer cells.