mRNA Expression Levels Of VEGF Research Articles

A study on the anti-tumor mechanism of total flavonoids from Radix Tetrastigmae against additional cell line based on COX-2-mediated Wnt/β-catenin signaling pathway.

This study is to explore the effect of total flavonoids from Radix Tetrastigmae (TF) against hepatic cancer and discuss the acting mechanism. Proliferation of HepG2 cells was promoted by PGE2 and Butaprost. Using AH6809 as the positive control, the inhibitory effect of TF on additional cell line was detected through a CCK-8 assay, the apoptosis rate was detected by flow cytometry, and the nuclear morphology of cells were observed by Hochest33258 staining. Then PCR was applied to determine the mRNA expression. The corresponding Protein expression were determined by Western Blot. The effects of TF on the body weight, tumor growth volume and tumor inhibition rate were observed in nude mice model of human hepatocellular carcinoma by vivo experiments. The results showed TF had an obvious inhibitory effect on HepG2 cells with a significant dose-dependent manner (P<0.01). Pyknosis was found under the fluorescence microscope after TF treatment for 24h by Hochest33258 staining. Typical features of apoptosis were observed in HepG2 cells treated with TF and the apoptosis rate increased with increase of concentration of the TF. mRNA expression levels of GSK-3β, Akt, VEGF, COX-2 and β-catenin were down regulated greatly by the TF in HepG2 cells. Moderate and high concentrations of TF led to an obvious down regulation of GSK-3β, p-GSK-3β, Akt, p-Akt, VEGF, COX-2 and β-catenin in HepG2 cells, while p-β-catenin was up regulated. The tumor inhibition rates with high, medium and low dose of TF were 64.07%, 53.64%, 46.69%, respectively. The inhibitory effect of total flavonoids on tumor growth in mice was better than that of CTX, and the inhibitory effect of TF on tumor growth was less than CTX. TF exhibited a significant inhibitory effect on HepG2 cells, promoting the apoptosis of HepG2 cells in a dose-dependent manner. TF was also regulatory of the COX-2-Wnt/β-catenin signaling pathway, which was presumed to be the working mechanism of Tetrastigmae.

Wheatgrass Extract Ameliorates Hypoxia-induced Mucin Gene Expression in A549 cells.

Background:Wheatgrass is known to have antioxidant, antiaging, and anti-inflammatory effect. However, its protective effect against hypoxia is not yet evaluated.Objective:In this study, we evaluated the protective and anti-inflammatory effect of wheatgrass against the hypoxia in airway epithelial cells.Materials and Methods:A549 human lung adenocarcinoma cells were incubated in a hypoxic condition (CO2 5%/O2 1%) for 24 hr in the presence of different concentration of wheatgrass 50, 75, 100, and 150 μg/mL, and the magnitude of each immunologic response produced by the A549 cells was compared. The mRNA expression level of mucin gene (MUC), 5A, 5B, 8, GM-CSF, TNF-α, and VEGF were evaluated by using real-time polymerase chain reaction. The MUC proteins level before and after knocking out the hypoxia-inducible factor (hif)-1α via short interfering (si) RNA transfection were assessed by immunoblot analysis. Accordingly, the involved cell signaling pathway was evaluated by immunoblot analysis.Results:The inflammatory cytokines (GM-CSF, TNF- α) and the expressions of MUC 5A, 5B, and 8 were augmented by hypoxia. The augmented MUC expression was decreased by the wheatgrass extract administration. Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass. Knockdown of hif-1α by siRNA reduced the mucin gene expression and which was more enhanced by wheatgrass extract.Conclusion:Theses results suggest that wheatgrass may be useful in the treatment of sinonasal disease by inhibiting mucus hypersecretion in airway epithelium.SUMMARY Wheatgrass extract decreases the hypoxia-induced MUC 5A, 5B and 8 expression.Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass.Wheatgrass inhibits p44/42 phosphorylation in hypoxia-exposed airway epithelial cells. Abbreviations used: A549: human lung adenocarcinoma cells, GM-CSF: granulocyte-macrophage colony stimulating factor, HIF: hypoxia inducible factor, IL: interleukin, MUC: mucin, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, TNF: tumor necrosis factors, VEGF: vascular endothelial growth factor, si RNA: short interfering RNA

Quercetin Inhibits Cell Migration and Invasion in Human Osteosarcoma Cells

Background/Aims: Osteosarcoma is a malignant tumor associated with high mortality; however, no effective therapies for the disease have been developed. Several studies have focused on elucidating the pathogenesis of osteosarcoma and have aimed to develop novel therapies for the disease. Quercetin is a vital dietary flavonoid that has been shown to have a variety of anticancer effects, as it induces cell cycle arrest, apoptosis, and differentiation and is involved in cell adhesion, metastasis and angiogenesis. Herein, we aimed to investigate the effects of quercetin on osteosarcoma migration and invasion in vitro and in vivo and to explore the molecular mechanisms underlying its effects on osteosarcoma migration and invasion. Methods: Cell viability, cell cycle activity and cell apoptosis were measured using CCK-8 assay and flow cytometry, and cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The mRNA and protein expression levels of several proteins of interest were assessed by real-time quantitative PCR and western blotting, respectively. Moreover, a nude mouse model of human osteosarcoma lung metastasis was established to assess the anti-metastatic effects of quercetin in vivo. Results: We noted no significant differences in cell cycle activity and apoptosis between HOS and MG63 cells and control cells. Treatment with quercetin significantly attenuated cell migration and invasion in HOS and MG63 cells compared with treatment with control medium. Moreover HIF-1α, VEGF, MMP2, and MMP9 mRNA and protein expression levels were significantly downregulated in HOS cells treated with quercetin compared with HOS cells treated with controls. Additionally, treatment with quercetin attenuated metastatic lung tumor formation and growth in the nude mouse model of osteosarcoma compared with treatment with controls. Conclusion: Our findings regarding the inhibitory effects of quercetin on cell migration and invasion suggest that quercetin may have potential as a therapy for human osteosarcoma.

Dynamic Perfusion Culture of Human Outgrowth Endothelial Progenitor Cells on Demineralized Bone Matrix In Vitro.

BackgroundThe aim of this study was to investigate the proliferation, differentiation, and tube formation of human outgrowth endothelial progenitor cells (OECs) cultured with porous demineralized bone matrix (DBM) under a dynamic perfusion system in vitro.Material/MethodsOECs were isolated, expanded, characterized, eGFP-transfected and seeded on DBM scaffold and cultured under static or dynamic perfusion conditions, and continuously observed under fluorescence microscope. DBM scaffolds were harvested on day six for RT-PCR and western blot assay to analyze the mRNA and protein expression level of CD34, VE-cadherin, and VEGF. Scanning electron microscope (SEM) was used to observe the tube formation of OECs seeded on DBM scaffolds.ResultsThe results showed the cell density of OECs on DBM was higher when exposed to shear stress generated by a dynamic perfusion system. Shear stress also markedly increased the expression level of VE-cadherin and VEGF and decreased the expression of CD34, at both mRNA and protein levels. SEM showed that the shear-stressed OECs formed tube-like structures inside the pores of DBM scaffolds.ConclusionsA dynamic perfusion system can be used as an innovative method for the rapid vascularization in tissue engineering, which can accelerate the proliferation and differentiation of OECs and the vascularization of implanted scaffolds.

Ginkgolide B enhances the differentiation of preosteoblastic MC3T3-E1 cells through VEGF: Involvement of the p38 MAPK signaling pathway.

Ginkgolide B (GB) is one of the ginkgolides isolated from the leaves of the Ginkgo biloba tree. Our previous study indicated that GB promotes the proliferation, migration and adhesion of endothelial progenitor cells, and the induction of angiogenesis through vascular endothelial factor (VEGF). In the present study, the effects of GB on the differentiation of MC3T3‑E1 cells and the signaling pathway involved were investigated invitro. The MC3T3‑E1 cell viability activities were assessed using an MTS assay. Measurements of alkaline phosphatase activity and Alizarin Red staining were used to identify osteoblastic differentiation of the MC3T3‑E1 cells. The mRNA and secretion levels of VEGF were detected using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis and enzyme-linked immunosorbent assays, respectively. The protein expression levels of phosphorylation‑associated markers were detected using western blot analysis and associated gene expression was determined using RT‑qPCR analysis. It was found that GB significantly promoted alkaline phosphatase activity and osteoblastic mineralization in the MC3T3‑E1 cells. In addition, the mRNA expression and secretion levels of VEGF in the MC3T3‑E1 cells were significantly increased in MC3T3‑E1 cells treated with GB. SB203580, a specific inhibitor of p38 mitogen‑activated protein (MAP) kinase, markedly suppressed the GB‑induced p38 kinase phosphorylation and GB‑induced synthesis of VEGF. PD98059, an inhibitor of the upstream kinase, which activates p44/p42 MAP kinase, had minimal effect on the GB‑induced phosphorylation of p44/p42 MAP kinase or the GB‑induced synthesis of VEGF. Taken together, these results indicated that GB promoted osteoblastic differentiation of the MC3T3‑E1 cells through VEGF, and that the p38, but not the p44/p42 MAP kinase signaling pathway, was involved in the GB‑induced synthesis of VEGF.

Cysteine‑rich61 RNA interference inhibits pathological angiogenesis via the phosphatidylinositol 3‑kinase/Akt‑vascular endothelial growth factor signaling pathway in endothelial cells.

Hypoxia is a key factor in the pathogenesis of angiogenesis, and cysteine‑rich61 (CCN1), an angiogenic factor, is involved in the development of pathological angiogenesis. The aim of the present study was to investigate the mechanism of CCN1 RNA interference (RNAi)‑induced inhibition of hypoxia‑induced pathological angiogenesis in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic conditionsinvitro. The effects of inhibiting phosphoinositide 3‑kinase (PI3K)/Akt signaling using LY294002 were investigated in hypoxic HUVECs. The proliferation and apoptosis of HUVECs under hypoxia were assessed using CCN1 RNAi. The CCN1‑PI3K/Akt‑vascular endothelial growth factor (VEGF) pathway was analyzed under hypoxic conditions using reverse transcription‑quantitative polymerase chain reaction and western blotting. CCN1 RNAi inhibited the proliferation and induced the apoptosis of the HUVECs under hypoxia, with hypoxia significantly increasing the mRNA and protein expression levels of CCN1, Akt and VEGF. By contrast, CCN1 RNAi reduced the mRNA and protein expression levels of CCN1, Akt and VEGF in the HUVECs (P<0.05). Furthermore, LY294002 reduced the mRNA and protein expression levels of CCN1 in the hypoxic cells (P<0.05). These data indicated that CCN1 inhibits apoptosis and promotes proliferation in HUVECs. Therefore, CCN1 RNAi may offer a novel therapeutic strategy, which may aid in the treatment of pathological angiogenesis via inhibition of the PI3K/Akt‑VEGF pathway.

A Comparative Animal Study of Tendon Grafts Healing After Remnant-Preserving Versus Conventional Anterior Cruciate Ligament Reconstruction.

BackgroundThe aim of this study was to determine if anterior cruciate ligament (ACL) reconstruction by remnant preservation promotes cell proliferation, vascularization, proprioception recovery, and improved biomechanical properties of the tendon grafts.Material/Methods75 New Zealand rabbits were randomly assigned into the control group (group A), conventional ACL reconstruction group (group B), ACL reconstruction using remnant preservation and graft through remnant sleeve technique group (group C), and ACL reconstruction using remnant preservation and remnant tensioning technique group (group D). The remnant and healing of tendon grafts in groups C and D were observed at 3, 6, and 12 weeks after surgery, and the mRNA expression levels of VEGF, NT-3 and GAP-43 in ACL (group A) or tendon graft samples (groups B, C, and D) were determined by real-time PCR. Tendon graft cell count, microvessel density (MVD), and proprioceptors were determined by H&E staining, CD34, and S-100 immunohistochemical staining. The biomechanical properties of the tendon graft at week 12 in groups B, C, and D were examined by using a tensile strength test.ResultsRemnant and tendon grafts were not healed at 3, 6, and 12 weeks after the operation in groups C and D. VEGF, NT-3, and GAP-43 mRNA expressions in groups B, C, and D were higher than those in group A (P<0.05), but no significant difference was observed between groups B, C, and D (P>0.05). Furthermore, tendon graft cell count, MVD, proprioception, and biomechanical properties showed no significant differences (P>0.05) among groups B, C, and D at various time points.ConclusionsThere was no significant difference in cell proliferation, vascularization, proprioception recovery, or biomechanical properties of the tendon grafts between remnant-preserving and conventional ACL reconstruction methods.

Effects and mechanisms of docosahexaenoic acid on the generation of angiopoietin-2 by rat brain microvascular endothelial cells under an oxygen- and glucose-deprivation environment.

ObjectiveThe aim of this study was to investigate the effects of docosahexaenoic acid (DHA) on the generation of angiopoietin-2 (Ang-2) by rat brain microvascular endothelial cells under an oxygen- and glucose-deprivation environment (OGD), and its relationship, if any, with cyclooxygenase 2 (COX-2) expression.MethodsAnnexin V and propidium iodide apoptosis assay was used to detect apoptosis. Enzyme linked immunosorbent assay was used to detect Ang-2, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and prostaglandin I2 (PGI2) content. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect Ang-2 and VEGF mRNA expression. Western blot was used to detect expression of COX-2 protein.ResultsDHA reduced the apoptosis rate (P = 0.026) and decreased the secretion of Ang-2, VEGF, PGE2, and PGI2 (P = 0.006, P = 0.000, P = 0.002, P = 0.004 respectively). The relative expression of Ang2 and Vegf mRNA, as well as COX-2 expression, also decreased (P = 0.000, P = 0.005, P = 0.007 respectively). These effects were antagonized by GW9662 (peroxisome proliferator-activated receptor-γ antagonist). COX-2 protein expression levels were positively correlated with Ang2 and Vegf mRNA expression levels (γ = 0.69, P = 0.038 and γ = 0.76, P = 0.032, respectively). Ang-2 and VEGF mRNA levels were positively correlated with Ang-2 (γ = 0.84, P = 0.012) and VEGF (γ = 0.71, P = 0.036) secretion levels respectively.ConclusionDHA reduced apoptosis induced by an OGD environment, thus decreasing Ang-2 and VEGF synthesis. This phenomenon was associated with a decrease in COX-2 protein expression, PGE2 and PGI2 secretion, and generation regulation via intracellular transcriptional pathways.

MiR-20b targets AKT3 and modulates vascular endothelial growth factor-mediated changes in diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of new-onset blindness. The roles of microRNAs in diabetic retinopathy are largely unknown. The aim of this study is to investigate the role of miR-20b in DR. Transfection of miR-20b mimic in high glucose (HG)-treated human retinal endothelial cells (HRECs) increased miR-20b expression and decreased the expression level of VEGF mRNA, while transfection of miR-20b inhibitor in control HRECs reduced the miR-20b expression with a corresponding increase of VEGF mRNA. In vitro functional assay showed that transfection of miR-20b mimic prevented HG-induced increase in transendothelial permeability and tube formation in HRECs. Transfection of miR-20b inhibitor or treatment of VEGF increased transendothelial permeability and tube formation in control HRECs. Luciferase reported assay showed that AKT3 is a target of miR-20b. Transfection of miR-20b mimic prevented the up-regulation of AKT3 induced by HG without changing the protein levels of other isoforms of AKT, and silencing of AKT3 caused decrease of VEGF mRNA and protein levels as well as prevented HG-induced increase in transendothelial permeability and tube formation. Finally, we showed that miR-20b was down-regulated in the retina and retinal endothelial cells in diabetic rats, with a correlated up-regulation of VEGF and AKT3. Intravitreal injection of miR-20b mimic in the diabetic rat significantly increased the miR-20b expression and decreased the expression levels of AKT3 and VEGF in the retina tissues, and intravitreal delivery of AKT3 siRNA in the diabetic rat significantly decreased the expressions of AKT3 and VEGF. Collectively, miR-20b is important for the regulation of VEGF-mediated changes in HRECs and rat retinal tissues under hyperglycemic conditions possibly via targeting AKT3.

Abstract 2485: Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function

Abstract Endothelial progenitor cells (EPCs) are bone marrow (BM) -derived stem cells to be committed and differentiated into mature endothelial cells. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor involved in TGF-β signaling pathway and participate in cell cycle arrest and anti-angiogenesis. RUNX3 is frequently deleted or transcriptionally silenced in a variety of cancers by epigenetic mechanisms. The aim of study was to determine the role of RUNX3 in EPC function and vasculogenesis. Here we found that RUNX3 homozygous knockout (Rx3-/-) embryos had vascular defects in embryonic day (ED) 10 and developmental abnormalities in ED 16.5 organs. The number of EPC colonies and circulating EPCs (CD34+/VEGFR2+ cells), migration and tube formation capacity of EPCs derived from RUNX3 heterozygous (Rx3+/-) mice were increased in compared to those wild type (WT) mice, suggesting that deletion of RUNX3 can enhance vasculogenesis. Expression level of VEGF, VEGFR2, SDF-1, CXCR4 and endothelial nitric oxide synthase (eNOS) mRNA was significantly up-regulated in Rx3+/- EPCs. Protein expression of VEGF, p-VEGFR2, p-Akt, p-ERK, p-SAPK/JNK and p-eNOS was also augmented in Rx3+/- EPCs. Finally, we found that recovery of blood flow was highly stimulated in Rx3+/- mice using hindlimb ischemia mouse models. Taken together, our study revealed that RUNX3 might inhibit vasculogenic processes in physiological ischemia by inhibition of EPC functions. Citation Format: Su Young Oh, Se-Hyung Lee, Sun Hee Lee, You Mie Lee. Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2485.

The protective role of autophagy in experimental osteoarthritis, and the therapeutic effects of Torin 1 on osteoarthritis by activating autophagy.

BackgroundRecent studies have shown that autophagy was associated with the development of osteoarthritis (OA), the purpose of this research was to determine the exact role of autophagy in OA and investigate effective therapeutic drugs to inhibit the pathological progression of OA.MethodsIn this study, a cellular OA model was generated by stimulating SW1353 cells with IL-1β and a rabbit OA model was established by intra-articular injection of collagenase, followed by treatment with Torin 1 or 3-Methyladenine (3-MA). The mRNA expression levels of VEGF, MMP-13 and TIMP-1 were determined by quantitative real-time PCR. The caitilage degeneration was examined by histological evaluation, chondrocytes degeneration and autophagosomes were observed by transmission electron microscopy. Expression levels of Beclin-1 and LC3 were evaluated by western blotting and immunofluorescence.ResultsThe degeneration of SW 1353 cells, cartilage and chondrocytes was related to the loss of autophagy in experimental OA. 3-MA increased the severity of degeneration of cells and cartilage by autophagy inhibition, while Torin 1 reduced that by autophagy activation.ConclusionsThe loss of autophagy is linked with the experimental OA and autophagy may play a protective role in the pathogenesis of OA. Treatment of Torin 1 can inhibit the degenerative changes of experimental OA by activating autophagy and it may be a useful therapeutic drug for OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-0995-x) contains supplementary material, which is available to authorized users.

Notch-1 signaling activates NF-κB in human breast carcinoma MDA-MB-231 cells via PP2A-dependent AKT pathway

Breast cancer has a high incidence in the world and is becoming a leading cause of death in female patients due to its high metastatic ability. High expression of Notch-1 and its ligand Jagged-1 correlates with poor prognosis in breast cancer. Our previous work has shown that Notch-1 signaling pathway upregulates NF-κB transcriptional activity and induces the adhesion, migration and invasion of human breast cancer cell line MDA-MB-231. However, the role of Notch-1 in NF-κB activation is still poorly understood. Here, we aim to understand the exact mechanism that Notch-1 regulates NF-κB activity. In MDA-MB-231 cells where Notch-1 is constitutively activated, the phosphorylation of p85 and AKT (Tyr308/Ser473) is upregulated, indicating PI3K/AKT pathway is activated. Notch-1 activation caused the increase of PP2A phosphorylation at Tyr307, indicating Notch-1 inhibits PP2A activity. NF-κB transcriptional activity was evaluated by dual-luciferase reporter assay, and the results showed that, while silencing of Notch-1, PP2A activity was upregulated and NF-κB activity was downregulated, whereas PP2A inhibitor okadaic acid (OA) restored NF-κB activity. Immunofluorescence and Western blots showed that OA treatment antagonized the decrease of p65 nuclear translocation caused by Notch-1 silencing. Moreover, OA treatment also upregulated MMP-2, MMP-9 and VEGF mRNA expression levels, indicating OA rescues Notch-1 silencing that caused low cell invasion. Taken together, our results suggest that Notch-1-activating PI3K/AKT/NF-κB pathway is PP2A dependent; PP2A may be a potential therapeutic target in breast cancer.

A small interfering RNA targeting vascular endothelial growth factor efficiently inhibits growth of VX2 cells and VX2 tumor model of hepatocellular carcinoma in rabbit by transarterial embolization-mediated siRNA delivery.

IntroductionHepatocellular carcinoma is currently the second leading cause of cancer-related deaths worldwide with an increasing incidence.ObjectiveThe objective of this study is to investigate the effect of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) on rabbit VX2 carcinoma cell viability in vitro and the effect of transarterial embolization (TAE)-mediated VEGF-siRNA delivery on the growth of rabbit VX2 liver-transplanted model in vivo.MethodsQuantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot technologies were used to detect the expression level of VEGF. TAE and computed tomography scan were used to deliver the VEGF-siRNA and detect the tumor volume in vivo, respectively. Microvessel density was detected by immunohistochemistry with CD34 antibody. A biochemical autoanalyzer was used to evaluate the hepatic and renal toxicity.ResultsThe designed VEGF-siRNAs could effectively decrease the expression levels of VEGF mRNA and protein in vitro and in vivo. In vitro, the viability of rabbit VX2 carcinoma cells was reduced by 38.5%±7.3% (VEGF-siRNA no 1) and 30.0%±5.8% (VEGF-siRNA no 3) at 48 hours after transfection. Moreover, in rabbit VX2 liver-transplanted model, the growth ratios of tumors at 28 days after TAE-mediated siRNA delivery were 155.18%±19.42% in the control group, 79.67%±19.63% in the low-dose group, and 36.09%±15.73% in the high-dose group, with significant differences among these three groups. Microvessel density dropped to 34.22±4.01 and 22.63±4.07 in the low-dose group and high-dose group, respectively, compared with the control group (57.88±5.67), with significant differences among these three groups. Furthermore, inoculation of VX2 tumor into the liver itself at later stage induced significant increase in alanine aminotransferase and aspartate aminotransferase, indicating an obvious damage of liver functions, while treatment of VX2 tumor via TAE-mediated VEGF-siRNA had no toxicity to the livers and kidneys of rabbits, and VEGF-siRNA had the ability to protect liver damage induced by tumor growth.ConclusionThis is the first study to demonstrate that targeting VEGF via TAE-mediated siRNA delivery may become a powerful new option for effective treatment of hepatocellular carcinoma in the clinic.

VEGF promotes gastric cancer development by upregulating CRMP4.

This study aimed to investigate the precise role of CRMP4 in gastric tumor growth and patient survival. The mRNA and protein expression levels of CRMP4, VEGF and VEGFR2 were validated by qRT-PCR and immunohistochemistry. We investigated the effects on tumor growth of overexpression and knockdown of CRMP4 both in vitro and in vivo by constructing stable gastric cell lines using lentiviral-mediated transduction and shRNA interference-mediated knockdown of CRMP4 expression. We further validated the role of the ERK/AKT signaling pathways in VEGF and CRMP4 expression using ERK and PI3K inhibitors. Increased expression of VEGF and CRMP4 were observed in gastric cancer tissues compared with tumor-adjacent tissue. We found that higher CRPM4 expression was associated with lymph node metastasis, TNM stage, tumor differentiation and poorer prognosis in gastric cancer patients. In HGC27 and SGC7901 gastric cancer cells, VEGF upregulated CRMP4 in time and dose-dependent manners. Overexpression of CRMP4 increased cell proliferation, migration and invasion, whereas knockdown of CRMP4 expression had opposite effects. VEGF activated CRMP4 expression in gastric cancer cells, and this effect was significantly inhibited by MAPK and PI3K inhibitors (PD98059 and LY294002). In mice, CRMP4 overexpression also resulted in increased tumor growth. These results suggest that increased CRMP4 expression mediated by the activation of VEGF signaling facilitates gastric tumor growth and metastasis, which may have clinical implications associated with a reduced survival rate in gastric cancer patients.

Recurrent feature after radiofrequency ablation therapy for hepatocelluar carcinoma: Risk of increasing malignant behavior.

213 Background: Radiofrequency ablation (RFA) is a widely used therapy for hepatocellular carcinoma (HCC). However, several reports demonstrated the aggressive local recurrence after RFA, suggesting the induction of further malignant transformation of HCC. Methods: Eighty-eight patients with HCC who underwent hepatic resection were included in this study. Hepatectomy was indicated for local recurrence of HCC after RFA (n = 10, RFA group) and for the HCC without prior RFA (n = 78, non-RFA group). Clinicopathological data and patient’s prognosis after hepatectomy were compared between the two groups. Expression levels of HIF-1, EpCAM, CD44, VEGF and EMT markers mRNA in the tumor tissues were examined. We also investigated miR-34a and miR-200c expressions in the two groups. Results: RFA group showed higher frequency of portal vein invasion and less tumor differentiation compared with the non-RFA group (p &lt; 0.05). Overall and disease-free survival rates in the RFA group were significantly worse than those in the non-RFA group (p &lt; 0.05). HIF-1 and EpCAM mRNA expression levels in the RFA group were significantly higher than those in the non-RFA group (p &lt; 0.05). The expressions of TGF-β, Twist and Snail-1 in RFA group were significantly higher than those in non-RFA group (P &lt; 0.05), and Vimentin expression in RFA group tended to be higher than that in non-RFA group (P = 0.07). Regarding to miRNA, miR-200c and miR-34a expressions in RFA group were significantly lower than those in non-RFA group (miR-200c: P = 0.04, miR-34a: P &lt; 0.01). Conclusions: Recurrent HCC after RFA showed the highly malignant potential through the expressions of HIF-1, cancer stem cell and EMT markers.

Expression of HIF-2α and VEGF in Cervical Squamous Cell Carcinoma and Its Clinical Significance.

CSCC is a systemic disease involving polygenic alteration and multiple steps, and HIF and VEGF are closely associated with tumorigenesis. Specimens surgically resected from 64 cases of CSCC and 22 cases of normal cervical tissue were selected randomly to detect the expression of HIF-2α and VEGF in CSCC for exploring their clinical significance; information regarding the age, lymph node metastasis, and FIGO staging were collected as well; expression of HIF-2α and VEGF was detected by qPCR and immunohistochemistry. We found that the expression of HIF-2α and VEGF mRNA in CSCC was significantly higher than that of normal cervical tissues and showed a positive correlation between them. The positive rates of HIF-2α and VEGF protein expression in CSCC and normal cervical tissues were 93.8% and 18.2%, respectively, with correlation between them. The expression of both HIF-2α and VEGF mRNA did not relate closely to age but the FIGO staging and lymph node metastasis. Compared with the counterpart control group, CSCC tissues with high FIGO staging and lymph node metastasis had a higher level of HIF-2α and VEGF mRNA expression. So, HIF-2α and VEGF were overexpressed in CSCC, which has a great clinical significance for its diagnosis.

Comparison of viability and antioxidant capacity between canine adipose-derived mesenchymal stem cells and heme oxygenase-1-overexpressed cells after freeze-thawing.

Allogenic adipose-derived mesenchymal stem cells (Ad-MSCs) are an alternative source for cytotherapy owing to their antioxidant and anti-inflammatory effects. Frozen-thawed allogenic Ad-MSCs can be used instantly for this purpose. However, the viability and function of frozen-thawed Ad-MSCs have not been clearly evaluated. The purpose of this study was to compare the viability and function of Ad-MSCs and heme oxygenase-1 (HO-1)-overexpressed Ad-MSCs in vitro after freeze-thawing. The viability, proliferation, antioxidant capacity and mRNA gene expression of growth factors were evaluated. Frozen-thawed cells showed significantly lower viability than fresh cells (77% for Ad-MSCs and 71% for HO-1 Ad-MSCs, P<0.01). However, the proliferation rate of frozen-thawed Ad-MSCs increased and did not differ from that of fresh Ad-MSCs after 3 days of culture. In contrast, the proliferation rate of HO-1-overexpressed Ad-MSCs was lower than that of Ad-MSCs. The mRNA expression levels of TGF-β, HGF and VEGF did not differ between fresh and frozen-thawed Ad-MSCs, but COX-2 and IL-6 had significantly higher mRNA expression in frozen cells than fresh cells (P<0.05). Fresh Ad-MSCs exhibited higher HO-1 mRNA expression than frozen-thawed Ad-MSCs, and fresh HO-1-overexpressed Ad-MSCs exhibited higher than fresh Ad-MSCs (P<0.05). However, there was no significant difference between fresh and frozen HO-1-overexpressed Ad-MSCs. The antioxidant capacity of HO-1-overexpressed Ad-MSCs was significantly higher than that of Ad-MSCs. Cryopreservation of Ad-MSCs negatively affects viability and antioxidant capacity, and HO-1-overexpressed Ad-MSCs might be useful to maximize the effect of Ad-MSCs for cytotherapy.

Expression of TRAP1 in gastric cancer tissue and its correlation with malignant biology.

To study the expression of tumor necrosis factor receptor-associated protein 1 (TRAP1) in gastric cancer tissue and its correlation with malignant biology. Gastric cancer tissue and adjacent normal tissue were collected, and mRNA content and protein content of TRAP1 were detected; gastric cancer cell lines SGC7901, BGC823, AGS and MGC803 were cultured, and mRNA contents and protein contents of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF were detected. mRNA and protein expression levels of TRAP1 in gastric cancer tissue were significantly higher than those in adjacent normal tissue, and mRNA and protein expression levels of TRAP1 in gastric cancer tissue with muscularis and serosa infiltration, lymph node metastasis, distant organ metastasis and TNM Ⅲ/Ⅳ stage were significantly higher than those in gastric cancer tissue with mucosa and submucosa infiltration, non-lymph node metastasis, non-distant organ metastasis and TNM Ⅰ/Ⅱ stage. mRNA and protein expression levels of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF in MGC803 were the highest, and mRNA and protein expression levels of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF in SGC7901 were the lowest. mRNA and protein expression levels of TRAP1 in gastric cancer cell lines were positively correlated with mRNA and protein expression of CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF. The expression of TRAP1 significantly increases in gastric cancer tissue; TRAP1 may regulate the malignant biology of cells by increasing the expression of CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF, thereby resulting in the occurrence and development of gastric cancer.

Inhibitory effect of lentivirus-mediated hTERTp-TK combined with hTERTp-tumstatin on human hepatocarcinoma HepG2 cells

To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo. Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively. The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05). The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.

Abstract 4953: Molecular and cellular localization profiles of tristetraprolin in colorectal cancer: Implications for tumor progression in diverse patient populations

Abstract Introduction: Colorectal cancer (CRC) and other solid tumors rely on production of pro-inflammatory cytokines and growth factors to support their rapid growth. Posttranscriptional control of these factors involves interplay between RNA stabilizing and destabilizing factors. We have observed that the protein tristetraprolin (TTP), a RNA destabilizer, is down-regulated in CRCs and that interleukin (IL)-8, a proinflammatory cytokine, is up-regulated. We hypothesized that loss of negative regulation by TTP selectively favors expression of growth factors and protooncogenes that promote tumor progression. Furthermore, new evidence suggests that TTP is associated estrogen receptor-α (ERα) and functions as a co-repressor of ERα activation interaction with histone deacetylases on the gene promoter; nuclear TTP is linked to this tumor suppressor function. Since CRCs of African Americans (AAs) often present worse outcomes relative to those of Caucasian Americans (CAs), we analyzed the expression of TTP and its targets and assessed its subcellular localization in primary CRCs of AA and CA patients and in cultured colon cancer cells. Its effects on disease progression were also determined. Methods: Paired normal and CRC tissues from 45 patients (AA = 26 and CA = 19), were evaluated by qPCR for mRNA expression levels of TTP, IL-8, and VEGF. Tissue sections were also assayed for immunophenotypic expression by immunohistochemistry, and the staining patterns (nuclear and cytoplasmic) were assessed. To determine TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116 and LoVo) were stimulated with TNF-α, and total RNA was analyzed for TTP, IL-8, VEGF, and cIAP2 expression; interaction of TTP and ERα was also assessed. Results: In 31 of 45 CRCs, there was down-regulated expression of TTP mRNA, and low TTP levels correlated with advanced tumor stage. Low levels of TTP were present in 21 of 26 AAs and in 12 of 19 CAs. There was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. There was an agreement between mRNA expression and immunophenotypic profiles for TTP and IL-8. Furthermore, there was reduced or a lack of nuclear staining of TTP in malignant cells relative to normal/benign epithelial cells. In cultured colon cancer cells, TTP mRNA levels inversely correlated with levels of IL-8, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with these cell survival factors. Conclusions: Reduced expression of TTP was noted in tumors, especially in CRCs of AAs, and a loss or decreased nuclear expression of TTP was observed in malignant cells. Lower TTP levels correlated with advanced tumor stage. These preliminary findings suggest that TTP negatively regulates the expression of cell survival factors in the NF-kB pathway. This work was supported by funds from the NIH/NCI (a career development supplement to grant U54-CA118948). Citation Format: Esther A. Suswam, Balananda-Dhurjati K. Putcha, Amit K. Tiwari, Trafina Jadhav, Kiera D. Walker, Lualhati Harkins, Samir Amer, Isam-Eldin Eltoum, Sejong Bae, Upender Manne. Molecular and cellular localization profiles of tristetraprolin in colorectal cancer: Implications for tumor progression in diverse patient populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4953. doi:10.1158/1538-7445.AM2015-4953