OBJECTIVETo explore the effect of colon cancer cell-derived interleukin-1α on the migration and proliferation of human umbilical vein endothelial cells as well as the role of IL-1α and IL-1ra in the angiogenesis process.

METHODSWestern blot was used to detect the expression of IL-1α and IL-1R1 protein in the colon cancer cell lines with different liver metastatic potential. We also examined how IL-1α and IL-1ra influence the proliferation and migration of umbilical vascular endothelial cells assessed by PreMix WST-1 assay and migration assay, respectively. Double layer culture technique was used to detect the effect of IL-1α on the proliferation and migration of vascular endothelial cells and the effect of IL-1ra on the vascular endothelial cells.

RESULTSWestern blot analysis showed that IL-1α protein was only detected in highly metastatic colon cancer HT-29 and WiDr cells, but not in the lowly metastatic CaCo-2 and CoLo320 cells.Migration assay showed that there were significant differences in the number of penetrated cells between the control (17.9±3.6) and 1 ng/ml rIL-1α group (23.2±4.2), 10 ng/ml rIL-1α group (31.7±4.5), and 100 ng/ml rIL-1α group (38.6±4.9), showing that it was positively correlated with the increasing concentration of rIL-1α (P<0.01 for all). The proliferation assay showed that the absorbance values were 1.37±0.18 in the control group, and 1.79±0.14 in the 1 ng/ml rIL-1α group, 2.14±0.17 in the 10 ng/ml rIL-1α group, and 2.21±0.23 in the 100 ng/ml rIL-1α group, showing a positive correlation with the increasing concentration of rIL-1α(P<0.01 for all). IL-1ra significantly inhibited the proliferation and migration of vascular endothelial cells (P<0.01). The levels of VEGF protein were (1.697±0.072) ng/ml, (3.507±0.064)ng/ml and (4.139±0.039)ng/ml in the control, HUVECs+ IL-1α and HUVECs+ HT-29 co-culture system groups, respectively, showing a significant difference between the control and HUVECs+ 10 pg/ml rIL-1α groups and between the control and HUVECs+ HT-29 groups (P<0.01 for both).

CONCLUSIONSOur findings indicate that colon cancer cell-derived IL-1α plays an important role in the liver metastasis of colon cancer through increased VEGF level of the colon cancer cells and enhanced vascular endothelial cells proliferation, migration and angiogenesis, while IL-1ra can suppress the effect of IL-1α and inhibit the angiogenesis in colon cancer.

Blotting, Western ; Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Coculture Techniques ; Colonic Neoplasms ; blood supply ; metabolism ; pathology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Interleukin 1 Receptor Antagonist Protein ; metabolism ; physiology ; Interleukin-1alpha ; metabolism ; physiology ; Liver Neoplasms ; secondary ; Neovascularization, Pathologic ; etiology

OBJECTIVETo construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.

METHODSGPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA.

RESULTSIn all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001).

CONCLUSIONGPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.

Apoptosis ; Carcinoma, Hepatocellular ; blood supply ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Gene Silencing ; Glypicans ; genetics ; Humans ; Liver Neoplasms ; blood supply ; metabolism ; pathology ; Neoplasm Invasiveness ; Neovascularization, Pathologic ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; beta Catenin ; metabolism

We previously demonstrated that there are acute and delayed phases of renal protection against renal ischemia and reperfusion (IR) injury with renal ischemic preconditioning (IPC). This study assessed whether hepatic IPC could also reduce distant renal IR injury through the blood stream-mediated supply of reactive oxygen species (ROS). Male C57BL/6 mice were randomly divided into four groups: group I, sham operated including right nephrectomy; group II (IR), left renal ischemia for 30 min and reperfusion injury; group III (IPC-IR), hepatic ischemia for 10 min followed by 10 min of reperfusion before left renal IR injury; group IV (MPG - IPC + IR), pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine (MPG) 15 min before hepatic IPC and left renal IR injury. Renal function, histopathologic findings, proinflammatory cytokines, and cytoprotective proteins were evaluated 15 min or 24 hr after reperfusion. Hepatic IPC attenuated the expression of proinflammatory cytokines, tumor necrosis factor alpha, intercellular adhesion molecule 1, and induced inducible nitric-oxide synthase, and the phosphorylation of Akt in the murine kidney. Renal function was better preserved in mice with hepatic IPC (group III) than groups II or IV. Hepatic IPC protects against distant renal IR injury through the blood stream-delivery of hepatic IPC-induced ROS, by inducing cytoprotective proteins, and by inhibiting inflammatory reactions.
Animals ; Intercellular Adhesion Molecule-1/genetics/metabolism ; *Ischemic Preconditioning ; Kidney/drug effects/metabolism/pathology/physiopathology ; Liver/blood supply/drug effects/physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type II/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism ; Reactive Oxygen Species/metabolism ; Reperfusion Injury/*metabolism/pathology/prevention & control ; Tiopronin/pharmacology ; Tumor Necrosis Factor-alpha/genetics/metabolism

OBJECTIVECelastrus orbiculatus Thunb. has been used for thousands of years in China as a remedy against cancer and inflammatory diseases. This study aims to investigate whether C. orbiculatus extract (COE) could inhibit angiogenesis, which is the pivotal step in tumor growth, invasiveness, and metastasis.

METHODSIn this study, the extract from the stem of C. orbiculatus was used. Mouse hepatic carcinoma cells (Hepa1-6) were treated with COE in different nontoxic concentrations (10, 20, 40, 80, and 160 μg/mL). The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively; the active fractions were further tested on C57BL/6 mice and human umbilical vein endothelial cells (HUVEC) for any antiangiogenic effects.

RESULTSCOE significantly inhibited proliferation and induced apoptosis in Hepa1-6 cells and inhibited VEGF expression at both mRNA and protein levels. Furthermore, this agent inhibited the formation of the capillary-like structure in primary cultured HUVEC in a dose-dependent manner. In vivo, COE significantly reduced the volume and weight of solid tumors with low adverse effects and decreased tumor angiogenesis.

CONCLUSIONSIn summary, COE could be used to treat hepatic carcinoma. The mechanisms of the antitumor activity of COE may be due to its effects against tumor angiogenesis by targeting the VEGF protein.

Administration, Oral ; Angiogenesis Inhibitors ; pharmacology ; therapeutic use ; Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; blood supply ; drug therapy ; pathology ; Celastrus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Collagen ; metabolism ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; metabolism ; Liver Neoplasms ; blood supply ; drug therapy ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; drug therapy ; pathology ; Neovascularization, Physiologic ; drug effects ; Phytotherapy ; Plant Extracts ; administration & dosage ; pharmacology ; therapeutic use ; Plant Stems ; chemistry ; Proteoglycans ; metabolism ; Signal Transduction ; drug effects ; Transcriptional Activation ; drug effects ; genetics ; Tumor Burden ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis ; metabolism

OBJECTIVETo study the clinicopathological features of diabetic microangiopathy in liver and diabetic hepatosclerosis (DHS) of elderly male with type 2 diabetes mellitus (T2DM).

METHODSOne hundred and twenty autopsy cases with T2DM (diabetic group) and contemporary 48 cases, non-diabetic and glucose tolerance abnormal, matched by gender and age (control group) were selected in the study. Cases with the cirrhosis and fibrosis of liver caused by other foregone etiological factors were excluded. The histopathological changes of microangiopathy in liver, hepatic portal areas and hepatic sinusoid were investigated by HE staining, histochemical and immunohistochemical stain methods. The clinical data of diagnostic DHS cases were analyzed.

RESULTS(1) Microangiopathy was observed in 54.2% (65/120) cases of diabetic group. Histological features: microangiopathy was found in interlobular arteries (especially in arteriole, the lumen diameter < 100 µm), which included endothelial denudation, eosinophilic material deposition in the tunica intima of artery, and eccentric intimal thickening. The smooth muscle fibers of tunica media were hyperplastic or atrophy. Fibroplasia and collagen deposition were found in the tunica adventitia of artery. Arterial lumina showed stenosis and occlusion. Microangiopathy was seen in 16.7% (8/48) cases of the control group. There was statistically significant difference between the two groups (χ(2) = 19.622, P < 0.01). (2) The fibrosis and sclerosis of portal areas were detected in 55.8% (67/120) cases of T2DM group. Hyaline collagen fiber tissues was deposited around interlobular arteries, interlobular veins and interlobular bile ducts, resulting in enlargement of the portal area and the secondary atrophy and disappearance of portal triad. The fibrosis and sclerosis of portal areas were detected in 22.9% (11/48) cases of the control group. There was a statistically significant difference between the two groups (χ(2) = 14.936, P < 0.01). (3) The pathological features of 14.2% (17/120) cases were consistent with the diagnosis of DHS. The fibrous tissue extended from fibrosis or sclerosis of portal areas, or eosinophilic material deposition in the hepatic sinusoid in non-zonal pattern. The results of histochemical staining showed collagen fiber deposition in hepatic sinusoid. Stainings for Collagen IV, SMA, CD34 were found in the hepatic sinusoid. The sclerosis of hepatic sinusoid was not detected in any case in the control group.Overall, 13/17 and 11/17 DHS cases had liver microangiopathy and portal areas sclerosis respectively. Diabetic nephropathy was seen in 10 of 17 DHS cases. Among the 17 cases, 7 cases showed ALP elevation, of which there were 3 cases with ALT and AST mild elevation.

CONCLUSIONSDiabetic microangiopathy is common in the liver of elderly men with T2DM. And DHS is associated with diabetic microangiopathy. Fibrosis and sclerosis of portal areas may be the early or concomitant changes of DHS on histological ground. DHS is one of the complications of T2DM.

Actins ; metabolism ; Aged ; Aged, 80 and over ; Alanine Transaminase ; blood ; Alkaline Phosphatase ; blood ; Antigens, CD34 ; metabolism ; Aspartate Aminotransferases ; blood ; Collagen Type IV ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; Diabetic Angiopathies ; blood ; etiology ; metabolism ; pathology ; Diabetic Nephropathies ; complications ; pathology ; Humans ; Liver ; blood supply ; pathology ; Liver Cirrhosis ; complications ; Male ; Middle Aged ; Sclerosis

To evaluate the changes induced in tumor tissue, the feeding artery, and neovascularization upon pingyangmycin-lipiodol emulsion treatment via transcatheter arterial chemoembolization (TACE) using the rabbit VX2 liver cancer model. The VX2 liver tumor model was established in 28 rabbits, and baseline tumor volume (V1, in mm3) was measured by spiral scan computed tomography (CT). Then, the rabbits were randomly divided into four groups (n = 7 each) and administered intraarterial therapies of: ultrafluid lipoidol embolization (group A); pingyangmycin (group B); pingyangmycin-lipiodol emulsion (group C); or saline (group D). All rabbits were sacrificed seven days later, and the response to therapy was determined by measuring the tumor volume (V2, in mm3), calculating the tumor growth rate, detecting expression of the vascular endothelial growth factor (VEGF) tumor biomarker, and performing histological analysis of the microvessel density (MVD) in the liver. Prior to therapy, the average V1 of the groups was statistically similar (A: 389.8+/-167.3, B: 404.1+/-184.9, C: 355.1+/-158.3, D: 378.1+/-189.0; (F = 0.257, P more than 0.05). In contrast, after therapy the average V2 of the groups was significantly different (A: 922.6+/-32.9, B: 665.9+/-99.9, C: 349.5+/-177.8, D: 1403.5+/-411.2; F = 26.23, P less than 0.05), as was the tumor growth ratio (A: 1.4, B: 0.6, C: -0.02, D: 2.7) and the mean positive ratio of VEGF (A: 57.1%, B: 42.9%, C: 28.6%, D: 100%; F = 8.407, P less than 0.05). MVD was highest in group D and lowest in group C (all, P less than 0.05). Bivariate correlation analysis revealed a positive correlation between VEGF expression and MVD (r = 0.743, P less than 0.01). Pingyangmycin exerts anti-tumor effects in the rabbit VX2 liver cancer model, but is more effective when administered as the combination therapy of pingyangmycin-lipiodol emulsion with TACE.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; therapeutic use ; Bleomycin ; administration & dosage ; analogs & derivatives ; therapeutic use ; Chemoembolization, Therapeutic ; methods ; Emulsions ; Ethiodized Oil ; administration & dosage ; therapeutic use ; Female ; Iodized Oil ; administration & dosage ; therapeutic use ; Liver Neoplasms, Experimental ; blood supply ; drug therapy ; pathology ; Male ; Microvessels ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Rabbits ; Random Allocation ; Tumor Burden ; drug effects ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUND/AIMS: Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats. METHODS: To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed. RESULTS: Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (P=0.615 and P=0.186). On TUNEL, numbers of apoptotic cells at 100x magnification (cells/field) were 31.8+/-24.2 in NC, 69.0+/-72.3 in TC, 80.2+/-63.1 in IP, and 21.2+/-20.8 in IC (P<0.05). When regeneration capacity was assessed by PCNA staining, PCNA-positive cells (cells/field) at 400x were 3.4+/-6.0 in NC, 16.9+/-69 in TC, 17.0+/-7.8 in IP and 7.4+/-7.6 in IC (P<0.05). CONCLUSIONS: Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.
Alanine Transaminase/blood ; Animals ; Apoptosis ; Aspartate Aminotransferases/blood ; Constriction ; Hepatectomy/methods ; Hepatic Artery ; *Ischemic Preconditioning ; Liver/blood supply ; Liver Cirrhosis, Experimental/chemically induced/complications/*pathology ; *Liver Regeneration ; Proliferating Cell Nuclear Antigen/metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/complications/enzymology/pathology ; Thioacetamide/toxicity

OBJECTIVETo investigate the sheltering effects of ω-3 polyunsaturated fatty acid (ω-3PUFA) and lymphatic drainage on distant organs in intestinal ischemia-reperfusion injury in rats.

METHODSForty-eight healthy Sprague-Dawley (SD) male rats (SPF grade) were randomly divided into 3 groups (16 rats in each group): normal diet group (N), enteral nutrition group (EN), enteral nutrition and ω-3PUFA group(PUFA group). Each group was divided into lymphatic drainage (I/R + D) group and no-drainage (I/R) group (n = 8). Each rats received gastrostomy. After given different nutrition for five days, the rats subjected to 60 min ischemia and 120 min reperfusion injury of the superior mesenteric artery. When the rats subjected to ischemia-reperfusion injury, drained intestinal lymph for 180 min in the I/R + D group. The serum level of alanine aminotransferase (ALT) and level of myeloperoxidase (MPO), nitric oxide (NO), total of nitric oxide synthase (tNOS), inducible nitric oxide synthase (iNOS) of lung were detected. The organ injury of lung and liver and the expression of high mobility group box 1(HMGB1, the endogenous ligand of TLR4) in these organs were investigated too.

RESULTSThe serum level of ALT in PUFA I/R + D and I/R group and EN I/R + D group were significantly lower than that in normal diet I/R group: (46 ± 20), (53 ± 15), (46 ± 21) and (100 ± 60) U/L (P < 0.05), respectively. The level of MPO, NO, tNOS, iNOS in lung in the I/R + D group were significantly lower than those in I/R group (P < 0.05): MPO (0.73 ± 0.15):(0.85 ± 0.10) unit/grams wet slice; NO (0.72 ± 0.51):(1.79 ± 1.32) µmol/gprot; tNOS (0.46 ± 0.15):(0.78 ± 0.27) U/mgprot; iNOS (0.06 ± 0.04):(0.11 ± 0.07) U/mgprot, respectively. The level of tNOS in PUFA I/R group was significantly lower than that in normal diet I/R group: (0.56 ± 0.13):(0.78 ± 0.27) U/mgprot (P < 0.05). MPO, NO, INOS levels in PUFA group were reduced compared with those in EN and normal diet group. HE stained sections and HMGB1 immunohistochemistry results showed that the organ injury in I/R group was severer than that in I/R + D group. The expression of HMGB1 increased in I/R group. The organ injury and the expression of HMGB1 in PUFA group were less than that in the other two main groups.

CONCLUSIONSLymphatic drainage can alleviate injury of distant organs after intestinal ischemia-reperfusion in rats. ω-3 polyunsaturated fatty acids can increase body resistance to injury and promote recovery.

Animals ; Disease Models, Animal ; Drainage ; Fatty Acids, Omega-3 ; pharmacology ; Intestines ; blood supply ; Liver ; metabolism ; pathology ; Lung ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; prevention & control

Angiogenesis Inhibitors ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Hepatocellular ; blood supply ; metabolism ; pathology ; therapy ; Chemoembolization, Therapeutic ; methods ; Endostatins ; therapeutic use ; Humans ; Liver Neoplasms ; blood supply ; metabolism ; pathology ; therapy ; Microvessels ; pathology ; Neovascularization, Pathologic ; pathology ; Vascular Endothelial Growth Factors ; metabolism

OBJECTIVETo assess the feasibility of usage of microbubbles conjugated with RGD peptides and contrast enhanced ultrasound (CEU) in detection of tumor angiogenesis.

METHODSLipid microbubbles (MB) were prepared, and the RGD peptides were covalently conjugated to the lipid shell of MB (MB(RGD)). Six nude mice with tumor created by dorsal inoculation of HepG2 tumor cells were used as the test group. Six nude mice without tumor were served as the control group. 10 minutes after bolus injection of MB and MB(RGD) randomly (30 min interval) via a tail vein catheter, CEU was performed on the tumors of the test group and the thigh skeletal muscles of control group. The video intensity (VI) of tumors and the skeletal muscles were measured. The tumors and the skeletal muscles were harvested for immunohistochemical examination.

RESULTSOnly a slight contrast enhancement of the tumor was seen with MB, and the VI was 5.33 ± 1.71. While a remarkable enhancement of the tumor was observed after injection of MB(RGD). The VI was up to 17.03 ± 3.58, 3.18 folds higher as compared with that obtained by injection of MB (P < 0.05). As expected, there were no obvious contrast enhancement of the skeletal muscles with both MB(RGD) and MB. There was a high expression of αvβ3-integrin in tumor neovascular endothelium, however, no apparent expression of αvβ3-integrin was observed in the skeletal muscle vascular endothelium.

CONCLUSIONCEU with MB(RGD) can be used to effectively evaluate the angiogenesis of tumors, and it may greatly contribute to the early judgement of the nature of tumor.

Animals ; Cell Line, Tumor ; Contrast Media ; Endothelium, Vascular ; diagnostic imaging ; metabolism ; Female ; Humans ; Integrin alphaVbeta3 ; metabolism ; Liver Neoplasms ; blood supply ; diagnostic imaging ; metabolism ; pathology ; Male ; Mice ; Mice, Nude ; Microbubbles ; Muscle, Skeletal ; blood supply ; Neoplasm Transplantation ; Neovascularization, Pathologic ; diagnostic imaging ; metabolism ; pathology ; Oligopeptides ; Ultrasonics ; methods ; Ultrasonography