NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma.

Tumor-associated macrophages (TAM) play pivotal roles in tumor progression and metastasis, but the contribution and regulation of different macrophage populations remain unclear. Here we show that Notch signaling plays distinct roles in regulating different TAM subsets in hepatocellular carcinoma (HCC). Myeloid-specific NOTCH blockade by conditional disruption of recombination signal binding protein Jκ (RBPj cKO) significantly delayed the growth of subcutaneously inoculated Lewis lung carcinoma (LLC), but accelerated orthotopically inoculated hepatic Hepa1-6 tumors in mice. In contrast to subcutaneous LLC, RBPj cKO significantly increased the number of TAMs in hepatic Hepa1-6 tumors despite impeded differentiation of monocyte-derived TAMs (moTAM). The dominating TAMs in orthotopic HCC manifested properties of Kupffer cells (KC) and hence are tentatively named KC-like TAMs (kclTAM). The increased proliferation of RBPj cKO kclTAMs was maintained even in Ccr2 -/- mice, in which moTAMs were genetically blocked. NOTCH signaling blockade accelerated proliferation of kclTAMs via enhanced ß-catenin-dependent WNT signaling, which also downregulated IL12 and upregulated IL10 expression by kclTAMs likely through c-MYC. In addition, myeloid-specific RBPj cKO facilitated hepatic metastasis of colorectal cancer but suppressed lung metastasis in mice, suggesting that the phenotype of RBPj cKO in promoting tumor growth was liver-specific. In patient-derived HCC biopsies, NOTCH signaling negatively correlated with WNT activation in CD68+ macrophages, which positively correlated with advanced HCC stages. Therefore, NOTCH blockade impedes the differentiation of moTAMs, but upregulates Wnt/ß-catenin signaling to promote the proliferation and protumor cytokine production of kclTAMs, facilitating HCC progression and hepatic metastasis of colorectal cancer. SIGNIFICANCE: These findings highlight the role of NOTCH and WNT signaling in regulating TAMs in hepatocellular carcinoma.

Expression of NEDD9 in hepatocellular carcinoma and its clinical significance.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) plays an integral role in natural and pathological cell biology. Overexpression of NEDD9 protein has been correlated with poor prognosis in various types of cancer. However, few available data address the precise function of the NEDD9 gene in hepatocellular carcinoma (HCC). In the present study, we investigated NEDD9 expression in 40 primary human HCC tissues compared with matched adjacent non-tumor hepatic tissues using RT-qPCR and western blot analysis. Immunohistochemistry was performed to analyze the correlations between NEDD9 expression and clinicopathological factors. Statistical analyses were applied to derive prognostic values of NEDD9 in HCC. The results showed that the NEDD9 mRNA and protein expression levels in HCC tissues were significantly higher than those in matched adjacent non-tumor hepatic tissues. High NEDD9 expression was correlated with larger tumor size, advanced tumor grade, metastasis, intrahepatic venous invasion and high UICC TNM stages in HCC patients. Patients with high NEDD9 expression levels exhibited poorer recurrence-free and overall survival than those with a low NEDD9 expression. Additionally, NEDD9 expression status was an independent prognostic factor for survival. This correlation remained significant in patients with early-stage HCC or with normal serum AFP levels. The results of this study suggest that NEDD9 may be a valuable prognostic biomarker for HCC, including early-stage and AFP-normal patients.

Notch signal protects non-parenchymal cells from ischemia/reperfusion injury in vitro by repressing ROS.

BACKGROUND: We have previously reported that Notch signaling pathway protects hepatocytes from ischemia/ reperfusion (I/R) injury by repressing reactive oxygen species (ROS) production. However, apart from hepatocytes, non-parenchymal cells including vascular endothelia cells, Kupffer cells and hepatic stellate cells are also reported to be involved in hepatic I/R injury. AIM: To clarify the role of Notch signaling in non-parenchymal cells subjected to I/R injury. MATERIALS AND METHODS: Human Umbilical Vein Endothelial Cells (HUVECs), mouse macrophage line RAW264.7 and rat hepatic stellate cell line HSC-T6 were cultured and subjected to I/R injury, respectively. Activation of Notch signaling was assessed by NICD western blot. Then, pharmacological inhibitor (γ-secretase inhibitor GSI) was used to block Notch signaling of related cell lines in vitro. Intracellular ROS was detected and analyzed by FACS and apoptosis was examined by TUNEL staining and Annexin V staining. RESULTS: Notch signaling responded to I/R injury and I/R injury induced activation of Notch signaling in nonparenchymal cells. Notch signal deficiency led to overproduction of ROS and aggravated cell death of non-parenchymal cells subjected to I/R injury. CONCLUSION: Notch signal protects non-parenchymal cells from I/R injury by repressing ROS.

The vasorelaxing effect of resveratrol on abdominal aorta from rats and its underlying mechanisms.

Evidence has indicated that resveratrol (Res) produces vasorelaxation and may decrease the coronary heart disease mortality. However, several pathways involved in the mechanism of vasorelaxation are still unclear. This study was designed, therefore, to test the probable ion channels or receptors involved in the mechanism. The abdominal aortic rings from the male Sprague-Dawley rats were perfused in the organ chambers filled with Kreb's solution, where the tension of each ring was measured. Treatment with L-NAME (a nitric oxide synthase inhibitor), glibenclamide and tetraethylammonium (TEA) significantly attenuated the vasorelaxing effect of Res. In lower concentration Res relaxed the ring in an endothelium-dependent manner, while in higher concentration the endothelium-independent manner could be observed. In calcium-free Kreb's solution, Res inhibited vasoconstriction induced by NE. With intracellular calcium depleted by thapsigargin, Res also inhibited vasoconstriction induced by Kreb's solution with high potassium via L-Ca(2+) channel. In a word, Res decreased both extracellular calcium influx and intracellular calcium release. These results suggest that: (1) Res may exert its relaxing effect on abdominal aorta by opening K(+) channel to hyperpolarize vascular smooth muscle.(2) Res relaxes the abdominal aorta in both endothelium-dependent and endothelium-independent manners. (3) Finally, Res attenuates both extracellular calcium influx and intracellular calcium release, which results in vasorelaxation.

Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis.

AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4',6-diamidino-2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1ß, il6, tnfα and tgfß, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.

Resveratrol attenuates ischemia/reperfusion injury in neonatal cardiomyocytes and its underlying mechanism.

This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a K(ATP) inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and K(ATP) may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.

Antioxidant activity and hepatoprotective effect of a polysaccharide from Bei Chaihu (Bupleurum chinense DC).

Chinese medicine plays a pivotal role in hepatoprotective treatment. In the present study, a water-soluble polysaccharide fraction (WBCP) was fractioned from the roots of Bupleurum chinense and purified by DEAE-cellulose and Surperdex 200 HR chromatography. The physicochemical properties, antioxidative and hepatoprotective activities of WBCP were evaluated in a rat model of hepatic injury caused by d-galactosamine (GalN). Hepatoprotective effect was evaluated by measuring aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities in the plasma of mice. Antioxidant activity was evaluated by measuring biochemical parameters in the mouse liver homogenate, such as glutathione reductase (GR), γ-glutamylcysteine synthetase (GCS), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities, as well as glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) levels. The results showed the oral administration of WBCP could significantly reduce the activity of AST, ALT, ALP and LDH, indicating that WBCP possesses hepatoprotective activity. Furthermore, there was general a statistically significant increase in the activities of GSH, GR, GCS, GST and SOD, and a loss in TBARS in the liver of WBCP-treated group compared with the control group. In addition, the elevated levels of pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α) in the serum of the experimental animals was significantly returned by WBCP treatment at the dose of 400 mg/kg. These results clearly demonstrated that WBCP possess promising hepatoprotective effects against GalN-induced liver damage, which may be mediated through augmentation of antioxidant defenses.

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma.

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4(+) and CD8(+) T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.

Prevention of pancreatic leakage after pancreaticoduodenectomy by modified Child pancreaticojejunostomy.

BACKGROUND: Pancreatic leakage after pancreaticoduodenectomy is associated with a morbidity and mortality. Different techniques have been used to make a safe anastomosis to the left pancreatic remnant. METHODS: We performed "modified Child pancreaticojejunostomy" for 31 patients, by which end-to-end pancreaticojejunal anastomosis was made with a two-layer polypropylene continuous running suture. RESULTS: In the patients who underwent pancreaticojejunostomy, the average operative time was 14.2 minutes. There was no pancreaticoenterostomy leakage in all patients, and no deaths occurred. CONCLUSIONS: In pancreaticojejunostomy, pancreatic anastomosis is time-saving and free from complications. Thus it is an improvement of pancreaticojejunostomy.

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis.

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.

Protective effects of tumor necrosis factor alpha antibody and ulinastatin on liver ischemic reperfusion in rats.

AIM: To study the protective effects of tumor necrosis factor alpha (TNF alpha) antibody and ulinastatin on liver ischemic reperfusion in rats. METHODS: One hundred and twenty male SD rats were randomly divided into four groups: normal control group, ischemic group, TNF alpha antibody group and TNF alpha antibody + ulinastatin group. The animals were killed at 0, 3, 6, 9, 12 h after ischemia for 60 min and followed by reperfusion. Serum alanine aminotransferase (ALT), malondialdehyde (MDA) and liver histopathology were observed. RESULTS: After ischemic reperfusion, the serum ALT and MDA were remarkably increased, and the hepatic congestion was obvious. Treatment of TNF alpha antibody and ulinastatin could significantly decrease serum ALT and MDA levels, and relieve hepatic congestion. CONCLUSION: Ulinastatin and TNF alpha antibody can suppress the inflammatory reaction induced by hepatic ischemic reperfusion, and have protective effects on rat hepatic ischemic reperfusion injury.

Relationship between urokinase-type plasminogen activator receptor and vascular endothelial growth factor expression and metastasis of gallbladder cancer.

AIM: To investigate the relationship of urokinase type plasminogen activator receptor (uPAR) and vascular endothelial growth factor (VEGF) expression with clinical and pathological characteristics of human gallbladder cancer. METHODS: uPAR and VEGF expressions in 68 gallbladder cancer tissues were detected with anti-receptor immunohistochemical stain. RESULTS: Expression rate of uPAR was 57.4% (39/68), and VEGF 51.5% (35/68) in gallbladder cancer tissues. Expression of both uPAR and VEGF was significantly related to metastasis, but not significantly correlated with differentiation stage and size of gallbladder cancer. CONCLUSION: Expression of uPAR and VEGF may be an invasive phenotype of gallbladder cancer and indicator for predicting prognoses, and uPAR expression is significantly correlated with the expression of VEGF.

Expression of PCNA and CD44mRNA in colorectal cancer with venous invasion and its relationship to liver metastasis.

AIM: To investigate the expression of proliferating cell nuclear antigen (PCNA) and CD44mRNA in colorectal cancer with venous invasion and its relationship with liver metastasis. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of PCNA and CD44mRNA in 31 cases of colorectal cancer with venous invasion. RESULTS: Positive expression rates of PCNA and CD44mRNA in colorectal cancer were higher than those without liver metastasis (P<0.05 and P<0.01). In case of colorectal cancer with liver metastasis, strongly positive rates of PCNA and CD44mRNA were 94.1% and 70.6%, respectively, significantly higher than those without liver metastasis. There was a positive relationship between the expressions of PCNA and CD44mRNA (r=0.67, P<0.05). CONCLUSION: Detection of PCNA and CD44mRNA expression in colorectal cancer may be useful for evaluating liver metastasis of cancer cells.

Argon supper-cryosurgery for patients with middle and late stage liver cancer.

OBJECTIVE: To assess the value of argon supper-cryosurgery for 42 patients with middle and late stage liver cancer. METHODS: Forty-two patients who had received argon supper-cryosurgery were analyzed retrospectively in terms of their clinical characteristics as well as the performance of argon supper-cryosurgery. RESULTS: All patients were ameliorated in symptoms shortly after the operation, including pain alleviation, psyche straightening up, alpha-fetoprotein descending or recovery. Jaundice occurred in 1 patient and intraabdominal hemorrhage in 2. The levels of aspartate transaminase and alanine transaminase in all patients were elevated 1 month after the operation, and normalized after protective therapy of the liver. No operative death was noted. CONCLUSIONS: Cold and heat reversed therapy of argon supper-cryosurgery can drastically destroy tumor tissue, especially the tumors which are too large to resect or close to the large vessels. It is applicable to increase the operative rate, decrease the operative death rate, and enlarge the therapeutic scope.