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ABSTRACT: Due to invasion and intrahepatic metastasis, the prognosis for patients with hepatocellular carcinoma (HCC) is poor. However, the mechanisms underlying these processes of HCC remain unclear. Cancer stem cells may be involved in early systemic dissemination and metastasis formation and side population (SP) cells isolated from diverse cancer cells possess stem cell-like properties. However, the mechanisms involved in migration and invasion of cancer stem cells are not well understood. In this study, we identified and isolated populations of SP cells from HCC cell lines using flow cyto-metry. SP cells showed higher levels of migration and invasion capability. Higher expression of miR-21 was observed in SP cells. Silencing of miR-21 led to a reduction in the migration and invasion of these cells and overexpression of miR-21 can increase in cell migration and invasion. Overexpression of miR-21 did not cause degradation of PTEN or RECK or PDCD4 mRNA but drastically inhibited its protein expression. Consistent with these results, silencing miR-21 increased the levels of PTEN, RECK and PDCD4 protein, respectively. The role of silencing miR-21 was partially attenuated by silencing of PTEN or RECK or PDCD4 mRNA. The results of this study revealed the aberrant expression of miR-21 in SP cells and showed that miR-21 regulates the expression of multiple target proteins that are associated with tumor dissemination. MiR-21 is a pro-metastatic miRNA in SP cells and raises the possibility that therapy of HCC may be improved by pharmaceutical strategies directed towards miR-21.
International Journal of Oncology 05/2013; · 2.40 Impact Factor
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ABSTRACT: Although Notch1 expression has been associated with progression or prognosis in various tumors, the role of Notch1 in hepatocellular carcinoma (HCC) remains unknown. This study sought to investigate the clinicopathological and prognostic relevance of Notch1 expression in HCC as well as the underlying mechanisms responsible. HCC tissues were stained with an anti-Notch1 antibody. The invasion capacities of cells were measured using Transwell cell culture chambers. Reverse transcription PCR and/or western blot were used to evaluate the expression levels of Notch1, matrix metalloproteinase (MMP)-2, and MMP-9. Notch1 expression was downregulated by RNA interference. The activity of MMP-2/MMP-9 was quantified by enzyme-linked immunosorbent assay, and cellular apoptosis was analyzed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Notch1 expression was mainly localized within the cytoplasm and at the cell membrane. High Notch1 expression correlated with tumor size, tumor grade, metastasis, venous invasion, and American Joint Committee on Cancer TNM stage (P < 0.05), and patients with high levels of Notch1 expression were at a significantly increased risk for shortened survival time (P < 0.05). In vitro, the downregulation of Notch1 expression decreased the invasion capacity of HCC cells via the regulation of MMP-2 and MMP-9. The results of the MTT assay showed that downregulation of Notch1 did not affect HCC cell viability. Notch1 may represent a novel candidate marker for patient prognosis as well a molecular target for HCC therapy.
Tumor Biology 11/2012; · 1.94 Impact Factor
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ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide; however, the prognosis of HCC patients remains poor. This poor prognosis is mainly attributed to the high rate of intrahepatic and distant metastasis. HCC often occurs in a hypoxic environment and hypoxia can activate metastatic programs, ultimately leading to tumor recurrence or metastasis. Thus, the discovery and subsequent development of novel agents to block HCC invasion and migration are the primary objectives of hepatic cancer research. The Notch1 signaling pathway might be involved in hypoxia-induced carcinoma metastasis. However, the mechanisms by which Notch1 mediates cell metastasis, particularly in hepatocellular carcinoma, are not yet entirely clear. The results of the present study show that hypoxia increases the invasion and migration capacities of different HCC cells. Activation of the Notch1 signaling pathway contributes to hypoxia-induced invasion and migration in HCC cells. The activated Notch1 signaling pathway can regulate Snail/E-cadherin through cyclooxygenase-2 (COX-2) under hypoxic conditions. The above results suggest that the Notch1/COX-2/Snail/E-cadherin pathway is possibly associated with hypoxia-induced invasion and migration in HCC cells. Thus, targeting Notch1 may be useful for devising novel preventive and therapeutic strategies for HCC.
Oncology Reports 10/2012; · 1.84 Impact Factor
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ABSTRACT: BACKGROUND: The Notch signaling pathway plays an important role in cancer, but the mechanism by which Notch1 participates in invasion and migration of hepatocellular carcinoma (HCC) cells is unclear. AIMS: Our purpose is to confirm the anti-invasion and anti-migration effects of the down-regulation of Notch1 in HCC cells. METHODS: The invasion and migration capacities of HCC cells were detected with Transwell cell culture chambers. The expressions of Notch1, Notch1 intracellular domain (N1ICD), E-cadherin, Snail, and cyclooxygenase-2 (COX-2) were analyzed by RT-PCR and/or western blotting. Notch1 and Snail were down-regulated by RNA interference, and COX-2 was inhibited by NS-398. Cell apoptosis was analyzed by MTT and flow cytometry. RESULTS: In HCC cells, Snail, Notch1, and COX-2 were up-regulated, and E-cadherin was down-regulated in mRNA and/or protein levels. The down-regulation of Snail or Notch1 or the inhibition of COX-2, respectively, can increase the mRNA and protein expressions of E-cadherin and decrease the invasion and migration capabilities of HCC cell. Down-regulated Notch1 or inhibited COX-2 can reduce the mRNA and protein expressions of Snail. The down-regulation of Notch1 can also reduce the protein expression of COX-2. However, exogenous PGE2 can reverse the role of down-regulated Notch1. The results of MTT and flow cytometry showed that down-regulated Notch1 did not affect HCC cell viability. CONCLUSIONS: Down-regulated Notch1 may be an effective approach to inactivating Snail/E-cadherin by regulating COX-2, which results in inhibiting the invasion and migration of HCC cells. The inhibitory effects of down-regulated Notch1 on cell invasion and migration were independent of apoptosis.
Digestive Diseases and Sciences 10/2012; · 2.12 Impact Factor
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ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common malignancies. The main cause of death in HCC patients is tumor progression with invasion and metastasis. However, the underlying mechanisms of HCC invasion and metastasis are still not fully understood. Some studies show that the Notch signaling pathway may participate in tumor invasion and metastasis. However, the mechanisms by which the Notch signaling pathway mediates tumor cell invasion, especially in hepatocellular carcinoma, are not yet known. In the current study, we investigated the anti-invasion effect of the downregulation of the Notch signaling pathway by DAPT in HCC cells. The Notch signaling pathway inhibitor could suppress invasion of HCC cells via the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathways, resulting in the downregulation of matrix metalloproteinase-2 and -9 (MMP-2 and -9) and vascular endothelial growth factor (VEGF). These observations suggested that inhibition of the Notch signaling pathway by DAPT would be useful for devising novel preventive and therapeutic strategies targeting invasion of HCC.
Oncology Reports 06/2012; 28(3):874-82. · 1.84 Impact Factor
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ABSTRACT: MicroRNA-10b (miR-10b) was recently reported to be dysregulated in some types of cancer and to play a role in invasion and metastasis. However, effects and potential mechanisms of action of miR-10b in the metastasis of hepatocellular carcinoma (HCC) have not been explored. In this study, we confirmed that miR-10b is highly expressed in metastatic HCC tissues and in metastatic HCC cell lines by qRT-PCR. Moreover, patients with higher miR-10b expression had significantly poorer overall survival, and high miR-10b expression was an independent predictor of poor prognosis. Inhibition of miR-10b reduced cell migration and invasion in MHCC97H cells, whereas over-expression of miR-10b in HepG2 cells increased cell migration and invasion. Bioinformatics and luciferase reporter assays revealed that miR-10b binds the 3'-UTR of CADM1 mRNA and represses its translation. Western blot and qRT-PCR showed that CADM1 is inhibited by miR-10b over-expression. Silencing of CADM1 resulted in substantially increased cell motility and invasion similar to that observed with over-expression of miR-10b in HepG2 cells. These results suggest that miR-10b may positively regulate the invasion and metastasis of HCC through targeting CADM1.
Tumor Biology 04/2012; 33(5):1455-65. · 1.94 Impact Factor
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ABSTRACT: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis.
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).
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 il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups.
MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.
World Journal of Gastroenterology 03/2012; 18(10):1048-58. · 2.47 Impact Factor
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Xiao Li,
Jun-Jie Li,
Jing-Yue Yang, De-Sheng Wang,
Wei Zhao,
Wen-Jie Song,
Wei-Min Li,
Jian-Feng Wang,
Wei Han,
Zhuo-Chao Zhang,
Yong Yu,
Da-Yong Cao,
Ke-Feng Dou
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ABSTRACT: Dendritic cells (DCs) release bioactive exosomes that play an important role in immune regulation. Because they express low levels of class I major histocompatibility complex (MHC) and co-stimulatory molecules, exosomes derived from donor immature DCs (imDex) prolong allograft survival by inhibiting T-cell activation. However, this effect is limited and does not induce immunological tolerance when imDex are administered alone. Thus, we tested the effect of combined treatment with donor imDex and low-dose rapamycin on inducing tolerance in a mouse cardiac transplantation model.
ImDex were obtained from the culture supernatant of immature DCs derived from donor mouse (C57BL/6) bone marrow and were injected with suboptimal doses of rapamycin into recipient mouse (BALB/c) before and after transplantation. The capacity of this treatment to induce immune tolerance was analyzed in vitro and in vivo using the mouse cardiac transplantation model.
Donor imDex expressed moderate levels of MHC class II and low levels of MHC class I and co-stimulatory molecules, but neither imDex nor subtherapeutic rapamycin dose alone induced cardiac allograft tolerance. Combined treatment with imDex and rapamycin, however, led to donor specific cardiac allograft tolerance. This effect was accompanied by decreased anti-donor antigen cellular response and an increased percentage of spleen CD4(+)CD25(+) T cells in recipients. Furthermore, this donor specific tolerance could be further transferred to naïve allograft recipients through injection of splenocytes, but not serum, from tolerant recipients.
Combined with immunosuppressive treatment, donor imDex can prolong cardiac allograft survival and induce donor specific allograft tolerance.
PLoS ONE 01/2012; 7(8):e44045. · 4.09 Impact Factor
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ABSTRACT: To investigate the biological effect of adenosine A2b receptor (A2bR) on the human hepatocellular carcinoma cell line HepG2, three A2bR siRNA constructs were transiently transfected into HepG2 cells. The results showed that A2bR siRNA reduced the levels of A2bR mRNA and protein. In order to further detect the function of A2bR, we established a stable hepatocellular carcinoma cell line (HepG2) expressing siRNA targeting the adenosine A2b receptor. Targeted RNAi significantly inhibited tumor cell growth in vitro, and flow cytometry (FCM) showed that significantly more cells expressing A2bR siRNA were in the G0/G1 phase compared to the untransfected group ((89.56% ± 3.15%) versus (56.19% ± 1.58%), P < 0.01). These results indicated that silencing the expression of adenosine A2b receptor in HepG2 cells can suppress cell growth effectively by blocking the cell cycle. Downregulation of adenosine A2b receptor gene expression with RNA interference could be a new approach to hepatocellular carcinoma therapy.
ISRN oncology. 01/2011; 2011:875684.
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Yong-Hui Liao,
Gui-He Zhang,
Dong Jia,
Peng Wang,
Nian-Song Qian,
Fei He,
Xiang-Tian Zeng,
Yong He,
Yan-Ling Yang,
Da-Yong Cao,
Yi Zhang, De-Sheng Wang,
Kai-Shan Tao,
Chang-Jun Gao,
Ke-Feng Dou
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ABSTRACT: Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging research implicates the participation of spinal glial cells in some neuropathic pain models. However, it remains unknown whether spinal glial cells are activated under type 2 diabetic conditions and whether they contribute to diabetes-induced neuropathic pain. In the present study, using a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The mechanical allodynia was significantly attenuated by intrathecally administrated l-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) did not have any effect on mechanical allodynia, which indicated that spinal astrocytic activation contributed to allodynia in db/db mice. Further study aimed to identify the detailed mechanism of astrocyte-induced allodynia in db/db mice. Results showed that spinal activated astrocytes dramatically increased interleukin (IL)-1β expression which may induce N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to enhance pain transmission. Together, these results suggest that spinal activated astrocytes may be a crucial component of mechanical allodynia in type 2 diabetes and "Astrocyte-IL-1β-NMDAR-Neuron" pathway may be the detailed mechanism of astrocyte-induced allodynia. Thus, inhibiting astrocytic activation in the spinal dorsal horn may represent a novel therapeutic strategy for treating DNP.
Brain research 10/2010; 1368:324-35. · 2.46 Impact Factor
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ABSTRACT: 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.
Cellular Immunology 06/2009; 259(1):13-20. · 1.97 Impact Factor
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ABSTRACT: Expression of Fas ligand (FasL) on the graft by gene transduction is expected to introduce apoptosis to lymphocytes to protect rejection, but the FasL-expressing graft cells may also induce apoptosis as the graft usually expresses Fas antigens. In this study, a strong antiapoptotic gene, bcl-2, was cotransfected with the FasL gene in rat liver graft to protect against Fas-mediated cell death and to prolong recipient survival.
Orthotopic liver transplantation was done in a strain combination of DA to LEW rats. After donor vascular isolation, adenovirus-mediated FasL and bcl-2 genes were cotransfected in the liver graft.
Intragraft expression of FasL mRNA was constitutively expressed after adenovirus-mediated transduction, although expression of FasL increased mildly in control grafts. Bcl-2 mRNA was highly expressed at 2 days after reperfusion. In contrast, lower expression of bcl-2 was observed in the control group. The average survival of the gene transferred allografts increased from (9.8+1.3) days to (18.5+8.7) days compared with the control group.
Our results indicate that rat liver allografts can be protected against host immune responses by adenovirus-mediated FasL and bcl-2 transfection, and that bcl-2 expression prevents the graft from Fas-mediated apoptosis.
Hepatobiliary & pancreatic diseases international: HBPD INT 12/2006; 5(4):505-10. · 1.08 Impact Factor
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ABSTRACT: High levels of adenosine accumulate in hypoxic tissues during the rapid growth of tumors, suggesting activation of adenosine receptors may facilitate tumor progress. The relevance of adenosine receptors to hepatocellular carcinoma (HCC), in particular the adenosine A(2b) receptor (A(2b)), is not yet fully understood. The aim of this study was to assess whether A(2b) was differentially expressed in normal and cancerous tissues and evaluate the clinicopathological correlation of A(2b) level in HCC. Expression of A(2b) in tumor cells was investigated by immunohistochemical staining. Protein analysis was done by Western blotting and evaluation of A(2b) mRNA expression levels utilized quantitative real-time PCR analysis of tissue samples of 64 hepatocellular carcinomas and in their paired adjacent normal tissues. Western blot data suggested that A(2b) was expressed predominantly in the cell membrane and cytoplasm of tumor cells and that the intensity of A(2b) protein expression was consistently higher in HCC than in adjacent normal tissues. Levels of A(2b) mRNA in HCC were significantly higher than in adjacent tissues, as measured by real-time PCR (P<0.001). With regard to venous invasion, satellite lesions and advanced pathologic Tumor-Node-Metastasis (pTNM) stage, the A(2b) level tended to be higher than that seen in negative cases (P<0.05). Our findings demonstrate that A(2b) expression is up-regulated in HCC, the expression level of A(2b) is correlated to tumor progression in HCC, and suggest that A(2b) may be a novel target for HCC therapeutic strategy.
Hepatology Research 09/2006; 36(1):56-60. · 2.20 Impact Factor
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ABSTRACT: To investigate the mechanism of enhancement of invasion of hepatocellular carcinoma (HCC) cells by lysophosphatidic acid (LPA).
Human HCC cells of the line SMMC7721 were cultured. LPA at different concentrations (2, 5, and 25 micromol/L) was added into the culture fluid. The Rho activity was detected with Rho activity detection kit. Western blotting was used to detect the protein expression of Rho. Adhesion test was conducted to calculate the adhesion percentage of the SMMC7721 cells. The invasion potential of the SMCC7721 cells was observed using transwell cell test.
LPA at the concentrations of 5, and 25 micromol/L increases the activity of Rho protein. When the concentration of LPA was 25 micromol/L the activity of Rho protein was 400 times that of the control group (P < 0.01). The Rho protein expression in the SMCC7721 cells increased when stimulated by LPA, peaked 20 approximately 25 hours after stimulation, and then gradually decreased. When the concentration of LPA was 25 micromol/L the Rho protein expression level was 242% higher than that of the control group. LPA at the concentration of 5 micromol/L and over increased the migratory and invading potential of the SMCC7721 cells and increased the adhesiveness of the SMCC7721 cells time-dependently.
LPA increases the migratory and invading potential of HCC cells through Rho signal transduction pathway.
Zhonghua yi xue za zhi 02/2006; 86(6):399-402.
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ABSTRACT: Intrahepatic extension is the main cause of liver failure and death in hepatocellular carcinoma patients. The small GTPase Rho and one of its effector molecules ROCK regulate cytoskeleton and actomyosin contractility, and play a crucial role in cell adhesion and motility. We investigated the role of small GTPase Rho in biological behaviors of hepatocellular carcinoma to demonstrate the importance of Rho in cancer invasion and metastasis.
Using Western blotting, we quantitated Rho protein expression in SMMC-7721 cells induced by Lysophosphatidic acid (LPA). Furthermore, we examined the role of Rho signaling in regulating the motile and invasive properties of tumor cells.
Rho protein expression was stimulated by LPA. Using the Rhotekin binding assay to assess Rho activation, we observed that the level of GTP-bound Rho was elevated transiently after the addition of LPA, and Y-27632 decreased the level of active Rho. LPA enhanced the motility of tumor cells and facilitated their invasion. Rho played an essential role in the migratory process, as evidenced by the inhibition of migration and motility of cancer cells by a specific inhibitor of ROCK, Y-27632.
The finding that invasiveness of hepatocellular carcinoma is facilitated by the Rho/Rho-kinase pathway is likely to be relevant to tumor progression and Y-27632 may be a new potential effective agent for the prevention of intrahepatic extension of human liver cancer.
World Journal of Gastroenterology 02/2004; 10(2):299-302. · 2.47 Impact Factor
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ABSTRACT: To investigate the hepatocellular apoptosis after hepatectomy in obstructive jaundice and biliary decompression rats.
After bile duct ligation for 7 days, rats were randomly divided into OB group in which the rats underwent 70% hepatectomy, OB-CD group in which the rats underwent hepatectomy accompanied by choledochoduodenostomy, CD-Hx group in which the rats underwent choledochoduodenostomy and then received 70% hepatectomy on the fifth day after biliary decompression. The control group (Hx group) only underwent hepatectomy.
The level of total serum bilirubin and serum enzymes was significantly lower in CD-Hx group than in OB-CD and OB groups on day 1, 3 and 5 after hepatectomy. The apoptotic index was significantly lower in CD-Hx group than in OB-CD and OB groups on day 3 and 5. The oligonucleosomal DNA fragments and Caspase-3 activity were also lower in CD-Hx group than in OB-CD and OB groups 3 days after hepatectomy, without differences between CD-Hx and Hx groups.
Hepatocellular apoptosis plays vital roles in jaundice rats, and biliary decompression is more effective in treatment of patients with severe jaundice before operation.
World Journal of Gastroenterology 01/2004; 9(12):2737-41. · 2.47 Impact Factor
