Angiogenesis In Human Hepatocellular Carcinoma Research Articles

Analysis and Validation of Human Targets and Treatments Using a Hepatocellular Carcinoma-Immune Humanized Mouse Model.

Background and AimsRecent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions.Approach and ResultsWe established cell line–based or patient‐derived xenograft–based humanized‐immune‐system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human‐specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real‐time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual‐combination, and triple‐combination treatment using N‐(1ʹ,2‐Dihydroxy‐1,2ʹ‐binaphthalen‐4ʹ‐yl)‐4‐methoxybenzenesulfonamide (C188‐9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL‐6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized‐immune‐system mice with HCC. In particular, intratumor human cluster of differentiation–positive (hCD14+) cells could produce IL‐33 through damage‐associated molecular pattern/Toll‐like receptor 4/activator protein 1, which up‐regulated IL‐6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL‐33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti‐HCC effect of C188‐9, bevacizumab, and pembrolizumab. The results showed that the anti‐HCC effect of triple‐combination therapy was superior to that of single or dual treatments.ConclusionsHumanized‐immune‐system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.

Effect of chemokine CXCL14 on in vitro angiogenesis of human hepatocellular carcinoma cells

The study was designed to investigate the effect of chemokine CXCL14 on in vitro angiogenesis of human hepatocellular carcinoma (HCC) cells. CXCL14 mRNA expression in HCC tissue samples and adjacent tissue samples was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). CXCL14 mRNA and protein expression in human normal hepatocyte HL-7702 and HCC cell line HepG2 were detected by qRT-PCR and western blot. In HepG2 cell line, the expression of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay method, cell viability was detected by CCK-8, cell proliferation was detected by colony formation assay, and cell migration as well as invasion ability was detected by Transwell assay. Moreover, human umbilical vein endothelial cell (HUVEC) tube formation assay was carried out to determine the cell ability of angiogenesis. Results showed that the overexpression of CXCL14 could inhibit angiogenesis, proliferation, migration, and invasion abilities of HCC cells.Highlights CXCL14 is lowly expressed in hepatocellular carcinoma tissues and cells. CXCL14 overexpression inhibits the angiogenesis of hepatocellular carcinoma cells. CXCL14 overexpression inhibits proliferation, invasion, and migration of hepatocellular carcinoma cells.

MicroRNA-3064-5p sponged by MALAT1 suppresses angiogenesis in human hepatocellular carcinoma by targeting the FOXA1/CD24/Src pathway.

Angiogenesis is critical for the development, progression, and metastasis of hepatocellular carcinoma (HCC), but the roles of miR-3064-5p in HCC angiogenesis are still unknown. In this study, the roles of miR-3064-5p in HCC angiogenesis were studied in 192 HCC patients, xenograft mouse models, and HCC cell lines. The results showed that miR-3064-5p expression was significantly decreased in HCC tissues and cells, and downregulated miR-3064-5p was associated with upregulated angiogenic potential of HCC. MiR-3064-5p inhibited proangiogenic VEGFA and angiogenin expressions but induced antiangiogenic endostatin and MMP12 expressions, finally leading to suppression of HCC angiogenesis, as shown by the decline in intratumoral microvessel density (MVD). Moreover, miR-3064-5p was inversely correlated with lncRNA MALAT1 and FOXA1. FOXA1 bound to and interacted with CD24 and then regulated Src phosphorylation. MiR-3064-5p played an antiangiogenic role by inhibiting the FOXA1/CD24/Src pathway, whereas oncogenic MALAT1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-3064-5p to alleviate the suppressive effect on the FOXA1 pathway. HCC patients with high miR-3064-5p, low MALAT1, or low FOXA1 expression had a better prognosis with longer overall survival and recurrence-free survival. In univariate and multivariate analyses, miR-3064-5p was identified as the independent prognostic predicator for HCC progression and patient survival. Taken together, miR-3064-5p exerts an antiangiogenic role by targeting the FOXA1/CD24/Src pathway but oncogenic lncRNA MALAT1 acts as a ceRNA to sponge miR-3064-5p. MiR-3064-5p is of great clinical significance and is a novel prognostic indicator and an attractive therapeutic target for HCC.

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self‐renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high‐resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)‐cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

EIF3C-enhanced exosome secretion promotes angiogenesis and tumorigenesis of human hepatocellular carcinoma.

Targeting tumor angiogenesis is a common strategy against human hepatocellular carcinoma (HCC). However, identification of molecular targets as biomarker for elevating therapeutic efficacy is critical to prolong HCC patient survival. Here, we showed that EIF3C (eukaryotic translation initiation factor 3 subunit C) is upregulated during HCC tumor progression and associated with poor patient survival. Expression of EIF3C did not alter proliferation and expression of other tumor progressive genes such as HIF1A, TGFβ1 and VEGF, but reduced cell migration in HCC cells. Nevertheless, expression of EIF3C in HCC cells significantly increase secretion of extracellular exosomes confirmed by increased exosomes labelling by PKH26 fluorescent dye, vesicles in exosome size detected by electronic microscopy and nanoparticle tracking analysis, and expression of divergent exosome markers. The EIF3C-increased exosomes were oncogenic to potentiate tumor angiogenesis via tube formation of HUVEC cells and growth of vessels by plugs assays on nude mice. Subcutaneous inoculation of EIF3C-exosomes mixed with Huh7 HCC cells not only promoted growth of vessels but also increased expression of EIF3C in tumors. Conversely, treatment of exosome inhibitor GW4869 reversed aforementioned oncogenic assays. We identified EIF3C activated expression of S100A11 involved in EIF3C-exosome increased tube formation in angiogenesis. Simultaneous high expression of EIF3C and S100A11 in human HCC tumors for RNA level in TCGA and protein level by IHC are associated with poor survival of HCC patients. Collectively, our results demonstrated that EIF3C overexpression is a potential target of angiogenesis for treatment with exosome inhibitor or S100A11 reduction to suppress HCC angiogenesis and tumorigenesis.

Deguelin suppresses angiogenesis in human hepatocellular carcinoma by targeting HGF-c-Met pathway.

Angiogenesis plays a crucial role in the development of human hepatocellular carcinoma (HCC). In the present study, we found a natural compound, deguelin, has a profound anti-angiogenesis effect on HCC. Deguelin suppressed vascular endothelial growth factor (VEGF)-induced human umbilical vascular endothelial cells (HUVECs) proliferation, migration, invasion, and capillary-like tube formation in vitro and reduced tumor angiogenesis in vivo. We discovered that VEGF receptor-mediated signal transduction cascades in HUVECs were inhibited by deguelin. Deguelin decreased the autocrine of VEGF in HCC cells in a time- and dose-dependent manner. Additionally, deguelin suppressed HGF-induced activation of the c-Met signaling pathway. Knocking down c-Met or inhibition of c-Met activation impaired HGF-mediated VEGF production. Importantly, we produced patient-derived hepatocellular carcinoma xenografts to evaluate the therapeutic effect of deguelin in vivo. Taken together, these results indicate that deguelin could inhibit HCC through suppression of angiogenesis on vascular endothelial cells and reduction of proangiogenic factors in cancer cells.

A new key player in VEGF-dependent angiogenesis in human hepatocellular carcinoma: dimethylarginine dimethylaminohydrolase 1

BackgroundAnti-angiogenic therapies, targeting VEGF, are a promising treatment for hepatocellular carcinoma (HCC). To enhance this potential therapy, identification of novel targets in this pathway is of major interest. Nitric oxide (NO) plays a crucial role in VEGF-dependent angiogenesis. NO production depends on arginine as substrate and asymmetric dimethylarginine (ADMA) as inhibitor. Dimethylarginine dimethylaminohydrolase 1 (DDAH-1) catabolizes ADMA and therefore regulates NO and VEGF expression. This study unravels additional mechanisms to improve VEGF targeting therapies.MethodsThe expression of DDAH-1 was examined in HCC specimen and non-tumorous background liver of 20 patients undergoing liver resection. Subsequently, arginine/ADMA balance, NO production, and VEGF expression were analyzed. The influence of hypoxia on DDAH-1 and angiogenesis promoting factors was evaluated in HepG2 cells and primary human hepatocytes.ResultsDDAH-1 expression was significantly induced in primary HCC tumors compared to non-tumorous background liver. This was associated with an increased arginine/ADMA ratio, higher NO formation, and higher VEGF expression in human HCC compared to non-tumorous liver. Hypoxia induced DDAH-1, iNOS, and VEGF expression in a time-dependent manner in HepG2 cells.ConclusionsOur results indicate that DDAH-1 expression is increased in human HCC, which is associated with an increase in the arginine/ADMA ratio and enhanced NO formation. Hypoxia may be an initiating factor for the increase in DDAH-1 expression. DDAH-1 expression is associated with promotion of angiogenesis stimulating factor VEGF. Together, our findings for the first time identified DDAH-1 as a key player in the regulation of angiogenesis in human HCC, and by understanding this mechanism, future therapeutic strategies targeting VEGF can be improved.

Role of the novel HSP90 inhibitor AUY922 in hepatocellular carcinoma: Potential for therapy.

The aim of the present study was to determine the correlation between hepatocellular carcinoma (HCC) and heat shock protein 90 (HSP90), involved in tumor angiogenesis, and to evaluate the effect of AUY922, a HSP90 inhibitor, in HCC. The expression of HSP90 and microvessel density (MVD) were measured in tissue samples from 76 patients with HCC by immunohistochemistry. Western blot analysis was performed to detect the expression of HSP90 in the HCC tissues and different HCC cell lines. The effects of time and concentration treatment with the AUY922 HSP90 inhibitor were investigated in HepG2 cells. Cell proliferation was measured using an MTT assay and a Transwell assay was performed to evaluate the migration of the HepG2 cells following treatment with different concentrations of AUY922. Positive staining of HSP90 was observed in 88.16% (67/76) of the HCC tissues, compared with 16.67% (4/24) of the normal tissues. The difference in the expression of HSP90 between the HCC and normal tissues was statistically significant (P<0.001). Tumors exhibiting positive expression of HSP90 had significantly higher MVD compared with the HSP90-negative counterparts (82.8±12.44 vs. 23.8±8.07, respectively; P<0.001). The expression levels of HSP90 were positively correlated with MVD in all the tissue samples (r_s=0.724; P<0.001). AUY922 inhibited the proliferation of the HepG2 cells in a time- and concentration-dependent manner, and the migration of HepG2 cells was distinctly suppressed following treatment with AUY922. These data suggested that the angiogenesis of human HCC may be mediated by HSP90, and that the specific HSP90 inhibitor, AUY922, has a therapeutic role in the treatment of HCC. Therefore, HSP90 may represent a selective target in molecularly targeted treatment of HCC.

The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance.

BackgroundA role for autophagy, a conserved cellular response to stress, has recently been demonstrated in human cancers. Aberrant expression of Beclin-1, an important autophagic gene, has been reported in various human cancers. In the present study, we investigated the significance and relationship between Beclin-1 expression and cell proliferation, apoptosis, microvessel density (MVD) and clinical pathological changes or prognosis in human hepatocellular carcinoma (HCC).MethodsA total of 103 primary HCC patients were involved in the study. Expression of Beclin-1, PCNA, NET-1, Bcl-2, Bax, Survivin in cancer cells and CD34 in stromal microvessels were evaluated immunohistochemically in tissue microarrays comprising 103 cases of HCC and 57 matched adjacent nontumor liver tissues. Correlations between clinicopathological characteristics and survival of HCC patients were explored.ResultsThe positive rate of Beclin-1 was significantly lower in HCC tissues than adjacent tissues (72.8 vs. 89.5%, χ2 = 6.085, P = 0.015). In HCC, Beclin-1 expression was negatively correlated with cirrhosis background (r = −0.216, P = 0.029), Edmondson grade (r = −0.249, P = 0.011), vascular invasion (r = −0.246, P = 0.012), PCNA (r = −0.242, P = 0.014), NET-1 (r = −0.245, P = 0.013), anti-apoptosis protein Bcl-2 (r = −0.245, P = 0.013) and MVD (r = −0.292, P = 0.003), and positively correlated with pro-apoptosis protein Bax (r = 0.242, P = 0.014).Significant differences in the 5-year survival rates were seen among patients with Beclin-1 strong positive (++) (59.1%, 13/22), moderate positive (+) (28.3%, 15/53) and weak negative expression (−) (14.6%, 7/28) (P = 0.043). Significant differences were detected between Beclin-1 (++) and either Beclin-1 (+) (P = 0.036) or Beclin-1 (−) groups (P = 0.008), but no significant difference between Beclin-1 (+) and Beclin-1 (−) groups (P = 0.281) was observed.Survival rates were positively related to high Beclin-1 co-expressed with low PCNA, NET-1, or Bcl-2, lower MVD, and high Bax. Univariate and multivariate Cox regression analysis revealed that Beclin-1 expression was an independent indicator for overall survival in HCC patients (P < 0.05).ConclusionsThe pathogenesis and progression of HCC are associated with reduced autophagy. The expression of Beclin-1 and Bax in HCC tissues may provide a synergistic effect towards inhibiting HCC proliferation, infiltration, metastasis and angiogenesis. Beclin-1 expression may be a valuable prognostic marker of HCC.

MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway

MicroRNA (miR)-26a can suppress tumor growth and metastasis of hepatocellular carcinoma (HCC). Since angiogenesis is important for tumor growth and metastasis, we investigated the possible roles of miR-26a in tumor angiogenesis. Down-regulation of miR-26a was found to correlate with an increased angiogenic potential of HCC. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit vascular endothelial growth factor A (VEGFA) expression in HCC cells and then suppress the promoting effects of HCC cells on in vitro proliferation, migration, and capillary tube formation of endothelial cells, as well as in vivo tumor angiogenesis of HCC. Hepatocyte growth factor (HGF) was identified as a target of miR-26a. HGF simulation antagonized the effects induced by miR-26a up-regulation. In contrast, silencing HGF induced similar effects to miR-26a. We further found that miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting HGF-hepatocyte growth factor receptor (cMet) and its downstream signaling pathway, in turn, suppressing VEGFA production in HCC cells and impairing VEGFR2-signaling in endothelial cells. HCC patients who had high miR-26a, low HGF, low VEGFA, or low microvessel density (MVD) in tumor tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, or in combination with HGF, was demonstrated to be an independent prognostic indicator for OS and TTR of HCC patients. miR-26a could suppress tumor angiogenesis of HCC through HGF-cMet signaling, and it is a new hopeful therapeutic target and prognostic marker for HCC.

BAG3 regulates epithelial–mesenchymal transition and angiogenesis in human hepatocellular carcinoma

Bcl2-associated athanogene 3 (BAG3) protein is a co-chaperone of heat-shock protein (Hsp) 70 and may regulate major physiological and pathophysiological processes. However, few reports have examined the role of BAG3 in human hepatocellular carcinoma (HCC). In this study, we show that BAG3 regulates epithelial–mesenchymal transition (EMT) and angiogenesis in HCC. BAG3 was overexpressed in HCC tissues and cell lines. BAG3 knockdown resulted in reduction in migration and invasion of HCC cells, which was linked to reversion of EMT by increasing E-cadherin expression and decreasing N-cadherin, vimentin and slug expression, as well as suppressing matrix metalloproteinase 2 (MMP-2) expression. In a xenograft tumorigenicity model, BAG3 knockdown effectively inhibited tumor growth and metastasis through reduction in CD34 and VEGF expression and reversal of the EMT pathway. In conclusion, BAG3 is associated with the invasiveness and angiogenesis in HCC, and the BAG3 gene may be a novel therapeutic approach against HCC.

Diagnostic and prognostic significance of peroxiredoxin 1 expression in human hepatocellular carcinoma

Peroxiredoxin 1 (Prdx1) is a member of the peroxiredoxin family of antioxidant enzymes and implicated in cell differentiation, proliferation, and apoptosis. The aim of the present study was to determine the expression and diagnostic and prognostic significance of Prdx1 in human hepatocellular carcinoma (HCC). Prdx1 expression was examined in 76 HCC patients and 20 healthy volunteers. The relationships between Prdx1 expression and clinicopathological features were analyzed. Receiver operating characteristics analysis was used to calculate the diagnostic accuracy of serum Prdx1, serum alpha-fetoprotein (AFP), and their combination. The prognostic impact of Prdx1 on overall survival (OS) and disease-free survival (DFS) of HCC patients was investigated. Prdx1-positive rate was significantly (p < 0.05) higher in HCC (77.1 %) than in adjacent non-tumorous liver tissues (18.4 %). Prdx1 immunoreactivity was positively correlated with tumor vascular endothelial growth factor expression and microvessel density. Prdx1 expression was significantly associated with tumor size, microvascular invasion, Edmondson grade, tumor capsula status, serum AFP, and tumor-node-metastasis stage. The combination of serum Prdx1 and AFP had a markedly higher area under the curve than serum Prdx1 alone. Positive Prdx1 expression was associated with unfavorable OS (p = 0.004) and DFS (p = 0.001). Multivariate analysis revealed intra-tumoral Prdx1 staining as an independent poor prognostic marker for OS (p = 0.006) and DFS (p = 0.002). Taken together, our data suggest that increased Prdx1 expression is associated with tumor angiogenesis and progression in HCC and serves as a promising biomarker for detection and prognosis of this malignancy.

Slug promoted vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

Activated amelogenin Y-linked (AMELY) regulation and angiogenesis in human hepatocellular carcinoma by biocomputation

In the present study, a comparison of the biological processes and gene ontology (GO) in human hepatocellular carcinoma (HCC) with high expression (fold change ≥2) of amelogenin Y-linked (AMELY)-activated upstream regulation networks with non-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) with low expression of activated networks was performed. The principle biological processes involved in non-tumor hepatitis/cirrhotic tissues include positive regulation of mismatch repair, regulation of transcription from RNA polymerase II promoters, negative regulation of cell-cell adhesion, protein ubiquitinatin and protein catabolism. The main biological processes involved in the development of HCC include positive regulation of calcium ion transport into the cytosol, cell proliferation, DNA replication, fibroblast proliferation, immune response, microtubule polymerization and protein secretion. Specific transcription from RNA polymerase II promoters, regulation of angiogenesis, cell growth, protein metabolism, Wnt receptor signaling pathways, negative regulation of endothelial cell differentiation, microtubule depolymerization, peptidase activity and progression through the cell cycle are also involved. Positive regulation of transcription is involved in both processes. An activated AMELY-coupled upstream positive regulation of immune response-mediated protein secretion to Wnt signaling and calcium into cytosol-induced regulation of cell growth and angiogenesis in HCC is proposed. The AMELY upstream regulation molecular network model was constructed with BUB1B, CST6, ESM1, HOXA5, LEF1, MAPT, MYBL2, NOTCH3, PLA2G1B, PROK1, ROBO1, SCML2 and UBE2C in HCC from a Gene Expression Omnibus (GEO) dataset by gene regulation network inference methods and our programming methods.

Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.

Late SV40 Factor (LSF) Enhances Angiogenesis by Transcriptionally Up-regulating Matrix Metalloproteinase-9 (MMP-9)

The transcription factor late SV40 factor (LSF) is overexpressed in human hepatocellular carcinoma (HCC) fostering a highly aggressive and metastatic phenotype. Angiogenesis is an essential component of cancer aggression and metastasis and HCC is a highly aggressive and angiogenic cancer. In the present studies, we analyzed the molecular mechanism of LSF-induced angiogenesis in HCC. Employing human umbilical vein endothelial cells (HUVEC) differentiation assay and chicken chorioallantoic membrane (CAM) assay we document that stable LSF overexpression augments and stable dominant negative inhibition of LSF (LSFdn) abrogates angiogenesis by human HCC cells. A quest for LSF-regulated factors contributing to angiogenesis, by chromatin immunoprecipitation-on-chip (ChIP-on-chip) assay, identified matrix metalloproteinase-9 (MMP-9) as a direct target of LSF. MMP-9 expression and enzymatic activity were higher in LSF-overexpressing cells and lower in LSFdn-expressing cells. Deletion mutation analysis identified the LSF-responsive regions in the MMP-9 promoter and ChIP assay confirmed LSF binding to the MMP-9 promoter. Inhibition of MMP-9 significantly abrogated LSF-induced angiogenesis as well as in vivo tumorigenesis, thus reinforcing the role of MMP-9 in facilitating LSF function. The present findings identify a novel target of LSF contributing to its oncogenic properties.

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Apoptotic and anti-angiogenic effects of Pulsatilla�koreana extract on hepatocellular carcinoma

Chemoprevention through the use of food and plants has emerged as a novel approach to control various malignancies including cancer. Pulsatilla koreana extract (PKE) has been used to treat malaria and dysentery. The functions and effect of PKE in cancer treatment have been reported but with less information. In this study, we investigated the effect of PKE on the progression of hepatocellular carcinoma (HCC) cells and its mechanism. PKE strongly suppressed the growth of HCC cells in a dose-dependent manner. Apoptosis by PKE was observed by DAPI and TUNEL staining and accompanied with increases of cleaved PARP and caspase-3 in Huh-7 cells. Also, PKE decreased the expression of hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF), and inhibited tube formation and migration of human umbilical vein endothelial cells (HUVECs). In addition, PKE potently suppressed in vivo neovascularization in a mouse Matrigel plug assay. Furthermore, in vivo study showed that PKE significantly inhibited tumor growth in a mouse xenograft model, and induced apoptosis by increasing the cleaved PARP and caspase-3. The expressions of Ki-67, VEGF, and CD31 in the tumor tissue were decreased by the treatment of PKE. Taken together, our study demonstrates that PKE not only induced apoptosis but also inhibited cell growth and angiogenesis of human HCC. We suggest that PKE is an effective chemotherapeutic candidate for cancer therapy against HCC.

Hepatocellular Carcinomas in Cirrhotic and Noncirrhotic Human Livers Share Angiogenic Characteristics

BackgroundThe antiangiogenic drug sorafenib has been shown to be an effective treatment for hepatocellular carcinoma (HCC) in patients with liver cirrhosis. It might also be effective in noncirrhotic HCC provided that the angiogenic properties of both tumor types are comparable. The aim of this study is to compare endothelial cell dynamics, microvessel density (MVD), and vessel maturation as indirect markers of angiogenesis in human HCC in cirrhotic and noncirrhotic livers.Materials and MethodsIn a tertiary care setting, 70 consecutive HCC tumors were analyzed for endothelial cell dynamics. CD34 was applied to identify tumor microvessels, double immunolabeling Ki67/CD34 and activated caspase-3/CD34 to assess endothelial cell proliferation and apoptosis, and α-smooth muscle actin/CD34 for pericyte coverage. These characteristics were compared in cirrhotic (n = 33) and noncirrhotic HCCs (n = 37). Microvessel density was correlated with radiological signs of hypervascularity as obtained with dynamic four-phase CT scans during the arterial and portal phase of contrast enhancement.ResultsMicrovessels in cirrhotic and noncirrhotic HCC were mainly mature. In both groups endothelial cell turnover was low and MVD was not different. There was no correlation between MVD and venous invasion, tumor size, and turnover of tumor cells or endothelial cells. MVD was negatively correlated with contrast washout in the portal venous phase of CT scanning. In transplanted patients, MVD was not correlated with survival, whereas in patients after liver resection a high MVD was associated with a better prognosis.ConclusionAngiogenic characteristics of HCC in cirrhotic and noncirrhotic livers have a remarkable similarity.