Sulforaphane exerts anti-angiogenesis effects against hepatocellular carcinoma through inhibition of STAT3/HIF-1?/VEGF signalling.
ABSTRACT: Angiogenesis plays an important role in hepatocellular carcinoma (HCC), the inhibition of which is explored for cancer prevention and treatment. The dietary phytochemical sulforaphane (SFN) is known for its anti-cancer properties in vitro and in vivo; but until now, no study has focused on the role of SFN in HCC tumor angiogenesis. In the present study, in vitro cell models using a HCC cell line, HepG2, and human endothelial cells, HUVECs, as well as ex vivo and in vivo models have been used to investigate the anti-tumor and anti-angiogenic effect of SFN. The results showed that SFN decreased HUVEC cell viability, migration and tube formation, all of which are important steps in angiogenesis. More importantly, SFN markedly supressed HepG2-stimulated HUVEC migration, adhesion and tube formation; which may be due to its inhibition on STAT3/HIF-1?/VEGF signalling in HepG2 cells. In addition, SFN significantly reduced HepG2 tumor growth in a modified chick embryo chorioallantoic membrane (CAM) assay, associated with a decrease of HIF-1? and VEGF expression within tumors. Collectively, these findings provide new insights into the inhibitory effect of SFN on HCC tumor angiogenesis as well as tumor growth, and indicate that SFN has potential for the prevention and treatment of HCC.
Project description:Eupafolin is a flavonoid extracted from the common sage herb which has been used in China as traditional medicine. Previous studies had reported that eupafolin had antioxidative, anti-inflammatory and antitumor effects. However, the function and the mechanism of eupafolin to exert its antitumor activity, especially its effect on tumor angiogenesis, have not been elucidated. Herein, we showed that eupafolin significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) in a dose-dependent manner. Meanwhile, the new blood microvessels induced by VEGF in the matrigel plug were also substantially suppressed by eupafolin. The results of HCC xenograft experiments demonstrated eupafolin remarkably inhibited tumor growth and tumor angiogenesis in vivo, suggesting the antitumor activity exerted by eupafolin was closely correlated with its potency on tumor angiogenesis. Mechanism investigations revealed that eupafolin significantly blocked VEGF-induced activation of VEGFR2 in HUVEC cells as well as its downstream signaling pathway. In addition to the effect on endothelial cells, through inhibiting Akt activity in tumor cells, VEGF secretion in HepG2 was dramatically decreased after eupafolin treatment. Our study was the first to report the activity of eupafolin against tumor angiogenesis as well as the underlying mechanism by which eupafolin to exert its anti-angiogenic activity.
Project description:Recent studies have revealed that natural plants-derived polysaccharides exhibit potent anti-tumor activity. Our earlier studies suggest that dandelion polysaccharide (DP) inhibits hepatocellular carcinoma (HCC) cell proliferation in vitro and in vivo. Here, we investigated the effects of DP on the angiogenesis of HCC and the potential molecular mechanisms by which DP regulates angiogenesis. Wound-healing and transwell invasion assays revealed that DP inhibited HUVECs migration and invasion in vitro, respectively. Tube formation assay, chick chorioallantoic membrane (CAM) assay, and immunohistochemistry (IHC) demonstrated that DP suppressed vasculogenesis in vitro and in vivo. Moreover, Western blot and immunofluorescence staining verified that DP treatment decreased the protein levels of some key factors involved in angiogenesis of HCC, such as hypoxia-inducible factor 1? (HIF-1?), vascular endothelial growth factor (VEGF), p-PI3K, and p-AKT. However, activation of PI3K/AKT pathway with insulin-like growth factor 1 (IGF-1) treatment attenuated the effect of DP on angiogenesis via lowering the expression of HIF-1? and VEGF. In summary, we found that DP treatment inhibited angiogenesis in vivo and in vitro through suppressing expression of VEGF and HIF-1a. Furthermore, we showed that the expression of VEGF and HIF1-? was modulated by PI3K/AKT signaling. Collectively, our study suggests that DP is a promising anti-cancer drug candidate for treating HCC.
Project description:AZD6244 (ARRY-142886), a highly selective MAPK-ERK kinase inhibitor, has shown excellent clinical efficacy in many tumors. However, the anti-tumor and anti-angiogenesis efficacy of AZD6244 on gastric cancer has not been well characterized. In this study, high p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. For absence of NRAS, KRAS and BRAF mutation, SGC7901 and BGC823 gastric cancer cells were relative resistance to AZD6244 in vitro. And such resistance was not attributed to the insufficient inhibition of ERK phosphorylation. However, tumor growth was significantly suppressed in SGC7901 xenografts by blockage of angiogenesis. This result was further supported by suppression of tube formation and migration in HUVEC cells after treatment with AZD6244. Moreover, the anti-angiogenesis effect of AZD6244 may predominantly attribute to its modulation on VEGF through p-ERK - c-Fos - HIF-1? integrated signal pathways. In conclusions, High p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. Targeting inhibition of p-ERK by AZD6244 suppress gastric cancer xenografts by blockage of angiogenesis without systemic toxicity. The anti-angiogenesis effect afford by AZD6244 may attribute to its modulation on p-ERK - c-Fos - HIF-1? - VEGF integrated signal pathways.
Project description:Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.
Project description:<h4>Background & aims</h4>microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC).<h4>Methods</h4>The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings.<h4>Results</h4>Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2? as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients.<h4>Conclusions</h4>miR-26a modulated angiogenesis of HCC through the PIK3C2?/Akt/HIF-1?/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.
Project description:The hypoxic microenvironment inside solid tumors, including hepatocellular carcinoma (HCC), is a major cause of tumor resistance to chemotherapy. The recently identified hypoxia-inducible factor (HIF)-2 executes the hypoxia response. Its expression feature and transcriptional targets indicate a possible dominance of HIF-2 in regulating genes in HCC. The aim of the present study was to determine whether transfection of siRNA targeting HIF-2? could enhance the efficacy of doxorubicin, the most commonly used drug in the treatment of HCC. Transfection of HIF-2 siRNA into human HCC cells downregulated the expression of HIF-2?, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-?, and cyclin D1, but had little effect on the expression of HIF-1?, fms-related tyrosine kinase-1 (Flt-1), the glucose transporter (GLUT)-1, and lactate dehydrogenase A (LDHA). Doxorubicin itself only downregulated VEGF expression. Furthermore, HIF-2 siRNA inhibited proliferation, induced cell cycle arrest at the G(0)/G(1) phase, and acted synergistically with doxorubicin to inhibit the growth of human HCC cells in vitro. Transfection of HIF-2 siRNA also downregulated tumoral expression of HIF-2?, VEGF, TGF-?, and cyclin D1 in vivo, and acted synergistically with doxorubicin to suppress the growth of HepG2 tumors established in immunodeficient mice by inhibiting cell proliferation, tumor angiogenesis and microvessel perfusion. The results of the present study suggest that targeting HIF-2? with siRNA warrants investigation as a potential strategy to enhance the efficacy of doxorubicin in the treatment of HCC.
Project description:The mechanisms underlying the role of chemokines in tumor angiogenesis is still not fully understood. In this study, we detected the influence of CCL19 on colorectal cancer (CRC) angiogenesis. The expression of CCL19 and CD31 in CRC tissues were detected by immunohistochemistry. Human CRC cell lines SW1116 and SW620 stably transfected with CCL19 lentivirus and CCL19 shRNA, and HUVEC stably transfected with CCR7 shRNA were used in our study. Our study showed that CCL19 was significantly low-expressed in CRC tissues and positively related to highly tumor microvessel density. In vitro, we observed that CCL19 high-expressed SW1116 supernatant was able to inhibit proliferation, migration, and sprouting responses of HUVEC, whereas CCL19 low-expressed SW620 supernatant can promote HUVEC angiogenesis. Additionally, we further demonstrated that these functions maybe achieved through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1?/VEGF-A pathway in a CCR7-dependent manner. Mice angiogenesis model also confirmed that elevated expression of CCL19 inhibit the angiogenesis of CRC in vivo. In summary, our results supported that CCL19 can inhibit CRC angiogenesis through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1?/VEGF-A pathway. This may be a novel therapeutic option for anti-vascular treatment in CRC.
Project description:Angiogenesis is considered as an important process in the development of malignancies and is associated with cancer progression and metastasis. Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and is recognized as a typical angiogenic tumor. Thus, it is of great importance to study the underlying mechanism of angiogenesis in HCC. The long non-coding RNA (lncRNA) ubiquitin conjugating enzyme E2C pseudogene 3 (UBE2CP3) has been reported as an oncogene that promotes tumor metastasis in HCC. However, the role and underlying mechanisms of UBE2CP3 in HCC angiogenesis are still unclear.We measured the expression levels of UBE2CP3 by in situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) in HCC patient samples. We also concomitantly used CD31/PAS double-staining to measure endothelial vessel (EV) density and used qRT-PCR to measure the CD31 mRNA level. HepG2 and SMMC-7721 cells were transfected with Lv-UBE2CP3 or Sh-UBE2CP3 virus to obtain stably over-expressing or knocking-down UBE2CP3 cell lines. The indirect effects of UBE2CP3 on ECs were studied by establishing a co-culture system using Transwell chambers with a 0.4-?m pore size. HCC cells and ECs in the co-culture system were separated, but the cytokines and growth factors were able to communicate with each other. Following exposed to HCC cells, ECs were collected for functional studies. Finally, we studied the function of UBE2CP3 in vivo by chick embryo chorioallantoic membrane (CAM) angiogenesis assays and nude mouse tumorigenicity assays.In this study, we found that UBE2CP3 expression was higher in HCC tissues than in para-tumor tissues and was up-regulated in tissues with high EV density. Functionally, we found that in the co-culture systems, HCC cells overexpressing UBE2CP3 promoted HUVEC proliferation, migration and tube formation via the activation of ERK/HIF-1?/p70S6K/VEGFA signalling, increasing the level of VEGFA in HCC cell supernatant. In addition, the opposite results appeared when the expression of UBE2CP3 in HCC cells was knocked down. Consistent with these results, CAM angiogenesis assays and nude mouse tumorigenicity assays showed that UBE2CP3 expression up-regulated EV density in vivo.Our study suggests that UBE2CP3 can enhance the interaction between HCC tumor cells and HUVECs and promote HCC tumorigenicity by facilitating angiogenesis.
Project description:BACKGROUND:Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. METHODS:Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). RESULTS:In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1? (HIF-1?) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. CONCLUSION:In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.
Project description: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, TGF?1 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.