Pancreatic Cancer Angiogenesis Research Articles

Clinical implications of EphB4 receptor expression in pancreatic cancer

In the study, we investigated the correlation between EphB4 receptor expression and the angiogenesis of pancreatic cancer. EphB4 receptor is unevenly distributed or distributed with small patches in pancreatic ductal cell cancer. While EphB4 receptor was not expressed in normal pancreatic tissues. It can be observed that the mRNA of EphB4 receptor is high expressed in all 15 cases of pancreatic ductal cell cancer tissues but not expressed in normal pancreatic tissues. Finally, positive correlation was observed between the mRNA expression level of EphB4 receptor and MVD. Immunohistochemical analysis showed that EphB4 receptor protein expression was related to tumor differentiation and clinical stage. It can be observed that MVD is relevant to histological differentiation and clinical stage. EphB4 receptor is correlated to the initiation, progression and tumor angiogenesis. In conclusion, EphB4 receptor maybe a promising targeted goal of antiangiogenic formation, which provides a new approach and method for tumor treatment.

Metronomic Ceramide Analogs Inhibit Angiogenesis in Pancreatic Cancer through Up-regulation of Caveolin-1 and Thrombospondin-1 and Down-regulation of Cyclin D1

AIMS: To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms. METHODS: Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice. RESULTS: Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine. CONCLUSIONS: Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.

Abstract 1611: EGCG inhibits growth of human pancreatic tumors orthotopically implanted in BALB/c Nude mice through activation of FKHRL1/FOXO3a transcription factor.

Abstract The forkhead transcription factors of the O class (FOXO) play a major role in cell proliferation, angiogenesis, metastasis and tumorigenesis. The objectives of this study were to examine whether FKHRL1/FOXO3a modulates antitumor activity of EGCG in pancreatic cancer model in vivo. PANC-1 cells were orthotopically implanted into BALB/c Nude mice and gavaged with EGCG after tumor formation. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. FKHRL1 nuclear translocation, and DNA binding were measured by immunohistochemistry and gelshift assay, respectively. The expression of PI3K, AKT, ERK, and FOXO3a and its target genes were measured by the Western blot analysis. EGCG-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FKHRL1/FOXO3a and expression of FOXO target genes. EGCG induced apoptosis by up-regulating Bim and activating caspase-3. EGCG inhibited markers of cell cycle (p27/KIP1), angiogenesis (CD31, VEGF, IL-6, IL-8 and HIF1α), and metastasis (MMP2 and MMP7). EGCG inhibited epithelial mesenchymal transition by upregulating the expression of E-cadherin and inhibiting the expression of N-cadherin, and Zeb1. These data suggest that EGCG inhibits pancreatic cancer orthotopic tumor growth, angiogenesis and metastasis which are associated with inhibition of PI3K/AKT and ERK pathways resulting in activation of FKHRL1/FOXO3a transcription factor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1611. doi:1538-7445.AM2012-1611

Abstract 3055: PD-0332991, a selective cyclin-dependent-kinase 4/6 Inhibitor, upregulates multiple genes promoting invasion and metastasis in pancreatic ductal adenocarcinoma

Abstract Deregulation of the Cyclin D-CDK4/6-Rb-E2F signaling pathway is among the most commonly found aberrations in human pancreatic ductal adenocarcinoma (PDAC). PD-0332991 is an orally available, highly specific and reversible inhibitor for Cdk4 and Cdk6, and demonstrates in vitro and in vivo growth inhibitory effects in multiple tumor types. However, the potential usefulness of PD-0332991 in PDAC is currently unknown. In this study, we examined the effects of PD-0332991 on multiple human pancreatic cancer cell lines, and we found that this agent inhibited Rb phosphorylation and E2F target gene expression, and induced G0/G1 cell cycle arrest. However, the growth inhibitory effect was only transient, due to the rapidly emerging compensatory changes of the G1/S machinery components, including upregulation of CyclinD1, Cdk4, and Cdk6, and downregulation of Rb, p107, and p130. Microarray analysis revealed that incubation with PD-0332991 upregulated multiple genes which promote pancreatic cancer invasion, metastasis, angiogenesis, and chemoresistance, including LAMC2, CYR61, SERPINE1, F3, ABCA1, ASNS, and DUSP1. In addition, using matrigel-coated transwell invasion chambers, we demonstrated that incubation with PD-0332991 enhanced the invasiveness of human pancreatic cancer cells. These observations suggest that PD-0332991 may have the potential to cause deleterious effects in PDAC by modulating gene expression in a manner that could promote cancer spread, metastasis, and chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3055. doi:1538-7445.AM2012-3055

Sp/KLF Family and Tumor Angiogenesis in Pancreatic Cancer

Tumor angiogenesis play a significant role in genesis, development and metastasis of pancreatic cancer though the process is different from angiogenesis in normal tissues. VEGF, one of the most important angiogenesis factors, is modulated by inflammation factors as well as transcriptional factors, such as members of Sp/KLF family. Recent research showed that VEGF related inflammation factors and Sp/KLF family members form a complex network structure, which causes genesis and development of tumor. Conceivably, fully understanding the mechanism linking VEGF related inflammation factors and Sp/KLF family members would promote the concept of tumor angiogenesis. Furthermore, it could also help to design effective strategies to target the key components of the network and control the development and progression of tumor.

Cytokine Network: New Targeted Therapy for Pancreatic Cancer

Increasing evidence has shown that cytokines have a role in tumor biology. The role of chemokines in tumor biology is important because these peptides may influence tumor growth, invasion, angiogenesis, and metastasis. In this review, we demonstrated the role of cytokines (Interleukin-1α, hepatocyte growth factor, Interleukin-8, stromal cell-derived factor-1 and CXC-chemokines/CXCR2 biological axis) in pancreatic cancer angiogenesis, especially from the standpoint of the interaction between tumor and its microenvironments. The cytokines are intimately related with cancer angiogenesis. Blocking these cytokines could attenuate pancreatic cancer associated angiogenesis and further considered as a novel anti-angiogenic target in pancreatic cancer.

Bone marrow‐derived proangiogenic cells in pancreatic cancer

Tumor-derived signals systemically induce an angiogenic switch that allows cancer cells to survive and grow. However, the vascular network in tumors is not well organized and fails to meet metabolic needs to maintain tissue homeostasis, resulting in significant hypoxia. Among various tumors, pancreatic ductal adenocarcinoma (PDAC) typically develops in an unusually disordered microenvironment, which contributes to its highly aggressive behavior. Since anti-vascular endothelial growth factor (VEGF) (Avastin) has failed to demonstrate a survival benefit in PDAC, we need to re-visit the basic biology of this disease and understand what makes it so refractory to the anti-angiogenic approaches that are clinically effective in other neoplasms. To address this issue, we specifically focused on the process of neovascularization where bone marrow-derived cells (BMDCs) play a role during pancreatic tumorigenesis. We have identified subsets of BMDCs that regulate key processes during development of the neovessels through paracrine Hedgehog signaling. Considering the importance of systemic responses occurring in tumor bearing hosts, we are currently using genetically engineered mice, which spontaneously develop PDAC, Pdx1-Cre;LSL-Kras(G12D);p53(lox/+) strain, to clarify critical events that can trigger aberrant angiogenesis in pancreatic cancer. These studies allow us to provide insights into the cellular and molecular mechanisms of pancreatic tumorigenesis and have an implication for the design of therapies against this difficult disease.

Effect of inhibition of signal transducer and activator of transcription 3 on angiogenesis of human pancreatic cancer cells in vitro

Objective To investigate the effect and mechanism of RNA interference ( RNAi)-mediated signal transducer and activator of transcription 3 ( STAT3 ) inhibition on human pancreatic cancer cells angiogenesis in vitro.Methods RNAi was used to inhibit STAT3 gene of SW1990 cells.Methyl thiazol tetrazolium (MTT) assay and flow cytometric analysis were performed to detect cell proliferation and cell cycle of human umbilical vein endothelial cell ( HUVEC),respectively.The migration ability of HUVECs was determined by cell migration assay.Enzyme linked immunosorbent assay (ELISA) was used to detect the protein expression of the vascular endothelial growth factor (VEGF).Results Inhibition of STAT3 with RNAi significantly inhibited the proliferation of HUVECs and decreased the migration ability of HUVECs invitro.The cell growth rate at 24,48,72 h in SW1990-RNAi group was (1.19 ±0.11)％,( 1.62 ± 0.15 ) ％,( 1.95 ± 0.18 ) ％,respectively.The percentage of HUVECs at G0/G1 phase in SW1990-RNAi group was (80.95 ± 7.49)％.Moreover,the VEGF protein expression in SW1990-RNAi cells was reduced by 60％.Conclusion Inhibition of STAT3 with RNAi can significantly inhibit the angiogenesis ability of pancreatic cancer cells through down-regulating VEGF,which may provide a novel strategy in preventing the angiogenesis of pancreatic cancer. Key words: RNA interference; Signal transducer and activator of transcription 3 ; Pancreatic carcinoma; Angiogenesis

The Expression and Clinical Significance of pSTAT3, VEGF and VEGF-C in Pancreatic Adenocarcinoma

Signal transducers and activators of transcription 3 (STAT3) is a central cytoplasmic transcription factor and regulates a number of pathways important in tumorigenesis including cell cycle progression, apoptosis, tumor angiogenesis, invasion and metastasis.This study aims to investigate the expression of pSTAT3, VEGF and VEGF-C in pancreatic adenocarcinoma and their relations to the clinicopathological features, tumor angiogenesis and prognosis. In the present study, the expression of pSTAT3, VEGF and VEGF-C and microvascular density (MVD) were examined via immunohistochemistry. The clinicopathological information was collected and patients were regularly followed up. The relationship between the parameters and the clinicopathological features were analyzed, and the univariate and multivariate prognostic factors were also analyzed. The expression of pSTAT3 in tumor tissues was significantly higher in contrast to that in normal tissues, and pSTAT3 was related to VEGF and VEGF-C expression, MVD, tumor size, lymphogenous status and TNM staging (P<0.05). Survival analysis suggested that tumor size, TNM staging, pSTAT3 and VEGF expression were risk factors of prognosis, but no independent factors were found. We concluded that pSTAT3, which was a risk factor of prognosis, was abnormally expressed in pancreatic adenocarcinoma and related to tumor size, TNM staging and lymphatic metastasis. pSTAT3 may promote tumor angiogenesis via up-regulating VEGF on protein and even gene levels, and enhance the early lymphatic metastasis through VEGF-C. Better understanding of STAT3 signaling pathways in angiogenesis may contribute to the development of novel therapeutic strategies in angiogenesis and metastasis of pancreatic cancer.

Metastasis: Genetics, Mechanism, and Diagnostic and Therapeutic Strategies

Metastasis: Genetics, Mechanism, and Diagnostic and Therapeutic Strategies

Impact of microvessel density on lymph node metastasis and survival after curative resection of pancreatic cancer

The roles of angiogenesis and the most prominent angiogenic vascular endothelial growth factor (VEGF) in diseases of the pancreas remain controversial. We compared microvessel density (MVD) and VEGF status in normal pancreatic, chronic pancreatic, and pancreatic cancer (PC) tissues to establish their prognostic relevance. Eighty samples of PC tissue, 32 samples of normal pancreatic tissue, and 20 samples of chronic pancreatitis (cP) tissue were immunostained with monoclonal anti-CD31 and polyclonal anti-VEGF antibody. The MVD was correlated with clinicopathological features and survival. Microvessel density was higher in PC than in cP (P < 0.001). Residual tumor status was highly predictive for survival (P < 0.001). After stratification for residual tumor status, we identified lymph node metastasis (LNM) in more than two lymph nodes (P < 0.04) and high MVD (P < 0.03) as risk factors for mortality. Multivariate analysis revealed only a high MVD (P = 0.03, odds ratio 0.441, 95% confidence interval 0.211-0.821) as an independent predictor of poor survival. Vascular endothelial growth factor was found over stromal cells in cP and over ductal adenocarcinoma cells in PC. Vascular endothelial growth factor expression status was not predictive of survival (P < 0.07). This study confirms the role of angiogenesis in PC and identifies MVD as an independent prognostic factor in patients with curatively resected PC.

Synergistic antitumor effect of adenovirus-mediated hING4 gene therapy and 125I radiation therapy on pancreatic cancer

Pancreatic cancer has a poor prognosis, even with surgery. ING4 is a member of the inhibitor of growth (ING) tumor suppressor family that has potent inhibitory effects on a variety of tumors; meanwhile, radiotherapy is a common adjunctive therapy for pancreatic cancer. Prior to this study, the effectiveness of a combination of ING4 gene-therapy and radiotherapy against pancreatic cancer had been unknown. In this study, we demonstrated that either ING4 or 125I radiotherapy treatment could induce Panc-1 pancreatic cancer cell growth suppression and apoptosis in vitro. Furthermore, both treatments inhibited tumor growth and angiogenesis of Panc-1 pancreatic cancer subcutaneously xenografted in vivo. Moreover, the combination therapy had a synergistic effect.

ShRNA silencing glycogen synthase kinase-3 beta inhibits tumor growth and angiogenesis in pancreatic cancer

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine protein kinase, plays a vital role in the tumorigenesis of many cancers, but its role in pancreatic cancer remains unknown. In this study, we showed that GSK-3β was aberrantly activated in pancreatic cancer. GSK-3β knockdown resulted in arrested proliferation and increased apoptosis in pancreatic cancer cell lines. Expression of Bcl-2 and vascular endothelial growth factor (VEGF) decreased significantly in a GSK-3β knockdown group. In a xenograft tumor model, GSK-3β knockdown inhibited tumor growth and angiogenesis. Our study showed that GSK-3β may become a promising therapeutic target for human pancreatic cancer.

3,5-Bis(2,4-Difluorobenzylidene)-4-piperidone, a Novel Compound That Affects Pancreatic Cancer Growth and Angiogenesis

Dysregulated Notch signaling plays an important role in the progression of cancer. Notch signaling affects tumor growth and angiogenesis through the actions of its ligand Jagged-1. In this study, we developed a novel compound 3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD) and determined that it inhibits cancer cell growth and its effects on Notch signaling. Intraperitoneal administration of DiFiD significantly suppressed growth of pancreatic cancer tumor xenografts. There was a reduction in CD31-positive blood vessels, suggesting that there was an effect on angiogenesis. In vitro, DiFiD inhibited the proliferation of various human and mouse pancreatic cancer cells while increasing activated caspase-3. Cell-cycle analyses showed that DiFiD induced G(2)-M arrest and decreased the expression of cell-cycle-related proteins cyclin A1 and D1 while upregulating cyclin-dependent kinase inhibitor p21WAF1. We next determined the mechanism of action. DiFiD reduced Notch-1 activation, resulting in reduced expression of its downstream target protein Hes-1. We further determined that the reduced Notch-1 activation was due to reduction in the ligand Jagged-1 and two critical components of the γ-secretase enzyme complex presenilin-1 and nicastrin. Ectopic expression of the Notch intracellular domain rescued the cells from DiFiD-mediated growth suppression. DiFiD-treated tumor xenografts also showed reduced levels of Jagged-1 and the γ-secretase complex proteins presenilin-1 and nicastrin. Taken together, these data suggest that DiFiD is a novel potent therapeutic agent that can target different aspects of the Notch signaling pathway to inhibit both tumor growth and angiogenesis.

Impact of Curcumin, Raspberry Extract, and Neem Leaf Extract on Rel Protein-Regulated Cell Death/Radiosensitization in Pancreatic Cancer Cells

Nuclear factor κB (NF-κB) plays an intrinsic role in promoting growth, angiogenesis, and metastasis in pancreatic cancer (PC) and serves as a mechanism underlying therapeutic resistance. Accordingly, we investigated the efficacy of bioactive phytochemicals in inhibiting radiotherapy (RT)-induced NF-κB activity, signaling, and NF-κB-dependent regulation of cell death. Panc-1, BxPC-3, and MIA PaCa-2 cells exposed to 10 Gy (single high dose [SDR]) or 2 Gy/d for 5 days (fractionated radiation [FIR]) with or without curcumin (CUR), neem leaf extract (NLE), or black raspberry extract (RSE) were analyzed. Radiotherapy profoundly induced NF-κB-DNA-binding activity with relatively robust activation after FIR. Curcumin, NLE, and RSE significantly inhibited both constitutive and RT-induced NF-κB. Furthermore, quantitative polymerase chain reaction profiling of 88 NF-κB pathway molecules demonstrated that CUR, NLE, and RSE comprehensively, yet differentially inhibited FIR/SDR-induced genes. Functionally, CUR, NLE, and RSE markedly conferred RT-inhibited cell viability/survival, robustly activated caspase-3/7 activity, and subsequent cell death. More importantly, NF-κB overexpression and silencing studies demonstrate that these compounds potentiate RT-induced cell death by targeting RT-induced NF-κB. These data strongly imply that CUR, NLE, and RSE may serve as effective "deliverables" to potentiate RT in PC cure and further throw light that these phytochemicals-induced cell killing may involve selective regulation of RT-induced NF-κB.

Abstract 1204: Fibroblast growth factor receptor 2 as a novel therapeutic target for cancer cell growth and angiogenesis in human pancreatic cancer

Abstract Background: Gene amplification or missence mutation of fibroblast growth factor receptor 2 (FGFR2) occurs in various cancer, and inhibition of activated mutation of FGFR2 induced apoptosis and growth inhibition of endometrial carcinoma. Therefore, FGFR2 has been considered as a potential therapeutic target in a variety of cancers. Due to alternative splicing, there are two isoforms of FGFR2, named FGFR2 IIIb and FGFR2 IIIc. Both FGFR2 isoforms are expressed in pancreatic ductal adenocarcinoma (PDAC). FGFR2 IIIb isoform, also known as keratinocyte growth factor receptor/KGFR, is expressed in approximately 40% of PDAC cases, and its expression correlates with VEGF-A expression and venous invasion. By contrast, FGFR2 IIIc isoform is expressed in 70% of PDAC cases and its expression correlates with retroperitoneal invasion, and increased growth rates and invasion of the cancer. These data suggests that both FGFR2 isoforms play crucial roles in promotion and progression of PDAC. However, it is not known whether FGFR2 can serve as a therapeutic target in PDAC. In the present study, we inhibited the expression of both FGFR2 isoforms by RNA silencing strategy to clarify the therapeutic effectiveness of FGFR2 knockdown in PDAC. Methods: FGFR2 IIIb and IIIc isoforms, and total FGFR2 expression levels were examined in six pancreatic cancer cell lines using real-time PCR analysis. FGFR2 short hairpin (sh) RNA targeting the common exon of IIIb and IIIc isoforms was prepared, and FGFR2 shRNA transfected PANC-1 cells (Sh cells) were established. Sham vectors were transfected into PANC-1 cells as negative controls (Sc cells). Decreased expression of FGFR2 mRNA and FGFR2 protein in the cell membrane was confirmed by real-time PCR and flow cytometry, respectively. The growth rates of Sh cells in vitro and in vivo were compared with Sc cells. Cell signaling was monitored by examining the activation of MAPK signaling pathways including JNK, p38 and ERK, and by assessing VEGF-A expression levels by immunofluorescence. Results: FGFR2, FGFR2IIIb and IIIc mRNA moieties were detected in all six pancreatic cancer cell lines. Sh cells showed decreased expression of FGFR2, FGFR2IIIb and IIIc mRNA, and FGFR2 protein in the cell membrane. The growth rates of Sh cells were lower than those of Sc cells in vitro and in the subcutaneous tissue of nude mice. Sh cells showed attenuation of phosphorylated of ERK pathway, but no alterations in JNK and p38 pathways, and expressed lower levels of VEGF-A. Conclusion: These findings indicate that FGFR2 signaling pathways exert important roles in pancreatic cancer cell growth and angiogenesis, and that inhibition of FGFR2 may serve as a therapeutic target in PDAC by suppressing cancer cell growth and angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1204. doi:10.1158/1538-7445.AM2011-1204

Dihydroartemisinin inhibits angiogenesis in pancreatic cancer by targeting the NF-κB pathway

Dihydroartemisinin (DHA) has recently shown antitumor activity in human pancreatic cancer cells. However, its effect on antiangiogenic activity in pancreatic cancer is unknown, and the mechanism is unclear. This study was aimed to investigate whether DHA would inhibit angiogenesis in human pancreatic cancer. Cell viability and proliferation, tube formation of human umbilical vein endothelial cells (HUVECs), nuclear factor (NF)-κB DNA-binding activity, expressions of vascular endothelial growth factor (VEGF), interleukin (IL)-8, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9 were examined in vitro. The effect of DHA on antiangiogenic activity in pancreatic cancer was also assessed using BxPC-3 xenografts subcutaneously established in BALB/c nude mice. DHA inhibited cell proliferation and tube formation of HUVECs in a time- and dose-dependent manner and also reduced cell viability in pancreatic cancer cells. DHA significantly inhibited NF-κB DNA-binding activity, so as to tremendously decrease the expression of NF-κB-targeted proangiogenic gene products: VEGF, IL-8, COX-2, and MMP-9 in vitro. In vivo studies, DHA remarkably reduced tumor volume, decreased microvessel density, and down-regulated the expression of NF-κB-related proangiogenic gene products. Inhibition of NF-κB activation is one of the mechanisms that DHA inhibits angiogenesis in human pancreatic cancer. We also suggest that DHA could be developed as a novel agent against pancreatic cancer.

STAT3-targeting RNA interference inhibits pancreatic cancer angiogenesis in vitro and in vivo

Signal transducers and activators of transcription 3 (STAT3) is a central cytoplasmic transcription factor that is activated by phosphorylation in response to extracellular signals and oncogenes. STAT3 regulates a number of pathways important in tumorigenesis including cell cycle progression, apoptosis, tumor angiogenesis, invasion and metastasis, and tumor cell evasion of the immune system. Our studies demonstrated that constitutively activated STAT3 plays an important role in the angiogenesis of pancreatic cancer. The objective of this study was to evaluate the potential use of RNA interference (RNAi) to knock down the STAT3 gene and the effect on angiogenesis of human pancreatic cancer cells in vitro and in vivo. We stably inhibited the expression of STAT3 and phosphorylated STAT3 (p-STAT3) using RNAi in the SW1990 pancreatic cancer cell line. Furthermore, RNAi for STAT3 inhibited STAT3-induced HUVEC cell migration and cell proliferation, and significantly suppressed the levels of secreted vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2) of SW1990 cells. In vivo experiments showed that RNAi for STAT3 significantly suppressed tumor growth and angiogenesis of SW1990 cells. Furthermore, silencing the STAT3 gene in SW1990 cells by RNAi also led to a decrease of VEGF and MMP-2 at the mRNA and protein levels. Collectively, these results demonstrate that the STAT3 signaling pathway plays an important role in the angiogenesis of pancreatic cancer and that knockdown of the STAT3 gene using the RNAi technique may be a novel therapeutic option for the treatment of pancreatic cancer.

Hispidulin, a small flavonoid molecule, suppresses the angiogenesis and growth of human pancreatic cancer by targeting vascular endothelial growth factor receptor 2‐mediated PI3K/Akt/mTOR signaling pathway

Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti-inflammatory and anti-oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs in a dose-dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF-induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice. To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF-triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal-regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis.

PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer

Purpose:Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.Experimental design:PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.Results:PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.Conclusion:Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.