Vasoactive intestinal peptide (VIP) increases vascular endothelial growth factor (VEGF) expression and secretion in human breast cancer cells

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.     

MICAL1 facilitates breast cancer cell proliferation via ROS‐sensitive ERK/cyclin D pathway

Molecule interacting with CasL 1 (MICAL1) is a multidomain flavoprotein mono‐oxygenase that strongly involves in cytoskeleton dynamics and cell oxidoreduction metabolism. Recently, results from our laboratory have shown that MICAL1 modulates reactive oxygen species (ROS) production, and the latter then activates phosphatidyl inositol 3‐kinase (PI3K)/protein kinase B (Akt) signalling pathway which regulates breast cancer cell invasion. Herein, we performed this study to assess the involvement of MICAL1 in breast cancer cell proliferation and to explore the potential molecular mechanism. We noticed that depletion of MICAL1 markedly reduced cell proliferation in breast cancer cell line MCF‐7 and T47D. This effect of MICAL1 on proliferation was independent of wnt/β‐catenin and NF‐κB pathways. Interestingly, depletion of MICAL1 significantly inhibited ROS production, decreased p‐ERK expression and unfavourable for proliferative phenotype of breast cancer cells. Likewise, MICAL1 overexpression increased p‐ERK level as well as p‐ERK nucleus translocation. Moreover, we investigated the effect of MICAL1 on cell cycle‐related proteins. MICAL1 positively regulated CDK4 and cyclin D expression, but not CDK2, CDK6, cyclin A and cyclin E. In addition, more expression of CDK4 and cyclin D by MICAL1 overexpression was blocked by PI3K/Akt inhibitor LY294002. LY294002 treatment also attenuated the increase in the p‐ERK level in MICAL1‐overexpressed breast cancer cells. Together, our results suggest that MICAL1 exhibits its effect on proliferation via maintaining cyclin D expression through ROS‐sensitive PI3K/Akt/ERK signalling in breast cancer cells.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Sodium butyrate induces differentiation of gastric cancer cells to intestinal cells via the PTEN/phosphoinositide 3‐kinase pathway

NaB (sodium butyrate) inhibits cell proliferation and induces differentiation in a variety of tumour cells. In this study, we aimed to determine whether NaB induced differentiation and regulated the expression of the mucosal factor MUC2 through the PTEN/PI3K (phosphoinositide 3‐kinase) pathway. BGC823 cells treated with NaB for 24–72 h showed marked inhibition of cell proliferation and alteration in cellular morphology. NaB treatment markedly increased the expression of PTEN and MUC2, but it decreased the expression of PI3K. These effects were enhanced by intervention with PI3K inhibitors and were reduced by intervention with PTEN siRNA. Hence, we conclude that NaB increased PTEN expression, promoted the expression of MUC2 and induced the differentiation of gastric cancer cells through the PTEN/PI3K signalling pathway.     

A proteomics approach to identifying novel protein targets involved in erinacine A–mediated inhibition of colorectal cancer cells’ aggressiveness

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A induction associated with cancer cells’ aggressive status and death. A proteomic approach was used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of HCT‐116 and DLD‐1 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Ten differentially displayed proteins were determined and validated in vitro and in vivo between the erinacine A‐treated and untreated groups. In addition, erinacine A time‐dependent induction of cell death and inhibitory invasiveness was associated with sustained phosphorylation of the PI3K/mTOR/p70S6K and ROCK1/LIMK2/Cofilin pathways. Furthermore, we demonstrated that erinacine A–induced HCT‐116 and DLD‐1 cells viability and anti‐invasion properties by up‐regulating the activation of PI3K/mTOR/p70S6K and production of ROS. Experiments involving specific inhibitors demonstrated that the differential expression of cofilin‐1 (COFL1) and profilin‐1 (PROF1) during erinacine A treatment could be involved in the mechanisms of HCT‐116 and DLD‐1 cells death and decreased aggressiveness, which occurred via ROCK1/LIMK2/Cofilin expression, with activation of the PI3K/mTOR/p70S6K signalling pathway. These findings elucidate the mechanism of erinacine A inhibiting the aggressive status of cells by activating PI3K/mTOR/p70S6K downstream signalling and the novel protein targets COF1 and PROF1; this could be a good molecular strategy to limit the aggressiveness of CRC cells.     

The role of the PI3K/AKT signalling pathway in the corneal epithelium: recent updates

Phosphatidylinositol 3 kinase (PI3K)/AKT (also called protein kinase B, PKB) signalling regulates various cellular processes, such as apoptosis, cell proliferation, the cell cycle, protein synthesis, glucose metabolism, and telomere activity. Corneal epithelial cells (CECs) are the outermost cells of the cornea; they maintain good optical performance and act as a physical and immune barrier. Various growth factors, including epidermal growth factor receptor (EGFR) ligands, insulin-like growth factor 1 (IGF1), neurokinin 1 (NK-1), and insulin activate the PI3K/AKT signalling pathway by binding their receptors and promote antiapoptotic, anti-inflammatory, proliferative, and migratory functions and wound healing in the corneal epithelium (CE). Reactive oxygen species (ROS) regulate apoptosis and inflammation in CECs in a concentration-dependent manner. Extreme environments induce excess ROS accumulation, inhibit PI3K/AKT, and cause apoptosis and inflammation in CECs. However, at low or moderate levels, ROS activate PI3K/AKT signalling, inhibiting apoptosis and stimulating proliferation of healthy CECs. Diabetes-associated hyperglycaemia directly inhibit PI3K/AKT signalling by increasing ROS and endoplasmic reticulum (ER) stress levels or suppressing the expression of growth factors receptors and cause diabetic keratopathy (DK) in CECs. Similarly, hyperosmolarity and ROS accumulation suppress PI3K/AKT signalling in dry eye disease (DED). However, significant overactivation of the PI3K/AKT signalling pathway, which mediates inflammation in CECs, is observed in both infectious and noninfectious keratitis. Overall, upon activation by growth factors and NK-1, PI3K/AKT signalling promotes the proliferation, migration, and anti-apoptosis of CECs, and these processes can be regulated by ROS in a concentration-dependent manner. Moreover, PI3K/AKT signalling pathway is inhibited in CECs from individuals with DK and DED, but is overactivated by keratitis.Subject terms: Growth factor signalling, Apoptosis, Extracellular matrix     

Reactive oxygen species stimulate VEGF production from C(2)C(12) skeletal myotubes through a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, the expression of which increases in skeletal muscle after exercise. Because exercise is also accompanied by increased intramuscular reactive oxygen species (ROS) generation, we tested the hypothesis that ROS stimulate VEGF production from skeletal myotubes. Differentiated C(2)C(12) skeletal myotubes exposed to ROS-producing agents exhibited a concentration-dependent increase in VEGF production, whereas undifferentiated myoblasts did not respond to oxidants. Moreover, conditioned medium from ROS-treated myotubes increased the bovine lung microvascular cell proliferation rate. To study the mechanism(s) involved in the stimulation of VEGF production by ROS, myotubes were pretreated with a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY-294002, before being exposed to hydrogen peroxide or pyrogallol. LY-294002 attenuated both Akt phosphorylation and VEGF production. In addition, oxidants increased nuclear factor-kappaB-dependent promoter activity in transiently transfected myotubes; however, pretreatment with the pharmacological inhibitor of nuclear factor-kappaB, diethyldithiocarbamate, did not affect the oxidant-stimulated VEGF release. We conclude that ROS induce VEGF release from myotubes via a PI3K/Akt-dependent pathway.     

Specific aquaporins facilitate Nox-produced hydrogen peroxide transport through plasma membrane in leukaemia cells

In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H₂O₂) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H₂O₂ transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H₂O₂ across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H₂O₂ was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H₂O₂ intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H₂O₂-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H₂O₂ transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation.     

The p110delta isoform of PI 3-kinase negatively controls RhoA and PTEN

Inactivation of PI 3-kinase (PI3K) signalling is critical for tumour suppression by PTEN. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. We report that the p110delta PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110delta in macrophages led to reduced Akt and Rac1 activation, but paradoxically to increased RhoA and PTEN activity. Partial inactivation of p190RhoGAP and a reduced binding of cytoplasmic RhoA to the cyclin-dependent kinase inhibitor p27 both contributed to the increased RhoA-GTP levels upon p110delta inactivation. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110delta inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110delta-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN.     

RNF7 promotes glioma growth via the PI3K/AKT signalling axis

RNF7 has been reported to play critical roles in various cancers. However, the underlying mechanisms of RNF7 in glioma development remain largely unknown. Herein, the expression level of RNF7 was examined in tissues by quantitative real-time PCR, Western blotting and immunohistochemistry. The effect of RNF7 on glioma progression was measured by performing CCK-8 and apoptosis assays, cell cycle-related experiments and animal experiments. The effect of RNF7 on PI3K/AKT signalling pathway was tested by Western blotting. First, we found that RNF7 was upregulated in tumour tissue compared with normal brain tissue, especially in high-grade glioma, and the high expression of RNF7 was significantly related to tumour size, Karnofsky Performance Scale score and a poor prognosis. Second, RNF7 overexpression facilitated tumour cell cycle progression and cell proliferation and suppressed apoptosis. Conversely, RNF7 knockdown suppressed tumour cell cycle progression and cell proliferation and facilitated apoptosis. Furthermore, follow-up mechanistic studies indicated that RNF7 could facilitate glioma cell proliferation and cell cycle progression and inhibit apoptosis by activating the PI3K/AKT signalling pathway. This study shows that RNF7 can clearly promote glioma cell proliferation by facilitating cell cycle progression and inhibiting apoptosis by activating the PI3K/AKT signalling pathway. Targeting the RNF7/PI3K/AKT axis may provide a new perspective on the prevention or treatment of glioma.     

Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/Nitric oxide pathway and modulates migration of brain microvascular endothelial cells

Endothelium of the cerebral blood microvessels, which constitutes the major component of the blood-brain barrier, controls leukocyte and metastatic cancer cell adhesion and trafficking into the brain parenchyma. In this study, using rat primary brain microvascular endothelial cells (BMEC), we demonstrate that the vascular endothelial growth factor (VEGF), a potent promoter of angiogenesis, up-regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) through a novel pathway that includes phosphatidylinositol 3 OH-kinase (PI3K), AKT, and nitric oxide (NO), resulting in the migration of BMEC. Upon VEGF treatment, AKT is phosphorylated in a PI3K-dependent manner. AKT activation leads to NO production and release and activation-deficient AKT attenuates NO production stimulated by VEGF. Transfection of the constitutive myr-AKT construct significantly increased basal NO release in BMEC. In these cells, VEGF and the endothelium-derived NO synergistically up-regulated the expression of ICAM-1, which was mediated by the PI3K pathway. This activity was blocked by the PI3K-specific inhibitor, wortmannin. Furthermore, VEGF and NO significantly increased BMEC migration, which was mediated by the up-regulation of ICAM-1 expression and was dependent on the integrity of the PI3K/AKT/NO pathway. This effect was abolished by wortmannin, by the specific ICAM-1 antibody, by the specific inhibitor of NO synthase, N(G)-l-monomethyl-arginine (l-NMMA) or by a combination of wortmannin, ICAM-1 antibody, and l-NMMA. These findings demonstrate that the angiogenic factor VEGF up-regulates ICAM-1 expression and signals to ICAM-1 as an effector molecule through the PI3K/AKT/NO pathway, which leads to brain microvessel endothelial cell migration. These observations may contribute to a better understanding of BMEC angiogenesis and the physiological as well as pathophysiological function of the blood-brain barrier, whose integrity is crucial for normal brain function.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110β/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.     

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.     

TLR-4 Signalling Accelerates Colon Cancer Cell Adhesion via NF-κB Mediated Transcriptional Up-Regulation of Nox-1

Surgery induced inflammation is a potent promoter of tumour recurrence and metastasis in colorectal cancer. The recently discovered family of Nox enzymes represent a major source of endogenous reactive oxygen species (ROS) and are now heavily implicated in tumour cell metastasis. Interestingly, Nox enzymes can be ‘purposefully’ activated by inflammatory cytokines and growth factors which are present in abundance in the peri-operative window. As colon cancer cells express Nox enzymes and Toll-like receptor 4 (TLR-4), we hypothesised that LPS may potentiate the ability of colon cancer cells to metastasise via Nox enzyme mediated redox signalling. In support of this hypothesis, this paper demonstrates that LPS induces a significant, transient increase of endogenous ROS in SW480, SW620 and CT-26 colon cancer cells. This increase in LPS-induced ROS activity is completely abrogated by a Nox inhibitor, diphenyleneiodonium (DPI), Nox1 siRNA and an NF-κB inhibitor, Dihydrochloride. A significant increase in Nox1 and Nox2 protein expression occurs following LPS treatment. Inhibition of NF-κB also attenuates the increase of Nox1 and Nox2 protein expression. The sub-cellular location of LPS-induced ROS generation lies mainly in the endoplasmic reticulum. LPS activates the PI3K/Akt pathway via Nox generated ROS and this signal is inhibited by DPI. This LPS activated Nox mechanism facilitates a significant increase in SW480 colon cancer cell adhesion to collagen I, which is inhibited by DPI, Nox1 siRNA and a PI3K inhibitor. Altogether, these data suggest that the LPS-Nox1 redox signalling axis plays a crucial role in facilitation of colon cancer cell adhesion, thus increasing the metastatic potential of colon cancer cells. Nox1 may represent a valuable target in which to prevent colon cancer metastasis.     

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.     

Proteomic studies of rat tibialis anterior muscle during postnatal growth and development

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.