Water extract of Korean red ginseng stimulates angiogenesis by activating the PI3K/Akt-dependent ERK1/2 and eNOS pathways in human umbilical vein endothelial cells

Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N(G)-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.     

A novel Src kinase inhibitor, M475271, inhibits VEGF-induced human umbilical vein endothelial cell proliferation and migration

Vascular endothelial growth factor (VEGF) was reported to be a potent proangiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that showed selective inhibition of Src kinase activity and tumor growth in vivo. Here, we examined the effect of M475271 on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration and their intracellular mechanisms. Our findings showed that M475271 pretreatment resulted in a significant inhibition of VEGF-induced HUVEC proliferation, [(3)H]thymidine incorporation, and migration. M475271 inhibited VEGF-induced Flk-1 and Src phosphorylation and their association. Confocal laser microscopic examination confirmed the inhibitory effect of M475271 on VEGF-induced Flk-1/Src association. M475271 inhibited VEGF-induced extracellular signal-regulated kinase1/2 (ERK1/2) and p38 but not Akt activation in a concentration-dependent manner. M475271, PI3-K inhibitor, and p38 inhibitor inhibited VEGF-induced HUVEC proliferation and migration. However, a MEK1/2 inhibitor inhibited VEGF-induced proliferation but not migration. These findings suggest that M475271 attenuates VEGF-induced HUVEC proliferation and migration through the inhibition of signaling pathways involving Src, ERK1/2, and/or p38. Taken together, these data indicate that M475271 may be a useful candidate for inhibition of endothelial cell proliferation and migration relevant to angiogenesis.     

Notoginsenoside Ft1 promotes angiogenesis via HIF-1α mediated VEGF secretion and the regulation of PI3K/AKT and Raf/MEK/ERK signaling pathways

Notoginsenoside Ft1 (Ft1) is a saponin isolated from Panax notoginseng, which has been used traditionally for the treatment of trauma injuries in East Asia. Here we show that Ft1 is a novel stimulator of angiogenesis. The results show that Ft1 induces proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells (HUVECs). Ft1 increases translocalization of hypoxia-inducible factor-1α (HIF-1α) from cytoplasm to nuclei, where it binds to the vascular endothelial growth factor (VEGF) promoter, increasing the expression of VEGF mRNA and the subsequent secretion of the growth factor. Ft1 induces the activation of PI3K/AKT and Raf/MEK/ERK signaling pathways. Pharmacological inhibition with LY294002, wortmanin or PD98059 reduces Ft1-induced angiogenesis, indicating the important role played by these pathways. In addition, Ft1 induces phosphorylation of the mammalian target of rapamycin (mTOR), and siRNA-mediated mTOR knockdown decreases tube formation, proliferation, transport of HIF-1α into nuclei and VEGF mRNA expression in response to Ft1. Finally, in vivo, Ft1 promotes the formation of blood vessels in Matrigel plug and wound healing in mice. Taken together, the present results reveal that Ft1 stimulates angiogenesis via HIF-1α-mediated VEGF expression, with PI3K/AKT and Raf/MEK/ERK signaling cascades concurrently participating in the process.     

Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways

Proliferation of Schwann cell in the injured peripheral nerve supports axonal regeneration and also is critical for the regeneration of injured nerves. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and synthesis of proliferating cell nuclear antigen (PCNA) and (ii) to activate mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositil-3 kinase (PI3K)/Akt signaling pathways in rat Schwann cells. CMCS was found to induce proliferation and PCNA synthesis in Schwann cells in a dose and time dependent manner. CMCS was shown to phosphorylate ERK1/2 and Akt in Schwann cell proliferation. The phosphorylation of ERK1/2 and Akt in Schwann cells was blocked by the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin. In addition, inhibition of the MEK/ERK or the PI3K/Akt signaling pathways significantly decreased the proliferative effects of CMCS in Schwann cells. Overall, the above results indicate that CMCS stimulates proliferation of Schwann cells by activating the intracellular signaling cascades of ERK1/2 and PI3K/Akt.     

Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.     

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.     

Taurine suppresses doxorubicin-triggered oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and inhibition of p53, p38-JNK

The objective of the present study was to investigate the signaling mechanisms involved in the beneficial role of taurine against doxorubicin-induced cardiac oxidative stress. Male rats were administered doxorubicin. Hearts were collected 3 weeks after the last dose of doxorubicin and were analyzed. Doxorubicin administration retarded the growth of the body and the heart and caused injury in the cardiac tissue because of increased oxidative stress. Similar experiments with doxorubicin showed reduced cell viability, increased ROS generation, intracellular Ca²⁺ and DNA fragmentation, disrupted mitochondrial membrane potential and apoptotic cell death in primary cultured neonatal rat cardiomyocytes. Signal transduction studies showed that doxorubicin increased p53, JNK, p38 and NFκB phosphorylation; decreased the levels of phospho ERK and Akt; disturbed the Bcl-2 family protein balance; activated caspase 12, caspase 9 and caspase 3; and induced cleavage of the PARP protein. However, taurine treatment or cardiomyocyte incubation with taurine suppressed all of the adverse effects of doxorubicin. Studies with several inhibitors, including PS-1145 (an IKK inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and LY294002 (a PI3-K/Akt inhibitor), demonstrated that the mechanism of taurine-induced cardio protection involves activation of specific survival signals and PI3-K/Akt as well as the inhibition of p53, JNK, p38 and NFκB. These novel findings suggest that taurine might have clinical implications for the prevention of doxorubicin-induced cardiac oxidative stress.     

Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6+/-0.7 microM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase-ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86+/-9% inhibited by 30 microM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.     

Pravastatin induces rat aortic endothelial cell proliferation and migration via activation of PI3K/Akt/mTOR/p70 S6 kinase signaling

The HMG-CoA reductase inhibitors (statins) have been shown to exert several vascular protective effects that are not related to changes in cholesterol profile, and these effects of statins are partly caused by the activation of angiogenesis. Endothelial cell (EC) proliferation and migration are crucial events for angiogenesis and statins are known to enhance these events. However, the molecular mechanism by which statins promote EC proliferation and migration is not fully understood. In this study, we show Akt and its downstream target mammalian target of rapamycin (mTOR) play an important role in pravastatin-induced EC proliferation and migration. We found that pravastatin significantly enhanced the proliferation and migration of rat aortic endothelial cells (rAECs). The addition of pravastatin to rAECs resulted in rapid phosphorylation of Akt and p70 S6 kinase (p70S6K). LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and p70S6K phosphorylation, whereas rapamycin, a specific inhibitor of mTOR, suppressed only p70S6K phosphorylation induced by pravastatin. Furthermore, both LY294002 and rapamycin inhibited pravastatin-induced rAEC proliferation and migration. Taken together, our findings indicate that pravastatin activates PI3K/Akt/mTOR /p70S6K signaling in this sequential manner and this pathway contributes to pravastatin-induced rAEC proliferation and migration.     

Targeted molecular strategies for cancer therapy based on the blockage of oncogenic pathways in human tumor cells

Constitutive activation of the extracellular signal-regulated kinase (ERK) and/or phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathways is associated with neoplastic transformation of a variety of human tumor cells. Treatment of such tumor cells with agents that specifically inhibit the activity of mitogen-activated protein kinase/ERK kinase (MEK) or PI3K completely suppresses their proliferation. However, treatment of cells with these inhibitors leads to only a modest increase in apoptotic cell death. Efficient induction of apoptosis is essential for the development of effective cancer chemotherapy. Therefore, we have examined whether specific blockade of these two signaling pathways affects the efficacy of the induction of apoptosis by various anti-cancer agents in tumor cells. We found that MEK inhibitors markedly enhance the efficacy of histone deacetylase inhibitors to induce the generation of reactive oxygen species and apoptosis. This effect was only observed in tumor cells in which the ERK pathway was constitutively activated. Furthermore, we found that PI3K inhibitors selectively and markedly enhanced the efficacy of the induction of apoptosis by doxorubicin. This latter effect was only detected in tumor cells in which the PI3K/Akt pathway was constitutively activated and in which the p53 pathway was functional. These combination treatments provide an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway or PI3K/Akt pathway is constitutively activated. This review gives an overview of the development of new targeted molecular strategies for cancer therapy based on the suppression of the activity of oncogenic pathways involved in the proliferation or survival of tumor cells.     

Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation.     

Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposure to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity.     

Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4): Protein kinase modulation and reactive oxygen species generation

While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G₂/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G₂/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21^waf1/cip1 and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G₂/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-l-cysteine fails to prevent ERK activation, G₂/M arrest, and differentiation induction. By contrast, N-acetyl-l-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G₂/M arrest, but uncoupled to the pro-apoptotic action of the drug.     

Stimulation of ERK2 by taurine with enhanced alkaline phosphatase activity and collagen synthesis in osteoblast-like UMR-106 cells

Taurine is present in a variety of tissues and exhibits many important physiological functions in the cell. Even though its functions are well documented in many tissues, its actions on bone cells are largely unknown. Considering a recent finding that taurine is present in the bone, we wished to determine if taurine could have any effects on osteoblast cells. Taurine (10 mM) stimulated alkaline phosphatase activity as well as collagen synthesis. Taurine also stimulated tyrosine phosphorylation of a number of cellular proteins including a 42-kDa protein. The 42-kDa protein was identified as extracellular signal regulated protein kinase 2 (ERK2). A mitogen-activated protein kinase kinase (MEK) inhibitor blocked the taurine-stimulated alkaline phosphatase activity and collagen synthesis. These results suggest that taurine could regulate osteoblast metabolism via ERK2 activation.     

Implication of NAG-1 in synergistic induction of apoptosis by combined treatment of sodium salicylate and PI3K/MEK1/2 inhibitors in A549 human lung adenocarcinoma cells

Aspirin is used as chemopreventive agents in a variety of human cancer cells including those of colon, lung, breast, and leukemia. Sodium salicylate (NaSal, the natural deacetylated form of aspirin) induced cell cycle arrest and apoptosis in a dose-dependent manner in A549 cells; high dose (20mM) of NaSal-induced apoptosis, whereas low dose (2-10mM) induced cell cycle arrest. We found that NaSal-activated Akt/PKB, ERK1/2, and p38MAPK signal cascades. Twenty micromolar of NaSal-induced apoptotic response of A549 cells was enhanced by the PI3K inhibitors (LY294002 and wortmannin) and in a less extent by the MEK1/2 inhibitors (U0126 and PD98059), whereas it was suppressed by the p38MAPK inhibitor (SB203580). Furthermore, simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could lower the dose of NaSal to induce apoptosis to 2mM in A549 lung cancer cells. Similar enhancement was observed in cells treated with 2mM NaSal and 100muM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrated that NAG-1 plays a key role in apoptosis by NaSal-based combined treatment. Collectively, our findings indicate that inhibition of the pro-survival Akt/PKB and ERK1/2 signaling may increase the chemopreventive effects of NaSal and combined treatment of two natural compounds (NaSal and genistein) results in a highly synergistic induction of apoptosis, thereby increasing the chemopreventive effects of NaSal against cancer.     

Phosphatidylinositol inhibits vascular endothelial growth factor-A--induced migration of human umbilical vein endothelial cells

Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 microg/ml) inhibited VEGF-A-induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 microg/ml inhibited the VEGF-A-induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.     

Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma.     

Nickel‐induced VEGF expression via regulation of Akt,ERK1/2, NFκB,and AMPK pathways in H460 cells

Prospective cohort studies have indicated that a highly nickel‐polluted environment may severely affect human health, resulting in such conditions as respiratory tract cancers. Such exposure can trigger vascular endothelial growth factor (VEGF) expression. However, the signal transduction pathways leading to VEGF induction by nickel compounds are not well understood. This study revealed the occurrence of VEGF induction in human non‐small‐cell lung cancer H460 cells exposed to NiCl₂. Moreover, exposing H460 cells to NiCl₂ activated extracellular signal‐regulated protein kinase (ERK), nuclear factor kappa B (NFκB), and protein kinase B (Akt) as well as downregulated AMP activated protein kinase (AMPK) expression. The mitogen‐activated protein kinase (MAPK) and ERK inhibitor significantly blocked NiCl₂‐induced ERK activation and VEGF production. Pretreating H460 cells with a PI3K/Akt inhibitor substantially inhibited NiCl₂‐induced VEGF expression and reduced Akt, ERK, and NFκB phosphorylation. Furthermore, 5‐aminoimidazole‐4‐carboxamide ribonucleoside‐induced AMPK activation improved VEGF expression in NiCl₂‐treated H460 cells significantly. These results indicate that NiCl₂ induces VEGF production through Akt, ERK, NFκB activation and AMPK suppression and mediates various types of pathophysiological angiogenesis.     

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.