绿茶提取物表没食子儿茶素-3-没食子酸酯对人卵巢癌HO-8910细胞增殖及Wnt/β-catenin信号通路相关基因表达的影响

目的研究绿茶提取物表没食子儿茶素-3-没食子酸酯(Epigallocatechin-3 gallste,EGCG)对人卵巢癌HO-8910细胞增殖及细胞内Wnt/β-catenin信号通路相关基因表达的影响,探讨EGCG抑制卵巢癌细胞生长的机制。方法用不同浓度的EGCG(10、20和40μg/ml)处理体外培养的人卵巢癌HO-8910细胞不同时间(24、48和72 h),采用MTT法检测细胞的增殖活力;流式细胞术检测细胞周期的变化;RT-PCR和Western blot分别检测细胞中β-catenin和下游靶基因CyclinD1 mRNA的转录水平和蛋白的表达水平。结果 EGCG可明显抑制HO-8910细胞的增殖活力,且抑制作用呈剂量-时间依赖性(P<0.05);40μg/ml EGCG干预后,HO-8910细胞主要阻滞于G0/G1期,且在48 h阻滞作用最为明显;EGCG可显著降低HO-8910细胞中β-catenin和CyclinD1基因mRNA的转录水平和蛋白的表达水平,且呈剂量-时间依赖性(P<0.01)。结论 EGCG可抑制HO-8910细胞的增殖,其机制可能与其抑制Wnt/β-catenin信号通路的活性有关,提示EGCG可能在卵巢癌的治疗中具有一定的应用前景。     

Short Hairpin RNA (shRNA) Ether �� go-go 1 (Eag1) Inhibition of Human Osteosarcoma Angiogenesis via VEGF/PI3K/AKT Signaling

Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers but the therapeutic potential of Eag1 in osteosarcoma remains elusive. In this study, we constructed an Ad5-Eag1-shRNA vector and evaluated its efficiency for Eag1 knockdown and its effects on osteosarcoma. Our results showed that Ad5-Eag1-shRNA had high interference efficiency of Eag1 expression and suppressed osteosarcoma growth both in vitro and in vivo. To explore the molecular mechanism underlying tumor growth inhibition induced by Eag1 silencing, the intratumoral microvessel density (MVD) was assessed by CD31 staining and the expression of vascular endothelial growth factor (VEGF) was detected by Western blot analysis. We found that Eag1 silencing led to decreased angiogenesis and VEGF expression in the xenograft model of osteosarcoma. Finally, we detected a time-dependent decrease in VEGF expression and considerably reduced phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation in osteosarcoma cells treated by Eag1 shRNA. Taken together, our results suggest that Eag1 silencing inhibits tumor growth and angiogenesis in osteosarcoma via the down regulation of VEGF/PI3K/AKT signaling.     

VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.     

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.     

In Vitro Antimetastatic Effect of Phosphatidylinositol 3-Kinase Inhibitor ZSTK474 on Prostate Cancer PC3 Cells

Tumor metastasis is the main cause of lethality of prostate cancer, because conventional therapies like surgery and hormone treatment rarely work at this stage. Tumor cell migration, invasion and adhesion are necessary processes for metastasis. By providing nutrition and an escape route from the primary site, angiogenesis is also required for tumor metastasis. Phosphatidylinositol 3-kinases (PI3Ks) are well known to play important roles in tumorigenesis as well as metastasis. ZSTK474 is a specific PI3K inhibitor developed for solid tumor therapy. In the present report, antimetastatic activities of ZSTK474 were investigated in vitro by determining the effects on the main metastatic processes. ZSTK474 exhibited inhibitory effects on migration, invasion and adhesive ability of prostate cancer PC3 cells. Furthermore, ZSTK474 inhibited phosphorylation of Akt substrate-Girdin, and the secretion of matrix metalloproteinase (MMP), both of which were reported to be closely involved in migration and invasion. On the other hand, ZSTK474 inhibited the expression of HIF-1α and the secretion of vascular endothelial growth factor (VEGF), suggesting its potential antiangiogenic activity on PC3 cells. Moreover, we demonstrated the antiangiogenesis by determining the effect of ZSTK474-reduced VEGF on tube formation of human umbilical vein endothelial cells (HUVECs). In conclusion, ZSTK474 was demonstrated to have potential in vitro antimetastatic effects on PC3 cells via dual mechanisms: inhibition of metastatic processes including cell migration, invasion and adhesion, and antiangiogenesis via blockade of VEGF secretion.     

Kaempferol Inhibits Zearalenone-Induced Oxidative Stress and Apoptosis via the PI3K/Akt-Mediated Nrf2 Signaling Pathway: In Vitro and In Vivo Studies

In this study, kaempferol (KFL) shows hepatoprotective activity against zearalenone (ZEA)-induced oxidative stress and its underlying mechanisms in in vitro and in vivo models were investigated. Oxidative stress plays a critical role in the pathophysiology of various hepatic ailments and is normally regulated by reactive oxygen species (ROS). ZEA is a mycotoxin known to exert toxicity via inflammation and ROS accumulation. This study aims to explore the protective role of KFL against ZEA-triggered hepatic injury via the PI3K/Akt-regulated Nrf2 pathway. KFL augmented the phosphorylation of PI3K and Akt, which may stimulate antioxidative and antiapoptotic signaling in hepatic cells. KFL upregulated Nrf2 phosphorylation and the expression of antioxidant genes HO-1 and NQO-1 in a dose-dependent manner under ZEA-induced oxidative stress. Nrf2 knockdown via small-interfering RNA (siRNA) inhibited the KFL-mediated defence against ZEA-induced hepatotoxicity. In vivo studies showed that KFL decreased inflammation and lipid peroxidation and increased H₂O₂ scavenging and biochemical marker enzyme expression. KFL was able to normalize the expression of liver antioxidant enzymes SOD, CAT and GSH and showed a protective effect against ZEA-induced pathophysiology in the livers of mice. These outcomes demonstrate that KFL possesses notable hepatoprotective roles against ZEA-induced damage in vivo and in vitro. These protective properties of KFL may occur through the stimulation of Nrf2/HO-1 cascades and PI3K/Akt signaling.     

Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.     

The Utilization of Bevacizumab in Patients with Advanced Ovarian Cancer: A Systematic Review of the Mechanisms and Effects

Most ovarian cancer cases are diagnosed at an advanced stage (III or IV), in which a primary debulking surgery combined with adjuvant systemic chemotherapy is the standard management. Since targeted therapy is less toxic to human cells than systemic chemotherapy, it has drawn much attention and become more popular. Angiogenesis is a critical process during the proliferation of ovarian cancer cells. Currently, many studies have put emphases on anti-angiogenetic medication, such as bevacizumab, the first and most investigated angiogenesis inhibitor that can exert anti-neoplastic effects. Bevacizumab is a recombinant humanized monoclonal antibody that has been approved for first-line maintenance treatment of advanced ovarian cancer. This review is a summary of current literature about the molecular mechanisms of actions, safety, and effects of bevacizumab for use in advanced epithelial ovarian cancer. Some common side effects of bevacizumab will be also discussed. As an inhibitor of angiogenesis, bevacizumab binds to circulating vascular endothelial growth factor (VEGF) and thereby inhibits the binding of VEGF to its receptors on the surface of endothelial cells. Neutralization of VEGF prevents neovascularization and leads to apoptosis of tumor endothelial cells and a decrease in interstitial fluid pressure within the tumors, which allows greater capacity for chemotherapeutic drugs to reach specific targeted sites. Grossly, bevacizumab has demonstrated some significant therapeutic benefits in many randomized trials in combination with the standard chemotherapy for advanced epithelial ovarian cancer. Based on the available evidence, a higher dosage and a longer duration of bevacizumab appear to achieve better therapeutic effects and progression-free survival. On the other hand, patients with more severe diseases or at a higher risk of progression seem to benefit more from bevacizumab use. However, many unknown aspects of bevacizumab, including detailed mechanisms of actions, effectiveness, and safety for the treatment of ovarian cancer, warrant further investigation.     

Effects of P4 Antagonist RU486 on VEGF and Its Receptors’ Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P₄) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P₄ receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P₄ antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P₄ in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.     

Molecular Mechanisms of (R,R)ZX-5 on NO Synthesis and Its Anti-Angiogenic Effect

(R,R)ZX-5 is a NO regulatory compound, which could significantly increase choroidal blood flow in New Zealand rabbit. The aim of this paper is to investigate the molecular mechanism of (R,R)ZX-5 promoting NO production. Besides this, we also investigated the antiangiogenic activity of (R,R)ZX-5. Analysis of Western blot showed that (R,R)ZX-5 up-regulated the expression of Akt, p-Akt (Thr473), eNOS and p-eNOS (Ser1177), down-regulated the expression of Cyclin D1 in human retinal endothelial cells and escalated the intracellular free Ca(2+) concentration. Additionally, (R,R)ZX-5 inhibited the growth of blood vessels in the chick chorioallantoic membrane model. It is concluded that (R,R)ZX-5 promotes choroidal blood flow through PI3K/Akt-eNOS and Akt-Ca(2+)-eNOS pathways. Additionally, (R,R)ZX-5 can inhibit angiogenesis.     

PLCε调节肾细胞癌血管生成的作用机制

目的探讨血管内皮生长因子(Vascular endothelial growth factor,VEGF)在PLCε-NF-κB信号通路中影响肿瘤血管生成的作用机制。方法将PLCε-shRNA表达质粒pGenesil-PLCε转染人肾透明细胞癌786-0细胞株,沉默磷脂酶Cε(Phos-pholipase C epsilon,PLCε)基因的表达,采用RT-PCR及Western blot检测转染细胞中PLCε、NF-κB和VEGF基因mRNA及蛋白水平表达的变化;应用NF-κB特异性抑制剂BAY11-7082处理细胞后,采用MTT法检测BAY11-7082对786-0细胞增殖的抑制作用,RT-PCR和Westernblot检测VEGF基因mRNA和蛋白水平表达的变化。结果重组质粒pGenesil-PLCε可明显抑制PLCε基因mRNA和蛋白水平的表达,抑制率分别为71.43%和50.01%,并明显下调NF-κB和VEGF基因mRNA和蛋白水平的表达;BAY11-7082可明显抑制786-0细胞增殖,且呈剂量和时间依赖性;应用BAY11-7082后,VEGF基因mRNA和蛋白的表达均被明显抑制。结论 PLCε可能通过抑制NF-κB基因的表达,从而抑制NF-κB依赖性基因VEGF的表达,进而影响肾细胞癌血管生成。     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.     

SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate–SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate–SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.     

Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin.     

Stimulations of strontium-doped calcium polyphosphate for bone tissue engineering to protein secretion and mRNA expression of the angiogenic growth factors from endothelial cells in vitro

The vascularization of tissue-engineered bone is the key problem needed solving before application of tissue-engineered bone in clinical practice. Meanwhile, endothelial cells are the major and important source of seed cells in bone tissue engineering, and significant on promoting vascularization in tissue-engineered bone. Vascularization (namely angiogenesis) is a process mainly controlled by several angiogenic growth factors (VEGF, bFGF and MMP-2) which can be secreted by endothelial cells. Therefore, the research on the stimulations of SCPP to the secretion of the angiogenic growth factors from endothelial cells is very important. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the protein secretion levels and mRNA expression of VEGF, bFGF and MMP-2 from cultured endothelial cells. As a control, we also researched the effect of HA on the mRNA expressions and protein secretion of angiogenic growth factors from cultured endothelial cells. We cultured endothelial cells with SCPP scaffolds containing various concentration of strontium and HA. The results obtained in the MTT and SEM tests indicated that endothelial cells on SCPP scaffold exhibited higher proliferation rate and were easy to get a good spread than them on CPP, the best state of growth and proliferation of cells could be observed on 8%SCPP. The results of ELISA demonstrated that the protein levels of VEGF, bFGF and MMP-2 from cultured endothelial cells increased with the increasing Sr doped in calcium polyphosphate in SCPP groups, the peaks appeared on 8%SCPP. All SCPP groups showed a better ability to stimulate the protein secretion of VEGF, bFGF and MMP-2 from endothelial cells relative to CPP group and HA group. The results of RT-PCR suggested that the 8%SCPP group exhibited a significantly higher mRNA expression of VEGF, bFGF and MMP-2 relative to CPP group and HA group. In conclusion, the results of this study demonstrated that 8%SCPP had obvious promotion for secretion and mRNA expression of angiogenic growth factors from cultured endothelial cells.