人endostatin体外诱导血管内皮细胞凋亡的实验研究

目的 探讨人endostatin抑制血管内皮细胞增殖的作用机制。方法 在含重组人endostatin蛋白和10%小牛血清的DMEM培养基中，培养人脐静脉内皮细胞ECV304。72h后，采用透射电镜，流式细胞术细胞周期分析和细胞核DNA的1.5%琼脂糖凝胶电泳，检测重组人endostatin蛋白作用后，血管内皮细胞的凋亡。结果 透射电镜下可见实验组ECV304细胞核染色质浓缩，边集，核碎裂及胞浆浓缩等，呈典型的凋亡细胞的形态学表现，流式细胞术细胞周期分析显示，在G1期峰前存在1个凋亡峰（20.6%)，1.5%琼脂糖凝胶电泳显示，细胞核DNA呈梯状，对照组ECV304细胞表达正常。结论 重组人endostatin蛋白可诱导血管内皮细胞凋亡，是其抑制血管内皮细胞增殖的原因之一。     

Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell line T24/83

Early reports indicated that ECV304 was a spontaneously-transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture. Many morphological, immunochemical, and genetic studies provided further evidence that ECV304 was a valuable biomedical research tool and could be used to study processes that include angiogenesis in vitro and signal transduction by a variety of G protein-coupled receptors. However, several distinct differences between ECV304 and HUVEC are now apparent and recent reports have indicated genetic similarity between ECV304 and T24/83, a human bladder cancer cell line. To further assess the utility of ECV304 as a human endothelial cell model, we compared the functional responses of ECV304 and T24/83 to a range of G protein-coupled receptor agonists. We also used DNA fingerprinting to karyotype both ECV304 and T24/83. Both ATP and uridine triphosphate (UTP) stimulated inositol phosphate metabolism in ECV304 without alteration of cAMP levels. Comparative data using selective P2Y receptor agonists indicated that this response, leading to calcium mobilization from intracellular stores, was predominantly mediated by the activation of P2Y2 receptors. Similar responses were recorded from both ECV304 and T24/83 cells. ECV304 expressed a relatively high basal activity of NOS that was reduced by L-NAME and stimulated by P2Y2 receptor agonists. In contrast, P2Y2 receptor activation did not induce prostaglandin synthesis in ECV304. Both ECV304 and T24/83 express receptors for adenosine, adrenaline, and calcitonin, which stimulate adenylate cyclase. Proliferation of ECV304 and T24/83 cells, measured by the incorporation of [3H]thymidine into DNA, was largely serum-independent. This was in contrast to parallel experiments with porcine and bovine aortic endothelial cells that indicated a marked serum-dependent increase in DNA synthesis. Genetic analysis confirmed that ECV304 and T24/83 are identical. ECV304 displays some endothelial characteristics and is useful for the study of receptor pharmacology. However, ECV304 is not of HUVEC origin and is therefore an inappropriate cell line to study endothelial cell biology.     

Induction of apoptosis by S-allylmercapto-L-cysteine, a biotransformed garlic derivative, on a human gastric cancer cell line

Epidemiological and experimental carcinogenesis studies provide evidence that certain components of garlic have anti-cancer activity. Although the biotransformed garlic derivative S-allylmercapto-L-cysteine (SAMC) has been reported to show an inhibitory effect on tumorigenesis, the mechanisms are poorly understood. The present study investigated the effect of SAMC on the growth of human gastric cancer SNU-1 cells. Upon treatment with SAMC, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis, including DNA fragmentation and an increase in the sub-diploid population. The anti-proliferative and apoptotic effect of SAMC was associated with the induction of Bax, p53, and caspase-9, rather than the induction of Bcl-2 and p21. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of SAMC, which mediates cell death. These results suggest that the apoptotic effect of SAMC on gastric cancer SNU-1 cells may be connected with caspase-3 activation through the induction of Bax and p53, rather then Bcl-2 and p21.     

血管内皮生长因子诱导的胶质瘤源性微血管内皮细胞体外三维成型特性

目的比较人脑胶质瘤源性微血管内皮细胞（GDMEC）和内皮细胞样细胞ECV304三维培养血管生成特性,探讨GDMEC在血管生成研究中的意义。方法采用免疫磁珠分选系统获得纯化的人脑GDMEC,以胶原为介质建立内皮细胞三维培养模型,比较观察GDMEC与ECV304细胞三维培养小管样结构（TLS）形成及不同浓度血管内皮生长因子（VEGF）对两种细胞TLS形成的诱导作用。结果所获GDMEC细胞纯度达98％,可连续传代培养。GDMEC的TLS形成数显著多于同数量、同时相点ECV304细胞的TLS形成数量。VEGF在体外对GDMEC有显著的促进TLS形成作用,且呈量-效和时-效关系;VEGF对ECV304细胞形成小管样结构的诱导作用弱。结论GDMEC在体外保持了内皮细胞特征和活跃的血管生成特性,对VEGF的反应性较ECV304好,因而GDMEC更适合体外血管生成研究。     

Anti-cancer natural products isolated from chinese medicinal herbs

In recent years, a number of natural products isolated from Chinese herbs have been found to inhibit proliferation, induce apoptosis, suppress angiogenesis, retard metastasis and enhance chemotherapy, exhibiting anti-cancer potential both in vitro and in vivo. This article summarizes recent advances in in vitro and in vivo research on the anti-cancer effects and related mechanisms of some promising natural products. These natural products are also reviewed for their therapeutic potentials, including flavonoids (gambogic acid, curcumin, wogonin and silibinin), alkaloids (berberine), terpenes (artemisinin, β-elemene, oridonin, triptolide, and ursolic acid), quinones (shikonin and emodin) and saponins (ginsenoside Rg3), which are isolated from Chinese medicinal herbs. In particular, the discovery of the new use of artemisinin derivatives as excellent anti-cancer drugs is also reviewed.     

Comparison of effects of epigallocatechin-3-gallate on hypoxia injury to human umbilical vein, RF/6A, and ECV304 cells induced by Na(2)S(2)O(4).

Hypoxia is related to the etiology of numerous pathological disease states, such as the formation of tumors or diverse retinopathies. Epigallocatechin-3-gallate (EGCG), a potent polyphenolic antioxidant and antiangiogenic compound found in green tea, has been shown to suppress the growth of blood vessels necessary for the growth of tumors and the induction of retinopathies. However, only a few studies have been carried focusing on the protective effects of EGCG on hypoxia-induced injury of cultured endothelial cells. The present study investigated the effects of EGCG on Na(2)S(2)O(4)-induced hypoxic injury in three types of cultured endothelial cells, primary isolates of normal human umbilical vein endothelial cells (HUVECs), and two transformed endothelial cells lines, RF/6A and ECV304. Our results indicated that Na(2)S(2)O(4) inhibited the growth of HUVE, RF/6A, and ECV304 cells in a dose-dependent manner; EGCG also exerted inhibitory effects on the growth of the three cell types, but the toxicity of EGCG to HUVECs was less than to RF/6A and ECV304 cells. The viability of HUVE, RF/6A, and ECV304 cells treated with EGGC were the lowest at 24, 24, and 36 h, respectively, and the IC(50) of EGCG were 420 +/- 8.0, 125 +/- 7.1, and 75 +/- 5.1 microM, respectively. Furthermore, EGCG, an efficient nontoxic agent, protected all three cell types from Na(2)S(2)O(4)-induced hypoxia injury, providing partial protection from hypoxia-induced injury in normal endothelial cells at 100, 30, and 10 microM for HUVE, RF/6A, and ECV304 cells, respectively.     

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.     

Silibinin’s regulation of proliferation and collagen gene expressions of rat pancreatic β-cells cultured on types I and V collagen involves β-catenin nuclear translocation

Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic β-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of β-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of β-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of β-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of β-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear β-catenin could be important for understanding fibrosis where collagen I and V may have differential effects.     

Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins

The p53 binding protein 2 (53BP2) has been identified independently as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). It was demonstrated that over-expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF-kappaB activation elicited by a physiological NF-kappaB inducer, interleukin-1beta (IL-1beta), and anti-apoptotic Bcl-2 family proteins on the 53BP2S-mediated apoptosis. We found that both NF-kappaB activation and Bcl-2 family proteins could prevent the 53BP2S-mediated depression of mitochondrial transmembrane potential, activation of caspase-9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti-apoptotic Bcl-2 family proteins. The regulatory network of the 53BP2S-mediated apoptosis cascade including its interacting proteins is discussed.     

High expression of human 15-lipoxygenase induces NF-kappaB-mediated expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and T-cell adhesion on human endothelial cells

Expression of 15-lipoxygenase (15-LO) is induced over 100-fold in early fatty streak lesions. 15-LO activity leads to the production of specific lipid hydroperoxides, which can have major effects on the expression of proinflammatory genes involved in atherogenesis. We have used retrovirus-mediated gene transfer to achieve stable high expression of 15-LO in human endothelial ECV304 cells. These cells were used to study the effects of 15-LO on the expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), activation of nuclear factor kappa B (NF-kappaB), and T-cell adhesion on endothelial cells. NF-kappaB activation was greatly potentiated by increased 15-LO activity in the stably transduced cells, and both VCAM-1 and ICAM-1 were significantly induced in these cells in response to tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) stimulation, as studied by flow cytometry. The induction of ICAM-1 was sensitive to antioxidants in a dose-dependent manner. The adherence of Jurkat T cells on the 15-LO-expressing endothelial cells was markedly induced after PMA stimulation. These results indicate that 15-LO activity may be involved in the early pathogenesis of atherosclerosis by inducing VCAM-1 and ICAM-1 expression and by increasing T-cell adhesion on the endothelium.     

Des-γ-carboxy prothrombin stimulates human vascular endothelial cell growth and migration

Des-γ-carboxy prothrombin (DCP) is an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells. Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. However, the role of DCP in the development of angiogenesis is not well understood. Herein, we report the effects of DCP on growth and migration of human vascular endothelial cells. DCP significantly stimulated the proliferation of HUVEC (ECV304) cells in a dose and time dependent manner, as measured by the MTT assay. A continuous rapid migration of ECV304 cells was observed in the presence of DCP measured by the scratch wound assay. The continuous rapid invasive activity, measured by transwell chamber assay also showed that DCP increased endothelial cells migration through the reconstituted extracellular matrix (Matrigel). Further, the tube formation of vascular endothelial cells on 3-D Matrigel showed an increased number of branch points of ECV304 cells induced by DCP in a dose dependent manner. The levels of vascular endothelial cell growth-related angiogenic factors and matrix metalloproteinase were also examined. DCP significantly stimulated the expression levels of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-2 (latent and active). Together, these data suggest that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities in angiogenesis of HCC. S. Cui and X.-J. Qu contributed equally to this work.     

Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8-inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-kappaB and cFLIP down-regulation attenuated NF-kappaB activation induced by TNF-alpha or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-alpha, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-kappaB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-kappaB activation in human HCCs.     

Exercise training attenuates age-induced changes in apoptotic signaling in rat skeletal muscle

The aging process in skeletal muscle is characterized by a loss of myocytes and reduction in cross-sectional area of the remaining myocytes, particularly in Type II (fast-twitch) muscle fibers. In multinucleated skeletal muscle, apoptosis may contribute to both fiber atrophy and loss of muscle fibers. Recent evidence suggests that the mitochondrial Bcl-2 family pathway may be a target of aging. Here the authors demonstrated that aging increased DNA fragmentation, cleaved caspase-3, and pro-apoptotic Bax in rat skeletal muscle. Twelve weeks of treadmill exercise training increased anti-apoptotic Bcl-2, while markedly reducing DNA fragmentation, and cleaved caspase-3, Bax, and Bax/Bcl-2 ratio in the white gastrocnemius and soleus muscles of old rats. Upstream anti-apoptotic NF-kappaB activity decreased in aging skeletal muscle, and increased with exercise training. Regulation of NF-kappaB activity with aging and exercise was not related to changes in NF-kappaB subunit protein levels. Instead, changes in post-translational activation of NF-kappaB occurred as a function of altered phosphorylation of IkappaB. These results indicate that treadmill exercise training attenuates fiber atrophy and pro-apoptotic signaling in aging skeletal muscle.     

创伤弧菌溶细胞素融合蛋白细胞毒活性相关分子机制研究

目的 研究创伤弧菌溶细胞素融合蛋白(rVVC)诱导人ECV304细胞(人脐静脉内皮细胞)凋亡过程中Caspase-3,-8,-9活性变化.方法 应用MTT法、Hochest33342/PI荧光双染、流式细胞术及DNA琼脂糖凝胶电泳分析rVVC对人ECV304细胞诱导凋亡的影响;比色法测定rVVC诱导人ECV304凋亡过程中Caspase-3,-8,-9活性变化.结果 MTT结果显示rVVC具有降低人ECV304细胞的存活率活性;浓度为2.0溶血单位(HU)/ml的rVVC作用人ECV304 12 h后,其诱导凋亡的活性高于对照组和浓度为0.5 HU/ml的 rVVC处理组,具有剂量依赖性;浓度为2.0 HU/ml rVVC处理组加40 μmol/L Caspase全酶抑制剂(Z-VAD-FMK)后凋亡率较2.0 HU/ml rVVC处理组有一定程度降低.浓度为2.0 HU/ml rVVC处理人ECV304细胞0.5 h后Caspase-3活性开始增高,于3 h达高峰,与对照组比较差异有统计学意义(P<0.01),Caspase-8,-9活性无明显变化.结论 rVVC对人ECV304具有凋亡诱导的生物学活性,Caspase-3可能与活性rVVC诱导的人ECV304凋亡有关.     

The role of nuclear factor kappaB on angiogenesis regulation through monocyte chemotactic protein-1 in myeloma

Multiple myeloma is malignant proliferation of plasma cells and plasmacytoid cells. Vascular endothelial growth factor (VEGF) is known to be one of the most important if not the main regulator of physiologic and pathologic angiogenesis which triggers growth, survival and migration of myeloma cells. It has been shown that circulating mature or bone marrow driven endothelial precursor cells play an important role in neovascularisation. In accordance with these observations, current therapeutic approaches to myeloma include VEGF inhibitors. Since angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes many products with a different function it would be of benefit to determine the key molecule produced by transformed plasma cells which stimulates bone marrow environment to produce their homing "milieu" secreting different cytokines such as VEGF, IL-6, and monocyte chemotactic protein-1 (MCP-1). This molecule could be nuclear factor kappa B (NF-kappaB). It has been confirmed that myeloma cells express and produce NF-kappaB. It has been established recently that by blocking NF-kappaB production MCP-1 secretion is reduced up to 60%. If so, this would also reduce production of IL-6 and VEGF, since MCP-1 upregulates VEGF and IL-6 production. This way one could make bone marrow bad environment for myeloma cells to settle, followed with no disease progression. Targeting to NF-kappaB intended to inhibits its activation with receptor antagonist would possibly significantly inhibit lipopolysaccharide-induced IL-6, MCP-1 and TNF-alpha. All of them being stimulators for VEGF secretion and indirectly activation of angiogenesis. To conclude, angiogenesis could be induced by myeloma cells themselves through NF-kappaB activation pathway and by inhibiting its activation we might prevent myeloma expansion in bone marrow and progression of the disease by decreased MCP-1 secretion.     

血小板对ECV304细胞PTA1表达和PDGF释放的影响

目的:探讨血小板对妊高征患者血清刺激下的ECV304细胞(ECV304)表达人血小板/T细胞活化抗原1(PTA1)和释放血小板衍生生长因子(PDGF)的影响。方法①用间接免疫荧光染色结合流式细胞仪分析观察血小板与妊高征患者血清刺激的ECV304细胞孵育前后PTA1的表达②用ELISA方法检测血小板与妊高征患者血清刺激的ECV304细胞孵育前后PDGF的量。结果:①血小板与妊高征患者血清刺激(24h、48h)的ECV304细胞孵育后PTA1表达的百分率(6.32%、4.13%)明显低于对照组(15.51%、5.64%)(P<0.05)。②妊高征患者血清刺激ECV304细胞8h、24h、48h释放PDGF的量(1593、2625、2175ng/L)明显高于正常孕妇组(235、405、133.5ng/L)差异显著(P<0.01)。③血小板与妊高征患者血清刺激的ECV304细胞孵育后,24h、48h细胞培养上清液中PDGF-B的量又进一步增加(3266、2360ng/L),与对照组比差异显著(P<0.05)。结论:ECV304细胞与血小板之间的黏附可能是通过PTA1分子发挥作用的,同时导致了PDGF的进一步释放。     

Disassembly of focal adhesions during apoptosis of endothelial cell line ECV304 infected with Orientia tsutsugamushi.

Many bacterial pathogens induce apoptosis in their host cells. We observed the cellular effect of ECV304 cells infected with Orientia tsutsugamushi. The infected cells became rounded and floated in culture supernatant. These floating cells as well as adherent cells exhibited typical features of apoptosis, such as DNA fragmentation and TUNEL staining. As many cells detached from growth substrate, we examined the focal adhesion using the immunofluorescence assay method and observed decreased focal adhesions in heavily infected cells. As endothelial cells could undergo apoptosis by the loss of focal adhesions, this change of focal adhesions may account for the Orientia-induced apoptosis.