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Rui Cheng,
Cen Li,
Chaoyang Li,
Ling Wei,
Lei Li,
Yang Zhang,
Yachao Yao,
Xiaoqiong Gu,
Weibin Cai,
Zhonghan Yang,
Jianxing Ma,
Xia Yang,
Guoquan Gao
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ABSTRACT: PURPOSE. Without therapeutic intervention, corneal neovascularization rapidly compromises visual acuity, and is a leading cause of blindness. Artesunate was reported to inhibit angiogenesis in tumors, although, the effects of artesunate on non-tumor angiogenesis have not been investigated. This study was designed to investigate the effect of artesunate on corneal neovascularization and delineate its underlying mechanism of action. METHODS. Rats with alkali-burned cornea were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in rat. Apoptotic cells were stained with AnnexinV and PI, and measured with flow cytometry analysis. Apoptosis-related and p38MAPK signaling were evaluated by Western blot analysis. RESULTS. Artesunate significantly inhibited corneal neovascularization and inflammation via specifically inducing apoptosis of vascular endothelial cells. In vascular endothelial cells, artesunate increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential, stimulated release of cytochrome C and cleavage of caspase9 and caspase3, suggesting that the mitochondrial apoptotic pathway was involved. Artesunate activated p38MAPK, and specific p38MAPK inhibitors suppressed artesunate-induced apoptosis in endothelial cells. Reactive oxygen species (ROS) levels were increased by artesunate. N-acetyl-L-cysteine blocked p38MAPK activation and protected endothelial cells from artesunate-induced apoptosis. Ferrous salt increased ROS levels and elevated the cytotoxic effect of artesunate on endothelial cells, while the iron chelating agent deferoxamine decreased ROS levels and artesunate-induced apoptosis. Artesunate had no effect on expression of Fas, Fas Ligand or caspase 8 cleavage. CONCLUSIONS. These results suggest that artesunate induces apoptosis of endothelial cells via an iron/ROS dependent p38MAPK-mitochondrial pathway.
Investigative ophthalmology & visual science 04/2013; · 3.43 Impact Factor
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ABSTRACT: PURPOSE:: Recent results showed that plasminogen kringle 5 (K5) has improved inhibitory effect on human umbilical vein endothelial cells (HUVECs) viability when 5 acidic amino acids in NH2 terminal outside kringle domain were replaced by 5 serine residues (mutant K5, mK5). This study was designed to identify the enhanced antiangiogenic activity of mK5 in corneal neovascularization (CNV). METHODS:: Alkali burn-induced CNV was induced and treated with K5 and mK5 for 11 days. CNV and inflammation were evaluated by the CNV area and the inflammatory index, respectively. At the end of treatment, the corneas were removed for terminal deoxynucleotidyl transferase dUTP nick end labeling detection and immunohistochemistry. The effects of mK5 and K5 on HUVECs apoptosis were tested by MTT, BrdU, and flow cytometry. The expression levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were detected by Western blot. RESULTS:: In a rat model of CNV induced by alkali, topical treatment with mK5 significantly decreased the neovascular area and inflammation compared with the wild-type K5-treated group. Meanwhile, mK5 and K5 specifically inhibited the HUVECs proliferation and induced vascular endothelial cell apoptosis in vitro and in vivo, and mK5 exerted higher apoptosis induction. Toward the mechanism of action, both mK5 and K5 significantly upregulated the expression of PEDF and mildly downregulated the expression of VEGF. The elevation of PEDF/VEGF ratio induced by mK5 was higher than that by K5. CONCLUSIONS:: These findings suggest that mK5 has more effective therapeutic potential in CNV than wild-type K5.
Cornea 01/2013; · 1.73 Impact Factor
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ABSTRACT: Abstract Aims: Diabetes is associated with nitrosative stress in multiple tissues. Overactivation of the Wnt pathway has been shown to play a pathogenic role in diabetic retinopathy (DR). The purpose of this study was to investigate whether nitrosative stress contributes to aberrant activation of Wnt signaling in diabetes. Results: Nitrosative stress induced by peroxynitrite (PN), 4-hydroxynonenal (HNE), or high glucose (HG) in retinal cells was assessed by a dichlorofluorescein fluorescence assay or by Western blot analysis and enzyme-linked immunosorbent assay of 3-nitrotyrosine (3-NT). These nitrosative stress inducers activated the canonical Wnt pathway, as shown by Western blot analysis of phosphorylated low-density lipoprotein receptor-related protein 6 (pLRP6), total and nuclear β-catenin levels, Luciferase reporter assay, and expression of the Wnt target genes intercellular adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF). Uric acid (UA), a PN scavenger, and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato Iron III Chloride (FeTPPS), a PN decomposition catalyst, suppressed Wnt signaling and ICAM-1 and VEGF overexpression induced by PN, HNE, and HG. Furthermore, UA and FeTPPS also inhibited Wnt signaling induced by the Wnt ligand. In streptozotocin-induced diabetic rats, retinal levels of 3-NT, β-catenin, nuclear β-catenin, pLRP6, VEGF, and ICAM-1 were markedly increased. UA treatment for 6 weeks ameliorated diabetes-induced Wnt signaling in the diabetic rat retina. The UA treatment also decreased inflammatory cell infiltration and extraverted serum albumin in the perfused retina of diabetic rats, suggesting decreased retinal inflammation and vascular leakage. Innovation and Conclusion: Nitrosative stress in diabetes contributes to Wnt pathway activation in the retina, and Wnt signaling may mediate the pathogenic effects of nitrosative stress in DR. Antioxid. Redox Signal. 00, 000-000.
Antioxidants & Redox Signaling 10/2012; · 8.20 Impact Factor
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Wei-Bin Cai,
Yang Zhang, Rui Cheng,
Zheng Wang,
Shu-Huan Fang,
Zu-Min Xu,
Xia Yang,
Zhong-Han Yang,
Jian-Xing Ma,
Chun-Kui Shao,
Guo-Quan Gao
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ABSTRACT: We had demonstrated that plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibited retinal neovascularization and hepatocellular carcinoma growth by anti-angiogenesis. The current study investigated the effects and the underlying mechanisms of K5 on both tumor growth and spontaneous pulmonary metastasis in Lewis lung carcinoma (LLC) implanted mouse model. Similarly, K5 could decrease expression of VEGF in LLC cells and grafted tissues and suppress tumor angiogenesis and growth. K5 had no direct effect on proliferation and apoptosis of LLC. However, K5 could significantly inhibit SDF-1α-induced chemotaxis movement of LLC cells and resulted in a great reduction of surface metastatic nodules and micrometastases in the lungs of LLC tumor-bearing mice. K5 also decreased expression of chemokine (C-X-C motif) receptor 4 (CXCR4) in LLC cells and grafted tissues. Furthermore, K5 down-regulated SDF-1α expression in metastatic lung tissues of LLC-bearing mice. Therefore, K5 may suppress tumor pulmonary metastasis through inhibiting SDF-1α-CXCR4 chemotaxis movement and down-regulation of VEGF. Moreover, the role of hypoxia inducible factor-1α (HIF-1α), a crucial transcriptional factor for both VEGF and CXCR4 expression, was evaluated. The siRNA of HIF-1α attenuated expression of VEGF and CXCR4 and inhibited LLC migration. K5 decreased HIF-1α protein level and impaired nuclear HIF-1α accumulation. These results showed for the first time that K5 inhibits LLC growth and metastasis via the dual effects of anti-angiogenesis and suppression of tumor cell motility by targeting the pivotal molecule, HIF-1α.
PLoS ONE 01/2012; 7(12):e53152. · 4.09 Impact Factor
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Zumin Xu,
Shuhuan Fang,
Yufang Zuo,
Yang Zhang, Rui Cheng,
Qingsong Wang,
Zhonghan Yang,
Weibin Cai,
Jianxing Ma,
Xia Yang,
Guoquan Gao
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ABSTRACT: Nasopharyngeal carcinoma (NPC), which has the highest incidence in South China, is mainly treated by radiotherapy. However, the survival rate remains low. Angiogenesis is closely correlated with progress of NPC. Thus, the combination of anti-angiogenesis with radiation is an attractive strategy for NPC treatment. A heterogenic xenografted human NPC nude mice model was established to investigate the effect of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic factor, and the combined effect of PEDF and radiotherapy on nasopharyngeal carcinoma. Pigment epithelium- derived factor remarkably suppressed the growth of NPC by 43.52% and decreased the tumor microvessel density (MVD). Pigment epithelium-derived factor had no effects on the proliferation and apoptosis of NPC cell lines by MTT and flow cytometry assay. However, PEDF decreased vascular endothelial growth factor (VEGF) in NPC cell lines by downregulation of hypoxia-inducible factor 1a, a crucial transcriptional factor for VEGF expression, as demonstrated by western blotting and immunofluorescent staining assay. Interestingly, irradiation alone could also effectively downregulate VEGF and MVD of xenografted tumor, which indicates that irradiation suppresses NPC not only by killing tumor cells but also through anti-angiogenesis. Furthermore, combined treatment of PEDF with irradiation enhanced the antitumor efficacy. The MVD and VEGF in the combined therapy were much less than in the treatment with PEDF or radiotherapy alone. Our observation demonstrated that the combination of PEDF with radiotherapy enhances the efficacy of the antitumor effect on NPC by the coordinated inhibition on angiogenesis, which implies the potential role of PEDF as an adjuvant agent for NPC treatment.
Cancer Science 06/2011; 102(10):1789-98. · 3.33 Impact Factor
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ABSTRACT: Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x(L) on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x(L) on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x(L) distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.
Apoptosis 06/2011; 16(8):846-55. · 4.07 Impact Factor
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ABSTRACT: Previous studies have shown that pigment epithelium-derived factor (PEDF) is an antitumor candidate with anti-oxidative, anti-inflammatory and anti-angiogenesis properties. However, the effect of PEDF on gastric carcinoma has not been elucidated. MTT assay and Annexin V/PI staining were performed. Immunohistochemistry was applied to detect microvessel density (MVD) of a xenograft model. The protein levels of vascular endothelial growth factor (VEGF) were examined by western blot analysis and hypoxia-inducible factor-1α (HIF-1α) translocation was investigated by immunofluorescence. Results showed that growth and angiogenesis of gastric carcinoma were suppressed after PEDF injection. PEDF could not directly suppress proliferation or induce apoptosis of gastric carcinoma cells. However, the expression of VEGF both in tumor tissues and gastric carcinoma cells was down-regulated by PEDF. The amount and nuclear translocation of HIF-1α, the transcription factor of VEGF, was also inhibited by PEDF. In conclusion, PEDF suppresses angiogenesis and growth of gastric carcinoma by down-regulating HIF-1α and VEGF and may have potential for gastric carcinoma treatment.
Oncology Reports 05/2011; 26(3):681-6. · 1.84 Impact Factor
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ABSTRACT: Pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor with multiple other functions, balances angiogenesis in the eye and blocks tumor progression. Cervical cancer, an angiogenesis-dependent tumor, is the second most common cancer in women without effective treatment. It has been reported that PEDF can inhibit several types of tumors, however, the potential of PEDF for the treatment of cervical carcinoma has not been well explored. The present study was designed to investigate the effect of recombinant PEDF on the neovascularization and growth of cervical carcinoma. We found for the first time that PEDF was downregulated apparently in human cervical carcinoma nests compared to either normal cervical epithelium or nonneoplastic peritumoral epithelium, suggesting potential anti-angiogenesis function by supplement of PEDF in cervical carcinoma. Intraperitoneal injection of PEDF in xenografted cervical carcinoma mice suppressed tumor growth with 68% reduction. Microvessel density in tumor tissues treated with PEDF was significantly decreased. PEDF dose-dependently inhibited proliferation and induced apoptosis of endothelial cells, but had no direct effect on proliferation and apoptosis of Hela cells under both normoxia and hypoxia. These results suggested that PEDF suppressed tumor growth by blocking angiogenesis instead of a direct cytotoxic effect on tumor cells. VEGF, a major angiogenic stimulator, was downregulated by PEDF in Hela cells by downregulation of HIF-1α, a crucial transcriptional factor for VEGF expression. Downregulation of VEGF expression in tumor cells through inhibiting HIF-1α, thus attenuating the paracrine effect of VEGF on endothelial cells, may represent a mechanism for the anti-angiogenic activity of PEDF.
Cancer biology & therapy 06/2010; 9(12):967-74. · 2.64 Impact Factor
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ABSTRACT: Pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor with multiple other functions, balances angiogenesis in the eye and blocks tumor progression. Retinoblastoma, an angiogenesis-dependent tumor, is the most common ocular cancer in children without effective treatment. It has been reported that PEDF can induce neuronal differentiation of retinoblastoma cells; however, its anti-angiogenic potential for inhibition of retinoblastoma growth in vivo has not been elucidated. The present study was designed to investigate the effect of PEDF on growth of retinoblastoma and the possible molecular mechanism. Soluble and non-fusion recombinant PEDF were generated in E. coli. Recombinant PEDF dose-dependently inhibited proliferation and induced apoptosis of endothelial cells. PEDF had no effects on the proliferation and apoptosis of retinoblastoma cell line SO-Rb50. Intraperitoneal injection of PEDF resulted in growth inhibition of heterotopic retinoblastoma xenografts at 68.78%. MVD in tumor tissues treated with PEDF was significantly decreased. These results suggested that PEDF suppressed tumor growth by blocking angiogenesis instead of a direct cytotoxic effect on tumor cells. Vascular endothelial growth factor (VEGF), a major angiogenic stimulator, was down-regulated by PEDF in both SO-Rb50 cells and retinoblastoma xenografts. Hypoxia-inducible factor (HIF)-1alpha, a crucial transcriptional factor for VEGF expression, was also down-regulated by PEDF both in vitro and in vivo. PEDF reduced HIF-1alpha nuclear translocation, which may be responsible for the down-regulation of VEGF. Down-regulation of VEGF expression in tumor cells through inhibiting HIF-1alpha, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a mechanism for the anti-angiogenic activity of PEDF.
Cancer Science 08/2009; 100(12):2419-25. · 3.33 Impact Factor
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ABSTRACT: Plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen, is an endogenous angiogenic inhibitor. We have previously shown that K5 inhibits ischemia-induced retinal neovascularization in a rat model. However, its anti-angiogenic potential and application in the treatment of neoplastic diseases have not been well investigated. Our present study was designed to test its effect on the neovascularization and growth of hepatocellular carcinoma, a typical hypervascular tumor. Recombinant human K5 was expressed in E. coli and purified by affinity chromatography. K5 inhibited proliferation and induced apoptosis of primary endothelial cells in dose-dependent manner, but no effect on pericytes from the same origin of endothelial cells, which suggested an endothelial cell-specific inhibition. Moreover, K5 had no effect on the proliferation and apoptosis of mouse HepA and human Bel7402 hepatoma cell lines even in the enhanced concentration range, which suggested K5 having no direct effect on tumor cells. Ventral injection of K5 significantly suppressed the tumor growth in graphed hepatocarcinoma mice model, which was established by injection of mouse HepA hepatoma cells. In xenografted hepatocarcinoma athymic mice model, which mimicked human tumors by injection of human Bel7402 hepatoma cells, K5 significantly suppressed the tumor growth. An average of 68% suppression of primary tumor growth was observed in the K5-treated mice compared with control group. K5 also inhibited intratumoral neovascularization in the two cancer models determined by micro vessel density (MVD) analysis. Injection of K5 significantly induced the cleavage of pro-caspase-3 in tumor tissues of grafted mouse model, which suggested K5 also induced apoptosis of tumor tissues and the decreased intratumoral microvascular density in K5 treated group may correlate with K5-induced endothelial cell apoptosis. These results suggest that tumor growth suppression of K5 depends on its anti-angiogenic activity and K5 could have therapeutic potential in hepatocellular carcinoma.
Cancer biology & therapy 05/2006; 5(4):399-405. · 2.64 Impact Factor
