Weibin Cai

PEDF inhibits growth of retinoblastoma by anti-angiogenic activity

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)‐1α, a crucial transcriptional factor for VEGF expression, was also down‐regulated by PEDF both in vitro and in vivo. PEDF reduced HIF‐1α nuclear translocation, which may be responsible for the down‐regulation of VEGF. Down‐regulation of VEGF expression in tumor cells through inhibiting HIF‐1α, 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 Sci 2009; 100: 2419–2425)

Combination of pigment epithelium-derived factor with radiotherapy enhances the antitumor effects on nasopharyngeal carcinoma by downregulating vascular endothelial growth factor expression and angiogenesis.

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 1α, 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 Sci 2011; 102: 1789–1798)

Kallikrein-binding protein inhibits growth of gastric carcinoma by reducing vascular endothelial growth factor production and angiogenesis.

Kallikrein-binding protein (KBP) has been identified as an endogenous angiogenic inhibitor. We previously showed that KBP inhibited rat retinal neovascularization by down-regulation of vascular endothelial growth factor (VEGF) in endothelial cells. However, its antiangiogenic potential for inhibition of gastric carcinoma and the effect on VEGF in tumor cells have not been elucidated. The present study was designed to investigate the effect of KBP on growth of gastric carcinoma and the possible molecular mechanism. Recombinant KBP dose dependently inhibited proliferation and induced apoptosis of endothelial cells, but no effect on proliferation and apoptosis of SGC-7901 gastric carcinoma cells. I.p. injection of KBP resulted in growth inhibition of both heterotopic and orthotopic gastric carcinoma xenografts at 61.4% and 52.3%, respectively. Microvessel density in tumor tissues treated with KBP was significantly decreased, suggesting that KBP suppressed tumor growth by antiangiogenesis. The expression and release of VEGF, a major angiogenic stimulator, were down-regulated by KBP in SGC-7901 cells and gastric carcinoma xenografts. RNA levels of VEGF in SGC-7901 cells were also decreased by KBP, thus suggesting the regulation at the transcriptional level. Therefore, hypoxia-inducible factor 1α (HIF-1α), a crucial transcriptional factor for VEGF expression, was examined in SGC-7901 cells treated by KBP. KBP reduced HIF-1α protein level and nuclear translocation, which may be responsible for the down-regulation of VEGF transcription. Down-regulation of VEGF expression and release in tumor cells through inhibiting HIF-1α, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a novel mechanism for the antiangiogenic and antitumor activity of KBP. [Mol Cancer Ther 2007;6(12):3297–306]

Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-xL subcellular distribution

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-xL 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-xL 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-xL 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.

The artemisinin derivative artesunate inhibits corneal neovascularization by inducing ROS-dependent apoptosis in vascular endothelial cells

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 nontumor 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 corneas were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in the rats. Apoptotic cells were stained with AnnexinV and propidine iodide (PI), and measured with flow cytometry analysis. Apoptosis-related and p38 mitogen-activated protein kinases (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 caspase 9 and 3, 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.

Dual regulation of adipose triglyceride lipase by pigment epithelium-derived factor: A novel mechanistic insight into progressive obesity

Both elevated plasma free fatty acids (FFA) and accumulating triglyceride in adipose tissue are observed in the process of obesity and insulin resistance. This contradictory phenomenon and its underlying mechanisms have not been thoroughly elucidated. Recent studies have demonstrated that pigment epithelium-derived factor (PEDF) contributes to elevated plasma FFA and insulin resistance in obese mice via the activation of adipose triglyceride lipase (ATGL). However, we found that PEDF downregulated adipose ATGL protein expression despite of enhancing lipolysis. Plasma PEDF and FFA were increased in associated with a progressive high-fat-diet, and those outcomes were also accompanied by fat accumulation and a reduction in adipose ATGL. Exogenous PEDF injection downregulated adipose ATGL protein expression and elevated plasma FFA, while endogenous PEDF neutralization significantly rescued the adipose ATGL reduction and also reduced plasma FFA in obese mice. PEDF reduced ATGL protein expression in a time- and dose-dependent manner in differentiated 3T3-L1 cells. Small interfering RNA-mediated PEDF knockdown and antibody-mediated PEDF blockage increased endogenous ATGL expression, and PEDF overexpression downregulated ATGL. PEDF resulted in a decreased half-life of ATGL and regulated ATGL degradation via ubiquitin-dependent proteasomal degradation pathway. PEDF stimulated lipolysis via ATGL using ATGL inhibitor bromoenol lactone, and PEDF also downregulated G0/G1 switch gene 2 (G0S2) expression, which is an endogenous inhibitor of ATGL activation. Overall, PEDF attenuated ATGL protein accumulation via proteasome-mediated degradation in adipocytes, and PEDF also promoted lipolysis by activating ATGL. Elevated PEDF may contribute to progressive obesity and insulin resistance via its dual regulation of ATGL.

SERPINA3K induces apoptosis in human colorectal cancer cells via activating the Fas/FasL/caspase-8 signaling pathway

SERPINA3K, also known as kallikrein‐binding protein (KBP), is a serine proteinase inhibitor with anti‐inflammatory and anti‐angiogenic activities. Our previous studies showed that SERPINA3K inhibited proliferation in a dose‐dependent manner and induced apoptosis of endothelial cells but had no influence on SGC‐7901 gastric carcinoma cells or HepG2 hepatocarcinoma cells. However, it is unknown whether SERPINA3K has a direct impact on other carcinoma cells and which mechanisms are involved. In this study, we report for the first time that SERPINA3K not only decreased cell viability but also induced apoptosis in the colorectal carcinoma cell lines SW480 and HT‐29. SERPINA3K‐induced apoptosis of SW480 and HT‐29 was rescued by interference with Fas ligand (FasL) small hairpin RNA. Moreover, SERPINA3K increased the expression of FasL and activated caspase‐8. Peroxisome proliferator‐activated receptor γ (PPARγ), a transcription factor of FasL, was also upregulated by SERPINA3K in a dose‐dependent manner. The upregulation effect of FasL induced by SERPINA3K was reversed after interference with PPARγ small interfering RNA. These results demonstrated that SERPINA3K‐induced SW480 and HT‐29 cell apoptosis was mediated by the PPARγ/Fas/FasL signaling pathway. Therefore, our study provides additional insight into the direct anti‐tumor function by inducing tumor cell apoptosis of SERPINA3K in colorectal tumors.

Pigment epithelium-derived factor inhibits angiogenesis and growth of gastric carcinoma by down-regulation of VEGF

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.

Nestin-mediated cytoskeletal remodeling in endothelial cells: novel mechanistic insight into VEGF-induced cell migration in angiogenesis

Nestin is highly expressed in poorly differentiated and newly formed proliferating endothelial cells (ECs); however, the role of this protein in angiogenesis remains unknown. Additionally, the cytoskeleton and associated cytoskeleton-binding proteins mediate the migration of vascular ECs. Therefore, the aim of the present study was to determine whether VEGF regulates the cytoskeleton, as well as other associated proteins, to promote the migration of vascular ECs. The coexpression of nestin and CD31 during angiogenesis in alkali-burned rat corneas was examined via immunohistochemical analysis. Western blot analyses revealed that the exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia promoted nestin expression in vitro. Additionally, nestin silencing via siRNA significantly inhibited many of the process associated with VEGF-induced angiogenesis, including tube formation and the migration and proliferation of HUVECs. Moreover, FITC-phalloidin labeling revealed that F-actin filaments were successfully organized into microfilaments in VEGF-treated cells, suggesting a network rearrangement accomplished via F-actin that contrasted with the uniform and loose actin filament network observed in the siRNA-nestin cells. The results of the present study highlight the key role played by nestin in activated HUVECs during angiogenesis. The inhibition of the ERK pathway suppressed the nestin expression induced by VEGF in the HUVECs. Therefore, our study provides the first evidence that nestin-mediated cytoskeleton remodeling in ECs occurs via filopodia formation along the cell edge, facilitating both filopodia localization and cell polarization and ultimately promoting HUVEC migration via VEGF induction, which may be associated with ERK pathway activation.

Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin.

Pigment epithelium-derived factor (PEDF) plays an important role in the tumor growth and metastasis inhibition. It has been reported that PEDF expression is significantly reduced in breast cancer, and associated with disease progression and poor patient outcome. However, the exact mechanism of PEDF on breast cancer metastasis including liver and lung metastasis remains unclear. The present study aims to reveal the impact of PEDF on breast cancer. The orthotopic tumor mice model inoculated by MDA-MB-231 cells stably expressing PEDF or control cells was used to assess liver and lung metastasis of breast cancer. In vitro, migration and invasion experiments were used to detect the metastatic abilities of MDA-MB-231 and SKBR3 breast cancer cells with or without overexpression of PEDF. The metastatic-related molecules including EMT makers, fibronectin, and p-AKT and p-ERK were detected by qRT-PCR, Western blot, and Fluorescent immunocytochemistry. PEDF significantly inhibited breast cancer growth and metastasis in vivo and in vitro. Mechanically, PEDF inhibited breast cancer cell migration and invasion by down-regulating fibronectin and subsequent MMP2/MMP9 reduction via p-ERK and p-AKT signaling pathways. However, PEDF had no effect on EMT conversion in the breast cancer cells which was usually involved in cancer metastasis. Furthermore, the study showed that laminin receptor mediated the down-regulation of fibronectin by PEDF. These results reported for the first time that PEDF inhibited breast cancer metastasis by down-regulating fibronectin via laminin receptor/AKT/ERK pathway. Our findings demonstrated PEDF as a dual effector in limiting breast cancer growth and metastasis and highlighted a new avenue to block breast cancer progression.