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BIOPHA-3379; No. of Pages 7 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
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Original article
Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway K.F. Huang a,b,*,1, X.P. Huang a, G.Q. Xiao a, H.Y. Yang a, J.S. Lin a,c,*,2, Y. Diao a,*,2 a
Institute of Molecular Medicine, Hua-qiao University, 362021 Quan Zhou, China Xiamen Medicine Research Institute, 361003 Xiamen, China c AgResearch, Invermay, Mosgiel, New Zealand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2014 Accepted 4 March 2014
Development of a novel angiogenesis inhibitor will be essential for the improvement of therapeutics against cancer. Kallistatin had been recognized as an endogenous angiogenesis inhibitor. Here, we demonstrated kallistatin’s strong anti-angiogenesis and anti-metastasis activity stimulated by breast cancer cells (MCF-7) and its mechanism of action in vitro. The anti-angiogenesis effect in vivo was evaluated by chicken chorioallantoic membrane (CAM) neovascularisation. Because of the underlying molecular mechanism of its anti-angiogenesis activity remains poorly understood. In this study, we examined whether the NF-kB signaling pathway was involved in the anti-angiogenesis and antimetastasis activity of kallistatin. Kallistatin significantly inhibited TNF-a-induced nuclear factor-kB activation in a dose-dependent manner. Addition of kallistatin inhibited TNF-a induced IkBa degradation; phosphorylation of IkBa kinase (IKK), nuclear factor-kB-p65 protein; and nuclear translocation of p65/50. Meanwhile, we investigated the effects of kallistatin on the expression of vascular endothelial growth factor (VEGF) and other angiogenesis-related gene in human umbilical vein endothelial cells (HUVECs). We found that kallistatin decreased the expression of VEGF and some angiogenesis-related genes, which promoted angiogenesis in cancer. Taken together, we suggested that kallistatin would inhibit tumor angiogenesis via inhibition of the NF-kB signaling pathway and finally abrogate NF-kB-dependent gene expression. All the results revealed that kallistatin would have potential as a novel. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Kallistatin NF-kB Angiogenesis Tumor
1. Introduction Kallistatin, a serine proteinase inhibitor, is first discovered and identified as a tissue kallikrein-binding protein and a unique serine proteinase inhibitor, and has emerged as a novel inhibitor of angiogenesis. Kallistatin shows a variety of biological effects in pathologic and physiologic responses, such as blood pressure regulation, anti-angiogenesis and anti-inflammation [1–6]. In previous studies, kallistatin gene delivery markedly inhibited human hepatocellular carcinomas relying on its anti-angiogenesis activities [7]. Adenoviral expression vector encoding human kallistatin significantly inhibited the angiogenesis of pre-established human
* Corresponding authors. Institute of Molecular Medicine, Hua-qiao University, 362021 Quan Zhou, China. E-mail addresses: [email protected] (K.F. Huang), [email protected] (J.S. Lin), [email protected], [email protected] (Y. Diao). 1 Tel./fax: +86 592 5957216. 2 Tel./fax: +86 595 22692516.
breast tumor xenografts in athymic mice [6]. However, the molecular mechanism by which kallistatin modulates angiogenesis has not been determined, so the exact mechanism for anti-angiogenesis activity of kallistatin remains to be elucidated. Angiogenesis is the formation of new blood vessels. In 1971, Folkman hypothesized that tumor growth is dependent on angiogenesis and subsequently experimental work demonstrated that for a tumor to grow beyond a size of 1–2 mm3 a substantial new blood supply must develop to support the increasing metabolic requirements [8,9]. Angiogenesis become an important feature of tumor metastasis and growth. As such, targeting tumor neovascularization is a favorable strategy for cancer therapy. A number of angiogenesis inhibitors including endostatin protein have been identified and used in preclinical experiment, and several inhibitors have also been applied in clinical trials [10,11]. Targeting angiogenesis is an exciting and attractive area in the treatment of cancer. The mechanisms of angiogenesis have been investigated since 1931 when Clark observed real-time capillary growth, and are still
http://dx.doi.org/10.1016/j.biopha.2014.03.005 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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not fully understood [12]. Many growth factors and cytokines have both inhibitory and stimulatory roles within sprouting angiogenesis, such as ICAM-1, VEGF, and MMPs and others. The most attractive investigated compound is vascular endothelial growth factor (VEGF), which is known to be a potent stimulator of angiogenesis [13–16]. NF-kB is a family of important transcriptional factors known to regulate a wide range of biological effects, including proliferation, metastasis, apoptosis and angiogenesis, via its downstream target genes [17,18]. This family is composed of five related transcription factors (p50, p65, p52, RelB, and c-Rel). The most important NF-kB dimmers are formed by p50 and p65 in NF-kB signaling pathway [19,20]. NF-kB regulates the expression of many genes whose products are involved in tumor angiogenesis such as angiogenesis (VEGF) and adhesion molecules (MMP-2, MMP-9, and ICAM-1) [21]. In this study, we first examined the anti-angiogenesis activity of kallistatin using a combination of in vitro and in vivo angiogenesis assays in order to evaluate the anti-angiogenesis activity of kallistatin. We examined whether the NF-kB signaling pathway was involved in the anti-angiogenesis and anti-metastasis activity of kallistatin. Kallistatin markedly inhibited angiogenesis induced by MCF-7 cells and the downstream angiogenesis-related genes of NF-kB signaling pathway. Furthermore, kallistatin significantly inhibited TNF-a induced IkBa degradation, phosphorylation of IkBa kinase (IKK), nuclear factor-kB-p65 protein, and nuclear translocation of p65/50. Here, we discussed the possible involvement of TNF-a induced NF-kB pathway in the anti-angiogenesis and anti-metastasis activity of the novel protein drug. 2. Materials and method 2.1. Materials Recombinant human kallistatin (rhKal) protein was purification by Hua-qiao University. Matrigel was purchased from BD Biosciences;3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and TNF-a were purchased from Sigma (USA); polyclonal antibodies against surviving, caspase-3, NFkB65, NF-kB50, ICAM-1, MMP-2,MMP-9,VEGF were purchased from Boster Biological Technology Inc. (Wuhan, China); polyclonal antibodies against phospho-IkB, IkBa, phospho-p65, p-IKK, IKK were obtained from KeyGEN Biotech (Nanjing, China); horseradish peroxidase (HRP) - conjugated anti-rabbit immunoglobulin (IgG) was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).
stained with Gimesa stain. Non-migrating cells on the upper surface of the filter were removed, and chemotaxis was quantified by counting the number of cells that migrated to the lower side of the filter using optical microscopes (100, Nikon, Tokyo, Japan). Six fields were counted for each assay. Each assay was conducted with three wells and similar results were repeated at least three times. 2.4. Tube formation assay MCF-7 cells were pretreated with rhKal (0, 40, 80 and 160 mg/ mL) for 24 h. Matrigel (BD Biosciences, USA) was thawed and mixed with an equal volume of serum-free 1640 containing 1 105 cells/mL of MCF-7 cells. The mixture was transferred into a 96-well plate and allowed to solidify and polymerize at 37 8C for 6 h. HUVEC s were harvested after trypsin treatment and suspended in serum-free medium before seeding and planted onto matrigel. After 6 h, the plate was examined for capillary tube formation under an inverted microscope and photographed and tubular structures were quantified by manually counting the tube numbers, and six randomly chosen fields were analyzed for each well. 2.5. Chicken chorioallantoic membrane (CAM) assay Anti-angiogenesis activity of rhKal on CAM was assayed as described previously [22]. Briefly, fertilized chicken eggs were incubated at 37 8C for 6 days. After this incubation, a small hole was punched on the broad side of the egg, and a window was carefully created through the eggshell. MCF-7cells (2 106 cells/embryo) were placed on the exposed CAM. Sterilized filter paper disk (5 5 mm) saturated with vehicle or with kallistatin (0, 4, 8, and 16 mg/egg) were placed on the CAMs. The eggs were then incubated at 37 8C for another 2 days. The neovascular zones under the disks were photographed. Angiogenesis was quantified by counting the number of blood vessel branch points. Ten eggs were used per group to ensure the reproducibility. 2.6. Rat aortic ring assay
HUVECs and MCF-7cells were cultured in 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), and maintained in a humidified atmosphere of 5% CO2 in air at 37 8C two passages weekly.
Rat aortic ring assay was performed as described previously with some modification [23]. The thoracic aorta was dissected from male Sprague-Dawley rats (6 weeks old) and cut into 1-mm long rings. The disks were treated as described previously [23]. After 3 days of growth, the culture medium was replaced with fresh serum-free medium, MCF-7 cells (3 105 cells/mL), which were pretreated with rhKal (0, 40, 80 and 160 mg/mL) for 24 h. Plates were then stored in incubator at 37 8C and 5% CO2. The media were changed each 2 days. After 7 days, the sprouting microvessels in five randomly chosen fields were counted and photographed under a microscope for each group.
2.3. Endothelial cell migration assay
2.7. NF-kB-dependent reporter gene expression assay
Transwell invasion assay was used to confirm the effect of rhKal on cell invasion activity. Briefly, the chamber was divided into two compartments by a polycarbonate filter with a pore size of 8 mm. The polycarbonate filter was coated with 0.2% gelatin. MCF-7 cells were seeded in 24-well plate and incubated. When a monolayer is formed, the cells were treated with rhKal (0, 40, 80 and 160 mg/mL) for 24 h. After that, the medium was replaced by a serum-free medium for 12 h, and then the culture plates were inserted with Transwell chambers. Then, 3 104 HUVECs suspended with control medium were loaded on the upper compartment of the chamber. After 4 h at 37 8C in 5% CO2, the filter was fixed and
The effect of rhKal on TNF-a-induced NF-kB-dependent luciferase reporter assays was performed by Luciferase Reporter Gene Assay Kit (Beyotime, China). Briefly, HUVECs (5 105 cells/ well) were plated in 6-well plates and transiently transfected by the lipofectin regeant with pNF-kB-TA-luc. We transfected the cells with 0.5 mg pNF-kB-TA-luc plasmid and 1 mg pGL6-TA control plasmid for 24 h. Then, we treated the cells with various dose of rhKal (0, 20, 40 and 60 mg/mL) and 10 ng/mL TNF-a for 24 h. The cells were collected and lysed in cell lysis solution. The luciferase was detected by the manufacturer (Spectra Max M3, MD, USA).
2.2. Cell culture
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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Fig. 1. Effect of recombinant human kallistatin (rhKal) on cell viability in breast cancer cells (MCF-7) cells. The rhKal had tiny effect on viability of MCF-7cells. Data represented survival rate as percentages of respective controls (n = 6).
2.8. Western-blotting A total of 1 106 cells were collected and lysed for 15 min in ice-cold lyses buffer (29 mM Tris–HCl, pH 7.4, 137 mM NaCl, 10% [w/v] glycerin, 1%[v/v]) (Triton X-100, 2 mM EDTA, 1 mM PMSF).
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After removing the cell debris by centrifugation at 16,000 g for 15 min, equal amounts of proteins were separated on a 12% SDSPAGE, transferred electrophoretically (Bio-Rad, USA) onto a nitrocellulose membrane (KeyGEN, Nanjing, China) and blocked with 5% non-fat milk powder (w/v) in tris-buffer saline Tween-20 (TBST, 10 mM Tris, 100 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature. The membranes were incubated with primary anti-human rabbit polyclonal antibodies (1:500) overnight at 4 8C, and with anti-b-actin rabbit polyclonal antibody (1:500) as a control. After washing with TBST five times, HRP-conjugated antirabbit secondary antibody (1:1000) were added and incubated at 37 8C for 1 h before another five-time washing. Labeled bands were developed with enhanced chemiluminescence detection system (Amersham Pharmacia, UK) and photographed with a Molecular Imager Gel Doc XR system (Bio-Rad, USA). Protein levels were quantified by density analysis using Quantity One software (Bio-Rad, USA). Relative protein expression levels were deduced from the ratio of the mean values of each band to that of b-actin.
Fig. 2. The anti-angiogenesis and anti-metastasis activity of recombinant human kallistatin (rhKal) in vitro and in vivo. A. Breast cancer cells (MCF-7) were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing human umbilical vein endothelial cells (HUVECs) migration. B. MCF-7 cells were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing HUVEC cells tube formation. C. MCF-7 cells were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing rat aortic ring microvessel sprouting. D. MCF-7 cells were placed on the exposed chicken chorioallantoic membrane (CAM) to induce angiogenesis in vivo. Sterilized filter paper disk (5 5 mm) saturated with vehicle or with kallistatin (4, 8, and 16 g/egg) were placed on the CAMs.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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2.9. Statistical analysis Assays were performed in duplicate and three independent experiments unless otherwise stated. Statistically significant differences between control and experimental groups were calculated by Dunnett’s test. Statistically significant is expressed as *P < 0.05, while highly statistically significant as **P < 0.01. 3. Results 3.1. The rhKal affected viability of HUVECs and MCF-7 cells The HUVECs were cultured in presence of rhKal at a range of concentrations (40–640 mg/mL in final) for different durations (48–72 h). The experiment included a group of cultured without rhKal as negative control. The proliferation inhibition rates were calculated from the loss of cell viability by assessment with MTT assay. The rhKal inhibited the growth of HUVECs in dose- and timedependent manners. In our previous research, the median inhibition concentration (IC50) values for 48 h, and 72 h of HUVEC cells were 189.6 mg/mL and 146.5 mg/mL, respectively. The rhKal had tiny effect on viability of MCF-7 cells (Fig. 1).
fewer HUVECs migrated to the opposite side of the filter in the Transwell chamber. The rhKal obviously suppressed migration of HUVECs induced by MCF-7 in a concentration-dependent manner. Then, tube formation assay was performed to evaluate the effects of rhKal on tube formation of HUVECs induced by MCF-7 cells on matrigel. As shown on Fig. 2B, MCF-7 cells promoted tube formation, and the number of tubes was much larger than that of the control group. The rhKal effectively reduced the quantity of endothelial tubes induced by MCF-7 in a concentration-dependent manner. We further evaluated the anti-angiogenesis effects of rhKal on the aorta sprout outgrowth assay in vitro, which mimics several stages in angiogenesis, including endothelial cell migration, proliferation, and tube formation. We utilized the model to evaluate the effect of rhKal on angiogenesis induced by MCF-7 cells. We found that new micro vessels began to grow when incubated for 5 days. After being cultivated for 7 days, the control group formed many microvessels. The MCF-7 cells could significantly promote the formation of micro vessels. The rhKal suppressed MCF-7 inducing formation of microvessel outgrowth from explants of rat aorta in a concentration-dependent manner (Fig. 2C). The above results suggested that the rhKal had ability to suppress MCF-7-induced migration and angiogenesis of HUVECs in vitro.
3.2. The rhKal suppressed MCF-7-induced angiogenesis in vitro 3.3. The rhKal suppressed MCF-7-induced angiogenesis in vivo Transwell chamber assay was used to evaluate the effect of rhKal on the migration of HUVEC s induced by MCF-7. As shown on Fig. 2A, MCF-7 cells significantly stimulated HUVECs migration. When MCF-7 cells were treated with 40, 80 and 160 mg/mL rhKal,
To evaluate whether anti-angiogenesis effects of rhKal could be recapitulated in vivo, we extended our studies by the CAM model, which provided a model for testing the effect of anti-angiogenesis
Fig. 3. Effect of recombinant human kallistatin (rhKal) on the protein expression of NF-kB pathway. A. Western-blot analysis of TNF-a-induced breast cancer cells (MCF-7) with cellular protein extracts with or without rhKal. B. Western-blot analysis of TNF-a-induced human umbilical vein endothelial cells (HUVECs) with cellular protein extracts with or without rhKal.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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agents on the process of new blood vessel formation. As shown on Fig. 2D, there is a normal pattern of blood vessels formed after 2-day incubation in the control group. More and thicker new blood vessels were formed in the MCF-7-induced group. When rhKal (4, 8, and 16 mg/egg) was added, the quantity of vessels was reduced. Quantitative analysis revealed that rhKal at 4, 8, and 16 mg/egg caused 14%, 27%, 49% reduction in the number of new vessels compared with the group induced by MCF-7 cells, respectively. These results displayed that rhKal inhibited the in vivo angiogenesis in a concentration-dependent manner.
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evaluated whether rhKal affected the TNF-a-induced p50 and p65 nuclear translocation. Western-blot assay was used to detect the effect of rhKal on TNF-a-induced nuclear translocation of p50and p65. As shown on Fig. 4A, we found that the rhKal could markedly suppress the TNF-a-induced p50 and p65 nuclear translocation. Finally, similar results were also observed by NF-kB-luciferase reporter gene assay. As shown on Fig. 4B, the rhKal significantly inhibited TNF-a induced NF-kB reporter gene activity in a dosedependent manner. All the results suggested that the rhKal strongly inhibited TNF-a induced NF-kB activity in MCF-7 and HUVEC cells.
3.4. The rhKal suppressed NF-kB pathway in response to TNF-a 3.5. The rhKal suppresses NF-kB-regulated gene expression The activation of NF-kB has been shown to promote angiogenesis. The aim of the study was to investigate the effects of rhKal on the NF-kB pathway. We investigated whether rhKal could affect the NF-kB signaling molecules stimulated by TNF-a. We first examined whether the NF-kB signaling pathway could be affected by rhKal on MCF-7 and HUVECs. As shown on Fig. 3A, B, the TNF-ainduced phosphorylation of IkBa was significantly inhibited by 200 mg/mL rhKal on MCF-7 and HUVEC cells. The TNF-a could markedly stimulate the activation of IkBa. Since rhKal suppressed the degradation and phosphorylation of IkBa, we tested the effect of rhKal on TNF-a-induced IKK activation, which is required for phosphorylation of IkBa. The results revealed that the TNF-ainduced activation of IKK was also suppressed by rhKal. The rhKal also suppressed the phosphorylation of NF-kB-p65 but not the expression of NF-kB-p65. Suppression of IkBa degradation should block the translocation of p50 and p65 to the cell nucleus. We next
NF-kB is known to regulate a variety of cell function, including proliferation, apoptosis and angiogenesis through regulating related gene expression [24]. In the above study, we found that the rhKal could effectively inhibit the angiogenesis and the activity of NF-kB pathway. We researched the effects of rhKal on NF-kBregulated gene expression treated with TNF-a. As shown on Fig. 4C, the rhKal inhibited angiogenesis-related gene expression in a dose-dependent manner, such as VEGF, MMP-2, MMP-9 and others. The results displayed that rhKal inhibited the angiogenesis via the expression of NF-kB-regulated gene. 4. Discussion Angiogenesis is the process by which new blood vessels are formed from pre-existing vasculature. Angiogenesis is a cascade
Fig. 4. Effect of recombinant human kallistatin (rhKal) on NF-kB activation and downstream protein. A. Western-blot analysis of p65 and p50 expression in cytosol and nuclear fractions of TNF-a-induced human umbilical vein endothelial cells (HUVEC). B. The rhKal inhibited TNF-a-induced NF-kB dependant reporter gene expression. HUVECs were transiently transfected with a NF-kB-luciferase reporter gene. After transfection, cells were pretreated with the indicated concentrations of rhKal for 24 h, and then incubated with 10 ng/mL TNF-a for another 24 h. C. The rhKal suppresses the expression of TNF-a-induced angiogenesis-related proteins. HUVEC cells were incubated with 10 ng/mL TNF-a and various dose of rhKal for the indicated concentrations.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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reaction, which includes many important cellular events, which is expected to be a promising target for treatment of various diseases, including cancer, diabetic retinopathy and rheumatoid arthritis [25]. In present, several anti-angiogenesis drugs were applied in clinical such as Bevacizumab [26,27], Sorafenib [28,29], Sunitinib [30,31] and others. The use of anti-angiogenesis drugs in cancer treatment strategy is still in its infancy, thus, studying of new antiangiogenesis drugs and molecular mechanisms underlying antiangiogenesis therapies are required for therapeutic improvements. Kallistatin, as an endogenous angiogenesis inhibitor, may have several advantages in the treatment of tumors. Intravitreal injection of kallistatin did not cause any toxicity to normal vasculature or serious inflammatory response [32]. Moreover, kallistatin has shown an obviously protective effect during acute phase inflammation and increases the survival rate of mice after endotoxin shock [33]. Kallistatin specifically induced apoptosis and inhibited growth of HUVECs, but had no direct effect on apoptosis and proliferation of tumor cells in the present research [34]. Comparing with the effect of kallistatin on angiogenesis; the mechanism underlying the anti-angiogenesis activity is not well understood. In this study, we showed that rhKal inhibited angiogenesis and metastasis in vitro and in vivo, which suggested that rhKal would be a suitable candidate for regulation of tumor angiogenesis. All the experiments demonstrated that rhKal had a strong antiangiogenesis activity and would be useful as an anti-cancer drug. Numerous pro-angiogenesis reagents have been identified as potential mediators of the angiogenic switch. The vascular endothelial growth factor (VEGF) is thought to be the most significant among them [35,36]. Matrix metalloproteinases (MMPs), a family of Zn-dependent end peptidases, are the major proteases participating in tumor cell migration, spreading, tissue metastasis and invasion, specifically MMP-2 and MMP-9, are important for migration in the process of tumor angiogenesis and metastasis [37,38]. We found that rhKal could inhibit the expression of many genes regulated by NF-kB pathway, such as MMP-2, MMP-9, ICAM-1 and VEGF. Meanwhile, rhKal inhibited IKK activation and TNF-a induced IkBa degradation, suppressed p65 nuclear translocation and phosphorylation. In summary, this study demonstrated that rhKal inhibited tumor angiogenesis, which was associated with blocking NF-kB pathway. The rhKal could be a useful and new therapeutic drug to control angiogenesis in tumor treatment. Further preclinical and clinical trials are required to research the full potential of this important protein drug. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study design was supported by the grants from the National Science Foundation of China (30973591, 81271691, 81270734); Natural Science Foundation of Fujian Province of China (2012D047); International Science & Technology Cooperation Program of China (2011DFG33320); Municipal Science and Technology Plan of Xiamen (3502Z20134043); Health department Foundation of Fujian (2013-2-110). References [1] Zhou GX, Chao L, Chao J. Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence. J Biol Chem 1992;267:25873–80.
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Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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[37] Itoh Y, Nagase H. Matrix metalloproteinases in cancer. Essays Biochem 2002;38:21–36. [38] Bernhard EJ, Gruber SB, Muschel RJ. Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc Natl Acad Sci U S A 1994;91: 4293–7.
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Original article
Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway K.F. Huang a,b,*,1, X.P. Huang a, G.Q. Xiao a, H.Y. Yang a, J.S. Lin a,c,*,2, Y. Diao a,*,2 a
Institute of Molecular Medicine, Hua-qiao University, 362021 Quan Zhou, China Xiamen Medicine Research Institute, 361003 Xiamen, China c AgResearch, Invermay, Mosgiel, New Zealand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2014 Accepted 4 March 2014
Development of a novel angiogenesis inhibitor will be essential for the improvement of therapeutics against cancer. Kallistatin had been recognized as an endogenous angiogenesis inhibitor. Here, we demonstrated kallistatin’s strong anti-angiogenesis and anti-metastasis activity stimulated by breast cancer cells (MCF-7) and its mechanism of action in vitro. The anti-angiogenesis effect in vivo was evaluated by chicken chorioallantoic membrane (CAM) neovascularisation. Because of the underlying molecular mechanism of its anti-angiogenesis activity remains poorly understood. In this study, we examined whether the NF-kB signaling pathway was involved in the anti-angiogenesis and antimetastasis activity of kallistatin. Kallistatin significantly inhibited TNF-a-induced nuclear factor-kB activation in a dose-dependent manner. Addition of kallistatin inhibited TNF-a induced IkBa degradation; phosphorylation of IkBa kinase (IKK), nuclear factor-kB-p65 protein; and nuclear translocation of p65/50. Meanwhile, we investigated the effects of kallistatin on the expression of vascular endothelial growth factor (VEGF) and other angiogenesis-related gene in human umbilical vein endothelial cells (HUVECs). We found that kallistatin decreased the expression of VEGF and some angiogenesis-related genes, which promoted angiogenesis in cancer. Taken together, we suggested that kallistatin would inhibit tumor angiogenesis via inhibition of the NF-kB signaling pathway and finally abrogate NF-kB-dependent gene expression. All the results revealed that kallistatin would have potential as a novel. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Kallistatin NF-kB Angiogenesis Tumor
1. Introduction Kallistatin, a serine proteinase inhibitor, is first discovered and identified as a tissue kallikrein-binding protein and a unique serine proteinase inhibitor, and has emerged as a novel inhibitor of angiogenesis. Kallistatin shows a variety of biological effects in pathologic and physiologic responses, such as blood pressure regulation, anti-angiogenesis and anti-inflammation [1–6]. In previous studies, kallistatin gene delivery markedly inhibited human hepatocellular carcinomas relying on its anti-angiogenesis activities [7]. Adenoviral expression vector encoding human kallistatin significantly inhibited the angiogenesis of pre-established human
* Corresponding authors. Institute of Molecular Medicine, Hua-qiao University, 362021 Quan Zhou, China. E-mail addresses: [email protected] (K.F. Huang), [email protected] (J.S. Lin), [email protected], [email protected] (Y. Diao). 1 Tel./fax: +86 592 5957216. 2 Tel./fax: +86 595 22692516.
breast tumor xenografts in athymic mice [6]. However, the molecular mechanism by which kallistatin modulates angiogenesis has not been determined, so the exact mechanism for anti-angiogenesis activity of kallistatin remains to be elucidated. Angiogenesis is the formation of new blood vessels. In 1971, Folkman hypothesized that tumor growth is dependent on angiogenesis and subsequently experimental work demonstrated that for a tumor to grow beyond a size of 1–2 mm3 a substantial new blood supply must develop to support the increasing metabolic requirements [8,9]. Angiogenesis become an important feature of tumor metastasis and growth. As such, targeting tumor neovascularization is a favorable strategy for cancer therapy. A number of angiogenesis inhibitors including endostatin protein have been identified and used in preclinical experiment, and several inhibitors have also been applied in clinical trials [10,11]. Targeting angiogenesis is an exciting and attractive area in the treatment of cancer. The mechanisms of angiogenesis have been investigated since 1931 when Clark observed real-time capillary growth, and are still
http://dx.doi.org/10.1016/j.biopha.2014.03.005 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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not fully understood [12]. Many growth factors and cytokines have both inhibitory and stimulatory roles within sprouting angiogenesis, such as ICAM-1, VEGF, and MMPs and others. The most attractive investigated compound is vascular endothelial growth factor (VEGF), which is known to be a potent stimulator of angiogenesis [13–16]. NF-kB is a family of important transcriptional factors known to regulate a wide range of biological effects, including proliferation, metastasis, apoptosis and angiogenesis, via its downstream target genes [17,18]. This family is composed of five related transcription factors (p50, p65, p52, RelB, and c-Rel). The most important NF-kB dimmers are formed by p50 and p65 in NF-kB signaling pathway [19,20]. NF-kB regulates the expression of many genes whose products are involved in tumor angiogenesis such as angiogenesis (VEGF) and adhesion molecules (MMP-2, MMP-9, and ICAM-1) [21]. In this study, we first examined the anti-angiogenesis activity of kallistatin using a combination of in vitro and in vivo angiogenesis assays in order to evaluate the anti-angiogenesis activity of kallistatin. We examined whether the NF-kB signaling pathway was involved in the anti-angiogenesis and anti-metastasis activity of kallistatin. Kallistatin markedly inhibited angiogenesis induced by MCF-7 cells and the downstream angiogenesis-related genes of NF-kB signaling pathway. Furthermore, kallistatin significantly inhibited TNF-a induced IkBa degradation, phosphorylation of IkBa kinase (IKK), nuclear factor-kB-p65 protein, and nuclear translocation of p65/50. Here, we discussed the possible involvement of TNF-a induced NF-kB pathway in the anti-angiogenesis and anti-metastasis activity of the novel protein drug. 2. Materials and method 2.1. Materials Recombinant human kallistatin (rhKal) protein was purification by Hua-qiao University. Matrigel was purchased from BD Biosciences;3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and TNF-a were purchased from Sigma (USA); polyclonal antibodies against surviving, caspase-3, NFkB65, NF-kB50, ICAM-1, MMP-2,MMP-9,VEGF were purchased from Boster Biological Technology Inc. (Wuhan, China); polyclonal antibodies against phospho-IkB, IkBa, phospho-p65, p-IKK, IKK were obtained from KeyGEN Biotech (Nanjing, China); horseradish peroxidase (HRP) - conjugated anti-rabbit immunoglobulin (IgG) was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).
stained with Gimesa stain. Non-migrating cells on the upper surface of the filter were removed, and chemotaxis was quantified by counting the number of cells that migrated to the lower side of the filter using optical microscopes (100, Nikon, Tokyo, Japan). Six fields were counted for each assay. Each assay was conducted with three wells and similar results were repeated at least three times. 2.4. Tube formation assay MCF-7 cells were pretreated with rhKal (0, 40, 80 and 160 mg/ mL) for 24 h. Matrigel (BD Biosciences, USA) was thawed and mixed with an equal volume of serum-free 1640 containing 1 105 cells/mL of MCF-7 cells. The mixture was transferred into a 96-well plate and allowed to solidify and polymerize at 37 8C for 6 h. HUVEC s were harvested after trypsin treatment and suspended in serum-free medium before seeding and planted onto matrigel. After 6 h, the plate was examined for capillary tube formation under an inverted microscope and photographed and tubular structures were quantified by manually counting the tube numbers, and six randomly chosen fields were analyzed for each well. 2.5. Chicken chorioallantoic membrane (CAM) assay Anti-angiogenesis activity of rhKal on CAM was assayed as described previously [22]. Briefly, fertilized chicken eggs were incubated at 37 8C for 6 days. After this incubation, a small hole was punched on the broad side of the egg, and a window was carefully created through the eggshell. MCF-7cells (2 106 cells/embryo) were placed on the exposed CAM. Sterilized filter paper disk (5 5 mm) saturated with vehicle or with kallistatin (0, 4, 8, and 16 mg/egg) were placed on the CAMs. The eggs were then incubated at 37 8C for another 2 days. The neovascular zones under the disks were photographed. Angiogenesis was quantified by counting the number of blood vessel branch points. Ten eggs were used per group to ensure the reproducibility. 2.6. Rat aortic ring assay
HUVECs and MCF-7cells were cultured in 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), and maintained in a humidified atmosphere of 5% CO2 in air at 37 8C two passages weekly.
Rat aortic ring assay was performed as described previously with some modification [23]. The thoracic aorta was dissected from male Sprague-Dawley rats (6 weeks old) and cut into 1-mm long rings. The disks were treated as described previously [23]. After 3 days of growth, the culture medium was replaced with fresh serum-free medium, MCF-7 cells (3 105 cells/mL), which were pretreated with rhKal (0, 40, 80 and 160 mg/mL) for 24 h. Plates were then stored in incubator at 37 8C and 5% CO2. The media were changed each 2 days. After 7 days, the sprouting microvessels in five randomly chosen fields were counted and photographed under a microscope for each group.
2.3. Endothelial cell migration assay
2.7. NF-kB-dependent reporter gene expression assay
Transwell invasion assay was used to confirm the effect of rhKal on cell invasion activity. Briefly, the chamber was divided into two compartments by a polycarbonate filter with a pore size of 8 mm. The polycarbonate filter was coated with 0.2% gelatin. MCF-7 cells were seeded in 24-well plate and incubated. When a monolayer is formed, the cells were treated with rhKal (0, 40, 80 and 160 mg/mL) for 24 h. After that, the medium was replaced by a serum-free medium for 12 h, and then the culture plates were inserted with Transwell chambers. Then, 3 104 HUVECs suspended with control medium were loaded on the upper compartment of the chamber. After 4 h at 37 8C in 5% CO2, the filter was fixed and
The effect of rhKal on TNF-a-induced NF-kB-dependent luciferase reporter assays was performed by Luciferase Reporter Gene Assay Kit (Beyotime, China). Briefly, HUVECs (5 105 cells/ well) were plated in 6-well plates and transiently transfected by the lipofectin regeant with pNF-kB-TA-luc. We transfected the cells with 0.5 mg pNF-kB-TA-luc plasmid and 1 mg pGL6-TA control plasmid for 24 h. Then, we treated the cells with various dose of rhKal (0, 20, 40 and 60 mg/mL) and 10 ng/mL TNF-a for 24 h. The cells were collected and lysed in cell lysis solution. The luciferase was detected by the manufacturer (Spectra Max M3, MD, USA).
2.2. Cell culture
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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Fig. 1. Effect of recombinant human kallistatin (rhKal) on cell viability in breast cancer cells (MCF-7) cells. The rhKal had tiny effect on viability of MCF-7cells. Data represented survival rate as percentages of respective controls (n = 6).
2.8. Western-blotting A total of 1 106 cells were collected and lysed for 15 min in ice-cold lyses buffer (29 mM Tris–HCl, pH 7.4, 137 mM NaCl, 10% [w/v] glycerin, 1%[v/v]) (Triton X-100, 2 mM EDTA, 1 mM PMSF).
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After removing the cell debris by centrifugation at 16,000 g for 15 min, equal amounts of proteins were separated on a 12% SDSPAGE, transferred electrophoretically (Bio-Rad, USA) onto a nitrocellulose membrane (KeyGEN, Nanjing, China) and blocked with 5% non-fat milk powder (w/v) in tris-buffer saline Tween-20 (TBST, 10 mM Tris, 100 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature. The membranes were incubated with primary anti-human rabbit polyclonal antibodies (1:500) overnight at 4 8C, and with anti-b-actin rabbit polyclonal antibody (1:500) as a control. After washing with TBST five times, HRP-conjugated antirabbit secondary antibody (1:1000) were added and incubated at 37 8C for 1 h before another five-time washing. Labeled bands were developed with enhanced chemiluminescence detection system (Amersham Pharmacia, UK) and photographed with a Molecular Imager Gel Doc XR system (Bio-Rad, USA). Protein levels were quantified by density analysis using Quantity One software (Bio-Rad, USA). Relative protein expression levels were deduced from the ratio of the mean values of each band to that of b-actin.
Fig. 2. The anti-angiogenesis and anti-metastasis activity of recombinant human kallistatin (rhKal) in vitro and in vivo. A. Breast cancer cells (MCF-7) were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing human umbilical vein endothelial cells (HUVECs) migration. B. MCF-7 cells were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing HUVEC cells tube formation. C. MCF-7 cells were pretreated with rhKal (0, 40, 80, and 160 mg/mL) for 24 h before inducing rat aortic ring microvessel sprouting. D. MCF-7 cells were placed on the exposed chicken chorioallantoic membrane (CAM) to induce angiogenesis in vivo. Sterilized filter paper disk (5 5 mm) saturated with vehicle or with kallistatin (4, 8, and 16 g/egg) were placed on the CAMs.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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2.9. Statistical analysis Assays were performed in duplicate and three independent experiments unless otherwise stated. Statistically significant differences between control and experimental groups were calculated by Dunnett’s test. Statistically significant is expressed as *P < 0.05, while highly statistically significant as **P < 0.01. 3. Results 3.1. The rhKal affected viability of HUVECs and MCF-7 cells The HUVECs were cultured in presence of rhKal at a range of concentrations (40–640 mg/mL in final) for different durations (48–72 h). The experiment included a group of cultured without rhKal as negative control. The proliferation inhibition rates were calculated from the loss of cell viability by assessment with MTT assay. The rhKal inhibited the growth of HUVECs in dose- and timedependent manners. In our previous research, the median inhibition concentration (IC50) values for 48 h, and 72 h of HUVEC cells were 189.6 mg/mL and 146.5 mg/mL, respectively. The rhKal had tiny effect on viability of MCF-7 cells (Fig. 1).
fewer HUVECs migrated to the opposite side of the filter in the Transwell chamber. The rhKal obviously suppressed migration of HUVECs induced by MCF-7 in a concentration-dependent manner. Then, tube formation assay was performed to evaluate the effects of rhKal on tube formation of HUVECs induced by MCF-7 cells on matrigel. As shown on Fig. 2B, MCF-7 cells promoted tube formation, and the number of tubes was much larger than that of the control group. The rhKal effectively reduced the quantity of endothelial tubes induced by MCF-7 in a concentration-dependent manner. We further evaluated the anti-angiogenesis effects of rhKal on the aorta sprout outgrowth assay in vitro, which mimics several stages in angiogenesis, including endothelial cell migration, proliferation, and tube formation. We utilized the model to evaluate the effect of rhKal on angiogenesis induced by MCF-7 cells. We found that new micro vessels began to grow when incubated for 5 days. After being cultivated for 7 days, the control group formed many microvessels. The MCF-7 cells could significantly promote the formation of micro vessels. The rhKal suppressed MCF-7 inducing formation of microvessel outgrowth from explants of rat aorta in a concentration-dependent manner (Fig. 2C). The above results suggested that the rhKal had ability to suppress MCF-7-induced migration and angiogenesis of HUVECs in vitro.
3.2. The rhKal suppressed MCF-7-induced angiogenesis in vitro 3.3. The rhKal suppressed MCF-7-induced angiogenesis in vivo Transwell chamber assay was used to evaluate the effect of rhKal on the migration of HUVEC s induced by MCF-7. As shown on Fig. 2A, MCF-7 cells significantly stimulated HUVECs migration. When MCF-7 cells were treated with 40, 80 and 160 mg/mL rhKal,
To evaluate whether anti-angiogenesis effects of rhKal could be recapitulated in vivo, we extended our studies by the CAM model, which provided a model for testing the effect of anti-angiogenesis
Fig. 3. Effect of recombinant human kallistatin (rhKal) on the protein expression of NF-kB pathway. A. Western-blot analysis of TNF-a-induced breast cancer cells (MCF-7) with cellular protein extracts with or without rhKal. B. Western-blot analysis of TNF-a-induced human umbilical vein endothelial cells (HUVECs) with cellular protein extracts with or without rhKal.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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agents on the process of new blood vessel formation. As shown on Fig. 2D, there is a normal pattern of blood vessels formed after 2-day incubation in the control group. More and thicker new blood vessels were formed in the MCF-7-induced group. When rhKal (4, 8, and 16 mg/egg) was added, the quantity of vessels was reduced. Quantitative analysis revealed that rhKal at 4, 8, and 16 mg/egg caused 14%, 27%, 49% reduction in the number of new vessels compared with the group induced by MCF-7 cells, respectively. These results displayed that rhKal inhibited the in vivo angiogenesis in a concentration-dependent manner.
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evaluated whether rhKal affected the TNF-a-induced p50 and p65 nuclear translocation. Western-blot assay was used to detect the effect of rhKal on TNF-a-induced nuclear translocation of p50and p65. As shown on Fig. 4A, we found that the rhKal could markedly suppress the TNF-a-induced p50 and p65 nuclear translocation. Finally, similar results were also observed by NF-kB-luciferase reporter gene assay. As shown on Fig. 4B, the rhKal significantly inhibited TNF-a induced NF-kB reporter gene activity in a dosedependent manner. All the results suggested that the rhKal strongly inhibited TNF-a induced NF-kB activity in MCF-7 and HUVEC cells.
3.4. The rhKal suppressed NF-kB pathway in response to TNF-a 3.5. The rhKal suppresses NF-kB-regulated gene expression The activation of NF-kB has been shown to promote angiogenesis. The aim of the study was to investigate the effects of rhKal on the NF-kB pathway. We investigated whether rhKal could affect the NF-kB signaling molecules stimulated by TNF-a. We first examined whether the NF-kB signaling pathway could be affected by rhKal on MCF-7 and HUVECs. As shown on Fig. 3A, B, the TNF-ainduced phosphorylation of IkBa was significantly inhibited by 200 mg/mL rhKal on MCF-7 and HUVEC cells. The TNF-a could markedly stimulate the activation of IkBa. Since rhKal suppressed the degradation and phosphorylation of IkBa, we tested the effect of rhKal on TNF-a-induced IKK activation, which is required for phosphorylation of IkBa. The results revealed that the TNF-ainduced activation of IKK was also suppressed by rhKal. The rhKal also suppressed the phosphorylation of NF-kB-p65 but not the expression of NF-kB-p65. Suppression of IkBa degradation should block the translocation of p50 and p65 to the cell nucleus. We next
NF-kB is known to regulate a variety of cell function, including proliferation, apoptosis and angiogenesis through regulating related gene expression [24]. In the above study, we found that the rhKal could effectively inhibit the angiogenesis and the activity of NF-kB pathway. We researched the effects of rhKal on NF-kBregulated gene expression treated with TNF-a. As shown on Fig. 4C, the rhKal inhibited angiogenesis-related gene expression in a dose-dependent manner, such as VEGF, MMP-2, MMP-9 and others. The results displayed that rhKal inhibited the angiogenesis via the expression of NF-kB-regulated gene. 4. Discussion Angiogenesis is the process by which new blood vessels are formed from pre-existing vasculature. Angiogenesis is a cascade
Fig. 4. Effect of recombinant human kallistatin (rhKal) on NF-kB activation and downstream protein. A. Western-blot analysis of p65 and p50 expression in cytosol and nuclear fractions of TNF-a-induced human umbilical vein endothelial cells (HUVEC). B. The rhKal inhibited TNF-a-induced NF-kB dependant reporter gene expression. HUVECs were transiently transfected with a NF-kB-luciferase reporter gene. After transfection, cells were pretreated with the indicated concentrations of rhKal for 24 h, and then incubated with 10 ng/mL TNF-a for another 24 h. C. The rhKal suppresses the expression of TNF-a-induced angiogenesis-related proteins. HUVEC cells were incubated with 10 ng/mL TNF-a and various dose of rhKal for the indicated concentrations.
Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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reaction, which includes many important cellular events, which is expected to be a promising target for treatment of various diseases, including cancer, diabetic retinopathy and rheumatoid arthritis [25]. In present, several anti-angiogenesis drugs were applied in clinical such as Bevacizumab [26,27], Sorafenib [28,29], Sunitinib [30,31] and others. The use of anti-angiogenesis drugs in cancer treatment strategy is still in its infancy, thus, studying of new antiangiogenesis drugs and molecular mechanisms underlying antiangiogenesis therapies are required for therapeutic improvements. Kallistatin, as an endogenous angiogenesis inhibitor, may have several advantages in the treatment of tumors. Intravitreal injection of kallistatin did not cause any toxicity to normal vasculature or serious inflammatory response [32]. Moreover, kallistatin has shown an obviously protective effect during acute phase inflammation and increases the survival rate of mice after endotoxin shock [33]. Kallistatin specifically induced apoptosis and inhibited growth of HUVECs, but had no direct effect on apoptosis and proliferation of tumor cells in the present research [34]. Comparing with the effect of kallistatin on angiogenesis; the mechanism underlying the anti-angiogenesis activity is not well understood. In this study, we showed that rhKal inhibited angiogenesis and metastasis in vitro and in vivo, which suggested that rhKal would be a suitable candidate for regulation of tumor angiogenesis. All the experiments demonstrated that rhKal had a strong antiangiogenesis activity and would be useful as an anti-cancer drug. Numerous pro-angiogenesis reagents have been identified as potential mediators of the angiogenic switch. The vascular endothelial growth factor (VEGF) is thought to be the most significant among them [35,36]. Matrix metalloproteinases (MMPs), a family of Zn-dependent end peptidases, are the major proteases participating in tumor cell migration, spreading, tissue metastasis and invasion, specifically MMP-2 and MMP-9, are important for migration in the process of tumor angiogenesis and metastasis [37,38]. We found that rhKal could inhibit the expression of many genes regulated by NF-kB pathway, such as MMP-2, MMP-9, ICAM-1 and VEGF. Meanwhile, rhKal inhibited IKK activation and TNF-a induced IkBa degradation, suppressed p65 nuclear translocation and phosphorylation. In summary, this study demonstrated that rhKal inhibited tumor angiogenesis, which was associated with blocking NF-kB pathway. The rhKal could be a useful and new therapeutic drug to control angiogenesis in tumor treatment. Further preclinical and clinical trials are required to research the full potential of this important protein drug. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study design was supported by the grants from the National Science Foundation of China (30973591, 81271691, 81270734); Natural Science Foundation of Fujian Province of China (2012D047); International Science & Technology Cooperation Program of China (2011DFG33320); Municipal Science and Technology Plan of Xiamen (3502Z20134043); Health department Foundation of Fujian (2013-2-110). References [1] Zhou GX, Chao L, Chao J. Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence. J Biol Chem 1992;267:25873–80.
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Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
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Please cite this article in press as: Huang KF, et al. Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-kB signaling pathway. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.03.005
