Arch. Pharm. Res. DOI 10.1007/s12272-014-0383-8
RESEARCH ARTICLE
Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3, ERK and AKT signaling pathways Ting Wang • Jian Liu • Xiao-Qin Xiao
Received: 23 November 2013 / Accepted: 29 March 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Cantharidin (CTD), a chemical compound secreted by blister beetles, has been shown with anti-tumor property in many cancer cells. In this study, our data showed that CTD exerts potent anti-angiogenesis activity in a dosedependent manner. CTD dose dependently suppressed human umbilical vascular endothelial cells proliferation, migration, and tube formation in vitro. Furthermore, CTD concentration dependently inhibited angiogenesis in chick embryo CAM model in vivo. At the molecular level, CTD abrogated VEGF-induced activation of STAT3 and suppressed the phosphorylation of JAK1 and ERK in a dosedependent manner. Furthermore, CTD blocked the phosphorylation of AKT in a time-dependent manner. Taken together, these findings clearly demonstrate for the first time that CTD can inhibit angiogenesis and may have applications in the development of new anti-angiogenesis drugs. Keywords Cantharidin (CTD) Angiogenesis Chick embryo CAM assay HUVEC
Ting Wang and Jian Liu have contributed equally to this work. T. Wang X.-Q. Xiao (&) Institutes of Combination of Chinese Traditional Medicine and Western Medicine, Hunan University of Chinese Medicine, Bachelor of Road, Changsha 410208, Hunan, China e-mail: [email protected] J. Liu Department of Emergency, The First Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan, China
Introduction Angiogenesis is the complex process in which new blood vessels are generated from preexisting ones. It involves cell proliferation, migration, and tube formation (Chen et al. 2012). In the development of solid tumors, angiogenesis forms a blood supply network for the tumor and thus plays a critical role in tumor growth and metastasis (Hanahan and Weinberg 2000; Shibuya 2008). The inhibition of angiogenesis, possibly in combination with other methods, may be an effective approach to the treatment of malignant tumors. Indeed, studies have shown that endogenous angiogenesis inhibitors can serve anti-cancer reagents (Folkman 2004). The identification and characterization of angiogenesis inhibitors with few side effects and the least tumor cell resistance are important steps in the development of new anti-cancer therapies. In the long history of Chinese medical practice, natural herbs and other products have typically been used as medicines, and many have shown certain effectiveness in empirical studies of the treatment of cancer, including lung cancer, leukemia, stomach cancer, liver cancer, and esophageal cancer (Liu et al. 2011). CTD, a natural toxin found in blister beetles, has been used to treat molluscum contagiosum virus infections and cancer in China and Vietnam (Moed et al. 2001; Wang 1989). Due to its toxic effects, precise dosage is crucial to the application of CTD, and inattentive overdose may easily have deadly consequences (Karras et al. 1996; Swingle et al. 2007). In order to take advantage of this ancient drug and make better use of it in modern medicine, it is important to first elucidate the mechanisms underlying its anti-cancer effects. For example, cucurbitacin E and usnic acid could inhibit tumor angiogenesis and growth by suppressing VEGFR2-mediated signaling pathways (Dong et al. 2010; Song et al.
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2012). In the present study, our data showed that CTD suppressed HUVEC proliferation, migration, and tube formation in vitro, inhibited angiogenesis in chick embryo CAM model in vivo. Moreover, CTD abrogated VEGFinduced activation of STAT3 and suppressed the phosphorylation of JAK1, AKT and ERK.
Materials and methods
mitomycin C for 2 h, the cells were scratched with a 1 ml pipette tip and rinsed three times with PBS. CTD of different concentrations in fresh medium with 10 ng/ml VEGF was added to the wells. After 8–12 h of incubation, the wound area was examined under an Olympus inverted microscope connected to a DXM1200 digital camera. Cell migration at different CTD concentrations was assessed using the percentage of the closed gap distance relative to control (with no CTD added). Each experiment was repeated three times.
Experimental materials Endothelial cell transwell migration assay The experimental materials and methods used here were similar to those used in our previously published studies (Dong et al. 2010; Song et al. 2012). In brief, HUVECs were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in completed endothelial cell medium at 37 °C in a humidified environment with 5 % CO2. Fertilized chicken eggs were purchased from Shanghai Poultry Breeding Co. Ltd (Shanghai, China). CTD was 98 % pure and obtained from Sigma-Aldrich (St. Louis, MO, USA), 10 mM solution of CTD was prepared in DMSO, and then diluted to desired concentrations (from 0.5 lM up to 10 lM) in cell culture medium. Cell proliferation assay A CellTiter96 AQueous One solution cell proliferation assay (MTS; Promega, Madison, WI, USA) was performed as describer previously (Song et al. 2012). HUVECs were seeded in 96-well plates (5 9 103 cells/well) and incubated for 12 h. They were then treated with CTD at different concentrations for 48–72 h then AQueous One solution was added, and absorbance was measured with a microplate reader (SpectraMax 190; Molecular Devices). Cell morphology assay Cell morphology was examined as describer previously (Dong et al. 2010). HUVECs were trypsinized and seeded in a 6-well plate. At 40–60 % confluence, culture medium was replaced with fresh medium CTD of different concentrations, and cells were incubated for another 36 h. HUVEC morphology was examined using an OLYMPUS phase contrast microscope. Endothelial cell wound-healing migration assay The endothelial cell wound-healing migration assay used was performed as described in previously papers (Dong et al. 2010). HUVECs were seeded in 6-well plates coated with 0.1 % gelatin Sigma-Aldrich (St. Louis, MO, USA) and cultured to confluency. After treatment with 2 lg/ml
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The endothelial cell transwell migration assay was performed as described previously (Yi et al. 2008). The insert of the transwell plate was coated with 0.1 % gelatin for 30 min and the transwell was then rinsed three times with PBS. Then fresh ECM containing 4 ng/ml VEGF was added to the lower chamber and HUVECs (4 9 104 cells/ well) were seeded in the top chamber. Next, cells were incubated with CTD of different concentrations for 4 h at 37 °C, 5 % CO2, and non-migrated cells on the upper surfaces of the membrane were gently scraped off with a cotton swab. The membrane containing migrated cells was fixed with 4 % paraformaldehyde for 20 min and stained with hematoxylin. Images were recorded using an Olympus inverted microscope, and migrated cells were counted manually. Cell invasion at different CTD concentrations was assessed using the number of migrated cells relative to control (no CTD added). Tube formation assay The tube formation assay was performed as described previously (Yi et al. 2008). Matrigel was thawed overnight at 4 °C. A pre-chilled 96-well plate was coated with 50 ll Matrigel and then incubated at 37 °C for 30 min. HUVECs (2 9 104 cells) were seeded onto Matrigel and incubated with CTD at different concentrations for 8–12 h 37 °C, 5 % CO2. Then endothelial cell tubular structures were examined under an Olympus inverted microscope. Tube formation at different concentrations of CTD was normalized to control (with no CTD added). Western blot analysis HUVECs were first starved in serum-free ECM for 4 h and then pretreated with CTD, followed by the stimulation with VEGF (10 ng/ml). The whole-cell extracts were prepared by lysis buffer supplement with different kinds of protein inhibitors (Chen et al. 2012). Equal protein aliquot of each lysate was subjected to SDS-PAGE (8 %), blotted onto PVDF membrane (Bio-Rad), probed with specific
Cantharidin inhibits angiogenesis
Luciferase reported gene assay The effect of CTD on VEGF-induced STAT3 dependent luciferase reporter assays was determined as described previously (Sandur et al. 2010). Briefly, after transfection of HMEC cells with STAT3 luciferased reporter gene (STAT3-luc, 0.5 lg) and 0.05 lg of the Renilla luciferase assay vector pRL (Promega) with lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, the cells were incubated with the indicated concentration of CTD for 24 h and then incubated with VEGF (10 ng/ml) for another 24 h. Cells were lysed in Reporter Lysis Buffer (Promega), and luciferase and Renilla activity was measured using a luminometer (Flexstation). Luciferase activity was normalized with Renilla activity in the cell lysate and expressed as an average of three independent experiments. Chick embryo CAM assay The CAM assay was performed as described in a previous study (Cho et al. 2009). Chick embryonic eggs were incubated for 5 days at 37 °C in 60 % relative humidity. Then a 1–2 cm2 window was opened at the blunt end of each egg and the shell membrane was removed to expose the CAM. A sterilized filter paper disk 5 mm diameter (Whatman, NJ, USA) soaked in CTD or DMSO was placed on the CAM. The window was then sealed and the eggs were returned to the incubator and incubated for another 2 days. The CAM microvessels were observed under a stereomicroscope, and the neovascularization was quantified using Image Pro Plus. Statistical analysis Data were presented as mean ± standard error. Statistical analysis was performed using Student’s t test. P \ 0.05 was considered statistically significant.
Results Inhibitory effects of CTD on HUVEC proliferation Cell proliferation is an essential step in angiogenesis, so the effect of CTD on HUVEC proliferation was assessed using a MTS assay. As shown in Fig. 1, CTD substantially inhibited HUVEC proliferation in a dose-dependent manner. At 0.5 lM CTD, the inhibition was already significant (P \ 0.001); at 1 lM, HUVEC viability dropped to about
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Fig. 1 Inhibitory effect of CTD on HUVEC proliferation. CTD inhibited HUVEC proliferation in a dose-dependent manner, as shown by an MTS assay. HUVECs (5 9 103 cells/well) were treated with different concentrations of CTD for 48 h before MTS measurement. ***P \ 0.001
50 % of control level; at 5 and 10 lM, HUVEC viabilities stabilized at about 30 % that of the control. Inhibitory effects of CTD on HUVEC migration and invasion Cell migration is also an important step in angiogenesis (Chen et al. 2012). In the present study, the effects of CTD on VEGF-induced HUVEC migration and invasion were assessed using a wound-healing migration assay and transwell migration assay. As illustrated in Fig. 2a, in the control group (without CTD added), 5 h after the scraping (denoted by the dotted lines), the gap was filled with migrated HUVECs. In contrast, HUVECs treated with 5 lM CTD showed only a few cells in the gap. Quantitative analysis (Fig. 2b) showed CTD suppressed HUVECs migration in a dose-dependent manner, with an IC50 of 2.5 lM. To rule out the possibility that this decrease in the number of migrated HUVECs in the gap could be accounted for by potential inhibitory effect of CTD on HUVEC growth, an MTS assay on the effect of CTD on cell viability in the same time frame (5 h) was performed. As shown in Fig. 2c, while CTD did reduce HUVEC viability, the magnitude of this effect was much smaller than that of the effect on the number of migrated cells. Even at 10 lM, about 60 % HUVECs still survived. This suggests that CTD directly inhibits the ability of HUVECs to migrate in the wound-healing migration assay. Similar results were obtained from the transwell migration assay that invasion of HUVECs treated with CTD was substantially inhibited relative to the control. Figure 2d shows that, at 5 h, the number of invaded HUVECs was drastically reduced when the cells were treated with 5 lM CTD. Quantitative analysis (Fig. 2e) showed this type of inhibitory effect to be dose dependent. At 1 lM, the inhibition was already significant (P \ 0.001).
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The number of invaded HUVECs dropped to less than 30 % when CTD concentration was 5 lM, and to less than 10 % when CTD concentration was 10 lM (P \ 0.001 in both cases), suggesting that the ability of HUVECs to invade the other chamber was suppressed with CTD treatment.
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Inhibitory effects of CTD on tube formation in HUVEC Another key step in angiogenesis is the formation of tubes of endothelial cells (Sandur et al. 2010). To investigate the effects of CTD on the formation of HUVEC capillary structures, HUVECs seeded on Matrigel and treated with different
Cantharidin inhibits angiogenesis b Fig. 2 Effects of CTD on HUVEC migration and invasion. a An
representative image of three independent experiments (woundhealing migration assay) is shown. HUVECs (10 9 103 cells/well) were treated with 10 ng/ml VEGF and with or without CTD (5 lM) for 5 h. Dotted lines denote the margins of scraping. b CTD inhibited HUVEC migration in a dose-dependent manner. **P \ 0.01, ***P \ 0.001. c Effects of CTD on HUVEC proliferation in 5 h. HUVECs (10 9 103 cells/well) were treated with different concentrations of CTD for 5 h before MTS measurement. ***P \ 0.001. d An representative image of three independent experiments (transwell invasion assay) is shown. HUVECs (40 9 103 cells/well) treated with 5 lM CTD were seeded in the upper chamber, and the bottom chamber was filled with ECM medium containing 4 ng/ml VEGF. HUVECs were allowed to migrate for 5 h. Cells with an irregular shape in images are cells that migrated into the lower chamber. e CTD inhibited HUVEC invasion in a dose-dependent manner. ***P \ 0.001
concentrations of CTD were examined. Figure 3a shows that, at 3 h, the number of tubes formed by HUVECs treated with 5 lM CTD was dramatically reduced relative to control cells. Quantitative analysis (Fig. 3b) further showed this inhibition to be dose-dependent. At 2.5 lM, total tube formation was less than 60 % that of control. It dropped to about 10 % of control levels at 5 lM, and to less than 5 % of control levels at 10 lM.
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A standard chick embryo CAM assay was performed to directly examine the effect of CTD on angiogenesis and vascular development in vivo (Cho et al. 2009). As shown in Fig. 4a, compared to control, CAM treated with CTD showed substantially fewer newly formed blood vessels. For quantitative analysis, new blood vessel branches in a circular area 5 mm in diameter surrounding the filter paper disc were counted. As shown in Fig. 4b, angiogenesis was dosedependently reduced with CTD treatment. Inhibitory effects of CTD on VEGF-induced phosphorylation of ERK and AKT and JAK1/STAT3 signaling pathway VEGF-induced ERK, JAK1/STAT3 and AKT signaling pathway are essential for angiogenesis (Wei et al. 2003; Fujio and Walsh 1999). These pathways have been demonstrated to regulate endothelial cell proliferation, migration, and tube formation (Chen et al. 2012). To figure out the molecular mechanism of CTD in anti-angiogenesis, we firstly examined the phosphorylation of AKT mediated by CTD in HMEC cells. Our data showed that the phosphorylation of AKT reached its peak within 40 min of VEGF stimulation in HMEC (Fig. 5a, left). Treatment with CTD significantly decreased the phosphorylation of AKT when compared with the VEGF -stimulated control (Fig. 5a, right). The results suggested that CTD suppressed VEGF-
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Fig. 3 Inhibitory effects of CTD on tube formation in HUVECs. a A representative image out of three independent experiments showing the inhibition on the formation of capillary structure in HUVECs of 5 lM CTD at 3 h. HUVECs (2 9 104 cells) were seeded in a 96 well plate coated with Matrigel and then treated with 5 lM CTD for 3 h. b CTD inhibited tube formation in HUVECs in a dose-dependent manner. HUVECs (2 9 104 cells) were seeded in a 96 well plate coated with Matrigel and then treated with indicated concentrations of CTD for 8 h. The tubes were counted. ***P \ 0.001
induced the phosphorylation of AKT. We next examined the effects of CTD on the phosphorylation of ERK, another pivotal molecular event during angiogenesis (Dai and Rabie 2007). The results showed that VEGF significantly induced the phosphorylation of ERK, while CTD concentration-dependently suppressed the VEGF-induced the phosphorylation of ERK (Fig. 5b). Similar results obtained
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T. Wang et al. Fig. 4 Inhibitory effects of CTD on angiogenesis in vivo by chick embryo CAM assay. a Representative images of control and CTD-treated (1 lg/ disc) CAMs. b Quantitative analysis of new blood vessel branching in the CAM assay (n = 10). ***P \ 0.001, *P \ 0.05
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from the analysis of JAK1 phosphorylation, which is also a well-established downstream events of VEGF stimulation (Chen et al. 2012). The results showed that CTD abrogated the VEGF-induced the phosphorylation of JAK1 (Fig. 5b). Furthermore, using the STAT3-luciferase assay (the STAT3-responsive elements linked to a luciferase reporter gene), the luciferase reporter gene activity of STAT3 was sharply increased when exposed to VEGF, whereas CTD suppressed STAT3 activity in a dose-dependent manner (Fig. 5a), suggesting that CTD can inhibit VEGF-induced activation of JAK1/STAT3 signaling pathway. Together, CTD has effect on VEGF-induced JAK1/STAT3 signaling pathway and phosphorylation of ERK and AKT.
Discussion In the long history of practice of traditional Chinese medicine, natural products including herbs and animal parts have been used. Among these, many have been proven to have anti-cancer therapeutic effects (Surh 2003). A
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series of recent studies previously combined in vitro and in vivo techniques to explore the effects of compounds extracted from Chinese medicine on angiogenesis, a complex process crucial to tumor growth and metastasis (Dong et al. 2010; Yi et al. 2008). In the present study, we examined the effects of CTD on angiogenesis. Both in vitro results with HUVECs and in vivo results with chick embryo CAM assays demonstrated that CTD also inhibited angiogenesis. This might underlie its anti-cancer effects. This is consistent with a previous in vitro study that reported that, in the highly metastatic ovarian carcinoma cell line HO-8910PM, CTD significantly suppresses cell invasion and metastasis (He et al. 2005). Norcantharidin (NCTD), the demethylated form of CTD, has been shown to be an anti-tumor angiogenesis agent by inhibiting HUVEC cell proliferation, migration, invasion, and tube formation in a dose-dependent manner (Zhang et al. 2013). Our data also showed that CTD has similar effect in angiogenesis. Furthermore, NCTD inhibits tumor angiogenesis via blocking VEGFR2/MEK/ERK signaling pathways, with little effect on the
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Fig. 5 Inhibitory effects of CTD on VEGF-induced STAT3-luciferase activity and the phosphorylation of JAK1, AKT and ERK. a CTD suppressed VEGF-induced the phosphorylation of AKT in a timedependent manner. HMEC cells were incubated with or without CTD (1 lM) for 6 h and then treated with 10 ng/ml VEGF for indicated times. Then cell lysates were subjected to immunoblotting with antibodies as indicated. b CTD inhibited VEGF-induced the phosphorylation of ERK and JAK1 in a dose-dependent manner. HMEC cells were incubated with indicated concentrations of CTD for 3 h and then treated with VEGF (10 ng/ml) for 20 min. Then cell lysates were subjected to immunoblotting with antibodies as indicated. c CTD inhibited VEGF-induced STAT3-luciferase activity in a dosedependent manner. After transfection of HMEC cells with STAT3 luciferased reporter gene (The STAT3-responsive elements linked to a luciferase reporter gene), the cells were incubated with the indicated concentration of CTD for 24 h and then incubated with VEGF (10 ng/ml) for another 24 h. Cell supernatants were collected and assayed for luciferase activity. *P \ 0.05, ***P \ 0.001
phosphorylation of AKT (Zhang et al. 2013). However, as a structure analog of NCTD, CTD suppressed VEGFinduced JAK1/STAT3 signaling pathway and phosphorylation of AKT and ERK, suggesting that the methyl group of CTD is critical for suppressing phosphorylation of AKT, whereas NCTD, which lacks this group, had no activity. How the methyl group of CTD modulates this role is unclear. It is possible that the methyl group of CTD in zerumbone is needed for suppressing the AKT activity.
This is in similar with the derivatives of alkylphosphocholine (Alam et al. 2012). Together, CTD which differs from NCTD, has a different molecular mechanism on angiogenesis. CTD has been reported as an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) (Li and Casida 1992; Honkanen 1993). Protein phosphorylation play an important role in regulating multiple cellular processes, including apoptosis and signal transduction pathways, but the biochemical target of CTD and the mechanism by which CTD inhibits cell growth and causes cell death remain elusive (Wera and Hemmings 1995; Huan et al. 2006). It has been suggested that the inhibitory effect of CTD on ovarian carcinoma cell line might involve down-regulation of NF-kappaB P65 subunit and VEGF (He et al. 2005). In anti-cancer therapy, multidrug resistance of cancer cells presents a serious obstacle to effective treatment (Beck et al. 1979). CTD has an excellent potential as an anti-cancer reagent because it is not involved in any multidrug-resistant phenotype in human leukemia cells (Efferth et al. 2002). In addition, its active concentration is rather low, below 2 lM in MTT assay (Efferth et al. 2002). This is consistent with the observation made in the present study that, at 1 lM, the inhibitory effects of CTD on HUVEC proliferation, migration, and tube formation were highly significant. In conclusion, the present study for the first time used both in vitro and in vivo techniques to demonstrate the inhibitory effects of CTD, a compound extracted from traditional Chinese anti-cancer medicine, on angiogenesis. Its natural source, lack of connection with multidrugresistant cancer cell lines, and low active dose make CTD a promising target for the development of anti-cancer drugs using modern technologies. However, further investigations into the mechanisms underlying its inhibition of angiogenesis must be performed in the near future.
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RESEARCH ARTICLE
Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3, ERK and AKT signaling pathways Ting Wang • Jian Liu • Xiao-Qin Xiao
Received: 23 November 2013 / Accepted: 29 March 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Cantharidin (CTD), a chemical compound secreted by blister beetles, has been shown with anti-tumor property in many cancer cells. In this study, our data showed that CTD exerts potent anti-angiogenesis activity in a dosedependent manner. CTD dose dependently suppressed human umbilical vascular endothelial cells proliferation, migration, and tube formation in vitro. Furthermore, CTD concentration dependently inhibited angiogenesis in chick embryo CAM model in vivo. At the molecular level, CTD abrogated VEGF-induced activation of STAT3 and suppressed the phosphorylation of JAK1 and ERK in a dosedependent manner. Furthermore, CTD blocked the phosphorylation of AKT in a time-dependent manner. Taken together, these findings clearly demonstrate for the first time that CTD can inhibit angiogenesis and may have applications in the development of new anti-angiogenesis drugs. Keywords Cantharidin (CTD) Angiogenesis Chick embryo CAM assay HUVEC
Ting Wang and Jian Liu have contributed equally to this work. T. Wang X.-Q. Xiao (&) Institutes of Combination of Chinese Traditional Medicine and Western Medicine, Hunan University of Chinese Medicine, Bachelor of Road, Changsha 410208, Hunan, China e-mail: [email protected] J. Liu Department of Emergency, The First Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan, China
Introduction Angiogenesis is the complex process in which new blood vessels are generated from preexisting ones. It involves cell proliferation, migration, and tube formation (Chen et al. 2012). In the development of solid tumors, angiogenesis forms a blood supply network for the tumor and thus plays a critical role in tumor growth and metastasis (Hanahan and Weinberg 2000; Shibuya 2008). The inhibition of angiogenesis, possibly in combination with other methods, may be an effective approach to the treatment of malignant tumors. Indeed, studies have shown that endogenous angiogenesis inhibitors can serve anti-cancer reagents (Folkman 2004). The identification and characterization of angiogenesis inhibitors with few side effects and the least tumor cell resistance are important steps in the development of new anti-cancer therapies. In the long history of Chinese medical practice, natural herbs and other products have typically been used as medicines, and many have shown certain effectiveness in empirical studies of the treatment of cancer, including lung cancer, leukemia, stomach cancer, liver cancer, and esophageal cancer (Liu et al. 2011). CTD, a natural toxin found in blister beetles, has been used to treat molluscum contagiosum virus infections and cancer in China and Vietnam (Moed et al. 2001; Wang 1989). Due to its toxic effects, precise dosage is crucial to the application of CTD, and inattentive overdose may easily have deadly consequences (Karras et al. 1996; Swingle et al. 2007). In order to take advantage of this ancient drug and make better use of it in modern medicine, it is important to first elucidate the mechanisms underlying its anti-cancer effects. For example, cucurbitacin E and usnic acid could inhibit tumor angiogenesis and growth by suppressing VEGFR2-mediated signaling pathways (Dong et al. 2010; Song et al.
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2012). In the present study, our data showed that CTD suppressed HUVEC proliferation, migration, and tube formation in vitro, inhibited angiogenesis in chick embryo CAM model in vivo. Moreover, CTD abrogated VEGFinduced activation of STAT3 and suppressed the phosphorylation of JAK1, AKT and ERK.
Materials and methods
mitomycin C for 2 h, the cells were scratched with a 1 ml pipette tip and rinsed three times with PBS. CTD of different concentrations in fresh medium with 10 ng/ml VEGF was added to the wells. After 8–12 h of incubation, the wound area was examined under an Olympus inverted microscope connected to a DXM1200 digital camera. Cell migration at different CTD concentrations was assessed using the percentage of the closed gap distance relative to control (with no CTD added). Each experiment was repeated three times.
Experimental materials Endothelial cell transwell migration assay The experimental materials and methods used here were similar to those used in our previously published studies (Dong et al. 2010; Song et al. 2012). In brief, HUVECs were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in completed endothelial cell medium at 37 °C in a humidified environment with 5 % CO2. Fertilized chicken eggs were purchased from Shanghai Poultry Breeding Co. Ltd (Shanghai, China). CTD was 98 % pure and obtained from Sigma-Aldrich (St. Louis, MO, USA), 10 mM solution of CTD was prepared in DMSO, and then diluted to desired concentrations (from 0.5 lM up to 10 lM) in cell culture medium. Cell proliferation assay A CellTiter96 AQueous One solution cell proliferation assay (MTS; Promega, Madison, WI, USA) was performed as describer previously (Song et al. 2012). HUVECs were seeded in 96-well plates (5 9 103 cells/well) and incubated for 12 h. They were then treated with CTD at different concentrations for 48–72 h then AQueous One solution was added, and absorbance was measured with a microplate reader (SpectraMax 190; Molecular Devices). Cell morphology assay Cell morphology was examined as describer previously (Dong et al. 2010). HUVECs were trypsinized and seeded in a 6-well plate. At 40–60 % confluence, culture medium was replaced with fresh medium CTD of different concentrations, and cells were incubated for another 36 h. HUVEC morphology was examined using an OLYMPUS phase contrast microscope. Endothelial cell wound-healing migration assay The endothelial cell wound-healing migration assay used was performed as described in previously papers (Dong et al. 2010). HUVECs were seeded in 6-well plates coated with 0.1 % gelatin Sigma-Aldrich (St. Louis, MO, USA) and cultured to confluency. After treatment with 2 lg/ml
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The endothelial cell transwell migration assay was performed as described previously (Yi et al. 2008). The insert of the transwell plate was coated with 0.1 % gelatin for 30 min and the transwell was then rinsed three times with PBS. Then fresh ECM containing 4 ng/ml VEGF was added to the lower chamber and HUVECs (4 9 104 cells/ well) were seeded in the top chamber. Next, cells were incubated with CTD of different concentrations for 4 h at 37 °C, 5 % CO2, and non-migrated cells on the upper surfaces of the membrane were gently scraped off with a cotton swab. The membrane containing migrated cells was fixed with 4 % paraformaldehyde for 20 min and stained with hematoxylin. Images were recorded using an Olympus inverted microscope, and migrated cells were counted manually. Cell invasion at different CTD concentrations was assessed using the number of migrated cells relative to control (no CTD added). Tube formation assay The tube formation assay was performed as described previously (Yi et al. 2008). Matrigel was thawed overnight at 4 °C. A pre-chilled 96-well plate was coated with 50 ll Matrigel and then incubated at 37 °C for 30 min. HUVECs (2 9 104 cells) were seeded onto Matrigel and incubated with CTD at different concentrations for 8–12 h 37 °C, 5 % CO2. Then endothelial cell tubular structures were examined under an Olympus inverted microscope. Tube formation at different concentrations of CTD was normalized to control (with no CTD added). Western blot analysis HUVECs were first starved in serum-free ECM for 4 h and then pretreated with CTD, followed by the stimulation with VEGF (10 ng/ml). The whole-cell extracts were prepared by lysis buffer supplement with different kinds of protein inhibitors (Chen et al. 2012). Equal protein aliquot of each lysate was subjected to SDS-PAGE (8 %), blotted onto PVDF membrane (Bio-Rad), probed with specific
Cantharidin inhibits angiogenesis
Luciferase reported gene assay The effect of CTD on VEGF-induced STAT3 dependent luciferase reporter assays was determined as described previously (Sandur et al. 2010). Briefly, after transfection of HMEC cells with STAT3 luciferased reporter gene (STAT3-luc, 0.5 lg) and 0.05 lg of the Renilla luciferase assay vector pRL (Promega) with lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, the cells were incubated with the indicated concentration of CTD for 24 h and then incubated with VEGF (10 ng/ml) for another 24 h. Cells were lysed in Reporter Lysis Buffer (Promega), and luciferase and Renilla activity was measured using a luminometer (Flexstation). Luciferase activity was normalized with Renilla activity in the cell lysate and expressed as an average of three independent experiments. Chick embryo CAM assay The CAM assay was performed as described in a previous study (Cho et al. 2009). Chick embryonic eggs were incubated for 5 days at 37 °C in 60 % relative humidity. Then a 1–2 cm2 window was opened at the blunt end of each egg and the shell membrane was removed to expose the CAM. A sterilized filter paper disk 5 mm diameter (Whatman, NJ, USA) soaked in CTD or DMSO was placed on the CAM. The window was then sealed and the eggs were returned to the incubator and incubated for another 2 days. The CAM microvessels were observed under a stereomicroscope, and the neovascularization was quantified using Image Pro Plus. Statistical analysis Data were presented as mean ± standard error. Statistical analysis was performed using Student’s t test. P \ 0.05 was considered statistically significant.
Results Inhibitory effects of CTD on HUVEC proliferation Cell proliferation is an essential step in angiogenesis, so the effect of CTD on HUVEC proliferation was assessed using a MTS assay. As shown in Fig. 1, CTD substantially inhibited HUVEC proliferation in a dose-dependent manner. At 0.5 lM CTD, the inhibition was already significant (P \ 0.001); at 1 lM, HUVEC viability dropped to about
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Fig. 1 Inhibitory effect of CTD on HUVEC proliferation. CTD inhibited HUVEC proliferation in a dose-dependent manner, as shown by an MTS assay. HUVECs (5 9 103 cells/well) were treated with different concentrations of CTD for 48 h before MTS measurement. ***P \ 0.001
50 % of control level; at 5 and 10 lM, HUVEC viabilities stabilized at about 30 % that of the control. Inhibitory effects of CTD on HUVEC migration and invasion Cell migration is also an important step in angiogenesis (Chen et al. 2012). In the present study, the effects of CTD on VEGF-induced HUVEC migration and invasion were assessed using a wound-healing migration assay and transwell migration assay. As illustrated in Fig. 2a, in the control group (without CTD added), 5 h after the scraping (denoted by the dotted lines), the gap was filled with migrated HUVECs. In contrast, HUVECs treated with 5 lM CTD showed only a few cells in the gap. Quantitative analysis (Fig. 2b) showed CTD suppressed HUVECs migration in a dose-dependent manner, with an IC50 of 2.5 lM. To rule out the possibility that this decrease in the number of migrated HUVECs in the gap could be accounted for by potential inhibitory effect of CTD on HUVEC growth, an MTS assay on the effect of CTD on cell viability in the same time frame (5 h) was performed. As shown in Fig. 2c, while CTD did reduce HUVEC viability, the magnitude of this effect was much smaller than that of the effect on the number of migrated cells. Even at 10 lM, about 60 % HUVECs still survived. This suggests that CTD directly inhibits the ability of HUVECs to migrate in the wound-healing migration assay. Similar results were obtained from the transwell migration assay that invasion of HUVECs treated with CTD was substantially inhibited relative to the control. Figure 2d shows that, at 5 h, the number of invaded HUVECs was drastically reduced when the cells were treated with 5 lM CTD. Quantitative analysis (Fig. 2e) showed this type of inhibitory effect to be dose dependent. At 1 lM, the inhibition was already significant (P \ 0.001).
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The number of invaded HUVECs dropped to less than 30 % when CTD concentration was 5 lM, and to less than 10 % when CTD concentration was 10 lM (P \ 0.001 in both cases), suggesting that the ability of HUVECs to invade the other chamber was suppressed with CTD treatment.
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Inhibitory effects of CTD on tube formation in HUVEC Another key step in angiogenesis is the formation of tubes of endothelial cells (Sandur et al. 2010). To investigate the effects of CTD on the formation of HUVEC capillary structures, HUVECs seeded on Matrigel and treated with different
Cantharidin inhibits angiogenesis b Fig. 2 Effects of CTD on HUVEC migration and invasion. a An
representative image of three independent experiments (woundhealing migration assay) is shown. HUVECs (10 9 103 cells/well) were treated with 10 ng/ml VEGF and with or without CTD (5 lM) for 5 h. Dotted lines denote the margins of scraping. b CTD inhibited HUVEC migration in a dose-dependent manner. **P \ 0.01, ***P \ 0.001. c Effects of CTD on HUVEC proliferation in 5 h. HUVECs (10 9 103 cells/well) were treated with different concentrations of CTD for 5 h before MTS measurement. ***P \ 0.001. d An representative image of three independent experiments (transwell invasion assay) is shown. HUVECs (40 9 103 cells/well) treated with 5 lM CTD were seeded in the upper chamber, and the bottom chamber was filled with ECM medium containing 4 ng/ml VEGF. HUVECs were allowed to migrate for 5 h. Cells with an irregular shape in images are cells that migrated into the lower chamber. e CTD inhibited HUVEC invasion in a dose-dependent manner. ***P \ 0.001
concentrations of CTD were examined. Figure 3a shows that, at 3 h, the number of tubes formed by HUVECs treated with 5 lM CTD was dramatically reduced relative to control cells. Quantitative analysis (Fig. 3b) further showed this inhibition to be dose-dependent. At 2.5 lM, total tube formation was less than 60 % that of control. It dropped to about 10 % of control levels at 5 lM, and to less than 5 % of control levels at 10 lM.
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A standard chick embryo CAM assay was performed to directly examine the effect of CTD on angiogenesis and vascular development in vivo (Cho et al. 2009). As shown in Fig. 4a, compared to control, CAM treated with CTD showed substantially fewer newly formed blood vessels. For quantitative analysis, new blood vessel branches in a circular area 5 mm in diameter surrounding the filter paper disc were counted. As shown in Fig. 4b, angiogenesis was dosedependently reduced with CTD treatment. Inhibitory effects of CTD on VEGF-induced phosphorylation of ERK and AKT and JAK1/STAT3 signaling pathway VEGF-induced ERK, JAK1/STAT3 and AKT signaling pathway are essential for angiogenesis (Wei et al. 2003; Fujio and Walsh 1999). These pathways have been demonstrated to regulate endothelial cell proliferation, migration, and tube formation (Chen et al. 2012). To figure out the molecular mechanism of CTD in anti-angiogenesis, we firstly examined the phosphorylation of AKT mediated by CTD in HMEC cells. Our data showed that the phosphorylation of AKT reached its peak within 40 min of VEGF stimulation in HMEC (Fig. 5a, left). Treatment with CTD significantly decreased the phosphorylation of AKT when compared with the VEGF -stimulated control (Fig. 5a, right). The results suggested that CTD suppressed VEGF-
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Fig. 3 Inhibitory effects of CTD on tube formation in HUVECs. a A representative image out of three independent experiments showing the inhibition on the formation of capillary structure in HUVECs of 5 lM CTD at 3 h. HUVECs (2 9 104 cells) were seeded in a 96 well plate coated with Matrigel and then treated with 5 lM CTD for 3 h. b CTD inhibited tube formation in HUVECs in a dose-dependent manner. HUVECs (2 9 104 cells) were seeded in a 96 well plate coated with Matrigel and then treated with indicated concentrations of CTD for 8 h. The tubes were counted. ***P \ 0.001
induced the phosphorylation of AKT. We next examined the effects of CTD on the phosphorylation of ERK, another pivotal molecular event during angiogenesis (Dai and Rabie 2007). The results showed that VEGF significantly induced the phosphorylation of ERK, while CTD concentration-dependently suppressed the VEGF-induced the phosphorylation of ERK (Fig. 5b). Similar results obtained
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T. Wang et al. Fig. 4 Inhibitory effects of CTD on angiogenesis in vivo by chick embryo CAM assay. a Representative images of control and CTD-treated (1 lg/ disc) CAMs. b Quantitative analysis of new blood vessel branching in the CAM assay (n = 10). ***P \ 0.001, *P \ 0.05
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from the analysis of JAK1 phosphorylation, which is also a well-established downstream events of VEGF stimulation (Chen et al. 2012). The results showed that CTD abrogated the VEGF-induced the phosphorylation of JAK1 (Fig. 5b). Furthermore, using the STAT3-luciferase assay (the STAT3-responsive elements linked to a luciferase reporter gene), the luciferase reporter gene activity of STAT3 was sharply increased when exposed to VEGF, whereas CTD suppressed STAT3 activity in a dose-dependent manner (Fig. 5a), suggesting that CTD can inhibit VEGF-induced activation of JAK1/STAT3 signaling pathway. Together, CTD has effect on VEGF-induced JAK1/STAT3 signaling pathway and phosphorylation of ERK and AKT.
Discussion In the long history of practice of traditional Chinese medicine, natural products including herbs and animal parts have been used. Among these, many have been proven to have anti-cancer therapeutic effects (Surh 2003). A
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series of recent studies previously combined in vitro and in vivo techniques to explore the effects of compounds extracted from Chinese medicine on angiogenesis, a complex process crucial to tumor growth and metastasis (Dong et al. 2010; Yi et al. 2008). In the present study, we examined the effects of CTD on angiogenesis. Both in vitro results with HUVECs and in vivo results with chick embryo CAM assays demonstrated that CTD also inhibited angiogenesis. This might underlie its anti-cancer effects. This is consistent with a previous in vitro study that reported that, in the highly metastatic ovarian carcinoma cell line HO-8910PM, CTD significantly suppresses cell invasion and metastasis (He et al. 2005). Norcantharidin (NCTD), the demethylated form of CTD, has been shown to be an anti-tumor angiogenesis agent by inhibiting HUVEC cell proliferation, migration, invasion, and tube formation in a dose-dependent manner (Zhang et al. 2013). Our data also showed that CTD has similar effect in angiogenesis. Furthermore, NCTD inhibits tumor angiogenesis via blocking VEGFR2/MEK/ERK signaling pathways, with little effect on the
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Fig. 5 Inhibitory effects of CTD on VEGF-induced STAT3-luciferase activity and the phosphorylation of JAK1, AKT and ERK. a CTD suppressed VEGF-induced the phosphorylation of AKT in a timedependent manner. HMEC cells were incubated with or without CTD (1 lM) for 6 h and then treated with 10 ng/ml VEGF for indicated times. Then cell lysates were subjected to immunoblotting with antibodies as indicated. b CTD inhibited VEGF-induced the phosphorylation of ERK and JAK1 in a dose-dependent manner. HMEC cells were incubated with indicated concentrations of CTD for 3 h and then treated with VEGF (10 ng/ml) for 20 min. Then cell lysates were subjected to immunoblotting with antibodies as indicated. c CTD inhibited VEGF-induced STAT3-luciferase activity in a dosedependent manner. After transfection of HMEC cells with STAT3 luciferased reporter gene (The STAT3-responsive elements linked to a luciferase reporter gene), the cells were incubated with the indicated concentration of CTD for 24 h and then incubated with VEGF (10 ng/ml) for another 24 h. Cell supernatants were collected and assayed for luciferase activity. *P \ 0.05, ***P \ 0.001
phosphorylation of AKT (Zhang et al. 2013). However, as a structure analog of NCTD, CTD suppressed VEGFinduced JAK1/STAT3 signaling pathway and phosphorylation of AKT and ERK, suggesting that the methyl group of CTD is critical for suppressing phosphorylation of AKT, whereas NCTD, which lacks this group, had no activity. How the methyl group of CTD modulates this role is unclear. It is possible that the methyl group of CTD in zerumbone is needed for suppressing the AKT activity.
This is in similar with the derivatives of alkylphosphocholine (Alam et al. 2012). Together, CTD which differs from NCTD, has a different molecular mechanism on angiogenesis. CTD has been reported as an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) (Li and Casida 1992; Honkanen 1993). Protein phosphorylation play an important role in regulating multiple cellular processes, including apoptosis and signal transduction pathways, but the biochemical target of CTD and the mechanism by which CTD inhibits cell growth and causes cell death remain elusive (Wera and Hemmings 1995; Huan et al. 2006). It has been suggested that the inhibitory effect of CTD on ovarian carcinoma cell line might involve down-regulation of NF-kappaB P65 subunit and VEGF (He et al. 2005). In anti-cancer therapy, multidrug resistance of cancer cells presents a serious obstacle to effective treatment (Beck et al. 1979). CTD has an excellent potential as an anti-cancer reagent because it is not involved in any multidrug-resistant phenotype in human leukemia cells (Efferth et al. 2002). In addition, its active concentration is rather low, below 2 lM in MTT assay (Efferth et al. 2002). This is consistent with the observation made in the present study that, at 1 lM, the inhibitory effects of CTD on HUVEC proliferation, migration, and tube formation were highly significant. In conclusion, the present study for the first time used both in vitro and in vivo techniques to demonstrate the inhibitory effects of CTD, a compound extracted from traditional Chinese anti-cancer medicine, on angiogenesis. Its natural source, lack of connection with multidrugresistant cancer cell lines, and low active dose make CTD a promising target for the development of anti-cancer drugs using modern technologies. However, further investigations into the mechanisms underlying its inhibition of angiogenesis must be performed in the near future.
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