PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1246–1251 (2014) Published online 17 February 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5126
Salicin, an Extract from White Willow Bark, Inhibits Angiogenesis by Blocking the ROS-ERK Pathways Chang-Seok Kong,1 Ka-Hyun Kim,2 Jae-Sun Choi,1,4 Ja-Eun Kim,1,3 Chan Park1,4 and Joo-Won Jeong1,2,4* 1
Department of Department of Department of 4 Department of 2 3
Biomedical Science, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Neuroscience, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Pharmacology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea
Salicin has been studied as a potent antiinflammatory agent. Angiogenesis is an essential process for tumor progression, and negative regulation of angiogenesis provides a good strategy for antitumor therapy. However, the potential medicinal value of salicin on antitumorigenic and antiangiogenic effects remain unexplored. In this study, we examined the antitumorigenic and antiangiogenic activity of salicin and its underlying mechanism of action. Salicin suppressed the angiogenic activity of endothelial cells, such as migration, tube formation, and sprouting from an aorta. Moreover, salicin reduced reactive oxygen species production and activation of the extracellular signal-regulated kinase pathway. The expression of vascular endothelial growth factor was also decreased by salicin in endothelial cells. When the salicin was administered to mice, salicin inhibited tumor growth and angiogenesis in a mouse tumor model. Taken together, salicin targets the signaling pathways mediated by reactive oxygen species and extracellular signal-regulated kinase, providing new perspectives into a potent therapeutic agent for hypervascularized tumors. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: salicin; angiogenesis; tumor progression; ROS; ERK.
INTRODUCTION The European Pharmacopoeia defines willow bark as the whole or fragmented dried bark of young branches or dried pieces of current year twigs of various species of the genus Salix (Nahrstedt et al., 2007). Willow bark has been used as a traditional medicine for the treatment of fever, pain, and inflammation (Hedner and Everts, 1998). Salicin, the major constituent of willow bark extract, is metabolized to salicylic acid in vivo and has been considered to play a role in antiinflammatory effects (Schmid et al., 2001). Willow bark suppresses inflammatory molecules and reduces oxidative stress in human endothelial cells (Freischmidt et al., 2012; Ishikado et al., 2012). Moreover, recent studies showed that willow bark extract has anticancer activity in various human cancers, including colon and lung cancers via induction of apoptosis or suppression of proliferation (Bonaterra et al., 2010; Hostanska et al., 2007). According to the recent study, salicin compound from Desmodium gangeticum reduced tumor volume in mice (Srivastava et al., 2013). However, the pharmacological effects of salicin on tumor angiogenesis and the related mechanisms remain unexplored. Angiogenesis is an essential process that supplies sufficient oxygen and nutrients needed for tumor growth (Carmeliet and Jain, 2000). Many growth factors and cytokines are secreted by tumor cells and recruit immune
* Correspondence to: Joo-Won Jeong, School of Medicine, Kyung Hee University, Seoul 130-701, Korea. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
cells to the tumor microenvironment, contributing to angiogenesis. Among various angiogenic factors, vascular endothelial growth factor (VEGF) plays a main role in modulating the steps of angiogenesis, including the migration and tube forming capacity of endothelial cells (Carmeliet, 2005). VEGF is mainly expressed in cancer cells and in activated endothelial cells (ECs) and promotes the angiogenic effects of ECs (Ferrara et al., 2003; Koch et al., 2011; Maxwell and Ratcliffe, 2002). Reactive oxygen species (ROS) are able to activate intracellular pathways, such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase, and p38 kinase, in endothelial cells and stimulate VEGF expression (Milkiewicz et al., 2007; Yoon et al., 2002). In this study, to investigate whether salicin has the antiangiogenic activity in tumor progression, we performed in vitro angiogenesis assays using endothelial cells and applied a mouse tumor model. Our results show that salicin inhibited tumor angiogenesis, suggesting that salicin may lead to the development of a therapeutic strategy for the treatment of solid tumors.
MATERIALS AND METHODS Reagents. Salicin (Fig. 1A) was purchased from SigmaAldrich. Matrigel was purchased from BD Pharmingen.
Cell culture and treatment. ECV304 cells, human umbilical vein endothelial cell-originated cells, were cultured Received 14 November 2013 Revised 23 December 2013 Accepted 10 January 2014
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Figure 1. Salicin decreases the angiogenic activities of endothelial cells. (A) The chemical structure of salicin. (B) ECV304 cells were exposed to the indicated concentrations of salicin for 24 h. Cell viability was examined via a MTT assay. The mean and standard deviation based on three independent experiments are shown. **p < 0.01 compared with the control groups. (C) A wounding migration assay was performed using ECV304 cells after treatment with salicin for 16 h. Scale bar = 100.0 μm. (D) The number of ECV304 cells that migrated beyond the reference line was counted in four independent experiments. **p < 0.01 compared with the control groups. (E) ECV304 cells were incubated on Matrigel for 8 h in the presence or absence of salicin. Then, the capillary-like structures were examined and photographed. Scale bar = 100.0 μm. (F) The total length of the tube-like structures was determined, and the data represent the mean ± standard deviation from three independent experiments. **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
in M199 supplemented with 10% fetal bovine serum and penicillin/streptomycin on 0.3% gelatin-coated dishes. RENCA cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone) and penicillin/streptomycin (Cellgro). The cells were maintained at 37 °C in a humidified 5% CO2 and 95% air incubator.
Wound healing migration assay. Confluent monolayers of cells plated on 24-well dishes were wounded using a micropipette tip and rinsed with phosphate-buffered saline (PBS) to remove detached cells. ECV304 cells were allowed to migrate for 16 h with 1 or 2 mM of salicin, followed by staining with GIEMSA. The number of cells that migrated beyond the reference line was counted. The experiments were independently repeated four times.
Tube formation assay. In total, 150 μL of Matrigel (10 mg/mL) was pipetted onto 48-well culture plates and polymerized for 30 min at 37 °C, and then 6.0 × 104 ECV304 cells were seeded on Matrigel and treated with 1 or 2 mM of salicin. After 8 h, morphologic changes of the cells were observed under a microscope and photographed. To quantify the tube formation of endothelial cells, the total length of tube-like structures was measured using Image J software (National Institute of Health, Bethesda, MD, USA). The experiments were independently repeated three times.
Measurement of reactive oxygen species. ECV304 cells were seeded on cover slips; H2O2 was added with or without 2 mM of salicin. After specific treatment for 1 h, the cells were incubated with 10 μM of 2′,7′dichlorofluorescein diacetate (DCFH-DA) (SigmaCopyright © 2014 John Wiley & Sons, Ltd.
Aldrich) for 30 min and washed twice with PBS. The ROS generation was detected using a fluorescence microscope with Fluorescein isothiocyanate filter. The experiments were independently repeated three times and, the fluorescence signal was measured using Image J software. Western blot analysis. Whole cell lysates were harvested using lysis buffer (40 mM Tris-HCl (pH7.4), 10 mM EDTA, 0.1% NP-40, and 120 mM NaCl) containing protease inhibitor. The lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). The antibodies used for western blotting were as follows: anti-pERK (Cell Signaling Technology), anti-ERK (Santa Cruz Biotechnology), anti-pAKT (Cell Signaling Technology), anti-AKT (Santa Cruz Biotechnology), and anti-α-tubulin (Santa Cruz Biotechnology). Animals and tumor mouse models. Male BALB/c mice (6 weeks old) were purchased from Daehan bio-link (Chungbuk, Korea), and we adhered to the Guide for Animal Experiments edited by the Korean Academy for Medical Sciences. In total, 2 × 106 RENCA cells (a BALB/c-derived renal carcinoma cell line) were mixed with 0.1 mL of Matrigel and injected subcutaneously and one tumor was induced per mouse. After 7 days, the four mice were injected intraperitoneally with either salicin (10 mg/kg) or equal volume of saline once a day for a week. Their body weights and the tumor volumes were measured using a digital balance and a Vernier caliper, respectively, every day. The tumor volume was calculated using the following equation: (mm3) = (0.5[width × length × height]). Four mice were used per each group. The experiments were independently repeated three times. Phytother. Res. 28: 1246–1251 (2014)
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Hemoglobin contents analysis. The RENCA tumors were isolated from the tumor-bearing BALB/c mice, photographed, and chopped into small pieces. The hemoglobin contents were measured using Drabkin’s reagent kit (Sigma-Aldrich) to quantify blood vessel formation. Small pieces of tumors were resuspended in 200 μL of DW and mechanically ground using a homogenizer. The homogenate was centrifuged for 5 min at 5000 rpm. 10 μL of supernatant was added to 100 μL of Drabkin’s reagent/Brij 35 solution, incubated 15 min at room temperature. The hemoglobin contents were determined using a microplate reader set to 540 nm, and the means were normalized to tumor weight. Twelve tumors were used per group.
Immunohistochemistry. The tumors were isolated from the animals, sectioned into 5 μm slices and placed on gelatin-coated slides. The sections were then incubated with PBS containing 0.3% Triton X-100 for 30 min and incubated with a primary antibody against CD31 (BD Pharmingen) at 4 °C overnight. The sections were incubated with biotinylated antibody, and then with the Elite ABC Kit (Vector Laboratories). Lastly, 3,3diaminobenzidine (Sigma-Aldrich) staining was used for the immunodetection of the tumor sections. The intensity of CD31 staining was measured using Image J software (Wayne Rasband).
Aortic ring sprouting assay. Aortas were excised from 6-week-old male Sprague–Dawley rats (Daehan BioLink, Chungbuk, Korea), and the fibroadipose tissue surrounding the aorta was removed. The aortas were sectioned into 1 mm slices, and then aortic rings were placed on Matrigel-coated wells, covered with an additional 50 μL Matrigel, and allowed to gel for 30 min at 37 °C. The equal volume of PBS or 3.5 mM of salicin were added to each well. Five days after treatment with salicin, microvessel outgrowth was photographed under a phase contrast microscope. The aortic ring sprouts were quantified using MultiScan-IP software (Interactive Technologies International). The experiments were independently repeated three times.
Statistical analysis. Analysis of variance tests were performed to assess significant differences between control and experimental groups. The level of significance was set at p < 0.01 or p < 0.05. Results are presented as the mean ± standard deviation.
RESULTS
migration is essential for angiogenesis, we next investigated the effects of salicin on the migratory properties of ECV304 cells using a wound healing migration assay. The migratory properties of ECV304 cells were reduced after treatment with salicin (Fig. 1C and D). Moreover, we found that salicin dose-dependently inhibited tubular formation of ECV304 cells (Fig. 1E and F). These results indicate that salicin significantly inhibits angiogenic activities of endothelial cells. Salicin inhibits reactive oxygen species production and the extracellular signal-regulated kinase pathway Reactive oxygen species play a critical role in tumor angiogenesis and regulate intracellular signaling pathways (Ushio-Fukai and Nakamura, 2008). To understand the mechanism of salicin on antiangiogenic and antitumorigenic effects, we determined the ROS levels in endothelial cells. As shown in Fig. 2A and B, oxidative stress was induced by treatment with H2O2 and intracellular ROS production was significantly inhibited by pretreatment with salicin in endothelial cells. During tumor angiogenesis, the ERK and/or AKT pathway is mainly activated by various stimuli (Chung et al., 2008; Dai et al., 2009; Park et al., 2009; Wang et al., 2008). Moreover, oxidative stress is one of the regulators of the ERK and AKT pathways (Hu et al., 2005; Wang et al., 2011; Wu et al., 2008). To examine whether decreased ROS by salicin can influence ERK and AKT activities, we determined the phosphorylation status of ERK and AKT. As shown in Fig. 2C, ERK (p42) phosphorylation was decreased by treatment with salicin, whereas AKT phosphorylation was not changed. When the expression of VEGF was tested, we found that salicin reduced the mRNA expression of VEGF in endothelial cells (Fig. 2D). From these results, we propose that decreased ROS levels by salicin inhibit the ERK signaling pathway in endothelial cells. Salicin inhibits tumor progression in mice To investigate the effects of salicin on tumor growth, we used a mouse tumor model with BALB/c mice and RENCA cells. Tumor growth was inhibited by the administration of salicin, and the tumor sizes gradually decreased (Fig. 3A). In this condition, administration of salicin did not affect the body weights of the mice (Fig. 3B), suggesting that this treated dose of salicin (10 mg/kg/day) was non-toxic for the mice. When the tumors were isolated from the mice, the tumor volume and weights in salicin-treated mice were significantly lower than those of the control mice (Fig. 3C and D), indicating that salicin had an inhibitory effect on the tumor growth.
Salicin decreases angiogenic activities of endothelial cells Salicin suppresses tumor angiogenesis To assess the antiangiogenic activity of salicin, in vitro angiogenesis models were used. ECV304 cells were treated with various concentrations of salicin for 24 h, and the viability was determined by a MTT assay. As shown in Fig. 1B, salicin had no effect on the cell viability up to 2 mM, whereas 5 mM of salicin induced cytotoxicity in ECV304 cells. Because endothelial cell Copyright © 2014 John Wiley & Sons, Ltd.
When the tumor tissues were stained with hematoxylin and eosin, we found that the density of tumor cells was decreased by treatment with salicin (Fig. 4A). To examine the level of angiogenesis during tumor progression, the hemoglobin contents of the tumors were measured. As shown in Fig. 4B, treatment with salicin significantly Phytother. Res. 28: 1246–1251 (2014)
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Figure 2. Salicin reduces oxidative stress and extracellular signal-regulated kinase (ERK) activation in endothelial cells. (A) ECV304 cells were treated with or without 2 mM of salicin for 1 h. Intracellular reactive oxygen species production was detected using 2′,7′-dichlorofluorescein diacetate. Scale bar = 200.0 μm. (B) The relative reactive oxygen species generation was measured in three independent experiments. The fluorescence intensity under control was set to 100%. **p < 0.01. (C) The levels of phospho-AKT, total AKT, phospho-ERK (p42), and total ERK (p42) were determined by western blotting using their corresponding antibodies. (D) Reverse transcriptase polymerase chain reaction was performed using specific primers for vascular endothelial growth factor (VEGF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
Figure 3. Salicin suppresses tumor progression in a mouse model. (A, B) BALB/c mice were injected subcutaneously with RENCA cells. After 7 days, the four mice were injected with salicin (10 mg/kg) intraperitoneally once a day for a week. The tumor volume and body weights were measured every day. The experiments were independently repeated three times with four mice per group. The mean ± standard deviation based on 12 mice per group are shown. *p < 0.05. (C) The mice were sacrificed on day 14, and the tumors were isolated from the mice. Scale bar = 0.5 cm. (D) The tumor volumes and weights were examined. The data are presented as the means ± standard deviation from 12 tumors per group. *p < 0.05, **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
decreased the level of hemoglobin within the tumors. Moreover, the expression of CD31, a specific marker for endothelial cells, was also decreased by salicin treatment (Fig. 4C and D), suggesting that salicin inhibits tumor progression by reducing angiogenesis within the tumor. To confirm the antiangiogenic effect of salicin, we performed a rat aortic ring sprouting assay. Salicin treatment reduced microvessel sprouting from rat aortic rings (Fig. 4E and F). Taken together, these Copyright © 2014 John Wiley & Sons, Ltd.
results indicate that salicin strongly decreases angiogenesis during tumor progression.
DISCUSSION In this study, we provide evidence that salicin has potent antiangiogenic and antitumor activities in vitro and Phytother. Res. 28: 1246–1251 (2014)
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Figure 4. Salicin reduces tumor angiogenesis. (A) The tumor sections were stained using hematoxylin and eosin. Scale bar = 100.0 μm. (B) The hemoglobin concentration within the tumors was measured using Drabkin’s reagent. Data are presented as the means ± standard deviation from 12 tumors per group. **p < 0.01 compared with the control groups. (C) Microvessels were visualized in tumor sections by staining with an antibody against CD31, a specific marker of endothelial cells. Scale bar = 100.0 μm. (D) Data represent the mean ± standard deviation from three independent experiments. p < 0.01 compared with the control groups. (E) Rat aortas grown in Matrigel were treated with or without 3.5 mM salicin. After 3–4 days, aortic rings were photographed under a microscope. Scale bar = 200.0 μm. (F) The experiments were independently repeated three times, and relative sprouting cell densities (% of control) were measured. **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
in vivo. Salicin is used as a precursor for the synthesis of salicylic acid and aspirin. According to several reports, aspirin and salicylic acid can inhibit VEGF signaling and angiogenesis (Borthwick et al., 2006; Khaidakov et al., 2010; Zhang et al., 2013). Aspirin is rapidly hydrolyzed to salicylate in contact with water. Salicylate is known to activate the p38 MAP kinase stress pathway and inhibit the ERK survival pathway (Wang and Brecher, 1999). Reactive oxygen species act as key regulators of cellular signaling induced by various stimuli and regulate various cellular events. Notably, increased intracellular ROS levels in tumors stimulate the induction of VEGF and promote angiogenic events, including cell proliferation, migration, cytoskeletal reorganization, and tube formation of endothelial cells (Luczak et al., 2004; Shono et al., 1996; Vepa et al., 1999; Yasuda et al., 1999). Many food-derived chemicals have demonstrated antiangiogenic activity by reducing oxidative stress and the relative signaling pathways (Gupta et al., 2010). Willow bark extract has also reduced oxidative stress and suppressed inflammatory reactions (Freischmidt et al., 2012; Ishikado et al., 2012). Collectively, we assumed that salicin suppressed tumor angiogenesis through reducing ROS production in the tumor microenvironment. Because activation of the HIF-1α-VEGF axis plays a critical role during tumor angiogenesis, inhibiting hypoxia-induced tumor angiogenesis is believed to be a potential strategy to attenuate tumorigenesis (Nordgren and Tavassoli, 2011). Moreover, many anticancer drugs inhibit the stability or the expression of HIF-1α, and inhibit the function of HIF-1 in tumor progression. When we tested whether salicin affected the level of HIF-1α in endothelial cells and RENCA cells, we found that salicin did not change the level of HIF-1α protein (data not shown). However, salicin inhibited VEGF expression (Fig. 4C), suggesting that Copyright © 2014 John Wiley & Sons, Ltd.
salicin decreased the expression of VEGF independent of HIF-1α protein expression. Sp1 is also a key modulator for VEGF expression and, as a consequence, is a potent regulator of angiogenesis. VEGF expression can be upregulated through the activation of various tyrosine kinase receptors, including the EGF family, insulin and IGF receptors, PDGF receptors, and FGF receptors (Pages and Pouyssegur, 2005). In ovarian cells, constitutively active forms of PI3-kinase increase the expression of VEGF, thus facilitating their expansion by up-regulating angiogenesis (Zhang et al., 2003). The ERK pathway has been to be essential for VEGF up-regulation in fibroblasts, and the PI3-kinase pathway is also implicated in VEGF expression in epithelial cells (Rak et al., 2000). From our data, salicin seems to decrease VEGF expression in a HIF-1α-independent manner by inactivating the ERK pathway. Activation of the ERK and AKT pathways are essential events for numerous cellular functions including angiogenesis and tumorigenesis (Jiang and Liu, 2009; Yang et al., 2008). Salicin markedly reduced the activation of ERK and VEGF expression in endothelial cells (Fig. 4B and C). Therefore, salicin may potentially prevent cancer development, and a key mechanism of inhibiting tumor angiogenesis by salicin might be, in part, through gene translation machineries. However, a more precise understanding of salicin’s mechanism of action in inhibiting the ERK pathway and its relevance in cancer treatment require further study. In conclusion, salicin, a natural product from foods, inhibits tumor progression and angiogenesis. Moreover, this inhibitory effect is mediated by reducing ROS production and ERK activation, suggesting that salicin can be a potent therapeutic agent for angiogenic disorders, such as cancer. The novel findings presented here provide new insight into the antiangiogenic Phytother. Res. 28: 1246–1251 (2014)
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properties of salicin and its potential use for cancer prevention and treatment.
Acknowledgements
funded by the Ministry of Education, Science and Technology (2012R1A1A2039164).
Conflict of Interest
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF)
The authors have no conflicts of interest to disclose.
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Salicin, an Extract from White Willow Bark, Inhibits Angiogenesis by Blocking the ROS-ERK Pathways Chang-Seok Kong,1 Ka-Hyun Kim,2 Jae-Sun Choi,1,4 Ja-Eun Kim,1,3 Chan Park1,4 and Joo-Won Jeong1,2,4* 1
Department of Department of Department of 4 Department of 2 3
Biomedical Science, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Neuroscience, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Pharmacology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea Anatomy and Neurobiology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea
Salicin has been studied as a potent antiinflammatory agent. Angiogenesis is an essential process for tumor progression, and negative regulation of angiogenesis provides a good strategy for antitumor therapy. However, the potential medicinal value of salicin on antitumorigenic and antiangiogenic effects remain unexplored. In this study, we examined the antitumorigenic and antiangiogenic activity of salicin and its underlying mechanism of action. Salicin suppressed the angiogenic activity of endothelial cells, such as migration, tube formation, and sprouting from an aorta. Moreover, salicin reduced reactive oxygen species production and activation of the extracellular signal-regulated kinase pathway. The expression of vascular endothelial growth factor was also decreased by salicin in endothelial cells. When the salicin was administered to mice, salicin inhibited tumor growth and angiogenesis in a mouse tumor model. Taken together, salicin targets the signaling pathways mediated by reactive oxygen species and extracellular signal-regulated kinase, providing new perspectives into a potent therapeutic agent for hypervascularized tumors. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: salicin; angiogenesis; tumor progression; ROS; ERK.
INTRODUCTION The European Pharmacopoeia defines willow bark as the whole or fragmented dried bark of young branches or dried pieces of current year twigs of various species of the genus Salix (Nahrstedt et al., 2007). Willow bark has been used as a traditional medicine for the treatment of fever, pain, and inflammation (Hedner and Everts, 1998). Salicin, the major constituent of willow bark extract, is metabolized to salicylic acid in vivo and has been considered to play a role in antiinflammatory effects (Schmid et al., 2001). Willow bark suppresses inflammatory molecules and reduces oxidative stress in human endothelial cells (Freischmidt et al., 2012; Ishikado et al., 2012). Moreover, recent studies showed that willow bark extract has anticancer activity in various human cancers, including colon and lung cancers via induction of apoptosis or suppression of proliferation (Bonaterra et al., 2010; Hostanska et al., 2007). According to the recent study, salicin compound from Desmodium gangeticum reduced tumor volume in mice (Srivastava et al., 2013). However, the pharmacological effects of salicin on tumor angiogenesis and the related mechanisms remain unexplored. Angiogenesis is an essential process that supplies sufficient oxygen and nutrients needed for tumor growth (Carmeliet and Jain, 2000). Many growth factors and cytokines are secreted by tumor cells and recruit immune
* Correspondence to: Joo-Won Jeong, School of Medicine, Kyung Hee University, Seoul 130-701, Korea. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
cells to the tumor microenvironment, contributing to angiogenesis. Among various angiogenic factors, vascular endothelial growth factor (VEGF) plays a main role in modulating the steps of angiogenesis, including the migration and tube forming capacity of endothelial cells (Carmeliet, 2005). VEGF is mainly expressed in cancer cells and in activated endothelial cells (ECs) and promotes the angiogenic effects of ECs (Ferrara et al., 2003; Koch et al., 2011; Maxwell and Ratcliffe, 2002). Reactive oxygen species (ROS) are able to activate intracellular pathways, such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase, and p38 kinase, in endothelial cells and stimulate VEGF expression (Milkiewicz et al., 2007; Yoon et al., 2002). In this study, to investigate whether salicin has the antiangiogenic activity in tumor progression, we performed in vitro angiogenesis assays using endothelial cells and applied a mouse tumor model. Our results show that salicin inhibited tumor angiogenesis, suggesting that salicin may lead to the development of a therapeutic strategy for the treatment of solid tumors.
MATERIALS AND METHODS Reagents. Salicin (Fig. 1A) was purchased from SigmaAldrich. Matrigel was purchased from BD Pharmingen.
Cell culture and treatment. ECV304 cells, human umbilical vein endothelial cell-originated cells, were cultured Received 14 November 2013 Revised 23 December 2013 Accepted 10 January 2014
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Figure 1. Salicin decreases the angiogenic activities of endothelial cells. (A) The chemical structure of salicin. (B) ECV304 cells were exposed to the indicated concentrations of salicin for 24 h. Cell viability was examined via a MTT assay. The mean and standard deviation based on three independent experiments are shown. **p < 0.01 compared with the control groups. (C) A wounding migration assay was performed using ECV304 cells after treatment with salicin for 16 h. Scale bar = 100.0 μm. (D) The number of ECV304 cells that migrated beyond the reference line was counted in four independent experiments. **p < 0.01 compared with the control groups. (E) ECV304 cells were incubated on Matrigel for 8 h in the presence or absence of salicin. Then, the capillary-like structures were examined and photographed. Scale bar = 100.0 μm. (F) The total length of the tube-like structures was determined, and the data represent the mean ± standard deviation from three independent experiments. **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
in M199 supplemented with 10% fetal bovine serum and penicillin/streptomycin on 0.3% gelatin-coated dishes. RENCA cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone) and penicillin/streptomycin (Cellgro). The cells were maintained at 37 °C in a humidified 5% CO2 and 95% air incubator.
Wound healing migration assay. Confluent monolayers of cells plated on 24-well dishes were wounded using a micropipette tip and rinsed with phosphate-buffered saline (PBS) to remove detached cells. ECV304 cells were allowed to migrate for 16 h with 1 or 2 mM of salicin, followed by staining with GIEMSA. The number of cells that migrated beyond the reference line was counted. The experiments were independently repeated four times.
Tube formation assay. In total, 150 μL of Matrigel (10 mg/mL) was pipetted onto 48-well culture plates and polymerized for 30 min at 37 °C, and then 6.0 × 104 ECV304 cells were seeded on Matrigel and treated with 1 or 2 mM of salicin. After 8 h, morphologic changes of the cells were observed under a microscope and photographed. To quantify the tube formation of endothelial cells, the total length of tube-like structures was measured using Image J software (National Institute of Health, Bethesda, MD, USA). The experiments were independently repeated three times.
Measurement of reactive oxygen species. ECV304 cells were seeded on cover slips; H2O2 was added with or without 2 mM of salicin. After specific treatment for 1 h, the cells were incubated with 10 μM of 2′,7′dichlorofluorescein diacetate (DCFH-DA) (SigmaCopyright © 2014 John Wiley & Sons, Ltd.
Aldrich) for 30 min and washed twice with PBS. The ROS generation was detected using a fluorescence microscope with Fluorescein isothiocyanate filter. The experiments were independently repeated three times and, the fluorescence signal was measured using Image J software. Western blot analysis. Whole cell lysates were harvested using lysis buffer (40 mM Tris-HCl (pH7.4), 10 mM EDTA, 0.1% NP-40, and 120 mM NaCl) containing protease inhibitor. The lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). The antibodies used for western blotting were as follows: anti-pERK (Cell Signaling Technology), anti-ERK (Santa Cruz Biotechnology), anti-pAKT (Cell Signaling Technology), anti-AKT (Santa Cruz Biotechnology), and anti-α-tubulin (Santa Cruz Biotechnology). Animals and tumor mouse models. Male BALB/c mice (6 weeks old) were purchased from Daehan bio-link (Chungbuk, Korea), and we adhered to the Guide for Animal Experiments edited by the Korean Academy for Medical Sciences. In total, 2 × 106 RENCA cells (a BALB/c-derived renal carcinoma cell line) were mixed with 0.1 mL of Matrigel and injected subcutaneously and one tumor was induced per mouse. After 7 days, the four mice were injected intraperitoneally with either salicin (10 mg/kg) or equal volume of saline once a day for a week. Their body weights and the tumor volumes were measured using a digital balance and a Vernier caliper, respectively, every day. The tumor volume was calculated using the following equation: (mm3) = (0.5[width × length × height]). Four mice were used per each group. The experiments were independently repeated three times. Phytother. Res. 28: 1246–1251 (2014)
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Hemoglobin contents analysis. The RENCA tumors were isolated from the tumor-bearing BALB/c mice, photographed, and chopped into small pieces. The hemoglobin contents were measured using Drabkin’s reagent kit (Sigma-Aldrich) to quantify blood vessel formation. Small pieces of tumors were resuspended in 200 μL of DW and mechanically ground using a homogenizer. The homogenate was centrifuged for 5 min at 5000 rpm. 10 μL of supernatant was added to 100 μL of Drabkin’s reagent/Brij 35 solution, incubated 15 min at room temperature. The hemoglobin contents were determined using a microplate reader set to 540 nm, and the means were normalized to tumor weight. Twelve tumors were used per group.
Immunohistochemistry. The tumors were isolated from the animals, sectioned into 5 μm slices and placed on gelatin-coated slides. The sections were then incubated with PBS containing 0.3% Triton X-100 for 30 min and incubated with a primary antibody against CD31 (BD Pharmingen) at 4 °C overnight. The sections were incubated with biotinylated antibody, and then with the Elite ABC Kit (Vector Laboratories). Lastly, 3,3diaminobenzidine (Sigma-Aldrich) staining was used for the immunodetection of the tumor sections. The intensity of CD31 staining was measured using Image J software (Wayne Rasband).
Aortic ring sprouting assay. Aortas were excised from 6-week-old male Sprague–Dawley rats (Daehan BioLink, Chungbuk, Korea), and the fibroadipose tissue surrounding the aorta was removed. The aortas were sectioned into 1 mm slices, and then aortic rings were placed on Matrigel-coated wells, covered with an additional 50 μL Matrigel, and allowed to gel for 30 min at 37 °C. The equal volume of PBS or 3.5 mM of salicin were added to each well. Five days after treatment with salicin, microvessel outgrowth was photographed under a phase contrast microscope. The aortic ring sprouts were quantified using MultiScan-IP software (Interactive Technologies International). The experiments were independently repeated three times.
Statistical analysis. Analysis of variance tests were performed to assess significant differences between control and experimental groups. The level of significance was set at p < 0.01 or p < 0.05. Results are presented as the mean ± standard deviation.
RESULTS
migration is essential for angiogenesis, we next investigated the effects of salicin on the migratory properties of ECV304 cells using a wound healing migration assay. The migratory properties of ECV304 cells were reduced after treatment with salicin (Fig. 1C and D). Moreover, we found that salicin dose-dependently inhibited tubular formation of ECV304 cells (Fig. 1E and F). These results indicate that salicin significantly inhibits angiogenic activities of endothelial cells. Salicin inhibits reactive oxygen species production and the extracellular signal-regulated kinase pathway Reactive oxygen species play a critical role in tumor angiogenesis and regulate intracellular signaling pathways (Ushio-Fukai and Nakamura, 2008). To understand the mechanism of salicin on antiangiogenic and antitumorigenic effects, we determined the ROS levels in endothelial cells. As shown in Fig. 2A and B, oxidative stress was induced by treatment with H2O2 and intracellular ROS production was significantly inhibited by pretreatment with salicin in endothelial cells. During tumor angiogenesis, the ERK and/or AKT pathway is mainly activated by various stimuli (Chung et al., 2008; Dai et al., 2009; Park et al., 2009; Wang et al., 2008). Moreover, oxidative stress is one of the regulators of the ERK and AKT pathways (Hu et al., 2005; Wang et al., 2011; Wu et al., 2008). To examine whether decreased ROS by salicin can influence ERK and AKT activities, we determined the phosphorylation status of ERK and AKT. As shown in Fig. 2C, ERK (p42) phosphorylation was decreased by treatment with salicin, whereas AKT phosphorylation was not changed. When the expression of VEGF was tested, we found that salicin reduced the mRNA expression of VEGF in endothelial cells (Fig. 2D). From these results, we propose that decreased ROS levels by salicin inhibit the ERK signaling pathway in endothelial cells. Salicin inhibits tumor progression in mice To investigate the effects of salicin on tumor growth, we used a mouse tumor model with BALB/c mice and RENCA cells. Tumor growth was inhibited by the administration of salicin, and the tumor sizes gradually decreased (Fig. 3A). In this condition, administration of salicin did not affect the body weights of the mice (Fig. 3B), suggesting that this treated dose of salicin (10 mg/kg/day) was non-toxic for the mice. When the tumors were isolated from the mice, the tumor volume and weights in salicin-treated mice were significantly lower than those of the control mice (Fig. 3C and D), indicating that salicin had an inhibitory effect on the tumor growth.
Salicin decreases angiogenic activities of endothelial cells Salicin suppresses tumor angiogenesis To assess the antiangiogenic activity of salicin, in vitro angiogenesis models were used. ECV304 cells were treated with various concentrations of salicin for 24 h, and the viability was determined by a MTT assay. As shown in Fig. 1B, salicin had no effect on the cell viability up to 2 mM, whereas 5 mM of salicin induced cytotoxicity in ECV304 cells. Because endothelial cell Copyright © 2014 John Wiley & Sons, Ltd.
When the tumor tissues were stained with hematoxylin and eosin, we found that the density of tumor cells was decreased by treatment with salicin (Fig. 4A). To examine the level of angiogenesis during tumor progression, the hemoglobin contents of the tumors were measured. As shown in Fig. 4B, treatment with salicin significantly Phytother. Res. 28: 1246–1251 (2014)
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Figure 2. Salicin reduces oxidative stress and extracellular signal-regulated kinase (ERK) activation in endothelial cells. (A) ECV304 cells were treated with or without 2 mM of salicin for 1 h. Intracellular reactive oxygen species production was detected using 2′,7′-dichlorofluorescein diacetate. Scale bar = 200.0 μm. (B) The relative reactive oxygen species generation was measured in three independent experiments. The fluorescence intensity under control was set to 100%. **p < 0.01. (C) The levels of phospho-AKT, total AKT, phospho-ERK (p42), and total ERK (p42) were determined by western blotting using their corresponding antibodies. (D) Reverse transcriptase polymerase chain reaction was performed using specific primers for vascular endothelial growth factor (VEGF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
Figure 3. Salicin suppresses tumor progression in a mouse model. (A, B) BALB/c mice were injected subcutaneously with RENCA cells. After 7 days, the four mice were injected with salicin (10 mg/kg) intraperitoneally once a day for a week. The tumor volume and body weights were measured every day. The experiments were independently repeated three times with four mice per group. The mean ± standard deviation based on 12 mice per group are shown. *p < 0.05. (C) The mice were sacrificed on day 14, and the tumors were isolated from the mice. Scale bar = 0.5 cm. (D) The tumor volumes and weights were examined. The data are presented as the means ± standard deviation from 12 tumors per group. *p < 0.05, **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
decreased the level of hemoglobin within the tumors. Moreover, the expression of CD31, a specific marker for endothelial cells, was also decreased by salicin treatment (Fig. 4C and D), suggesting that salicin inhibits tumor progression by reducing angiogenesis within the tumor. To confirm the antiangiogenic effect of salicin, we performed a rat aortic ring sprouting assay. Salicin treatment reduced microvessel sprouting from rat aortic rings (Fig. 4E and F). Taken together, these Copyright © 2014 John Wiley & Sons, Ltd.
results indicate that salicin strongly decreases angiogenesis during tumor progression.
DISCUSSION In this study, we provide evidence that salicin has potent antiangiogenic and antitumor activities in vitro and Phytother. Res. 28: 1246–1251 (2014)
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Figure 4. Salicin reduces tumor angiogenesis. (A) The tumor sections were stained using hematoxylin and eosin. Scale bar = 100.0 μm. (B) The hemoglobin concentration within the tumors was measured using Drabkin’s reagent. Data are presented as the means ± standard deviation from 12 tumors per group. **p < 0.01 compared with the control groups. (C) Microvessels were visualized in tumor sections by staining with an antibody against CD31, a specific marker of endothelial cells. Scale bar = 100.0 μm. (D) Data represent the mean ± standard deviation from three independent experiments. p < 0.01 compared with the control groups. (E) Rat aortas grown in Matrigel were treated with or without 3.5 mM salicin. After 3–4 days, aortic rings were photographed under a microscope. Scale bar = 200.0 μm. (F) The experiments were independently repeated three times, and relative sprouting cell densities (% of control) were measured. **p < 0.01 compared with the control groups. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
in vivo. Salicin is used as a precursor for the synthesis of salicylic acid and aspirin. According to several reports, aspirin and salicylic acid can inhibit VEGF signaling and angiogenesis (Borthwick et al., 2006; Khaidakov et al., 2010; Zhang et al., 2013). Aspirin is rapidly hydrolyzed to salicylate in contact with water. Salicylate is known to activate the p38 MAP kinase stress pathway and inhibit the ERK survival pathway (Wang and Brecher, 1999). Reactive oxygen species act as key regulators of cellular signaling induced by various stimuli and regulate various cellular events. Notably, increased intracellular ROS levels in tumors stimulate the induction of VEGF and promote angiogenic events, including cell proliferation, migration, cytoskeletal reorganization, and tube formation of endothelial cells (Luczak et al., 2004; Shono et al., 1996; Vepa et al., 1999; Yasuda et al., 1999). Many food-derived chemicals have demonstrated antiangiogenic activity by reducing oxidative stress and the relative signaling pathways (Gupta et al., 2010). Willow bark extract has also reduced oxidative stress and suppressed inflammatory reactions (Freischmidt et al., 2012; Ishikado et al., 2012). Collectively, we assumed that salicin suppressed tumor angiogenesis through reducing ROS production in the tumor microenvironment. Because activation of the HIF-1α-VEGF axis plays a critical role during tumor angiogenesis, inhibiting hypoxia-induced tumor angiogenesis is believed to be a potential strategy to attenuate tumorigenesis (Nordgren and Tavassoli, 2011). Moreover, many anticancer drugs inhibit the stability or the expression of HIF-1α, and inhibit the function of HIF-1 in tumor progression. When we tested whether salicin affected the level of HIF-1α in endothelial cells and RENCA cells, we found that salicin did not change the level of HIF-1α protein (data not shown). However, salicin inhibited VEGF expression (Fig. 4C), suggesting that Copyright © 2014 John Wiley & Sons, Ltd.
salicin decreased the expression of VEGF independent of HIF-1α protein expression. Sp1 is also a key modulator for VEGF expression and, as a consequence, is a potent regulator of angiogenesis. VEGF expression can be upregulated through the activation of various tyrosine kinase receptors, including the EGF family, insulin and IGF receptors, PDGF receptors, and FGF receptors (Pages and Pouyssegur, 2005). In ovarian cells, constitutively active forms of PI3-kinase increase the expression of VEGF, thus facilitating their expansion by up-regulating angiogenesis (Zhang et al., 2003). The ERK pathway has been to be essential for VEGF up-regulation in fibroblasts, and the PI3-kinase pathway is also implicated in VEGF expression in epithelial cells (Rak et al., 2000). From our data, salicin seems to decrease VEGF expression in a HIF-1α-independent manner by inactivating the ERK pathway. Activation of the ERK and AKT pathways are essential events for numerous cellular functions including angiogenesis and tumorigenesis (Jiang and Liu, 2009; Yang et al., 2008). Salicin markedly reduced the activation of ERK and VEGF expression in endothelial cells (Fig. 4B and C). Therefore, salicin may potentially prevent cancer development, and a key mechanism of inhibiting tumor angiogenesis by salicin might be, in part, through gene translation machineries. However, a more precise understanding of salicin’s mechanism of action in inhibiting the ERK pathway and its relevance in cancer treatment require further study. In conclusion, salicin, a natural product from foods, inhibits tumor progression and angiogenesis. Moreover, this inhibitory effect is mediated by reducing ROS production and ERK activation, suggesting that salicin can be a potent therapeutic agent for angiogenic disorders, such as cancer. The novel findings presented here provide new insight into the antiangiogenic Phytother. Res. 28: 1246–1251 (2014)
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properties of salicin and its potential use for cancer prevention and treatment.
Acknowledgements
funded by the Ministry of Education, Science and Technology (2012R1A1A2039164).
Conflict of Interest
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF)
The authors have no conflicts of interest to disclose.
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