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Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation
Acta Histochemica 114 (2012) 94–100
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Acta Histochemica journal homepage: www.elsevier.de/acthis
Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation Mehmet S¸ahin a , Emel S¸ahin b , Saadet Gümüs¸lü c,∗ a b c
Health Sciences Research Centre, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey Central Laboratory, Clinical Biochemistry Unit, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey Department of Biochemistry, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey
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Article history: Received 28 December 2010 Received in revised form 4 March 2011 Accepted 6 March 2011
Keywords: Angiogenesis Lycopene Apigenin Cancer HUVEC
a b s t r a c t Angiogenesis is the formation process of new blood vessels from preexisting vessels. Solid tumors need angiogenesis for growth and metastasis. The suppression of tumor growth by inhibition of neoangiogenic processes represents a potential approach to cancer treatment. Lycopene has powerful antioxidant capacities and anticarcinogenic properties. The aim of this study was to investigate the effects of lycopene on angiogenesis in vitro. For this reason, we measured in vitro angiogenesis in human umbilical vein endothelial cells including parameters of cell proliferation, tube formation, cell migration. Lycopene and apigenin were observed to block the endothelial cell proliferation in a dose-dependent manner. In addition, they significantly decreased the capillary-like tube lengths, tube formation and endothelial cell migration. This study provides indications that apigenin and lycopene, which are considered as chemopreventive agents, to be effective in vitro on endothelial cells and angiogenesis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Angiogenesis, the formation of new blood vessels, plays several roles in various human pathologies including: rheumatoid arthritis, diabetic retinopathy, atherosclerosis, psoriasis, and chronic airway inflammation. One of the most important roles of angiogenesis is to support tumor growth, which is dependent on nutritional support derived from the local blood supply (Sahin et al., 2009). Targeting inhibition of angiogenesis represents a potential approach in the treatment of solid tumors and such antiangio-
Abbreviations: BHT, butylated hydroxytoluene; DMSO, dimethyl sulfoxide; EBM-2, endothelial basal medium; EGM-2, endothelial growth medium; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Hep3B, a human hepatoma cell line; HUVEC, human umbilical vein endothelial cells; IGF1, insulin-like growth factor; KB-1, a cell line derived from a human oral cavity tumor; LNCaP, an androgen-sensitive human prostate adenocarcinoma cell line; MCF-7, a breast cancer cell line; MDA-MB-231, an estrogen-independent human breast cancer cell line; MMP, matrix metalloproteinase; MTT, (3 (4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide); PC3, a metastatic cell line derived from advanced androgen independent prostate cancer; RPMI, Roswell Park Memorial Institute cell culture medium; SK-Hep1, an immortal and metastatic cell line derived from a liver adenocarcinoma; THF, tetrahydrofuran; VEGF, vascular endothelial growth factor. ∗ Corresponding author. Tel.: +90 2422496896; fax: +90 2422274495. E-mail addresses: [email protected] (M. S¸ahin), [email protected] (E. S¸ahin), [email protected] (S. Gümüs¸lü). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.03.004
genic strategies inhibiting the growth of endothelial cells may be more advantageous than targeting cancer cells. The inhibition of tumor growth requires a chronic inhibition of vascular organization, defined as ‘angiostasis’ (Hanahan and Folkman, 1996). Long-term treatment is necessary to achieve angiostasis and it is necessary to identify and characterize angiostatic molecules showing low or no toxicity. Current dietary guidelines to combat chronic diseases including cancer, recommend increased intake of plant foods that are rich in antioxidants. The role of dietary antioxidants, including carotenoids and flavonoids in disease prevention has received much attention (Kim et al., 1998; Agarwal and Rao, 2000). Apigenin is a flavonoid with anti-inflammatory, anticarcinogenic and free radical scavenger properties (Kim et al., 1998). Lycopene, one of more than 600 carotenoids, is a natural pigment synthesized by plants and photosynthetic microorganisms. Several epidemiological studies have strongly implied that consumption of foods containing high concentrations of lycopene may reduce the risk of certain types of cancer (Gann et al., 1999; Giovannucci et al., 2002). The mechanism of lycopene action is still under investigation. Lycopene was shown to induce apoptosis in a dose-dependent manner in prostate cancer cells (Hantz et al., 2005). Similarly, treatment of human colon carcinoma cells with lycopene at 2.0 or 4.0 M was found to be able to induce apoptosis (Salman et al., 2007). There have been several reports that lycopene can induce cell cycle arrest. It has been reported that the growth of Hep3B human hepatoma cells was inhibited 20–50% by lycopene (Park et al., 2005). A simi-
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Fig. 1. Lycopene inhibited tube formation produced by human umbilical vein endothelial cells. Representative micrographs of HUVECs cultured for 24 h on Matrigel in the presence of the indicated compound are illustrated. Serum-starved cells were seeded to each well of 24-well plate as ∼2.5 × 104 cells in EGM-2 complete medium. HUVEC cells were incubated in (A) THF (Control), (B) Lyc 1 M, (C) Lyc 5 M and (D) Lyc 10 M. Scale bar = 250 m.
lar study with the human prostate cancer cell lines, LNCaP and PC3, also found that lycopene induced mitotic arrest at the G0/G1 phase (Ivanov et al., 2007). With respect to anti-invasive and anti-metastatic activities, lycopene (5–10 M) has been shown to decrease the activities of the gelatinolytic matrix metalloproteinases, MMP-2 and MMP-9, and to inhibit adhesion, invasion and migration of SK-Hep1 cells, which is a highly invasive human hepatoma cell line (Hwang and Lee, 2006). Lycopene at similar concentrations was also found to induce the metastasis suppressor gene nm23-H1 (Huang et al., 2005). Although there have been several studies based on the antioxidant and anticarcinogenic activities of lycopene, its role on endothelial cells and angiogenesis has remained unclear. The aim of this study was to investigate whether lycopene is effective on endothelial cells and angiogenesis inhibition at different concentrations, and to compare the effects of lycopene with apigenin with regard to antiangiogenic activities. Materials and methods Reagents Human umbilical vein endothelial cells (HUVECs) and MatrigelTM were purchased from BD Biosciences (BD-354234; Bedford, MA, USA). EGM-2® BulletKit® (CC-3162, EBM2 + supplements) for HUVEC culture was purchased from Lonza (Walkersville, MD, USA). Lycopene and apigenin were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Cell culture Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 complete medium consisting of EBM-2 basal medium supplemented with hydrocortisone, ascorbic acid, heparin, GA-1000 and 2% FBS plus growth factors (epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1)) except for serum starvation in which FBS concentration was reduced to 0.1% and no growth factors were added. Cell culture flasks or well plates, pre-coated with gelatin, were used for culture of HUVECs (Sigma–Aldrich, Irvine, Ayrshire, UK). All cells were grown in a humidified atmosphere, 95% air and 5% CO2 at 37 ◦ C and passaged every 4–6 days. Cells from the fourth to the sixth passage were used for experiments. Lycopene was dissolved in tetrahydrofuran (THF; stabilized with 0.025% BHT) and applied to the cells at 0 M, 1 M, 5 M and 10 M concentrations. Lycopene was prepared according to methods reported by Martin et al. (2000) and Lin et al. (2007) to increase its stability and to facilitate its uptake by cells. Apigenin was dissolved in dimethyl sulfoxide (DMSO) and added at 5 mg/l (18.5 M) and 10 mg/l (37 M) concentrations. The final concentration of DMSO and THF did not exceed respectively 0.2% (v/v) and 0.1% (v/v) in any case.
In vitro angiogenesis assay MatrigelTM (BD-354234, Bedford, MA, USA) was thawed at 4 ◦ C, and 250 l were quickly added to each well of a 24-well plate and allowed to solidify for 1 h at 37 ◦ C. Once solid, HUVECs, serum-
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starved for 24 h, were added to each well (∼2.5 × 104 cells/well) in EGM-2 complete medium. After cells had adhered to the Matrigel, DMSO, THF, apigenin or lycopene was added, and cells were incubated at 37 ◦ C for 24 h. The tube formation of HUVECs was recorded with an Olympus IX81 Photomicroscope (Olympus, Tokyo, Japan) with a ×4 objective. Tube lengths of three different fields of images were assayed with the UTHSCSA ImageTool Version 3.0 software programme (University of Texas Health Science Center, San Antonio, TX, USA). Experiments were repeated five times and the results were expressed as mean ± standard deviation (SD). Cell proliferation assay The effects of lycopene and apigenin on the viability of cells were determined by MTT (3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide) assay as described previously with some modifications (Mosmann, 1983). MTT was purchased from AppliChem (Darmstadt, Germany). MTT stock solution (5 mg/ml) in RPMI-1640 without phenol red (Sigma–Aldrich, St. Louis, MO, USA) was diluted (1:10) for experiments. The cells were plated at ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24well plates were serum-starved for 24 h. After 24 h, HUVECs were treated with DMSO, THF, apigenin, or lycopene and incubated at 37 ◦ C for 24 h. After medium was exchanged with 500 l MTT solution, cells were incubated at 37 ◦ C for 3 h. At the end of the incubation period, the converted dye was solubilized with 500 l acidic isopropanol (0.04 M HCl in absolute isopropanol). Absorbance of the converted dye was measured at 570 nm with background subtraction at 650 nm by colorimetric plate reader (Model No.: 1500; Thermo Labsystem, Finland). Experiments were repeated five times and the results were expressed as mean ± SD. Cell migration assay We used a Millipore QCMTM Endothelial Migration Assay Kit containing fibronectin coated 24-well Boyden Chamber System with 3 m pore width (Millipore ECM201, Temecula, CA, USA). Experiments were carried out according to manufacturer’s kit assay procedures. Briefly, migrated cells at the bottom plate were dyed with CyQUANT® GR fluorescent dye (Millipore, Temecula, CA, USA). This green-fluorescent dye was assayed with a fluorescence plate reader using 480/520 nm filter set (Type: 374; Thermo Fluoroskan Ascent FL, Finland). Experiments were repeated five times and results were expressed as mean ± SD of five different experiments. Wound healing assay For another cell migration assay, a wound healing assay was applied as horizontal migration. The experiment was applied using methods of Schleef and Birdwell (1982) with some modifications. After full-confluent HUVECs were starved of serum and growth factors for 24 h, cells in 12-well plate were scratched with a sterile 200 l plastic pipette tip across monolayer of cells. Cells were treated with DMSO, THF, apigenin or lycopene. When HUVECs in control groups had almost completely migrated (approximately 12–14 h), cells were dyed with 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylene indocarbocynanine perchlorate (Dil) (Aldrich 468495) and photographed by integrated Olympus DP70 digital camera (Olympus, Japan) on an inverted microscope (Olympus IX81S1F-2, Japan) with x40 magnification. Dil were prepared as stock solution (1 mg/ml) in DMSO and diluted as 1:200 in growth medium for experiments. Migration distances of ten different fields at photographs were assayed with UTHSCSA ImageTool Version 3.0 software programme (University of Texas Health Science Center, San Antonio, TX, USA). Experiments were repeated five times.
Fig. 2. Inhibition of the in vitro angiogenesis of human umbilical vein endothelial cells by apigenin. Representative micrographs of HUVECs cultured for 24 h on Matrigel as mentioned in material and methods section in the presence of the indicated compound are illustrated. HUVECs were incubated with (A) DMSO (Control), (B) Apigenin (5 g/ml) and (C) Apigenin (10 g/ml). Scale bar = 250 m.
Statistical analysis Data were expressed as mean ± standard deviation (SD). Comparison of groups according to the parameters was performed using the Student’s t-test and test of one-way ANOVA (Tukey’s Multiple Comparison) in Prism Program of GraphPad Software (San Diego, CA). P values less than 0.05 were considered statistically significant. Results Lycopene and apigenin inhibit capillary-like tube formation In this study, we found that 1 M, 5 M and 10 M concentrations of lycopene have significantly suppressed the endothelial tube formations (7102 ± 497, 4448 ± 426, 2718 ± 287, respectively) on Matrigel matrix as compared with controls (14574 ± 575) in which the cells were treated with THF (p < 0.001) (Fig. 1, Fig. 3A).
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Fig. 3. Tube lengths of capillary-like structures are affected by the doses of lycopene in (A) and apigenin in (B). *** Shows p < 0.001.
We found that the inhibition of tube lengths and organization was dose-dependent. In apigenin-treated HUVECs, capillary-like tube formation was also dramatically inhibited at 5 g/ml (18.5 M) or 10 g/ml (37 M) concentrations (8098 ± 479, 3842 ± 461, respectively) as compared with control (16431 ± 502, p < 0.001) (Figs. 2 and 3 Figs. 2 and 3B).
Similarly, migration of cells towards each other was found to be suppressed (or migration length increase) both group treated with lycopene (610.17 ± 36.17) and apigenin (670.62 ± 39.39) as compared to controls THF (68.41 ± 15.36) and DMSO (99.46 ± 19.39) (Fig. 6A–D) (p < 0.001).
Lycopene and apigenin suppress cell proliferation
Discussion
Treatment of cells with three different concentrations of lycopene (Fig. 4A) and two different concentrations of apigenin (Fig. 4B) decreased the proliferation of endothelial cells in a dosedependent manner. As compared with controls, percentage of the proliferating cells in groups treated with 1 M, 5 M and 10 M concentrations of lycopene (63.34 ± 2.34, 41.53 ± 2.17, 30.24 ± 1.96 expressed as the percentage of control cells, respectively) was found to be significantly decreased (p < 0.001). Percentage of proliferation was seen to be dose-dependent. Difference between doses was found significant (p < 0.001) (Fig. 4A). Apigenin was also shown to be effective against endothelial proliferation at 5 g/ml and 10 g/ml concentrations (54.24 ± 2.33 and 38.15 ± 2.38, respectively) compared with control groups (expressed as 100) (p < 0.001). In addition, there was a significant difference among the doses, as well (p < 0.001) (Fig. 4B).
The critical role of tumor angiogenesis in cancer progression was postulated about 40 years ago in pioneering studies by Folkman et al. (1971). However, only in recent years has the knowledge of endothelial cell physiology and tumor angiogenesis provided the necessary background to develop effective antiangiogenic strategies. Developing antiangiogenic strategies against the growth of endothelial cells may be more advantageous than directly targeting cancer cells in shrinkage of tumors. The endothelial cell, which is a cell type common to all solid tumors, represents a preferential target for therapy. Even though every cancer is virtually a unique disease, the tumor endothelium is a relatively uniform, normal cell type (Tosetti et al., 2002). Another importance of this approach is the apparent inability by the endothelial cells to counteract therapy through development of multi-drug resistance mechanisms, due to the low mutagenesis rate of this normal cell type (Boehm et al., 1997). Chronic inhibition of vascular recruitment may be required to block tumor growth; thus, long-term treatment may be necessary (Hanahan and Folkman, 1996). Therefore, we need to identify and characterize angiostatic molecules endowed with low or no toxicity. Substantial efforts have been dedicated to identifying natural and synthetic compounds that can be used to either prevent insurgence of primary tumors in subjects at high risk to develop cancer or prevent tumor relapse after surgical removal. Cancer chemoprevention involves the use of agents to slow the progression of carcinogenesis, reverse, or inhibit it, with the aim of lowering the risk of developing invasive or clinically significant disease. Chemopreventive drugs must be devoid of toxicity and well tolerated since they must be used over extended periods (Hanahan and Folkman, 1996). In this study, we investigated the effects of lycopene on endothelial cells. Because of its chemical structure, lycopene is a molecule that has very powerful antioxidant activity. As described earlier, growth of endothelial cells occur under tight regulation. However, conditions around the cancerous tissue change the regulation to constitute new vessels. Under these conditions, endothelial cells increasingly proliferate, migrate towards chemoattractants produced by cancer cells, and finally generate a new capillary network. In this study, we have investigated whether these steps are affected
Lycopene and apigenin block endothelial cell migration Endothelial cells migrate towards the chemo-attractants during angiogenesis. In this system endothelial cells migrate through the pores and move to the bottom of the plate containing EGM-2 medium and serum. Lycopene at 1 M, 5 M and 10 M concentrations suppressed endothelial cell migration through pores compared with controls (56.02 ± 3.11, 52.10 ± 2.44, 42.06 ± 3.24, respectively) (Fig. 5A). Values of fluorescence intensity in control groups were expressed as 100%. We did not find any significant difference between 1 and 5 M lycopene. However, differences among other doses were found as significant (p < 0.001). It was shown 5 g/ml and 10 g/ml apigenin concentrations decreased fluorescence intensity of cells (57.98 ± 3.22, 49.72 ± 2.84, respectively) as compared with control groups (expressed as 100%) (p < 0.001). 10 g/ml apigenin was more effective than 5 g/ml (p < 0.01) (Fig. 5B). Lycopene and apigenin decrease directional migration Additionally, we have also used another migration assay method named “wound healing assay” showing directional migration. We only assayed 10 M lycopene and 10 g/ml apigenin for this assay.
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Fig. 4. Percentage proliferation of endothelial cells. HUVECs were plated at ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24-well plate were serum-starved for 24 h. After this period, cells in growth medium were treated with DMSO, THF, apigenin and lycopene. After incubated at 37 ◦ C for 24 h, cells were treated with MTT solution and the converted dye was measured at 570 nm. (A) Percentage of proliferating cells exposed to lycopene. Values represent mean ± SD of five different experiments: (a) C vs. Lyc (1 M), (b) C vs. Lyc (5 M), (c) Lyc (1 M) vs. Lyc (5 M), (d) C vs. Lyc (10 M), (e) Lyc (1 M) vs. Lyc (10 M) and (f) Lyc (5 M) vs. Lyc (10 M). (B) Percentage of living cells exposed to apigenin: (a) C vs. Api (5 g/ml), (b) C vs. Api (10 g/ml) and (c) Api (5 g/ml) vs. Api (10 g/ml). (C) Control; Lyc: lycopene; Api: apigenin.
by lycopene. MatrigelTM matrix used in our study is an extracellular matrix provided from a cancer type and containing an agent essential for angiogenesis. In addition, we added EGM containing growth factors such as VEGF and FGF important for the angiogenesis process of cells. Under these environmental conditions, while we observed widespread tube formation in cells treated with DMSO or THF alone, capillary-like tubular formation appeared to be suppressed in cells treated with lycopene. In our experiment, lycopene was applied at concentrations of 0, 1, 5 and 10 M. We observed that lycopene significantly inhibited in vitro angiogenesis in a dosedependent manner. Apigenin which is used for positive control is known to display antiangiogenic effects in a variety of studies (Trochon et al., 2000; Kim, 2003; Erdogan et al., 2007). We also demonstrated that apigenin inhibited tube formation in a dose-dependent manner. The proliferation rate of endothelial cell increases during angiogenesis. Therefore, we investigated the effects of lycopene via the MTT method on cells in medium containing serum and growth factors and observed lycopene significantly suppressed cell proliferation for all doses used. While lycopene at the most effective dose 10 M decreased proliferation by 69.76%, 5 M and 1 M lycopene inhibited proliferation by 58.47% and 36.66%, respectively. Differences among the concentrations are statistically significant. Ivanov et al. (2007) suggested treatment of LNCaP prostate carcinoma cells with
lycopene decreased the cell proliferation. In another study, it was shown that lycopene suppressed the proliferative capacity of several malignant cell lines (Salman et al., 2007). Moreover, in cell culture system, lycopene was demonstrated to strongly inhibit IGF1-mediated proliferation of human endometrial, breast and lung cancer cells (Karas et al., 2000). Lycopene is also known to inhibit proliferation of human oral cavity tumor (KB-1) (Livny et al., 2002) and human breast cancer cells (MCF-7 and MDA-MB-231) (Prakash et al., 2001). Even if the types of the cells are different, the general suppressive effects of lycopene on proliferation are consistent with our study. We revealed that apigenin at both 5 g/ml and 10 g/ml also significantly inhibited cell proliferation 45.76% and 61.85%, respectively. As regards the migration assay, another important parameter for in vitro angiogenesis, we used two different methods to assay endothelial cell migration. One of the methods is based on cell movements towards the chemoattractant. In this experiment, we assayed migration of cells from the upper micro-pore (3 m) plate coated with fibronectin to the bottom plate containing serum and growth factors. Control of the experiment involves using albumin coated plates instead of fibronectin. Consequently, we observed all concentrations of lycopene, as compared with THF, inhibited cell migration. Similarly, apigenin also significantly suppressed the migration. Another method, named wound healing assay, is based
Fig. 5. Lycopene (A) and apigenin (B) treatments suppress the migration of endothelial cells from fibronectin-coated plates with pores (3 m) to bottom plate containing serum and growth factors. Migrated cells were dyed and assayed with fluorescence plate reader. Data were given as relative percent fluorescence intensity according to controls (DMSO or THF). No significant difference between 1 and 5 M lycopene. ***p < 0.001, **p < 0.01.
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Fig. 6. The fluorescence microphotographs of wound healing assayed cells treated with (A) THF (Control), (B) lycopene (10 M), (C) DMSO (Control) and (D) Apigenin (10 g/ml). After full-confluent HUVECs were starved with serum and growth factors for 24 h, cells in 12-well plate were scratched with sterile 200 l plastic pipette tip. Cells were treated with the indicated compound. When HUVECs in control groups almost completely migrated, cells were stained with Dil fluorescence dye as mentioned in material and methods section. Microphotographs were taken under fluorescence microscope at ×40 magnification. Reciprocal distance (pixel) between cells at ten different fields of each of five independent experiments was calculated as mean ± SD. Scale bar = 100 m. ***p < 0.001.
on mimicking the cell migration during wound healing in vivo. Apigenin and lycopene in this assay significantly decrease the movement of cells. Hwang and Lee (2006) suggested that lycopene at 5 and 10 M inhibited the adhesion, invasion and migration of a highly invasive human hepatoma cell line, SK-Hep1. Moreover, it was found that the metastasis suppressor gene nm23-H1 was induced in these cells (Huang et al., 2005). The studies performed with SK-Hep1 are similar to our study in terms of anti-migration properties of lycopene. Our study is consistent with other reported studies defending the protective effects of flavonoids and carotenoids against chronic diseases (Kim et al., 1998; Agarwal and Rao, 2000). In our study, it was revealed apigenin and lycopene showed similar effects under the same experimental conditions. It may be concluded that lycopene is more effective than apigenin when comparing their concentrations. Various epidemiological and experimental studies have revealed that lycopene is an important agent to prevent from cancer, however, effects of lycopene on endothelial cells or angiogenesis could not be found in the literature. In conclusion, the present results suggest that lycopene has some antiangiogenic properties, though additional studies are needed to demonstrate the effects of lycopene on molecular mechanisms in endothelial cells.
Conflict of interest The authors have no conflict of interest.
Acknowledgements Funding for this study was provided by grants from the Akdeniz University Scientific Research Project Unit (Project Number: 2006.03.0122.005). Also this study was supported by Akdeniz University Health Sciences Institute.
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Acta Histochemica journal homepage: www.elsevier.de/acthis
Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation Mehmet S¸ahin a , Emel S¸ahin b , Saadet Gümüs¸lü c,∗ a b c
Health Sciences Research Centre, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey Central Laboratory, Clinical Biochemistry Unit, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey Department of Biochemistry, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey
a r t i c l e
i n f o
Article history: Received 28 December 2010 Received in revised form 4 March 2011 Accepted 6 March 2011
Keywords: Angiogenesis Lycopene Apigenin Cancer HUVEC
a b s t r a c t Angiogenesis is the formation process of new blood vessels from preexisting vessels. Solid tumors need angiogenesis for growth and metastasis. The suppression of tumor growth by inhibition of neoangiogenic processes represents a potential approach to cancer treatment. Lycopene has powerful antioxidant capacities and anticarcinogenic properties. The aim of this study was to investigate the effects of lycopene on angiogenesis in vitro. For this reason, we measured in vitro angiogenesis in human umbilical vein endothelial cells including parameters of cell proliferation, tube formation, cell migration. Lycopene and apigenin were observed to block the endothelial cell proliferation in a dose-dependent manner. In addition, they significantly decreased the capillary-like tube lengths, tube formation and endothelial cell migration. This study provides indications that apigenin and lycopene, which are considered as chemopreventive agents, to be effective in vitro on endothelial cells and angiogenesis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Angiogenesis, the formation of new blood vessels, plays several roles in various human pathologies including: rheumatoid arthritis, diabetic retinopathy, atherosclerosis, psoriasis, and chronic airway inflammation. One of the most important roles of angiogenesis is to support tumor growth, which is dependent on nutritional support derived from the local blood supply (Sahin et al., 2009). Targeting inhibition of angiogenesis represents a potential approach in the treatment of solid tumors and such antiangio-
Abbreviations: BHT, butylated hydroxytoluene; DMSO, dimethyl sulfoxide; EBM-2, endothelial basal medium; EGM-2, endothelial growth medium; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Hep3B, a human hepatoma cell line; HUVEC, human umbilical vein endothelial cells; IGF1, insulin-like growth factor; KB-1, a cell line derived from a human oral cavity tumor; LNCaP, an androgen-sensitive human prostate adenocarcinoma cell line; MCF-7, a breast cancer cell line; MDA-MB-231, an estrogen-independent human breast cancer cell line; MMP, matrix metalloproteinase; MTT, (3 (4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide); PC3, a metastatic cell line derived from advanced androgen independent prostate cancer; RPMI, Roswell Park Memorial Institute cell culture medium; SK-Hep1, an immortal and metastatic cell line derived from a liver adenocarcinoma; THF, tetrahydrofuran; VEGF, vascular endothelial growth factor. ∗ Corresponding author. Tel.: +90 2422496896; fax: +90 2422274495. E-mail addresses: [email protected] (M. S¸ahin), [email protected] (E. S¸ahin), [email protected] (S. Gümüs¸lü). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.03.004
genic strategies inhibiting the growth of endothelial cells may be more advantageous than targeting cancer cells. The inhibition of tumor growth requires a chronic inhibition of vascular organization, defined as ‘angiostasis’ (Hanahan and Folkman, 1996). Long-term treatment is necessary to achieve angiostasis and it is necessary to identify and characterize angiostatic molecules showing low or no toxicity. Current dietary guidelines to combat chronic diseases including cancer, recommend increased intake of plant foods that are rich in antioxidants. The role of dietary antioxidants, including carotenoids and flavonoids in disease prevention has received much attention (Kim et al., 1998; Agarwal and Rao, 2000). Apigenin is a flavonoid with anti-inflammatory, anticarcinogenic and free radical scavenger properties (Kim et al., 1998). Lycopene, one of more than 600 carotenoids, is a natural pigment synthesized by plants and photosynthetic microorganisms. Several epidemiological studies have strongly implied that consumption of foods containing high concentrations of lycopene may reduce the risk of certain types of cancer (Gann et al., 1999; Giovannucci et al., 2002). The mechanism of lycopene action is still under investigation. Lycopene was shown to induce apoptosis in a dose-dependent manner in prostate cancer cells (Hantz et al., 2005). Similarly, treatment of human colon carcinoma cells with lycopene at 2.0 or 4.0 M was found to be able to induce apoptosis (Salman et al., 2007). There have been several reports that lycopene can induce cell cycle arrest. It has been reported that the growth of Hep3B human hepatoma cells was inhibited 20–50% by lycopene (Park et al., 2005). A simi-
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Fig. 1. Lycopene inhibited tube formation produced by human umbilical vein endothelial cells. Representative micrographs of HUVECs cultured for 24 h on Matrigel in the presence of the indicated compound are illustrated. Serum-starved cells were seeded to each well of 24-well plate as ∼2.5 × 104 cells in EGM-2 complete medium. HUVEC cells were incubated in (A) THF (Control), (B) Lyc 1 M, (C) Lyc 5 M and (D) Lyc 10 M. Scale bar = 250 m.
lar study with the human prostate cancer cell lines, LNCaP and PC3, also found that lycopene induced mitotic arrest at the G0/G1 phase (Ivanov et al., 2007). With respect to anti-invasive and anti-metastatic activities, lycopene (5–10 M) has been shown to decrease the activities of the gelatinolytic matrix metalloproteinases, MMP-2 and MMP-9, and to inhibit adhesion, invasion and migration of SK-Hep1 cells, which is a highly invasive human hepatoma cell line (Hwang and Lee, 2006). Lycopene at similar concentrations was also found to induce the metastasis suppressor gene nm23-H1 (Huang et al., 2005). Although there have been several studies based on the antioxidant and anticarcinogenic activities of lycopene, its role on endothelial cells and angiogenesis has remained unclear. The aim of this study was to investigate whether lycopene is effective on endothelial cells and angiogenesis inhibition at different concentrations, and to compare the effects of lycopene with apigenin with regard to antiangiogenic activities. Materials and methods Reagents Human umbilical vein endothelial cells (HUVECs) and MatrigelTM were purchased from BD Biosciences (BD-354234; Bedford, MA, USA). EGM-2® BulletKit® (CC-3162, EBM2 + supplements) for HUVEC culture was purchased from Lonza (Walkersville, MD, USA). Lycopene and apigenin were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Cell culture Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 complete medium consisting of EBM-2 basal medium supplemented with hydrocortisone, ascorbic acid, heparin, GA-1000 and 2% FBS plus growth factors (epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1)) except for serum starvation in which FBS concentration was reduced to 0.1% and no growth factors were added. Cell culture flasks or well plates, pre-coated with gelatin, were used for culture of HUVECs (Sigma–Aldrich, Irvine, Ayrshire, UK). All cells were grown in a humidified atmosphere, 95% air and 5% CO2 at 37 ◦ C and passaged every 4–6 days. Cells from the fourth to the sixth passage were used for experiments. Lycopene was dissolved in tetrahydrofuran (THF; stabilized with 0.025% BHT) and applied to the cells at 0 M, 1 M, 5 M and 10 M concentrations. Lycopene was prepared according to methods reported by Martin et al. (2000) and Lin et al. (2007) to increase its stability and to facilitate its uptake by cells. Apigenin was dissolved in dimethyl sulfoxide (DMSO) and added at 5 mg/l (18.5 M) and 10 mg/l (37 M) concentrations. The final concentration of DMSO and THF did not exceed respectively 0.2% (v/v) and 0.1% (v/v) in any case.
In vitro angiogenesis assay MatrigelTM (BD-354234, Bedford, MA, USA) was thawed at 4 ◦ C, and 250 l were quickly added to each well of a 24-well plate and allowed to solidify for 1 h at 37 ◦ C. Once solid, HUVECs, serum-
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starved for 24 h, were added to each well (∼2.5 × 104 cells/well) in EGM-2 complete medium. After cells had adhered to the Matrigel, DMSO, THF, apigenin or lycopene was added, and cells were incubated at 37 ◦ C for 24 h. The tube formation of HUVECs was recorded with an Olympus IX81 Photomicroscope (Olympus, Tokyo, Japan) with a ×4 objective. Tube lengths of three different fields of images were assayed with the UTHSCSA ImageTool Version 3.0 software programme (University of Texas Health Science Center, San Antonio, TX, USA). Experiments were repeated five times and the results were expressed as mean ± standard deviation (SD). Cell proliferation assay The effects of lycopene and apigenin on the viability of cells were determined by MTT (3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide) assay as described previously with some modifications (Mosmann, 1983). MTT was purchased from AppliChem (Darmstadt, Germany). MTT stock solution (5 mg/ml) in RPMI-1640 without phenol red (Sigma–Aldrich, St. Louis, MO, USA) was diluted (1:10) for experiments. The cells were plated at ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24well plates were serum-starved for 24 h. After 24 h, HUVECs were treated with DMSO, THF, apigenin, or lycopene and incubated at 37 ◦ C for 24 h. After medium was exchanged with 500 l MTT solution, cells were incubated at 37 ◦ C for 3 h. At the end of the incubation period, the converted dye was solubilized with 500 l acidic isopropanol (0.04 M HCl in absolute isopropanol). Absorbance of the converted dye was measured at 570 nm with background subtraction at 650 nm by colorimetric plate reader (Model No.: 1500; Thermo Labsystem, Finland). Experiments were repeated five times and the results were expressed as mean ± SD. Cell migration assay We used a Millipore QCMTM Endothelial Migration Assay Kit containing fibronectin coated 24-well Boyden Chamber System with 3 m pore width (Millipore ECM201, Temecula, CA, USA). Experiments were carried out according to manufacturer’s kit assay procedures. Briefly, migrated cells at the bottom plate were dyed with CyQUANT® GR fluorescent dye (Millipore, Temecula, CA, USA). This green-fluorescent dye was assayed with a fluorescence plate reader using 480/520 nm filter set (Type: 374; Thermo Fluoroskan Ascent FL, Finland). Experiments were repeated five times and results were expressed as mean ± SD of five different experiments. Wound healing assay For another cell migration assay, a wound healing assay was applied as horizontal migration. The experiment was applied using methods of Schleef and Birdwell (1982) with some modifications. After full-confluent HUVECs were starved of serum and growth factors for 24 h, cells in 12-well plate were scratched with a sterile 200 l plastic pipette tip across monolayer of cells. Cells were treated with DMSO, THF, apigenin or lycopene. When HUVECs in control groups had almost completely migrated (approximately 12–14 h), cells were dyed with 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylene indocarbocynanine perchlorate (Dil) (Aldrich 468495) and photographed by integrated Olympus DP70 digital camera (Olympus, Japan) on an inverted microscope (Olympus IX81S1F-2, Japan) with x40 magnification. Dil were prepared as stock solution (1 mg/ml) in DMSO and diluted as 1:200 in growth medium for experiments. Migration distances of ten different fields at photographs were assayed with UTHSCSA ImageTool Version 3.0 software programme (University of Texas Health Science Center, San Antonio, TX, USA). Experiments were repeated five times.
Fig. 2. Inhibition of the in vitro angiogenesis of human umbilical vein endothelial cells by apigenin. Representative micrographs of HUVECs cultured for 24 h on Matrigel as mentioned in material and methods section in the presence of the indicated compound are illustrated. HUVECs were incubated with (A) DMSO (Control), (B) Apigenin (5 g/ml) and (C) Apigenin (10 g/ml). Scale bar = 250 m.
Statistical analysis Data were expressed as mean ± standard deviation (SD). Comparison of groups according to the parameters was performed using the Student’s t-test and test of one-way ANOVA (Tukey’s Multiple Comparison) in Prism Program of GraphPad Software (San Diego, CA). P values less than 0.05 were considered statistically significant. Results Lycopene and apigenin inhibit capillary-like tube formation In this study, we found that 1 M, 5 M and 10 M concentrations of lycopene have significantly suppressed the endothelial tube formations (7102 ± 497, 4448 ± 426, 2718 ± 287, respectively) on Matrigel matrix as compared with controls (14574 ± 575) in which the cells were treated with THF (p < 0.001) (Fig. 1, Fig. 3A).
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Fig. 3. Tube lengths of capillary-like structures are affected by the doses of lycopene in (A) and apigenin in (B). *** Shows p < 0.001.
We found that the inhibition of tube lengths and organization was dose-dependent. In apigenin-treated HUVECs, capillary-like tube formation was also dramatically inhibited at 5 g/ml (18.5 M) or 10 g/ml (37 M) concentrations (8098 ± 479, 3842 ± 461, respectively) as compared with control (16431 ± 502, p < 0.001) (Figs. 2 and 3 Figs. 2 and 3B).
Similarly, migration of cells towards each other was found to be suppressed (or migration length increase) both group treated with lycopene (610.17 ± 36.17) and apigenin (670.62 ± 39.39) as compared to controls THF (68.41 ± 15.36) and DMSO (99.46 ± 19.39) (Fig. 6A–D) (p < 0.001).
Lycopene and apigenin suppress cell proliferation
Discussion
Treatment of cells with three different concentrations of lycopene (Fig. 4A) and two different concentrations of apigenin (Fig. 4B) decreased the proliferation of endothelial cells in a dosedependent manner. As compared with controls, percentage of the proliferating cells in groups treated with 1 M, 5 M and 10 M concentrations of lycopene (63.34 ± 2.34, 41.53 ± 2.17, 30.24 ± 1.96 expressed as the percentage of control cells, respectively) was found to be significantly decreased (p < 0.001). Percentage of proliferation was seen to be dose-dependent. Difference between doses was found significant (p < 0.001) (Fig. 4A). Apigenin was also shown to be effective against endothelial proliferation at 5 g/ml and 10 g/ml concentrations (54.24 ± 2.33 and 38.15 ± 2.38, respectively) compared with control groups (expressed as 100) (p < 0.001). In addition, there was a significant difference among the doses, as well (p < 0.001) (Fig. 4B).
The critical role of tumor angiogenesis in cancer progression was postulated about 40 years ago in pioneering studies by Folkman et al. (1971). However, only in recent years has the knowledge of endothelial cell physiology and tumor angiogenesis provided the necessary background to develop effective antiangiogenic strategies. Developing antiangiogenic strategies against the growth of endothelial cells may be more advantageous than directly targeting cancer cells in shrinkage of tumors. The endothelial cell, which is a cell type common to all solid tumors, represents a preferential target for therapy. Even though every cancer is virtually a unique disease, the tumor endothelium is a relatively uniform, normal cell type (Tosetti et al., 2002). Another importance of this approach is the apparent inability by the endothelial cells to counteract therapy through development of multi-drug resistance mechanisms, due to the low mutagenesis rate of this normal cell type (Boehm et al., 1997). Chronic inhibition of vascular recruitment may be required to block tumor growth; thus, long-term treatment may be necessary (Hanahan and Folkman, 1996). Therefore, we need to identify and characterize angiostatic molecules endowed with low or no toxicity. Substantial efforts have been dedicated to identifying natural and synthetic compounds that can be used to either prevent insurgence of primary tumors in subjects at high risk to develop cancer or prevent tumor relapse after surgical removal. Cancer chemoprevention involves the use of agents to slow the progression of carcinogenesis, reverse, or inhibit it, with the aim of lowering the risk of developing invasive or clinically significant disease. Chemopreventive drugs must be devoid of toxicity and well tolerated since they must be used over extended periods (Hanahan and Folkman, 1996). In this study, we investigated the effects of lycopene on endothelial cells. Because of its chemical structure, lycopene is a molecule that has very powerful antioxidant activity. As described earlier, growth of endothelial cells occur under tight regulation. However, conditions around the cancerous tissue change the regulation to constitute new vessels. Under these conditions, endothelial cells increasingly proliferate, migrate towards chemoattractants produced by cancer cells, and finally generate a new capillary network. In this study, we have investigated whether these steps are affected
Lycopene and apigenin block endothelial cell migration Endothelial cells migrate towards the chemo-attractants during angiogenesis. In this system endothelial cells migrate through the pores and move to the bottom of the plate containing EGM-2 medium and serum. Lycopene at 1 M, 5 M and 10 M concentrations suppressed endothelial cell migration through pores compared with controls (56.02 ± 3.11, 52.10 ± 2.44, 42.06 ± 3.24, respectively) (Fig. 5A). Values of fluorescence intensity in control groups were expressed as 100%. We did not find any significant difference between 1 and 5 M lycopene. However, differences among other doses were found as significant (p < 0.001). It was shown 5 g/ml and 10 g/ml apigenin concentrations decreased fluorescence intensity of cells (57.98 ± 3.22, 49.72 ± 2.84, respectively) as compared with control groups (expressed as 100%) (p < 0.001). 10 g/ml apigenin was more effective than 5 g/ml (p < 0.01) (Fig. 5B). Lycopene and apigenin decrease directional migration Additionally, we have also used another migration assay method named “wound healing assay” showing directional migration. We only assayed 10 M lycopene and 10 g/ml apigenin for this assay.
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Fig. 4. Percentage proliferation of endothelial cells. HUVECs were plated at ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24-well plate were serum-starved for 24 h. After this period, cells in growth medium were treated with DMSO, THF, apigenin and lycopene. After incubated at 37 ◦ C for 24 h, cells were treated with MTT solution and the converted dye was measured at 570 nm. (A) Percentage of proliferating cells exposed to lycopene. Values represent mean ± SD of five different experiments: (a) C vs. Lyc (1 M), (b) C vs. Lyc (5 M), (c) Lyc (1 M) vs. Lyc (5 M), (d) C vs. Lyc (10 M), (e) Lyc (1 M) vs. Lyc (10 M) and (f) Lyc (5 M) vs. Lyc (10 M). (B) Percentage of living cells exposed to apigenin: (a) C vs. Api (5 g/ml), (b) C vs. Api (10 g/ml) and (c) Api (5 g/ml) vs. Api (10 g/ml). (C) Control; Lyc: lycopene; Api: apigenin.
by lycopene. MatrigelTM matrix used in our study is an extracellular matrix provided from a cancer type and containing an agent essential for angiogenesis. In addition, we added EGM containing growth factors such as VEGF and FGF important for the angiogenesis process of cells. Under these environmental conditions, while we observed widespread tube formation in cells treated with DMSO or THF alone, capillary-like tubular formation appeared to be suppressed in cells treated with lycopene. In our experiment, lycopene was applied at concentrations of 0, 1, 5 and 10 M. We observed that lycopene significantly inhibited in vitro angiogenesis in a dosedependent manner. Apigenin which is used for positive control is known to display antiangiogenic effects in a variety of studies (Trochon et al., 2000; Kim, 2003; Erdogan et al., 2007). We also demonstrated that apigenin inhibited tube formation in a dose-dependent manner. The proliferation rate of endothelial cell increases during angiogenesis. Therefore, we investigated the effects of lycopene via the MTT method on cells in medium containing serum and growth factors and observed lycopene significantly suppressed cell proliferation for all doses used. While lycopene at the most effective dose 10 M decreased proliferation by 69.76%, 5 M and 1 M lycopene inhibited proliferation by 58.47% and 36.66%, respectively. Differences among the concentrations are statistically significant. Ivanov et al. (2007) suggested treatment of LNCaP prostate carcinoma cells with
lycopene decreased the cell proliferation. In another study, it was shown that lycopene suppressed the proliferative capacity of several malignant cell lines (Salman et al., 2007). Moreover, in cell culture system, lycopene was demonstrated to strongly inhibit IGF1-mediated proliferation of human endometrial, breast and lung cancer cells (Karas et al., 2000). Lycopene is also known to inhibit proliferation of human oral cavity tumor (KB-1) (Livny et al., 2002) and human breast cancer cells (MCF-7 and MDA-MB-231) (Prakash et al., 2001). Even if the types of the cells are different, the general suppressive effects of lycopene on proliferation are consistent with our study. We revealed that apigenin at both 5 g/ml and 10 g/ml also significantly inhibited cell proliferation 45.76% and 61.85%, respectively. As regards the migration assay, another important parameter for in vitro angiogenesis, we used two different methods to assay endothelial cell migration. One of the methods is based on cell movements towards the chemoattractant. In this experiment, we assayed migration of cells from the upper micro-pore (3 m) plate coated with fibronectin to the bottom plate containing serum and growth factors. Control of the experiment involves using albumin coated plates instead of fibronectin. Consequently, we observed all concentrations of lycopene, as compared with THF, inhibited cell migration. Similarly, apigenin also significantly suppressed the migration. Another method, named wound healing assay, is based
Fig. 5. Lycopene (A) and apigenin (B) treatments suppress the migration of endothelial cells from fibronectin-coated plates with pores (3 m) to bottom plate containing serum and growth factors. Migrated cells were dyed and assayed with fluorescence plate reader. Data were given as relative percent fluorescence intensity according to controls (DMSO or THF). No significant difference between 1 and 5 M lycopene. ***p < 0.001, **p < 0.01.
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Fig. 6. The fluorescence microphotographs of wound healing assayed cells treated with (A) THF (Control), (B) lycopene (10 M), (C) DMSO (Control) and (D) Apigenin (10 g/ml). After full-confluent HUVECs were starved with serum and growth factors for 24 h, cells in 12-well plate were scratched with sterile 200 l plastic pipette tip. Cells were treated with the indicated compound. When HUVECs in control groups almost completely migrated, cells were stained with Dil fluorescence dye as mentioned in material and methods section. Microphotographs were taken under fluorescence microscope at ×40 magnification. Reciprocal distance (pixel) between cells at ten different fields of each of five independent experiments was calculated as mean ± SD. Scale bar = 100 m. ***p < 0.001.
on mimicking the cell migration during wound healing in vivo. Apigenin and lycopene in this assay significantly decrease the movement of cells. Hwang and Lee (2006) suggested that lycopene at 5 and 10 M inhibited the adhesion, invasion and migration of a highly invasive human hepatoma cell line, SK-Hep1. Moreover, it was found that the metastasis suppressor gene nm23-H1 was induced in these cells (Huang et al., 2005). The studies performed with SK-Hep1 are similar to our study in terms of anti-migration properties of lycopene. Our study is consistent with other reported studies defending the protective effects of flavonoids and carotenoids against chronic diseases (Kim et al., 1998; Agarwal and Rao, 2000). In our study, it was revealed apigenin and lycopene showed similar effects under the same experimental conditions. It may be concluded that lycopene is more effective than apigenin when comparing their concentrations. Various epidemiological and experimental studies have revealed that lycopene is an important agent to prevent from cancer, however, effects of lycopene on endothelial cells or angiogenesis could not be found in the literature. In conclusion, the present results suggest that lycopene has some antiangiogenic properties, though additional studies are needed to demonstrate the effects of lycopene on molecular mechanisms in endothelial cells.
Conflict of interest The authors have no conflict of interest.
Acknowledgements Funding for this study was provided by grants from the Akdeniz University Scientific Research Project Unit (Project Number: 2006.03.0122.005). Also this study was supported by Akdeniz University Health Sciences Institute.
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