Journal of Pediatric Surgery (2013) 48, 2460–2465

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Antiangiogenic effect of propranolol on the growth of the neuroblastoma xenografts in nude mice☆ Ting Xu, Xianmin Xiao ⁎, Shan Zheng, Jicui Zheng, Haitao Zhu, Yi Ji, Shaobo Yang Department of Surgery of Children’s Hospital, Fudan University, Shanghai, P.R. China Received 22 August 2013; accepted 26 August 2013

Key words: Propranolol; Neuroblastoma; Angiogenesis; Vascular endothelial growth factor; Microvessel density

Abstract Background: Propranolol has been reported to display an antiangiogenic effect on infantile hemangiomas and also some adult cancers. Little is known, however, about whether propranolol has such effect on pediatric malignancies. Methods: Nude mice bearing BE(2) C neuroblastoma xenografts were injected intraperitoneally with propranolol and divided into groups of PROP-2 (n = 11), -5 (n = 11), and -10 (n = 10) according to the treating dosages of 2, 5, and 10 mg kg− 1 day−1, respectively. The tumor volume and body weight were recorded every other day. All mice were sacrificed on day 9, and the levels of angiogenic factors were measured in harvested xenografts by immunohistochemistry and western blotting. Results: The tumor volume and weight of PROP-2 (0.72 ± 0.28 cm3, 0.59 ± 0.21 g) and PROP-5 (0.81 ± 0.35 cm3, 0.61 ± 0.25 g) were significantly decreased when compared with those of CTL (1.22 ± 0.58 cm3, 0.93 ± 0.15 g; P b 0.01). The tumor microvessel density (MVD) scores that PROP2, -5, and -10 groups had (49.28 ± 17.53, 52.45 ± 17.11, and 51.09 ± 13.18 pixels per picture, respectively) were lower than those from the control group (65.29 ± 17.33 pixels per picture, P b 0.01). Furthermore, vascular endothelial growth factor (VEGF), metalloproteinase-2 (MMP-2), and metalloproteinase-9 (MMP-9) levels were significantly lower in the groups with propranolol treated dosage of 5 and 10 mg kg− 1 day− 1 than in the control group. Conclusions: Propranolol can exhibit an inhibitory effect on the tumor growth and angiogenic factors expression in neuroblastoma xenografts, which may provide some knowledge to the role of β-blockers in the management of NB. © 2013 Elsevier Inc. All rights reserved.

Neuroblastoma (NB) is a neural crest-derived embryonic malignancy of the postganglionic sympathetic nervous system [1]. It is the most common childhood extracranial ☆ Statement of Financial Support: Key Clinical Discipline of the Chinese Ministry of Health in 2010–2012 and the National Natural Science Foundation of China, grant no.81072069. ⁎ Corresponding author. Department of Surgery, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, P.R. China. E-mail address: [email protected] (X. Xiao).

0022-3468/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2013.08.022

solid tumor, which accounts for approximately 15% of all childhood cancer mortalities [2]. The treatment methods used in the management of NB include surgery, chemotherapy, radiotherapy, and biotherapy. In the past few decades, the outcome in these patients with favorable biological features has improved substantially due to advances in multimodal treatments, while the outcome in children with a high-risk clinical phenotype remains poor with a long-term survival rate less than 40% [3,4]. Therefore, there is an

Antiangiogenic effect of propranolol urgent need for innovative therapeutic options for these poor children. Propranolol, a non-selective β-adrenergic receptor blocker, was traditionally prescribed for many disorders in cardiac system. The newly discovered effect of propranolol was first observed by Leaute-Labreze et al, when propranolol achieved the growth arrest of capillary hemangiomas in infants who received it for cardiac complications [5]. Since the spectacular effect of propranolol on hemangiomas, many physicians from other hospitals utilized it to treat children with problematic hemangiomas and the outcomes of most cases were good [6]. The possible mechanisms of therapeutic effect of propranolol on hemangiomas are complex, which may due to vasoconstriction, inhibition of angiogenesis, and induction of apoptosis in capillary endothelial cells [7,8]. Propranolol was also shown to inhibit the growth factorinduced proliferation, chemotactic motility and differentiation of normal cultured human umbilical vein endothelial cells, as well as their formation of capillary-like structures in a dose-dependent manner [9]. Moreover, propranolol was found to have an inhibitory effect in tumor progression, metastasis, and the secretion of angiogenic cytokines in several kinds of cancers. The metastasis in melanoma and lung carcinoma mice model was reduced by the propranolol administration [10]. The production of angiogenic factors, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2), was significantly decreased by propranolol treatment in human leukemic cells and nasopharyngeal carcinoma cells [11,12]. Based on these studies, we hypothesized that propranolol can inhibit tumor-associated angiogenesis and therefore arrest the growth of malignant solid tumors. In the present study, we investigated the effect of propranolol on tumor angiogenesis and growth in a NB nude mice model.

2. Methods 2.1. Cell culture and animals BE(2)-C, a NB cell line purchased from American Type Culture Collection (ATCC), was cultured in Dulbecco's Modified Eagle Medium 1:1 Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin G, and 100 μg/ml streptomycin(Invitrogen). Cells were grown in 75 cm2 flasks in a fully humidified incubator containing 5% CO2 at 37 °C. Four six-week-old athymic nude mice (BALB/c-nu/nu) were obtained from Slac Laboratory Animal Co, Ltd (Shanghai, China), and housed in laminar-flow cabinets under specific pathogen-free conditions. The study was approved by the Institutional Animal Care and Use Committee of Fudan University. Tumor establishment BE(2)-C cells (5 × 105 cells in 100 μl PBS) were injected subcutaneously on left hind flank

2461 of the animal according to an established protocol [13]. Palpable xenografts tumors developed within 4–6 days. Then, mice with xenografts were randomly divided into four groups. Animals in the control group (CTL, n = 12) only received a comparable amount of saline. Mice in the other three groups received a daily i. p. injection of propranolol (Sigma-Aldrich) at the following dosage of 2, 5, 10 mg/kg (accordingly as PROP-2, n = 11; PROP-5, n = 11; and PROP-10, n = 10) dissolved in saline per day. Tumor volume was measured every other day with an external caliper, and calculated using the formula: 1/2 × length × width2 [14]. The body weight of each mouse was monitored every other day too. The mice were sacrificed at the end of 9 days' treatment. The xenografts were removed and weighed and then snap-frozen in liquid nitrogen. Paraffin-embedded tumor blocks were prepared for further analysis.

2.2. Immunohistochemistry Paraffin-embedded, 4 μm sections were deparaffinized in xylene and rehydrated through a graded series of ethanol in distilled water. Tumor microvessel density (MVD) was evaluated by staining with anti-CD34 antibody (1:300, Abcam) incubated overnight at 4 °C. Omission of primary antibody served as a negative control. Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide (H2O2) in PBS for 15 min, followed by incubation with the secondary antibody for 2 h at room temperature. Diaminobenzidine was used as a substrate to develop a brown precipitate. Tumor areas were examined with optical microscopy. Four random 100 × fields on each section were captured using a digital camera (Olympus DP72, Japan) and analyzed with Image J. Colors were separated, and the 8-bit binary image for the brown channel was transferred to grayscale followed by automatic thresholding that clearly identified the CD34 positive endothelial cells. Fixed light intensity settings were used throughout the analysis. MVD scores were calculated as the number of CD34 positive pixels per picture and the mean MVD scores of 10 sections was recorded for each group.

2.3. Western blotting Tumor tissues 80 mg were collected in RIPA buffer (50 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) on ice for 15 minutes. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, Inc.). After denaturation in boiling water for 5 min, samples (20 μg) were loaded onto the SDS-polyacrylamide gradient gels (Bio-Rad, Hercules, USA). Proteins were resolved by electrophoresis and transferred to polyvinyldinefluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked with 5% non-fat milk and then separately incubated overnight at 4 °C with the appropriate primary antibody:

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anti-human MMP-9, anti-human MMP-2 (both 1:1500 dilutions, Cell signaling), and anti-human VEGF (1:500 dilutions, Santa-Cruz). After incubation for 2 hours with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:3000 dilutions, Pierce, Thermo) at room temperature. Corresponding membranes were probed with anti-human GAPDH antibody (1:10000 dilutions, Kangcheng Co. Ltd, China) to ensure equal loading of protein. Bands were visualizing using an enhanced ECL chemiluminescence system (Millipore) according to the manufacturer's directions, imaged on a Bio-Rad Molecular Imager ChemiDocTM × RST with Image LabTM software (USA), and analyzed by Image J. The experiments were repeated three times.

2.4. Statistical analysis Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago). Data were expressed as mean ± SEM, and one-way analysis of variance (ANOVA) in conjunction with least significant difference test was performed to analyze experiments. The level of significance was set at P b 0.05.

3. Results 3.1. Propranolol inhibited BE(2)-C xenografts growth Tumor bearing mice were treated with different dose of propranolol or saline for 9 days. Mice appeared healthy during the treatment, with no significant loss of body weight (Table 1) and no signs of toxicity or metastasis by visual inspection. The growth curves of BE(2)-C xenografts with different doses of propranolol are shown in Fig. 1. Propranolol treatments with 2 and 5 mg kg − 1 day − 1 (PROP-2 and PROP-5) were associated with significant decrease in tumor volume (Fig. 1). On day 9, tumor volume in PROP-2 and PROP-5 become significantly smaller than that in the CTL group (Fig. 1). The animals were sacrificed on day 9, with xenografts harvested and weighed. The tumor weight in PROP-2 and PROP-5 were 36.6% and 34.4%

Table 1

lighter than that of the CTL group, respectively (P b 0.05, Table 1).

3.2. Propranolol decreased MVD and angiogenic factors expression in BE(2)-C xenografts The effect of propranolol on MVD and specific proteins expression including VEGF, MMP-2 and MMP-9 in the xenografts were examined by immunohistochemical staining of tumor sections for CD34 and Western blotting. Fig. 2 shows that tumor sections from the PROP-2, -5, and -10 groups had lower MVD (49.28 ± 17.53, 52.45 ± 17.11, and 51.09 ± 13.18 pixels per picture, respectively) than those from the CTL group (65.29 ± 17.33 pixels per picture, P b 0.01). Furthermore, VEGF, MMP-2, and MMP-9 protein levels were significantly lower in the xenografts from the PROP-5 and -10 groups compared with those from the CTL group (Fig. 3).

4. Discussion Accumulating studies have demonstrated that β-adrenergic antagonists can inhibit tumor cell proliferation in vitro, and block stress-induced enhancement of tumor progression or metastasis without affecting primary tumor growth in vivo [15]. Recently, several clinical studies provided evidence that propranolol might impact tumor development and improve the cancer-specific mortality in breast cancer [16]. The patients receiving β-blockers for hypertension were less likely to have advanced breast cancers [17]. A series of in vitro experiments showed that the non-selective β-blocker propranolol inhibited norepinephrine-induced tumor progression, metastasis, and the secretion of angiogenic cytokines. When chemotherapeutic drugs accompanied by propranolol could have a synergistic effect in inhibiting the proliferation of some tumor cell lines [18]. Several in vivo studies have demonstrated that propranolol can reduce metastasis of PC-3 prostate cancer cells [19], prevent surgical stress-induced tumor growth in ovarian carcinoma [20], and modulate pancreatic cancer progression with high specificity [21]. To our knowledge, however, the effect of propranolol pediatric malignant tumors is not yet been explored to date.

Effects of propranolol on mice and xenograft weight.

Groups

No. of animals

Mice body weight (g) Day 1

CTL PROP-2 PROP-5 PROP-10

12 11 11 10

20.76 20.10 20.91 20.43

Day 9 ± ± ± ±

1.39 1.28 1.21 1.83

⁎ P b 0.01, the xenograft weight compared with the CTL group.

22.93 22.52 22.85 22.32

± ± ± ±

1.38 1.21 1.25 1.14

Xenograft weight (g)

P-value

0.93 0.59 0.61 0.76

0.002 ⁎ 0.005 ⁎ 0.352

± ± ± ±

0.15 0.21 0.25 0.21

Antiangiogenic effect of propranolol

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Fig. 1 Effect of propranolol on BE(2)-C neuroblastoma xenografts growth in nude mice. A, Representative size and appearance of xenograft nodules in the CTL, PROP-2, -5 and -10 groups on day 9. B, Growth curves of xenografts showing the mean volume (with SEM) of CTL (n = 12), PROP-2 (n = 11), PROP-5 (n = 11), and PROP-10 (n = 10) groups (*P b 0.05, compared with the CTL group).

We conducted the NB xenograft experiments by subcutaneously injecting BE(2)-C cells into nude mice to observe the impact of propranolol on NB in vivo. The BE(2)-C cell line was chosen because it contains an amplified MYCN locus and represents the most aggressive NB phenotype with high tumor vessel density. In the current study, we found that tumor bearing mice treated with proper dosage (2 and 5 mg kg− 1 day− 1) of propranolol seemed to exhibit a slower growth speed of the NB xenografts when compared to the CTL group. An obvious tumor weight suppression was recorded in the groups of PROP-2 and PROP-5 when compared with the CTL group on the end day of this experiment, but not in PROP-10. Our findings were also consistent with the results of other experiments that lower dosage of propranolol could have better therapeutic effect

Fig. 2 Effect of propranolol on tumor angiogenesis in vivo. Quantified MVD scores of xenografts of PROP-2, PROP-5, and PROP-2 groups (*P b 0.05, compared with CTL).

[21,22], and the dosage of propranolol used in the treatment in infantile hemangiomas was 1–3 mg kg− 1 day− 1. It is well known that the growth and metastasis of solid tumors depend on angiogenesis. In NB, Métier et al. were the first to demonstrate that tumor angiogenesis was associated with clinical phenotypes; high vascular index was correlated strongly with disseminated disease, unfavorable histology and poor survival [23]; therefore, tumor vasculature is an attractive target for therapy. Antiangiogenic agents thereby may be used to hinder neovascularization and prevent dissemination of this pediatric cancer, and many of these kinds of agents were under preclinical test. VEGF is an endothelial cell-specific mitogen that plays a crucial role in the angiogenesis and growth of nearly all human tumors [24]. In NB, the major stimulators of angiogenesis include, but are not limited to, the VEGF family members. Tumor cells can constitutively produce VEGF, which can promote angiogenesis by recruiting endothelial cells through various mechanisms. Furthermore, VEGF is also a paracrine/ autocrine growth factor for malignant cells that express the VEGF receptor. In our study, the Western blotting results showed that the expression of VEGF in the PROP-5 and PROP-10 were significantly lower than the CTL group. The values of MVD calculated in all propranolol treated groups were also lower than that of CTL group. Our findings suggested that propranolol could have a role in decreasing the level of angiogenesis in these experimental tumors. As a result, the growth of the NB xenografts was suppressed. Matrix metalloproteinase (MMP), a multigene family of proteolytic endopetidases, play an important role in tumor growth, metastasis, and angiogenesis [25–27]. MMPs take

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Fig. 3 Western blotting of VEGF, MMP-2, and MMP-9 protein expression in BE(2)-C xenografts treated with or without propranolol. (A) Representative images of MMP-9, MMP-2, and VEGF protein expression in all groups. (B) The quantitative evaluation histogram of MMP-2 protein expression in all groups. (C) The quantitative evaluation histogram of VEGF protein expression in all groups. (D) The quantitative evaluation histogram of MMP-9 protein expression in all groups. Data are presented as mean ± SEM. (*P b 0.05, compared with CTL).

part in important steps of angiogenesis, such as remodeling of the basement membrane and degradation of extracellular matrix components. The key members of the MMP family participating in tumor angiogenesis are MMP-2, MMP-9, and MMP-14 [25,28,29]. MMP-2 up-regulation is strongly associated with clinically advanced disease [29]. MMP-9 can regulate tumor angiogenesis by degradation of extracellular components [30]. In our study, the expression of MMP-2 and MMP-9 in the group of PROP-5 and PROP-10 were significantly decreased compared with the CTL group, which might also involve in the process of antiangiogenesis. Numerous antiangiogenic molecules have been described to date, most of which are being tested in preclinical or clinical trials for adult cancers with only a small fraction showing antiangiogenic activity in patients [31]. Direct angiogenic inhibitors are not currently used in NB therapy protocols, although some preclinical studies evaluating antiangiogenic agents have been conducted [32]. However, it is becoming increasingly clear that the “classic” antiangiogenic compounds result in tumor resistance development [33]. Furthermore, the side effects in cardiovascular system have been observed with several antiangiogenic drugs [34]. In contrast, a systematic review recently revealed a low rate of adverse events in the treatment of complicated hemangiomas with propranolol. Unlike the classic anti-

angiogenic molecules, all of these adverse effects, such as sleep changes, cardiac and gastrointestinal symptoms, can disappear after the propranolol withdrawal [6,35]. All these evidences would suggest propranolol a safe and promising candidate of antiangiogenic drugs. Taken together, we believe that the proper dosages of propranolol, especially 5 mg kg− 1 day− 1, could have an inhibitory effect both on the tumor growth and angiogenic factors expression in our experimental system. Due to study limitations, the mortality of the experimental animals was not observed, nor were the long-term effects of propranolol treatment investigated. Although further studies are needed to better understand the possible mechanisms of propranolol induced tumor suppression, our preliminary results may provide some knowledge to the role of β-blockers in the management of NB.

Acknowledgments This work was supported by the Key Clinical Discipline of the Chinese Ministry of Health in 2010–2012, and the National Natural Science Foundation of China, grant no.81072069.

Antiangiogenic effect of propranolol

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Antiangiogenic effect of propranolol on the growth of the neuroblastoma xenografts in nude mice.

Propranolol has been reported to display an antiangiogenic effect on infantile hemangiomas and also some adult cancers. Little is known, however, abou...
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