Melittin Suppresses VEGF-A-Induced Tumor Growth by Blocking

Article pubs.acs.org/jnp

Melittin Suppresses VEGF-A-Induced Tumor Growth by Blocking VEGFR‑2 and the COX-2-Mediated MAPK Signaling Pathway Jeong-Eun Huh,†,# Jung Won Kang,‡,# Dongwoo Nam,‡ Yong-Hyeon Baek,§ Do-Young Choi,‡ Dong-Suk Park,*,§ and Jae-Dong Lee*,‡ †

Oriental Medicine Research Center for Bone and Joint Disease, East-West Bone & Joint Research Institute, Kyung Hee University, 149, Sangil-dong, Gangdong-gu, Seoul, 134-727, Korea ‡ Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul, 130-701, Korea § Department of Acupuncture and Moxibustion, Kyung Hee University Hospital at Kangdong, 149, Sangil-dong, Gangdong-gu, Seoul, 134-727, Korea ABSTRACT: Melittin (1) is a major polypeptide in honey bee venom that has been used traditionally against chronic inflammation and cancer. However, its molecular mechanism has not been determined. In this study, the antitumor effect of 1 was compared with that of NS398, a cyclooxygenase-2 (COX-2) inhibitor, in vivo and in vitro. Subcutaneous injection of 1 at 0.5 and 5 mg/kg suppressed significantly vascular endothelial growth factor (VEGF)-A-transfected highly metastatic Lewis lung cancer (VEGF-A-hm LLC) tumor growth by 25% and 57%, respectively. Also, 1 inhibited significantly the number of vessels around VEGF-A-hm LLC cells. The results were superior to those obtained in the mice treated with NS398. Compound 1 dose-dependently inhibited proliferation and tube formation in human umbilical vein endothelial cells (VEGF-A-HUVECs), without affecting cell viability in native HUVECs. In addition, 1 decreased the expression of VEGF receptor-2 (VEGFR-2), COX-2, and prostaglandin E2 (PGE2) in VEGF-A-transfected HUVECs. These effects were accompanied by a reduction of the phosphorylation of extracellular signal-regulated kinase 1/2 and c-jun Nterminal kinase, whereas it increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580 abolished the downregulation of COX-2 and VEGFR-2 and the inhibition of cell proliferation by 1. The antitumor activity of 1 may be associated with antiangiogenic actions via inhibiting VEGFR-2 and inflammatory mediators involved in the MAPK signaling pathway.

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signal-regulated kinases (ERK 1/2) and stress signaling process such as the c-jun, N-terminal kinase (JNK), and the p38 MAPK pathway.5 NS398, a COX-2-specific inhibitor of nonsteroidal antiinflammatory drugs (NSAIDs), inhibits the cyclooxygenase (COX) pathways. COX enzymes play a role in inflammation and cancer progression in the pulmonary microenvironment. There are two isotypes of COX enzymes, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and cell types, while COX-2 is inducible by stimuli such as cytokines, growth factors, and inflammatory mediators.6 In particular, the major COX-2 metabolite, prostaglandin E2 (PGE2), signals through four G-protein-coupled receptors, triggering downstream signaling cascades, such as the MAPK pathway,6,7 leading to the activation of downstream gene expression. High levels of COX-2 and PGE2 are associated with the progression of nonsmall-cell lung cancer, including in some premalignant lesions, and are associated with proliferation, invasion, apoptosis resistance, suppression of immune response, and poor patient prognosis independent of cancer stage.8−12 Inhibition of COX-

ngiogenesis, the process involving the growth of new blood vessels from pre-existing vessels, occurs under a variety of physiological and pathological conditions, including embryonic development, wound healing, chronic inflammation, and tumor progression and metastasis.1 Any significant increase in tumor mass over 2−3 mm in size must be preceded by an increase in the vascular supply to deliver nutrients and oxygen to the tumor cells and the neovascularization of endothelial cells.1,2 Thus, blockage of angiogenesis is considered to be an important therapeutic and preventive target for cancer. Vascular endothelial growth factor (VEGF) plays a key role in the regulation of the angiogenic process and promotes angiogenesis by induction of the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).3 Of the VEGF families of proteins, VEGF-A isoforms such as VEGF165 and VEGF121 are secreted and have mitogenic action on the endothelial cells through binding to membrane receptors for tyrosine kinases expressed on endothelial cells, including VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 is the most important receptor for VEGF-A signaling in vascular endothelial cells.3,4 Tyrosine phosphorylation in the intracellular domain further activates the mitogen-activated protein kinase (MAPK) signaling pathway, including extracellular © 2012 American Chemical Society and American Society of Pharmacognosy

Received: June 26, 2012 Published: October 30, 2012 1922

dx.doi.org/10.1021/np300446c | J. Nat. Prod. 2012, 75, 1922−1929

Journal of Natural Products

Article

Figure 1. Structure of melittin (1), a peptide consisting of 26 amino acids with the sequence GIGAVLKVLTTGLPALISWIKRKRQQ (molecular formula C131H229N39O31, molecular weight 2846.5).

effects by inducing apoptosis in human leukemic cells, MG63 sarcoma cells, and hepatocellular carcinoma and by reducing tumor cell metastasis via the suppression of tumor cell motility and migration.19−23 However, the precise molecular details of the antitumor and anti-inflammatory activities of 1 have not been determined. In the present study, it was investigated as to whether 1 exerts an antitumor effect through antiangiogenesis and antiinflammation based on the role of VEGFR-2 and the COX-2mediated MAPK signaling pathway. With respect to COX-2 targeting action, NS398, a known selective inhibitor of COX-2, was used for comparison of the efficacy.

2 by nonsteroidal anti-inflammatory drugs result in inhibition of angiogenesis and down-regulation of angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF)-2.13,14 Melittin (1) is the principal active component of the venom of the European honey bee (Apis mellifera L.; Apidae) and is used for the treatment of arthritis, atherosclerosis, and cancer in traditional medicine.15,16 This compound has shown potent suppressive effects on the inflammatory responses of BV2 microglia cells, Raw 264.7 macrophage cells, and synoviocytes obtained from rheumatoid arthritis patients.17,18 A recent study showed that binding of 1 to the sulfhydryl group of I kappa B kinases (IKKs) resulted in reduced IKK activities, IkappaB release, nuclear factor-kappaB (NF-κB) activity, and generation of inflammatory mediators.17 Furthermore, lipopolysaccharide (LPS)-induced nitric oxide (NO) and PGE2 production occurs via JNK pathway-dependent inactivation of NF-κB, suggesting that inactivation of the JNK pathways may also contribute to the anti-inflammatory and antiarthritic effects of 1 and honey bee venom.18 Also, 1 has been reported to have antitumor

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RESULTS AND DISCUSSION Melittin (1) Inhibits Tumor Growth in VEGF-A-hm LLCBearing Mice. To examine whether the antiangiogenic action of 1 translated to antitumor activity in vivo, C57BL/6 mice were teated with subcutaneous (sc) implants of VEGF-Ainduced hm LLC allografts with sc injections of 1 (0.5 and 5 1923



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(5 mg/kg) showed about a 22% inhibition, compared with a control (Figure 3), showing that the antiangiogenic effect of 1

mg/kg/day) or NS398 (5 mg/kg) every other day for 18 days starting after tumor inoculation. Tumors grew rapidly and reached an average volume of 1180 mm3 18 days after the inoculation of VEGF-A-hm LLC cells. Compound 1 significantly inhibited VEGF-A-induced tumor growth by 25% (0.5 mg/kg/day) and 57% (5 mg/kg/day) over the study period, while NS398 (5 mg/kg) inhibited growth by about 42%, as compared with a control (Figure 2A). Compound 1 also

Figure 3. Melittin (1) inhibits the angiogenesis in VEGF-A-transfected hm LLC cells induced angiogenesis in vivo. (A) Mice were intradermally inoculated with VEGF-A-transfected hm LLC (VEGFA-hm LLC) cells and administered subcutaneously with 1 (0.5, 5 mg/ kg) for 7 days from day 3 post tumor inoculation. Tumor-inoculated sites were isolated from mice 10 days after tumor inoculation. The macroscopic observation of neo-vessel formation is shown. (B) Tumor-supplying vessels were counted. Values represent means ± SEM (n = 7) (***p < 0.001 compared with control).

was greater than that of NS398. No acute side effects such as hair loss, lethargy, and mortality were detected. Tumor angiogenesis occurs as a cascade of molecular and cellular events. The process is initiated by production of angiogenic growth factors from tumor, stromal, and infiltrating inflammatory cells, and the tumor microenvironment plays a critical role in tumor growth, invasion, metastasis, and angiogenesis.24,25 Antiangiogenic therapy for cancer aims to sustain release in which tumor cell proliferation and tumor expansion are stalled by inhibiting tumor-related angiogenesis. Therefore, inhibition of angiogenesis may prevent cancers from becoming malignant.26,27 In this study, the antiangiogenic effect by 1 against VEGF-A-induced tumor angiogenesis was confirmed in an intradermal model. These results suggest that compound 1 exerts its antitumor activity primarily through antiangiogenesis in VEGF-A-hm LLC cells. Melittin (1) Does Not Decrease Cytotoxicity in HUVECs. The cytotoxic effects of 1 and NS398 on HUVECs were determined initially. The cell viability of these cells treated with 1, 5, 10, 20, and 40 μM of 1 was 103.8 ± 1.3%, 98.9 ± 1.1%, 99.4 ± 0.7%, 98.5 ± 0.5%, and 96.5 ± 2.3% compared with the controls, respectively. Therefore, nontoxic concentrations were used in subsequent experiments on 1 (Figure 4B). Melittin (1) Inhibits VEGF-A-Induced Proliferation and Tube Formation of HUVECs. VEGF-A is one of the most potent angiogenic factors and is involved in proliferation and differentiation of a variety of normal and malignant cells and tissues.28,29 To determine whether 1 affects VEGF-A-activated endothelial cell proliferation and capillary-like tube formation crucial for angiogenic response, highly producing VEGF-AHUVECs were used. Compound 1 significantly inhibited the VEGF-A-induced proliferation in a dose-dependent manner with an IC50 of 18 μM (Figure 4C). This inhibitory effect was

Figure 2. Melittin (1) inhibits tumor growth in VEGF-A-transfected hm LLC-bearing mice. VEGF-A-transfected hm LLC (VEGF-A-hm LLC) cells were injected subcutaneously into the right flanks of C57BL/6 mice. Twelve days after tumor inoculation, the tumor size established ∼60 mm3, and mice were injected subcutaneously with 1 (0.5 mg/kg or 5 mg/kg) or NS398 (5 mg/kg). (A) Tumor volumes were measured with a caliper every three days for 24 days after tumor implantation and calculated according to the formula 0.52 × L (longest perpendicular length) × W2 (width). (B) Tumor weight. Values represent means ± SEM (n = 10) (*p < 0.05, **p < 0.01, and ***p < 0.001 compared with control).

suppressed tumor weight by 60% (0.5 mg/kg/day) and 91% (5 mg/kg/day) (Figure 2B). No acute side effects such as body weight loss, lethargy, hair loss, and mortality were evident. It is noteworthy that an effective dose of 0.5 or 5 mg/kg of 1 would exert an antitumor effect without toxicity in the VEGF-Ainduced hm LLC tumor model. The experimental design did not exclude a direct antiangiogenic effect of 1 on tumor cells. Thus, additional studies are required to elucidate the cause− effect relationship between the antitumor and antiangiogenic effects of 1. Melittin (1) Suppresses VEGF-A-hm LLC Tumor CellInduced Angiogenesis. To verify that antiangiogenesis was involved in the hm LLC growth inhibition by 1 in mice, the inhibitory activity of 1 was examined against angiogenesis induced in vivo by tumor cells. VEGF-A-hm LLC cells were inoculated intradermally onto the back skin of mice, and sc administration of 1 was carried out at doses of 0.5 or 5 mg/kg/ day for 7 days. Compound 1 significantly reduced the number of vessels oriented to the VEGF-A-induced tumor cell mass by 52% (0.5 mg/kg/day) and 59% (5 mg/kg/day), while NS398 1924



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Figure 4. Melittin (1) inhibits VEGF-A-induced proliferation of endothelial cells. (A) The expression of VEGF-A was analyzed in the medium of VEGF-A-transfected HUVECs by Western blotting. High levels of expression of VEGF-A on HUVECs were established by transfection with the pcDNA3.1 expression vector containing mouse VEGF-A or pcDNA3.1 vector alone (mock), as mentioned in the Experimental Section. Western blot analysis showed positive bands for VEGF-A (58 kDa). (B) Effect of 1 or NS398 on viability of nontransfected HUVECs (native HUVECs). Native HUVECs were treated with 1 or NS398 for 48 h in M199 serum-free medium. Viable cells were examined in an MTT assay. Data were obtained from three independent experiments in triplicate, and bars represent the means ± SEM (***p < 0.001 compared with control). (C) Effect of 1 or NS398 on VEGF-A-transfected HUVECs (VEGF-A-HUVECs). VEGF-A-HUVECs were exposed to 1 or NS398 in M199 serum free medium for 48 h, and then cell proliferation was assessed by BrdU incorporation assays. The absorbance was read by an ELISA reader. Data were obtained from three independent experiments in triplicate, and the bars represent means ± SEM (***p < 0.001 compared with control).

not due to the cytotoxicity of 1 in VEGF-A-HUVECs, because this compound 1 did not show any significant cytotoxic effects on native HUVECs (Figure 4B). Next, the effect of 1 on capillary differentiation of VEGF-A-HUVECs seeded on Matrigel was examined. Melittin (1) significantly reduced the formation of capillary-like structures in VEGF-A-HUVECs in a dose-dependent manner with an IC50 of approximately 13 μM (Figure 5A), but did not affect the native HUVECs (Figure 5B). In comparison, NS398 showed a moderate inhibitory effect on VEGF-A-induced proliferation of HUVECs (Figure 4C) and a significant effect on tube formation of VEGF-AHUVECs, with an IC50 value of approximately 20 μM (Figure 5C). The angiogenesis process involves the proliferation, migration, and tube formation of endothelial cells and can be initiated by angiogenic cytokines such as VEGF.30−32 In the present experiments, 1 markedly suppressed cell proliferation and tube formation in a dose-dependent manner, suggesting that melittin (1) possesses antiangiogenic property. NS398, a selective COX-2 inhibitor, also inhibited cell proliferation and capillary-like tube formation, supporting the important role of the COX-2 pathway as a target in angiogenesis inhibition by 1. Melittin (1) Decreases VEGFR-2 and Inflammatory Mediator in VEGF-A-Transfected HUVECs. To determine whether the observed antiangiogenic attributes of 1 were associated with an attenuation of VEGFR-2 phosphorylation or COX-2 targets, Western blot analysis was performed. Compound 1 downregulated VEGFR-2, VEGF-A, and COX-

2 expression in VEGF-A transfected HUVECs (Figure 6A), but did not affect VEGFR-2, VEGF-A, and COX-2 expression in mock-transfected HUVECs (Figure 6B). PGE2, a product of COX-2 and VEGF, has been linked to cancer.25,33,34 Therefore, the levels of secreted PGE2 and VEGF were measured using an enzyme-linked immunosorbant assay (ELISA). Likewise, 1 significantly decreased VEGF production and PGE2 release in a concentration-dependent manner compared with the control (Figure 6C and D). VEGF or COX-2 expression is strictly correlated with angiogenesis in endothelial cells.34,35 Presently, VEGF protein expression in tumors was also consistent with that of endothelial cells and with VEGFR-2; the latter is a potential key target of antiangiogenic tumor therapy.27,34−37 COX-2 is also highly expressed in endothelial cells within tumors, and it plays a critical role in regulating tumor growth as well as inflammatory and angiogenic processes in tumor tissues.34,35,37,38 The angiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism, including PGE2.34,37 Mechanistically, 1 significantly attenuated the expression of COX-2 as well as VEGFR-2 and decreased the production of VEGF and secretion of PGE2 in HUVECs. These results provide mechanistic insights into how 1 targets multiple facets of vascular endothelial angiogenic signaling through the VEGF and COX-2 axes. Melittin (1) Targets VEGFR-2 and the COX-2 Signaling Pathway. The VEGF-A/VEGFR-2 or VEGF-A/COX-2mediated MAPK signaling pathway is a potential key target 1925



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Figure 5. Melittin (1) inhibits VEGF-A-induced tube formation of endothelial cells. Native HUVECs were treated with different concentrations of 1 (B), or VEGF-A-HUVECs were mixed with the indicated concentrations of 1 (A) or NS398 (C) and then plated on diluted growth factor-reduced matrigel (5 mg/mL) for 18 h. After washing and fixation, cells were observed under a microscope (100×) and photographed. The tube area from a randomly chosen field was quantified using an image analysis system. This figure is a representation of three similar experiments in triplicate. Data are presented as means ± SEM (###p < 0.001 compared with mock-transfected endothelial cells. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with VEGF-A-transfected endothelial cells).

MAPK pathways. This study is the first report of the decrease by 1 of the VEGF-A-activated MAPK signaling pathway in HUVECs. In summary, melittin (1) suppressed VEGF-A-induced LLC tumor growth and tumor-associated angiogenesis in vivo. This polypeptide inhibited VEGF-A-induced proliferation and capillary differentiation (tube formation) without cytotoxicity associated with VEGFR-2 and COX-2 via the MAPK signaling pathway. Taken together, these findings suggest that 1 may be a cancer chemopreventive agent, as a target inhibitor with antiangiogenic and anti-inflammatory activities, which would have an advantage in cancer therapy when compared with a selective COX-2 inhibitor drug such as NS398. Additional studies are warranted to confirm the antitumor efficacy of 1 and to determine its pharmacokinetics, cellular uptake, and metabolism.

of angiogenic tumor therapy.34,35,39 To understand better how 1 inhibits VEGF-A-induced angiogenesis, the potential involvement of the MAPK pathway was investigated by Western blotting. 1 diminished the phosphorylation of ERK 1/2 in a dose-dependent manner in VEGF-A-transfected HUVECs. Interestingly, 1 completely blocked VEGF-Ainduced phosphorylation of JNK at a concentration of 1 μM. In contrast, 1 increased the phosphorylation of p38 in a concentration-dependent manner in VEGF-A-HUVECs (Figure 7A). The p38-specific inhibitor SB203580 effectively blocked the downregulation of phosphorylation of p38 in VEGF-A-transfected HUVECs (Figure 7B), restored the expression of COX-2 or VEGF-A by 1 (Figure 7C), and induced the antiproliferative activity by 1 in VEGF-Atransfected HUVECs at nontoxic concentrations (Figure 7D). The ERK pathways are activated by growth factors and angiogenic cytokines, such as VEGF, while the JNK and p38 pathways are activated by environmental stress in addition to growth factor signaling.40 These pathways in endothelial cells are critical in the angiogenic response cascades, and individual MAPK branches separately drive endothelial cell proliferation, migration, invasion, and differentiation during angiogenesis.40,41 The observation that 1 suppressed the phosphorylation of ERK and JNK, while increasing the p38 MAPK pathway in VEGF-A-HUVECs, favors the suggestion that these downstream components might be targeted by 1 for antiangiogenic cellular responses. Considering that p38 MAPK is generally activated by genotoxic agents or apoptosis,40 activation of p38 MAPK by 1 may mediate the apoptosis of endothelial cells during the antiangiogenic process. Our data also showed that the p38 MAPK inhibitor blocked antiproliferative activity and decreased COX-2 and VEGF expression by 1 in VEGF-AHUVECs, supporting a critical role of p38 MAPK in the inhibition of COX-2 and angiogenesis by 1 with the other

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EXPERIMENTAL SECTION

General Experimental Procedures. Melittin (1) (>90% pure), NS398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide), (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), heparin, gelatin, and mouse anti-β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). PGE2 was purchased from R&D Systems (Minneapolis, MN, USA). Endothelial-basal media (EBM), Eagle’s minimal essential medium (EMEM), fetal bovine serum (FBS), penicillin−streptomycin, and Lipofectamine 2000 were obtained from Gibco (Grand Island, NY, USA). A 5-bromo-2′deoxyuridine (BrdU) colorimetric assay kit was purchased from Roche (Stanhofer, Mannheim, Germany). Growth factor reduced matrigel and mouse anti-COX-2 were purchased from Becton-Dickinson (San Jose, CA, USA). Mouse anti-VEGF-A and anti-VEGFR-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-phospho-ERK-1/2, phospho-p38, phospho-JNK, ERK, p38, and JNK were from Cell Signaling Technology (Danvers, MA, USA). Nonfat dry milk was purchased from Bio-Rad (Hercules, CA, USA). The enhanced chemiluminescence detection system and 1926



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mouse embryo tissue and then 1 μg of total RNA were reverse transcribed into single-stranded cDNA using Superscript II reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Amplification of VEGFA164 cDNA was carried out in each reaction by polymerase chain reaction (PCR). The primer sequences of mouse VEGF-A164 (GenBank accession no. NM_001025366.2) were 5′-TACCTCCACCATGCCAAGTG-3′ (forward) and 5′-TGCTGTAGGCTCATCTC3′ (reverse). The reaction mixture was first denatured at 50 °C for 30 min and 94 °C for 2 min. The PCR conditions were 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s for 30 cycles, followed by 72 °C for 10 min. Mouse VEGF-A164 cDNA fragment was inserted into the pDrive plasmid (Qiagen, Valencia, CA, USA) and subcloned into the pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) eukaryotic expression vector. The full-length sequence was confirmed by enzymatic digestion and dideoxy sequencing. hm LLC cells or HUVECs were transfected with the pcDNA3.1 expression vector containing mouse VEGF-A164 (VEGF-A-hm LLC or VEGF-A-HUVECs) or pcDNA3.1 vector alone (mock-hm LLC or mock HUVECs) as a control. The cell lines were maintained in an atmosphere of 5% CO2 at 37 °C in EMEM supplemented with 10% FBS. After 48 h of transfection, cells were trypsinized and replated in cell culture medium containing 800 μg/mL of G418. G418-resistant clones were selected and then expanded for in vivo and in vitro study. Tumor Growth in VEGF-A-Induced hm LLC-Bearing Mice. VEGF-A-hm LLC cells at a density of 1 × 106 cells/100 μL were delivered into the right flank of C57BL/6 mice by sc injection. Starting 12 days after tumor inoculation, the mice were given sc injections of 1 (0.05 or 5 mg/kg) or NS398 above the thoracic vertebra every other day. Tumor volume was estimated (in ca. 60 mm3) twice weekly as the product of two-dimensional caliper measurements and calculated according to the following formula: 0.52 × L (longest perpendicular length) × W2 (width). All animal experiments were carried out according to international accredited guidelines and approved by medical ethical regulations of the Kyung Hee University Medical Center (KHMC-IACUC-2010008). Tumor Cell-Induced Angiogenesis. Mice were inoculated intradermally with VEGF-A-hm LLC cells (5 × 105 cells in 50 μL) in the back. Compound 1 and NS398 were administered by sc injections of 0.5 and 5 mg/kg per day starting on day 3 after tumor cell inoculation. Ten days after the tumor inoculation, the mice were sacrificed and the tumor-inoculated skin was separated from the underlying tissues. Angiogenesis was quantified by counting only the vessels directly supplying the tumor under a dissecting microsope. Cell Viability. The mitochondrial-dependent reduction of MTT to formazan was used to measure cell respiration as an indicator of cell viability. HUVECs were seeded onto 0.1% gelatin-coated 96-well microplates at a density of 1 × 104 cells per well in 100 μL of complete M199 medium. After incubation at 37 °C in a humidified incubator for 24 h, cells were treated with different concentrations of 1 (1, 5, 10, 20, or 40 μM) in serum-free M199 basal medium for 48 h at 37 °C in a CO2 incubator. After incubation, 20 μL of MTT (stock solution; 5 mg/mL) was added to each well, and the samples were incubated for a further 3 h. The medium was then removed, and 2-propanol was added to dissolve the formazan. After centrifugation at 5000g for 5 min, 100 μL of supernatant from each sample was transferred to 96well plates and absorbance was read at 570 nm. Cell Proliferation BrdU Assay. VEGF-A-HUVECs were seeded at a density of 5 × 103 cells/well in 96-well plates. After 24 h, the medium was removed and replaced with basal medium, and the samples were exposed to various concentrations of 1 (5, 10, or 20 μM) or NS398 (5, 10, or 20 μM) with or without VEGF-A. For inhibition of the p38 MAPK activity, cells were pretreated with the p38 inhibitor SB203580 for 30 min prior to addition of 1. After 48 h of incubation at 37 °C, 10 μL of BrdU was added to each well, and the samples were incubated for a further 6 h at 37 °C. Cells were fixed, and anti-BrdUperoxide dismutase was added and then detected by the 3,5,3′,5′tetramethylbenzidine substrate reaction. The reaction was quantified using an ELISA reader at 480−650 nm.

Figure 6. Melittin (1) inhibits VEGF-A-induced expression of VEGFR-2 and reduces the secretion of PGE2 in VEGF-A-transfected HUVECs. (A, B) Expression of VEGFR-1, VEGFR-2, and VEGF-A in VEGF-A-transfected HUVECs and mock-transfected HUVECs. Cells were treated with 1 (1, 5, and 10 μM) for 24 h. The level of VEGFR-1, VEGFR-2, and VEGF-A expression was assessed by Western blotting. (C) VEGF production inhibited by 1 in VEGF-A-transfected HUVECs. (D) PGE2 release reduced by 1 in VEGF-A-transfected HUVECs. The cells were exposed to 1 (1, 5, and 10 μM) in M199 containing 5% FBS for 24 h, and then VEGF production or PGE2 release in culture supernatant was measured by ELISA (###p < 0.001 compared with mock-transfected HUVECs). Results are expressed as means ± SEM of three experiments performed in triplicate (***p < 0.001 compared with VEGF-A-transfected HUVECs). nitrocellulose membrane were from Amersham Life Science (Arlington Heights, IL, USA). Cell Culture. Mouse Lewis lung carcinoma (LLC) cells that were originated spontaneously from mouse lung were kindly provided by Dr. Ikuo Saiki (Toyama Medical and Pharmaceutical University, Toyama, Japan). A highly metastatic subline of LLC (hm LLC) was generated by three rounds of serial in vivo passages and recovered from spontaneous lung metastasis after subcutaneous inoculation of LLC cells.36 hm LLC cells were maintained as monolayer cultures in EMEM supplemented with 10% FBS, 100 units/mL antibiotic− antimycotic, and 2.2 g/L sodium bicarbonate. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical vein using type II collagenase.36 Transfection with VEGF-A of hm LLC and HUVECs. To establish VEGF-A-transfected cell lines, total RNA isolated from 1927



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Figure 7. Melittin (1) inhibits VEGF-A-induced angiogenesis through MAPK pathways in VEGF-A-transfected HUVECs. (A) Effect of 1 on MAPK signaling in VEGF-A-transfected HUVECs. (B) Effect of the p38-specific inhibitor SB203580 on phosphorylation of p38 in VEGF-A-transfected HUVECs. (C) Effect of the p38-specific inhibitor SB203580 on COX-2 and VEGF-A expression increased by 1 co-treatment of VEGF-A-transfected HUVECs. (D) Effect of p38-specific inhibitor SB203580 on cell proliferation co-treatment with 1 in VEGF-A-transfected HUVECs (***p < 0.001 compared with VEGF-A control and ap < 0.05 compared with 1). Results are expressed as means ± SEM of three experiments carried out in triplicate. Tube Formation Assay on Matrigel. Unpolymerized matrigel was added to 24-well plates with a total volume of 250 μL in each well and allowed to polymerize for 1 h at 37 °C. VEGF-A-HUVECs, mock HUVECs, or native HUVECs were plated at a density of 1 × 105 cells on the matrigel, and various concentrations of 1 (5, 10, or 20 μM) or NS398 (5, 10, or 20 μM) were added. After an 18 h incubation in a 5% CO2 atmosphere at 37 °C, the cells were photographed and analyzed to determine the extent of tube formation. Western Blot Analysis. VEGF-A-HUVECs exposed to 1, 5, or 10 μM 1 were harvested and washed with cold PBS. For inhibition of the p38 MAPK, VEGF-A-HUVECs were pretreated with the p38 inhibitor SB203580 for 30 min prior to addition of 1. Cells were lysed with lysis buffer (Invitrogen) with protease inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL pepstatin A, and 1 mM 4-(2aminoethyl)benzenesulfonyl fluoride) and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). Protein concentration was measured by a commercial protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard (Bio-Rad). The proteins (10 μg/lane) were size-fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-C nitrocellulose membranes for 2 h at 300 mA using a transfer system. Membranes were blocked with 5% nonfat dry milk in TBS-T (0.05% Tween-20, 130 mM NaCl, and 25 mM Tris base). The blocked membranes were then individually immunoblotted with primary antibody (1:1000 dilution) against VEGFR-2, VEGF-A, COX-2, phospho-ERK-1/2, phospho-JNK, phospho-p38, ERK, JNK, p38, and β-actin. The blots were incubated with horseradish peroxidase (HRP)-conjugated antibodies and an enhanced chemiluminescence detection system.

Measurement of VEGF. The culture supernatant of VEGF-Atransfected HEVECs was individually collected, and the level of VEGF was measured using specific enzyme immunoassay kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Determination of PGE2 Activity. Prostaglandin synthesis/release was determined by an ELISA kit (Cayman, Ann Arbor, MI, USA), according to the manufacturer’s instructions. Statistical Analysis. The results were expressed as means ± SEM calculated from the specified numbers of determination. Statistically significant differences between experimental and control groups were detected by Student’s t-test. Tumor volume, weight, and immunohistochemistry data were analyzed using ANOVA followed by Duncan’s multiple range test.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-440-6792 or +82-2-958-9208. Fax: 82-2-440-6699. E-mail: [email protected] or [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the contribution of Prof. Y. G. Park, Department of Pathology, College of Medicine, Kyung 1928



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(36) Huh, J. E.; Baek, Y. H.; Lee, M. H.; Choi, D. Y.; Park, D. S.; Lee, J. D. Cancer Lett. 2010, 292, 98−110. (37) Cheng, A. S.; Chan, H. L.; To, K. F.; Leung, W. K.; Chan, K. K.; Liew, C. T.; Sung, J. J. Int. J. Oncol. 2004, 24, 853−860. (38) Iniguez, M. A.; Rodriguez, A.; Volpert, O. V.; Fresno, M.; Redondo, J. M. Trends Mol. Med. 2003, 9, 73−78. (39) Kim, H. N.; Kim, H.; Kong, J. M.; Bae, S.; Kim, Y. S.; Lee, N.; Cho, B. J.; Lee, S. K.; Kim, H. R.; Hwang, Y. I.; Kang, J. S.; Lee, W. J. J. Cell Biochem. 2011, 112, 894−901. (40) Wada, T.; Penninger, M. Oncogene 2004, 23, 2838−2849. (41) Binion, D. G.; Otterson, M. F.; Rafiee, P. Gut 2008, 57, 1509− 1517.

Hee University. This work was supported by a grant from the Kyung Hee University in 2011 (KHU-20110063).

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