molecules Article
Antiangiogenic Potential of Microbial Metabolite Elaiophylin for Targeting Tumor Angiogenesis Haet Nim Lim 1,† , Jun-Pil Jang 2,† , Jang Mi Han 1 , Jae-Hyuk Jang 2 , Jong Seog Ahn 2, * and Hye Jin Jung 1, * 1 2
* †
Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, Tangjeong-myeon, Asan-si, Chungnam 336-708, Korea;
[email protected] (H.N.L.);
[email protected] (J.M.H.) Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Korea;
[email protected] (J.-P.J.);
[email protected] (J.-H.J.) Correspondence:
[email protected] (J.S.A.);
[email protected] (H.J.J.); Tel.: +82-43-240-6160 (J.S.A.); +82-41-530-2354 (H.J.J.) These authors contributed equally to this work.
Received: 4 December 2017; Accepted: 9 February 2018; Published: 2 March 2018
Abstract: Angiogenesis plays a very important role in tumor progression through the creation of new blood vessels. Therefore, angiogenesis inhibitors could contribute to cancer treatment. Here, we show that a microbial metabolite, elaiophylin, exhibits potent antiangiogenic activity from in vitro and in vivo angiogenesis assays. Elaiophylin dramatically suppressed in vitro angiogenic characteristics such as proliferation, migration, adhesion, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF) at non-toxic concentrations. In addition, elaiophylin immensely inhibited in vivo angiogenesis of the chorioallantoic membrane (CAM) from growing chick embryos without cytotoxicity. The activation of VEGF receptor 2 (VEGFR2) in HUVECs by VEGF was inhibited by elaiophylin, resulting in the suppression of VEGF-induced activation of downstream signaling molecules, Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, nuclear factor-κB (NFκB), matrix metalloproteinase (MMP)-2 and -9 which are closely associated with VEGF-induced angiogenesis. We also found that elaiophylin blocked tumor cell-induced angiogenesis both in vitro and in vivo. Elaiophylin downregulated the expression of VEGF by inhibiting hypoxia inducible factor-1α (HIF-1α) accumulation in tumor cells. To our knowledge, these results for the first time demonstrate that elaiophylin effectively inhibits angiogenesis and thus may be utilized as a new class of natural antiangiogenic agent for cancer therapy. Keywords: angiogenesis; cancer therapy; elaiophylin; vascular endothelial growth factor receptor 2 (VEGFR2); hypoxia inducible factor-1α (HIF-1α)
1. Introduction Angiogenesis plays a very important role in tumor progression through the creation of new blood vessels. The new blood vessels grow and infiltrate into the tumor, resulting in the supply of essential oxygen and nutrients to the tumor and cancer metastasis [1–3]. Thus, it is extremely important to block angiogenesis of the tumor. Vascular endothelial growth factor (VEGF), also known as VEGF-A, is recognized as the major mediator of angiogenesis in cancer [4]. VEGF binds to and activates VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1 has higher affinity for VEGF, whereas its tyrosine kinase activity is approximately 10-fold weaker than that of VEGFR2. For this reason, the major proangiogenic signal is generated from VEGFR2 activated by VEGF. Through the binding to its primary receptor VEGFR2 on the cell surface, VEGF stimulates the major hallmarks of angiogenesis, including endothelial cell Molecules 2018, 23, 563; doi:10.3390/molecules23030563
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Molecules 2018, 22, x FOR PEER REVIEW 2 of 13 survival, proliferation, invasion and migration as well as vascular permeability and inflammation. The binding of VEGF to VEGFR2 promotes receptor dimerization, allowing autophosphorylation surface, VEGF stimulates the major hallmarks of angiogenesis, including endothelial cell survival, of intracellular tyrosine residues. The activation of VEGFR2 contributes to the phosphorylation of proliferation, invasion and migration as well as vascular permeability and inflammation. The binding multiple downstream signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 of VEGF to VEGFR2 promotes receptor dimerization, allowing autophosphorylation of intracellular (ERK1/2), c-Jun N-terminal (JNK), p38 and nuclear factor-κB that subsequently promote tyrosine residues. The kinase activation of VEGFR2 contributes to the(NFκB) phosphorylation of multiple the proangiogenic cellular molecules, responses such [5]. Therefore, the blockade of the signalkinase transduction mediated downstream signaling as Akt, extracellular signal-regulated 1/2 (ERK1/2), by VEGF/VEGFR2 has been considered as a powerful therapeutic strategy for inhibiting angiogenesis. c-Jun N-terminal kinase (JNK), p38 and nuclear factor-κB (NFκB) that subsequently promote the Hypoxia-inducible is a the heterodimeric factormediated composed proangiogenic cellular factor-1 responses(HIF-1) [5]. Therefore, blockade of thetranscription signal transduction by of VEGF/VEGFR2 has been considered asand a powerful therapeutic strategy for inhibiting angiogenesis. an oxygen-regulated α-subunit (HIF-1α) a constitutively expressed β-subunit (HIF-1β) [6]. HIF-1 is Hypoxia-inducible factor-1 (HIF-1) a heterodimeric transcription of an overexpressed in many human cancers andis activates the transcription of factor many composed downstream targets oxygen-regulated α-subunit (HIF-1α) and a constitutively expressed β-subunit (HIF-1β) [6]. HIF-1 is on that contribute to tumor angiogenesis, including VEGF [7,8]. HIF-1 activity in tumors depends overexpressed in many human cancers and activates the transcription of many downstream targets the availability of HIF-1α subunit, the expression of which increases under hypoxic conditions and that contribute to tumor angiogenesis, including VEGF [7,8]. HIF-1 activity in tumors depends on the through the activation of oncogenes and/or inactivation of tumor suppressor genes. The increased availability of HIF-1α subunit, the expression of which increases under hypoxic conditions and HIF-1α levelsthe have been correlated with theinactivation promoted of tumor and aggressive tumor through activation of oncogenes and/or tumorangiogenesis suppressor genes. The increased growth, causing poor prognosis and treatment failure in a number of cancers [9]. Given considerable HIF-1α levels have been correlated with the promoted tumor angiogenesis and aggressive tumor clinical and experimental evidence,and HIF-1α has been believed as a promising target forconsiderable treating tumors. growth, causing poor prognosis treatment failure in a number of cancers [9]. Given clinical and experimental evidence, HIF-1α has been glycosylated believed as a promising target formacrolide treating tumors. Elaiophylin (Figure 1A) is a C2 symmetry 16-membered that was (Figure 1A) is a C2melanosporus symmetry glycosylated 16-membered that was originally originallyElaiophylin isolated from Streptomyces [10]. Previous studiesmacrolide have revealed that elaiophylin isolated from Streptomyces melanosporusanticancer, [10]. Previous studiesand haveimmunosuppressive revealed that elaiophylin possesses possesses antibacterial, antihelminthic, antiviral activities [11–17]. antibacterial, antihelminthic, anticancer, antiviral and immunosuppressive activities [11–17]. More recently, elaiophylin was identified as an autophagy inhibitor, with antitumor effect More in human recently, elaiophylin was identified as an autophagy inhibitor, with antitumor effect in human ovarian cancer cells [18]. However, angiogenesis inhibition by elaiophylin has never been explored. ovarian cancer cells [18]. However, angiogenesis inhibition by elaiophylin has never been explored. In this study, the antiangiogenic activity of elaiophylin was evaluated for the first time on both In this study, the antiangiogenic activity of elaiophylin was evaluated for the first time on both in in vitro endothelial in vivo vivochicken chickenembryo embryo chorioallantoic membrane model. Moreover, vitro endothelialcell cellmodel model and and in chorioallantoic membrane model. Moreover, the underlying mechanism responsible tumor angiogenesis by elaiophylin the underlying mechanism responsiblefor forthe the suppression suppression ofoftumor angiogenesis by elaiophylin was was also investigated. also investigated.
Figure 1. The effect of elaiophylin on the viability of human umbilical vein endothelial cells
Figure 1. The effect of elaiophylin on the viability of human umbilical vein endothelial cells (HUVECs). (HUVECs). (A) Chemical structure of elaiophylin; (B) The effect of elaiophylin on the viability of (A) Chemical structure of elaiophylin; (B) The effect of elaiophylin on the viability of HUVECs. HUVECs. Cells were treated with various concentrations of elaiophylin (0–1000 nM) for 72 h, and cell Cellsviability were treated with various concentrations elaiophylin (0–1000 nM) for 72 cell viability was measured by the MTT colorimetricof assay; (C) The effect of elaiophylin onh, theand cytotoxicity was measured by the MTT colorimetric assay; (C) The effect of elaiophylin on the cytotoxicity of HUVECs. Cells were treated with elaiophylin (0–400 nM) and incubated for 72 h. Cell cytotoxicity of HUVECs. Cells were treated (0–400 nM) and incubated for 72toh.DMSO-treated Cell cytotoxicity was measured by the trypanwith blue elaiophylin assay. Data were presented as percentage relative control (% of Eachblue value represents mean ± SE from independent experiments. was measured bycontrol). the trypan assay. Data the were presented asthree percentage relative to DMSO-treated control (% of control). Each value represents the mean ± SE from three independent experiments.
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2. Results 2. Results 2.1. The Effect of Elaiophylin on the Viability of HUVECs 2.1. The Effect of Elaiophylin on the Viability of HUVECs First, to determine the appropriate treatment dose of elaiophylin with no cytotoxicity for First, to assays, determine the appropriate treatment dose ofwere elaiophylin with no cytotoxicity for angiogenesis various concentrations of elaiophylin applied to human umbilical vein angiogenesis assays, variousforconcentrations of elaiophylin applied to human umbilical vein endothelial cells (HUVECs) 72 h and viability assays werewere carried out using the MTT colorimetric endothelial cells (HUVECs) for 72 h and viability assays were carried out using the MTT colorimetric assay and trypan blue exclusion method. Elaiophylin inhibited the viability of HUVECs with an IC50 assay trypannM, blue but exclusion inhibited of HUVECs with antreatment IC50 value valueand of 439.4 didn’tmethod. cause Elaiophylin the cytotoxic effect the on viability HUVECs up to 200 nM of 439.4 1B,C). nM, but didn’t cause cytotoxic effect on HUVECs up toHUVECs 200 nM treatment (Figure at 1B,C). (Figure Therefore, the the in vitro angiogenesis assays using were performed the Therefore, the in vitro angiogenesis assays using HUVECs were performed at the treatment treatment concentrations of less than 200 nM of elaiophylin. concentrations of less than 200 nM of elaiophylin. 2.2. The Effect of Elaiophylin on the Angiogenesis In Vitro 2.2. The Effect of Elaiophylin on the Angiogenesis In Vitro Angiogenesis is a very complex process in which several key steps are involved. The steps Angiogenesis very complex process several key steps are involved. The steps include include stimulationisofa endothelial cells (ECs)in bywhich angiogenic factors, enzymatic degradation of capillary stimulation of endothelial cells (ECs) byproliferation, angiogenic factors, enzymatic degradation of of ECs capillary basement membrane by activated ECs, migration and tubulogenesis [2,3]. basement membrane by activated ECs, proliferation, migration and tubulogenesis ECs [2,3].were We We thus investigated whether elaiophylin affects these key stages. Serum starvedofHUVECs thus investigated whether affects these key stages. Serum starvedof HUVECs stimulated by VEGF with orelaiophylin without elaiophylin and proangiogenic phenotypes HUVECs were stimulated by VEGFtowith or without proangiogenic phenotypes ofangiogenesis HUVECs were observed. Notably, demonstrate thatelaiophylin the effects and of elaiophylin on VEGF-induced are observed. Notably, to demonstrate thatperformed the effectsthe of elaiophylin VEGF-induced angiogenesis are not just due to its cytotoxicity, we first trypan blue on exclusion experiment. The viability not just due tostimulated its cytotoxicity, we first performed the85% trypan blue experiment. Thetreatment viability of HUVECs by VEGF was more than even atexclusion 200 nM of elaiophylin of HUVECs by VEGF more thaneffectively 85% even atinhibited 200 nM ofthe elaiophylin treatmentproliferation, (Figure 2A). (Figure 2A).stimulated At subtoxic doses,was elaiophylin VEGF-induced At subtoxic doses, to elaiophylin effectively inhibited the VEGF-induced migration, migration, adhesion laminin, invasion and tube formation of HUVECs in aproliferation, dose-dependent manner adhesion to laminin, invasionwe and tube formation of HUVECs in a dose-dependent mannerproliferation (Figure 2B– (Figure 2B–F). In addition, found that elaiophylin can suppress bFGF-stimulated F). In addition, we found that that it elaiophylin suppress bFGF-stimulated of HUVECs, of HUVECs, suggesting might alsocan affect angiogenesis mediatedproliferation by other growth factors suggesting (Figure 2G).that it might also affect angiogenesis mediated by other growth factors (Figure 2G).
2.3. The Effect of Elaiophylin on the Angiogenesis In Vivo antiangiogenic activity activity of of elaiophylin elaiophylin was was also also validated validated in in vivo using a chorioallantoic The antiangiogenic membrane (CAM) assay. Coverslips containing vehicle alone or elaiophylin the membrane (CAM) assay. Coverslips containing vehicle alone or elaiophylin were were placedplaced on theon CAM CAM surface and angiogenesis were observed under The a microscope. inhibition of surface and angiogenesis zones werezones observed under a microscope. inhibition of The neovascularization neovascularization on control coverslips 19% (nelaiophylin = 21), whereas elaiophylin much more potently on control coverslips was 19% (n = 21),was whereas much more potently inhibited the inhibited the angiogenesis of the CAM (82% at 0.1 µg/egg, n = 28) without toxicity against pre-existing angiogenesis of the CAM (82% at 0.1 μg/egg, n = 28) without toxicity against pre-existing vessels vessels (Figure 3).together, Taken together, these results that elaiophylin has a inhibitory potent inhibitory effect (Figure 3). Taken these results suggestsuggest that elaiophylin has a potent effect on the on the angiogenesis vitro angiogenesis both inboth vitroinand in and vivo.in vivo.
Figure 2. Cont.
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Figure Theeffect effect ofofelaiophylin on the endothelial growth factor (VEGF)-induced angiogenesis Figure 2. 2. The elaiophylin onvascular the vascular endothelial growth factor (VEGF)-induced in vitro. (A) effect The effect of elaiophylin on the cytotoxicity ofgrowth HUVECs stimulated by VEGF; (B–F) The Figure 2. The of elaiophylin on the vascular endothelial factor (VEGF)-induced angiogenesis angiogenesis in vitro. (A) The effect of elaiophylin on the cytotoxicity of HUVECs stimulated by inhibitory effect of elaiophylin on theon growth (B); migration (C); adhesion (D); invasion (E)(B–F) and tubein vitro. (A) The effect of elaiophylin the cytotoxicity of HUVECs stimulated by VEGF; The (D); VEGF; (B–F) The inhibitory effect of elaiophylin on the growth (B); migration (C); adhesion forming ability (F)elaiophylin of HUVECs by VEGF. Dotted black lines indicate the edge(E) of and the gap inhibitory effect of oninduced the growth (B); migration (C); adhesion (D); invasion tube-at invasion (E) and tube-forming ability (F) of HUVECs induced byonVEGF. Dotted black lines indicate 0 h. * p
