Background
Leptin is an adipocyte-derived hormone that plays a major role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure via hypothalamic-mediated effects [
1,
2]. Besides its anorexigenic function, leptin regulates several physiological processes, including angiogenesis [
3‐
5]. Human endothelium and primary cultures of human endothelial cells express the leptin receptor, ObR [
6,
7].
In vitro studies demonstrated that leptin can stimulate growth and survival of endothelial cells as well as induce their migration and organization into capillary-like tubes [
6‐
9].
In vivo, leptin is able to induce complete angiogenesis in the chick choriallantoic membrane assay [
6] and disc angiogenesis system [
10] as well as promote neovascularization in corneas of normal, but not ObR-deficient Zucker fa/fa, rats [
7] or normal mice [
11]. In addition to its own effects, leptin synergizes with vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) in the stimulation of blood vessel growth and vascular permeability [
11].
Proangiogenic and mitogenic functions of leptin have been implicated in development and progression of different neoplasms. Multiple studies demonstrated that leptin is able to stimulate survival [
12‐
14], proliferation [
15‐
17], migration and invasiveness [
18‐
22] of several cancer cell types. In addition, leptin might also contribute to tumor neoangiogenesis. Exposure of cancer cells to hypoxic conditions and/or elevated concentrations of growth factors, such as insulin, can activate production of endogenous leptin, raising intratumoral levels of this hormone [
23‐
28]. Proangiogenic effects of leptin can be further potentiated by its ability to upregulate the expression of other angiogenic factors, such as VEGF, bFGF, interleukin 1-β, and leukemia inhibitory factor in cancer cells [
18,
29‐
31].
New evidence suggests leptin can be involved in the development of brain tumors [
13,
22,
32‐
35]. Initial work documented the presence of leptin and ObR transcripts in various human intracranial tumors [
34]. Other reports demonstrated that rat glioma tissues and cell lines express leptin mRNA [
33,
36], and that in rat C6 cells leptin can increase survival [
13,
32,
33] and enhance migration and invasion of these cells [
22].
We recently demonstrated that both leptin and ObR proteins are overexpressed in human brain tumors relative to normal brain tissue, and that leptin/ObR expression levels positively correlate with the degree of malignancy. The highest levels of leptin and ObR were found in glioblastoma multiforme (GBM), where both proteins were coexpressed with activated forms of serine/threonine protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3). Interestingly, the greatest amounts of all these proteins were detected in perivascular areas and in groups of cells invading the adjacent brain parenchyma [
35].
In ObR-positive glioblastoma cell lines LN18 and LN229, leptin stimulates cell proliferation and induces STAT3 and Akt pathways as well as inactivates the cell cycle suppressor Rb [
35]. Furthermore, leptin-dependent phosphorylation of STAT3 in LN18 and LN229 cells can be inhibited with Aca1, a novel ObR antagonist [
37].
Until present, no studies addressed the potential angiogenic role of leptin in human GBM. Considering that glioma progression from lower-grade tumors to highly malignant GBM is characterized by increasing intratumoral expression of leptin [
35] as well as induction of angiogenesis [
38,
39], we investigated angiogenic properties (induction of tube formation) of GBM-derived leptin using endothelial cell models and specific ObR antagonists. The effects were compared with that produced by VEGF, the best characterized angiogenic factor.
Discussion
Malignant astrocytic gliomas, especially GBMs, are characterized by poor prognosis and low patient survival rates [
51]. Although these tumors rarely metastasize, they almost always recur locally because of their inherent tendency for diffuse infiltration [
52,
53]. In particular, a strong induction of angiogenesis marks the transition from lower-grade tumors to more aggressive and lethal GBMs [
39]. Therefore, despite advanced clinical approaches with surgery, radiotherapy and chemotherapy [
54‐
56], inhibition of angiogenesis might represent a key strategy in the treatments of gliomas.
Recent preclinical data demonstrated that anti-VEGF agents (e.g. ceradinib, bevacizumab) can transiently normalize the elevated permeability and interstitial pressure of brain tumor vessels, enhancing in this way the penetration of concurrently administered drugs [
38‐
40,
52,
57,
58]. Besides direct VEGF or VEGFR2 inhibition for glioblastoma, clinical studies are being conducted or planned with agents targeting further downstream or alternative pathways frequently altered in brain tumors, including the mTOR/Akt and EGFR pathways [
39].
Nevertheless, the success with the existing compounds in the management of brain tumors is very limited. It is likely that combination of therapeutic agents targeting different pathways, especially angiogenic pathways, will produce more significant clinical effects. In this context, we focused on leptin, a multifunctional hormone that is able to exert angiogenic activity in different
in vitro and in vivo model systems [
6‐
8,
10,
11,
18,
29‐
31].
Leptin has been implicated in neoplastic processes, especially in obesity-related cancers, where the hormone has been shown to stimulate cancer cells growth, survival [
12,
14,
15,
28,
59], resistance to different chemotherapeutic agents [
60,
61] as well as migration, invasion and angiogenesis [
18‐
21,
29,
62].
In the central nervous system (CNS) leptin regulates several physiological brain functions, including hippocampal and cortex-dependent learning, memory and cognitive function, neuronal stem cells maintenance, and neuronal and glial development [
63,
64]. In addition, recent research suggests the potential role of this hormone in the progression of brain tumors [
35]. We previously demonstrated that the expression of leptin and ObR in human brain tumor tissues correlates with the degree of malignancy, and the highest levels of both markers are detected in GBM. Specifically, and in relevance to the present study, leptin and ObR were expressed in over 80% and 70% of 15 GBM tissues analyzed [
35]. Other studies demonstrated leptin mRNA expression in rat glioma tissues and cell lines [
33,
36]. Because leptin and ObR in human brain tumors are commonly coexpressed, leptin effects are likely to be mediated by autocrine pathways. Using in vitro models, we found that LN18 and LN229 ObR-positive GBM cells respond to leptin with cell growth and induction of the oncogenic pathways of Akt and STAT3, as well as inactivation of the cell cycle suppressor Rb [
35]. However, the potential role of intratumoral leptin in glioma progression, especially in the regulation of angiogenesis, has never been addressed. Here we investigated if the hormone can be expressed by human GBM cell cultures, if it can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. The results were compared with that induced by the best-characterized angiogenic regulator, VEGF.
Our data demonstrated that conditioned media produced by both LN18 and LN229 GBM cell lines enhanced HUVEC tube formation and proliferation. These data are in agreement with previous reports showing that GBM cultures express VEGF and other factors that can induce HUVEC angiogenesis [
65‐
67].
We found variable levels of leptin and VEGF mRNA in LN18 and LN229 cell lines cultured under SFM conditions. In general, the abundance of VEGF transcripts in both cell lines was significantly greater that that of leptin mRNA. Secreted leptin and VEGF proteins were found in LN18 CM, while in LN229 CM, leptin was undetectable and VEGF was present at low levels. The reason for lack or minimal presence of these proteins in LN229 CM, despite quite prominent expression of the cognate mRNAs, is unclear. It is possible that it is due to limited sensitivity of ELISA assays unable to detect proteins below the minimal threshold level. We speculate that LN229 cells might produce proteins binding VEGF and leptin, thereby converting them into ELISA-unrecognizable complexes. Alternatively, LN229 CM might contain proteases degrading the angiogenic proteins.
In order to clarify if LN18 CM angiogenic and mitogenic effects are, at least in part, related to leptin secreted by these cells, we used specific ObR inhibitor, Aca1. We have previously demonstrated that this antagonist binds ObR in vitro, inhibits leptin-induced signaling at pM-low nM concentrations in different types of cancer cells, including LN18 and LN229 cells, while its derivative Allo-aca is able to reduce the growth of hormone-receptor positive breast cancer xenografts and enhance survival of animals bearing triple-negative breast cancer xenogranfts [
37,
68]. Furthermore, All-aca also inhibits leptin activity in some animal models of rheumatoid arthritis [
69]. Interestingly, we also detected CNS activity of Aca1, suggesting that the peptide has the ability to pass the blood-brain barrier [
37,
68,
70].
In the present work, we found that Aca 1 can abrogate leptin-induced tube formation and mitogenesis of HUVEC at 10 and 25 nM concentrations, respectively. Notably, the peptide alone did not affect cell growth and did not modulate the ability of HUVEC to organize into tube-like structures, suggesting that it acts as a competitive antagonist of ObR. Next, we demonstrated that Aca1 at 10-50 nM concentrations was able to antagonize tube formation and growth effects of LN18 CM. The anti-angiogenic effects of 25 and 50 nM Aca1 were comparable to that obtained with 1 μM SU1498, while anti-mitotic activity of 25 and 50 nM Aca1 was comparable to the action of 5 μM SU1498. Furthermore, the combination of low doses of Aca1 (10 nM) and SU1498 (1 or 5 μM) produced greater inhibition of CM effects than that obtained with single antagonists.
Interestingly, Aca1 or SU1498 appeared to differentially affect the morphology of HUVEC cultures. While Aca1 reverted the organized ES phenotype to the initial appearance of dispersed cell culture, SU1498 disrupted ES structures, reduced cell-matrix attachment and induced cell aggregation. This might suggest that the inhibitors affect different cellular mechanism and that leptin and VEGF control HUVEC biology through different pathways.
Taken together, our data indicated that GBM cells are able to induce endothelial cells proliferation and organization in capillary-like structures through, at least in part, leptin- and VEGF-dependent mechanisms. Thus, leptin might contribute to the progression of GBM through the stimulation of new vessel formation. Leptin action can be direct or indirect, through upregulation of VEGF expression. Indeed, we observed that leptin can transiently increase VEGF mRNA levels in GBM cells at 6-8 h of treatment (data not shown). In this context, effective reduction of tube formation and mitogenic activity of endothelial cells by ObR antagonist, especially in the combination with VEGFR2 inhibitor, suggest that targeting both leptin and VEGF pathways might represent a new therapeutic strategy to treat GBM.
Methods
Cell lines and growth conditions
All cell lines were obtained from ATCC (Manassas, VA). Glioblastoma cell lines LN229 and LN18 were cultured in low glucose-Dulbecco modified Eagle's Medium (DMEM) (Cellgro Mediatech, Manassas, VA) containing 5% fetal bovine serum (Cellgro Mediatech, Manassas, VA). Human Umbilical Vein Endothelial Cells (HUVEC) were maintained in Vascular Cell Basal Medium (VCBM), supplemented with the Vascular Cell Growth kit BBE, both purchased from ATCC.
ObR and VEGFR inhibitors
The ObR antagonist, Aca1, is a short leptin-based peptidomimetic (H-Thr-Glu-Nva-Val-Ala-Leu-Ser-Arg-Aca-NH2) whose sequence is based on leptin/ObR binding site III. The process of peptide design, screening and development has been reported by us before [
37,
71,
72]. The efficacy of Aca1 and its derivative Allo-aca
in vitro and
in vivo has been described in detail previously [
37,
68]. SU1498, the antagonist of VEGFR2 was purchased from Calbiochem, USA.
Conditioned medium (CM) preparation
Subconfluent LN18 and LN229 cell cultures were placed in SFM (DMEM low glucose supplemented with 0.42 g/mL bovine serum albumin, 1 μM FeSO4 and 2 mM L-glutamine) for 24 or 48 h, and then the CM was collected, centrifuged at 2000 rpm for 5 min, and the supernatants frozen at -80°C until use. The number of cells in cultures used for CM production was counted.
Proliferation assays
HUVEC (1.5 × 104) were plated in 24-well plates and allowed to adhere overnight in the growth medium. Then the cells were treated for 24 h with either 200 ng/mL leptin (R&D Systems, Minneapolis, MN) in presence or absence of 10, 25 or 50 nM Aca1, or with 50 ng/ml VEGF in presence or absence of 1 or 5 μM SU1498 or left untreated as control. For assays with GBM-derived CM, HUVEC were seeded as described above, and allowed to adhere overnight. Then the culture medium was replaced with SFM (negative control) or CM mixed with HUVEC growth medium (1:1) with or without Aca1 (10, 25, 50 nM) and/or SU1498 5 μM. At conclusion of proliferation assays, the cells were counted under the microscope with trypan-blue exclusion. Each experiment was performed in triplicate and repeated at least three times.
The tube formation assay was based on procedures described by Park et al and Feng et al. [
9,
42]. For the tube-like formation assays, 24-wells plates were coated with 300 μl of 2 mg/mL collagen I (BD Biosciences, Bedford, MA) prepared according to manufacturer's instructions. Where appropriate, leptin (100 ng/mL) and/or Aca1 (10, 25, 50 nM) and/or VEGF (50 ng/ml) and/or SU1498 (1, 5, 10 μM) were added to the collagen I prior to polymerization. Then, 8 × 10
4 of HUVEC suspended in 1 ml of HUVEC growth medium containing various treatments were plated on the top of the collagen layers. For tube formation assay performed with CM, HUVEC were seeded in 1 ml of SFM (negative control) or GBM-derived CM mixed (1:1) with HUVEC growth medium, containing or not Aca1 and/or SU1498. After 8 and 24 h for assays performed in HUVEC growth medium and CM, respectively, the HUVEC were stained with Giemsa (diluted 1:10 in distilled water) for 15 min. The number of ES, representing tube-like formation capability of HUVEC, was scored by two observers in 10 fields using a contrast phase microscope (Olympus CKX FA) with 10× magnification.
Quantitative Real Time PCR (qRT-PCR)
Subconfluent cultures of LN18 and LN229 cells were placed in SFM for 24 and 48 h, and then RNA was isolated using Trizol reagent (Invitrogen), according to manufacturer's instructions. A total of 10 μg of RNA was reverse transcribed in 100 μl of reaction volume using the High-Capacity cDNA Archive (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Seven μl of the RT products were used to amplify leptin and VEGF sequences using the Hs00174877_m1 and the Hs00900054_m1 TaqMan probes (Applied Biosystems), respectively. To normalize qRT-PCR reactions, parallel reactions were run on each sample for β-actin. Changes in the target mRNA content relative to β-actin mRNA were determined using a comparative CT method to calculate changes in CT, and ultimately fold and percent change. An average CT value for each RNA was obtained for replicate reactions.
Western blot (WB) analysis
Subconfluent cultures (80% of density) of HUVEC were placed in SFM for 1 h, pretreated for 1 h with ObR or VEGFR inhibitors, and then treated with 200 ng/mL leptin or 50 ng/mL VEGF for 15 min or left untreated. Next, the cells were lysed in a buffer containing 1% NP40, 50 mM HEPES pH 7.5, 250 mM NaCl, 5 mM EDTA pH 8.0, 0.1% SDS, protease inhibitors 1× (Complete Mini EDTA-free protease inhibitors, Roche, Germany) and phosphatase inhibitors (10 mM Na3Vo4 and 50 mM NaF). The expression of proteins was analyzed in 50-70 μg of total cell lysates. The following antibodies (Ab) were used for WB: for phospho-STAT3, STAT3 Tyr705, D3A7 mAb, 1:1000 and for total STAT3, STAT3 79D7 mAb, 1:1000, all purchased from Cell Signaling, MA, USA; for glyceraldehyde-3-phosphate dehydrogenase 6C5 (GAPDH), 1:1000 (Santa Cruz Biotechnology, CA, USA).
Leptin and VEGF detection by ELISA
CM obtained from 2-3 plates of 80% confluent GBM cultures was collected, as described above. The concentrations of leptin and VEGF in CM (obtained using WB lysis buffer without 0.1% SDS) were measured using leptin and VEGF Human Quantikine ELISA Kits (R&D Systems). The standard curve was created using purified leptin or VEGF. The concentrations of leptin or VEGF are expressed as pg/mL/9 × 106 LN18 cells and pg/mL/6 × 106 LN229 cells. All detected concentrations were within the range of the standard curve. All measurements were done in triplicate and the experiments were repeated three times.
Statistical analysis
All experiments were done at least in triplicates and data analyzed by Student's t-test. Differences with p values of ≤ 0.05 were considered significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RF carried out the majority of experiments, analyzed and elaborated data, and drafted the manuscript, MB carried out experiments, LO prepared the ObR antagonist and analyzed data, ES designed the study, analyzed data and edited the final manuscript. All authors read and approved the final manuscript.