Open Access

Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2‑mediated signaling pathways in lung cancer

  • Authors:
    • Fajiu Li
    • Jie Huang
    • Dongyuan Ji
    • Qinghua Meng
    • Chuanhai Wang
    • Shi Chen
    • Xiaojiang Wang
    • Zhiyang Zhu
    • Cheng Jiang
    • Yi Shi
    • Shuang Liu
    • Chenghong Li
  • View Affiliations

  • Published online on: April 28, 2017     https://doi.org/10.3892/ol.2017.6103
  • Pages: 89-96
  • Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Tumor angiogenesis is essential during lung cancer development and targeting angiogenesis may possess a potential therapeutic value. The present study demonstrates that azithromycin, a Food and Drug Administration‑approved antibiotic drug, is a novel tumor angiogenesis inhibitor. Azithromycin inhibits capillary network formation of human lung tumor associated‑endothelial cells (HLT‑ECs) in vitro and in vivo. It significantly inhibits HLT‑EC adhesion and vascular endothelial growth factor (VEGF)‑induced proliferation of HLT‑ECs in a dose‑dependent manner without affecting migration. In addition, azithromycin induces apoptosis of HLT‑ECs even in the presence of VEGF. Notably, azithromycin inhibits proliferation and induces apoptosis in multiple lung cancer cell lines to a significantly reduced extent compared with in HLT‑ECs, suggesting that HLT‑ECs are more susceptible to azithromycin treatment. In a lung tumor xenograft model, azithromycin significantly inhibits tumor growth and its anti‑tumor activities are achieved by suppressing angiogenesis. Notably, the inhibitory effects of azithromycin on angiogenesis are associated with its ability to suppress VEGF‑induced activation of VEGF receptor 2 (VEGFR2), phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt), focal adhesion kinase, and disruption of focal adhesion assembly and actin stress fiber formation in HLT‑ECs. The present study identifies that azithromycin targets VEGFR2‑mediated focal adhesion and PI3K/Akt signaling pathways in HLT‑ECs, leading to the suppression of angiogenesis and lung tumor growth.

Introduction

Lung cancer is one of the leading causes of cancer associated mortality worldwide, with a 5-year survival rate of ~20% (1). The conventional treatment of cytotoxic chemotherapy for lung cancer appears to have reached an effectiveness plateau and new treatment options are required (2,3). Angiogenesis, the growth of new blood vessels from pre-existing ones, is a fundamental step in the transition of tumors between the dormant and malignant stages (4,5). Vascular endothelial growth factor (VEGF), through activation of VEGF receptor 1 (VEGFR1) and 2 (VEGFR2), is a potent pro-angiogenic factor and a key mediator of angiogenesis in malignant tissues (6). Previous studies have revealed that increased levels of VEGF and high microvessel density are associated with poor prognosis in lung cancer patients (7,8). In addition, the anti-VEGF monoclonal antibody bevacizumab has been tested to improve tumor response and progression-free survival in patients with non-small cell lung cancer (NSCLC) (3,9). Tumor angiogenesis has been the target of major drug discovery programs and angiogenesis inhibitors have been evaluated in clinical trials for lung cancer treatment for the last decade (2,3,1012).

Azithromycin is a Food and Drug Administration-approved antibiotic and the primary drug for the treatment of chlamydia and mycoplasma pneumonia (13). It destroys bacteria by inhibiting protein synthesis through reversible binding to the 50S subunit of bacterial ribosomes (14). With the exception of its antimicrobial activity, azithromycin exerts anti-inflammatory effects by suppressing the secretion of pro-inflammatory cytokines, including interluekin-8 (15). Previously, azithromycin was reported to inhibit the proliferation of cervical and gastric tumor cells (16,17) and enhance the effects of chemotherapy in NSCLC, however little is known about the underlying mechanisms of its anti-cancer activities (18).

In the present study, the effect of azithromycin in lung tumor angiogenesis was investigated using in vitro and in vivo angiogenesis models, and a lung tumor xenograft model. The present results demonstrated that azithromycin potently inhibits angiogenesis in vitro and in vivo by inducing apoptosis and inhibiting the spreading and proliferation of human lung tumor associated-endothelial cells (HLT-ECs). Azithromycin also effectively inhibits lung tumor growth by suppressing angiogenesis. Finally, the inhibitory effects of azithromycin on angiogenesis are identified to be attributed to its inhibition of VEGFR2-mediated downstream signaling pathways.

Materials and methods

HLT-EC, reagents and drugs

Primary HLT-EC was purchased from Cell Biologics, Inc. (Chicago, IL, USA) and grown in Complete Human Endothelial Cell Medium (Cell Systems Corporation, Kirkland, WA, USA). HLT-EC used for the experiments were from passages 2–4 and starved in endothelial cell medium (ECM, served as the basal medium; Cell Systems Corporation) for 3 h prior to being treated with drugs. Human lung cancer PC-9, A549, NCI-H69, DMS-53, H157 and EBC-1 cell lines, were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 1 mM sodium pyruvate, 2 mM L-glutamine and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). Recombinant human VEGF165 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Azithromycin (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) was dissolved in dimethyl sulfoxide.

In vitro capillary network formation

The HLT-ECs (2×104/well), together with various concentrations of azithromycin, were seeded onto solidified Matrigel matrix (BD Biosciences, Franklin Lakes, CA, USA) in a 96-well plate. Following 6 h of incubation at 37°C, capillary network formation was analyzed under light microscopy (Zeiss GmbH, Jena, Germany). The total tube length was quantified using NIH Image J 1.32 software (National Institutes of Health, Bethesda, MD, USA).

Transwell migration assay

A Transwell migration assay (Cell Biolabs, Inc., San Diego, CA, USA) was performed using 6.5 mm diameter polycarbonate filters pre-coated with 0.1% gelatin. The HLT-ECs (4×104/well) were seeded into the upper chambers of the Transwell plates, and subsequently treated with azithromycin. ECM with or without 20 ng/ml VEGF was placed into the lower chamber. Following 4–6 h of incubation at 37°C, cells spreading on the upper surfaces of the filter (non-migrated cells) were wiped away with cotton swabs, and the migrated cells on the lower surface of the filter were fixed and stained with 0.4% Giemsa (Sigma-Aldrich; EMD Millipore).

Cell spreading assay

HLT-ECs together with various concentrations (1, 5 and 10 µM) of azithromycin were seeded onto a 20X diluted Matrigel-coated 96-well-plate for 2 h. The attached cells were then photographed under light microscopy. Images were taken using magnification ×20 under phase-contrast.

Measurement of proliferation and apoptosis

HLT-ECs were treated with azithromycin in ECM medium with or without 20 ng/ml VEGF. Lung cancer cells were treated with azithromycin in DMEM medium. Following 2 days of treatment, cellular proliferation activity was measured by the CellTiter 96® aqueous one solution cell proliferation assay kit (Promega Corporation, Madison, MI, USA). To measure cell apoptosis, cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and then analyzed on a Beckman Coulter FC500 flow cytometer. The percentage of Annexin V-positive cells was determined by CXP software analysis (Beckman Coulter, Inc., Brea, CA, USA).

Denaturing SDS-PAGE and western blot (WB) analysis

HLT-ECs were treated with azithromycin in endothelial cell medium with or without 20 ng/ml VEGF for 30 min and subsequently lysed using a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Inc.) to extract total protein. Frozen tumor tissues were homogenized using a polytron homogenizer in ice-cold RIPA buffer for 10 min. Equal amounts (10 µg) of protein from cell extracts or tumor tissues were resolved using denaturing 10–15% SDS-PAGE and analyzed by WB analysis. Antibodies (dilution, 1:1,000) used in WB analyses included anti-p-VEGFR2 (no. 2478), anti-VEGFR2 (no. 2479), anti-VEGF (no. 2463), anti-hypoxia-inducible factor (HIF; no. 3716), anti-phosphorylated (p)-focal adhesion kinase (FAK; no. 3283), anti-FAK (no. 3285), anti-p-phosphatidylinositol 3-kinase (PI3K; no. 4228), anti-PI3K (no. 4292), anti-p-protein kinase B (Akt; no. 4060), anti-Akt (no. 9272) and anti-actin (no. 4967; all from Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were incubated with primary antibodies at 4°C overnight.

Directed in vivo angiogenesis assay

The in vivo angiogenesis in Matrigel plug was determined using the directed in vivo angiogenesis assay kit (Trevigen, Inc., Gaithersburg, MD, USA) (19). The angioreactors were filled with basement membrane extracts alone, in combination with VEGF (20 ng/ml)/basic fibroblast growth factor (bFGF; 30 ng/ml) or with VEGF/bFGF in combination with 1, 5 or 10 µM azithromycin, and incubated at 37°C for 1 h to allow gelling. The angioreactors containing total 20 µl volume were implanted under the skin of right flank of severe combined immunodeficiency mice for two weeks. All work was conducted with the formal approval of the Jianghan University Animal Care Committee (Jianghan, China) and strictly followed the ethical guidelines on the care and use of animals. Male SCID mice with 20–25 g were purchased from Animal Resources Centre Australia (Murdoch, WA, Australia) and housed in specific pathogen-free rooms in animal holding unit of Jianghan University. A maximum of 5 mice were maintained in a cage with water bottle and overhead food hopper. A total of 25 mice at 6-week old were used. The invaded endothelial cells were isolated and labeled with FITC-lectin. The quantification of FITC-lectin labeled endothelial cells was performed using a SPECTRAmax microplate spectrofluorometer (excitation 485 nm and emission 510 nm; Molecular Devices, LLC, Sunnyvale, CA, USA).

Lung xenograft mouse tumor in SCID mice and blood vessel analysis

All procedures were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee. SCID mice at 6 weeks old were purchased from Animal Resources Centre Australia (Murdoch, WA, Australia). In total 20 male mice (20–25 g) were used in the present study. Mice were housed in pathogen-free room with temperature ~25°C. A maximum of 5 were maintained in a cage with water bottle and overhead food hopper. A 14/10 h light/dark cycle is used. Lung tumor A549 cells at 1×106/100 µl PBS were injected into the flank of each mouse. When the tumor volume reached ~200 mm3, the mice were treated with vehicle control (20/80%, DMSO/Saline) or intraperitoneal azithromycin at 20 mg/kg daily (n=10 per group). Tumor length and width was measured every three days and the volume was calculated as: Length × width2 × 0.5. For tumor blood vessel analysis, tumor frozen section slides (5 µm) were fixed with 4% paraformaldehyde (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) and stained with 1:200-diluted anti-CD31 antibody (no MA3100) followed by 1:500-diluted Alex-fluor 488-conjugated 2nd antibody (no A-11034; Molecular Probes; Thermo Fisher Scientific, Inc.). The nuclei were stained with 4, 6-DAPI (Molecular Probes; Thermo Fisher Scientific, Inc.). Blood vessels were viewed with ×40 magnification under confocal microscopy (Zeiss GmbH). For vessel quantification, the average number of vessels per microscopic field from 3 microscopic fields per tumor section were analyzed.

Analysis of actin stress fibers and focal adhesions in HLT-ECs

HLT-ECs were treated with azithromycin in ECM medium with or without 20 ng/ml VEGF for 60 min at 37°C. Cells were fixed with 4% formalin for 10 min and washed with 1X PBS. Cells were then permeabilized with 0.5% Triton X-100 for 10 min and stained with 1:500 diluted anti-paxillin antibody (no sc-373880) and 1:500 diluted FITC-conjugated immunoglobulin G (no sc-20052; both from Santa Cruz Biotechnolgy, Inc., Dallas, TX, US). Tetramethylrhodamine-conjugated phalloidin (Sigma-Aldrich; EMD Millipore) was used to stain the actin cytoskeleton.

Statistical analysis

All data are expressed as the mean ± standard deviation to indicate data variability. Statistical analyses were performed using Student's unpaired t-test. A one way analysis of variance was used for comparison in instances of multiple parameters. All statistical analyses were performed using the Graphpad Prism v6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Azithromycin effectively inhibits lung tumor angiogenesis in vitro and in vivo

To investigate the effects of azithromycin in lung tumor angiogenesis, in vitro and in vivo angiogenesis assays were performed using HLT-ECs isolated from human lung tumors. ECs can rapidly align and form tubular structures within 6 h when cultured on complete Matrigel matrix, which is rich in extracellular matrix and various growth factors, including VEGF and bFGF (20).

Azithromycin was revealed to effectively inhibit capillary network formation of HLT-ECs on complete Matrigel matrix in a dose-dependent manner (Fig. 1A and B). The effector concentration for half maximal response of azithromycin was as low as ~3 µm (Fig. 1B). It was then examined whether azithromycin could inhibit angiogenesis in vivo using a modified basic Matrigel plug angiogenesis assay (19). Basic Matrigel together with drugs were retained in a silicon tube and implanted subcutaneously into the mouse for two weeks. As shown in Fig. 1C, a mixture of VEGF and bFGF induced potent angiogenesis compared with the control (basic Matrigel alone). When azithromycin was added together with VEGF/bFGF, it suppressed VEGF/bFGF-induced angiogenesis in a dose-dependent manner, as assessed by measuring the number of ECs using fluorescently labeled EC-binding lectin (Fig. 1D). Collectively, the present results demonstrate that azithromycin acts as an angiogenesis inhibitor in vitro and in vivo.

Azithromycin suppresses spreading, proliferation and induces apoptosis of HLT-ECs without affecting HLT-EC migration

Capillary network formation is a multi-step and dynamic process involving cell attachment to matrix, migration, spreading, morphogenesis and apoptosis (21). Therefore, migration, spreading, proliferation and apoptosis assays were performed using HLT-ECs to understand how azithromycin interfered with capillary network formation of lung tumor ECs. VEGF is the most important EC specific angiogenic growth factor, stimulating various aspects of angiogenesis (22); therefore, the effects of azithromycin on VEGF-stimulated angiogenesis were investigated.

Azithromycin was revealed to exhibit no effect on VEGF-induced HLT-ECs migration (Fig. 2A). By contrast, azithromycin significantly inhibited HLT-EC spreading to diluted complete Matrigel [composed of laminin, collage type IV, heparan sulfate proteoglycans and entactin (23); Fig. 2B]. VEGF is known to stimulate proliferation of ECs and protect ECs against apoptosis (22). Results consistently demonstrated that there were more proliferating and less apoptotic HLT-ECs in the presence of VEGF compared with the control (basic medium without VEGF; Fig. 2C and D). Notably, azithromycin inhibited VEGF-induced proliferation of HLT-ECs and induced apoptosis in a dose-dependent manner, even in the presence of VEGF (Fig. 2C and D). Compared with HLT-ECs, azithromycin at the same concentration (10 µm) inhibited proliferation and induced apoptosis of multiple lung cancer cell lines, including PC-9, A549, NCI-H69, DMS-53, H157 and EBC-1, to a significantly reduced extent (Fig. 3A and B), suggesting that HLT-ECs were more susceptible compared with lung cancer cells to azithromycin treatment.

Azithromycin inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2 (VEGFR2) -mediated signaling pathways

To understand the molecular basis of azithromycin-mediated anti-angiogenic effects, the action of azithromycin on the phosphorylation of VEGFR2 was firstly examined, since VEGFR2 is the crucial and main receptor mediating VEGF-stimulated angiogenic activities (24). Azithromycin inhibited VEGFR2 activation by suppressing phosphorylation of VEGFR2 at Tyr1175 at 1, 5 and 10 µm in HLT-ECs (Fig. 4A), which was consistent with its in vitro and in vivo functions. As a consequence of the deactivation of VEGFR2, suppressed phosphorylation of PI3K, Akt and FAK was observed in azithromycin-treated HLT-ECs (Fig. 4A and B).

FAK is an essential component of the focal adhesion complex that attaches cells to the extracellular matrix, and is important for cell spreading and survival (25). Activated FAK then activates and recruits paxillin into the focal adhesion complex, followed by an accumulation of actin stress fibers to form proper focal adhesion complexes. Consistent with inhibition of HLT-EC spreading and FAK activation (Figs. 2B and 4A), azithromycin effectively blocked VEGF-induced paxillin recruitment into focal adhesions, as shown by the reduced paxillin staining (Fig. 4C) and VEGF-induced stress fiber formation (Fig. 4D).

The above experiments associated with each other to reveal that inhibition of HLT-EC spreading, proliferation and survival by azithromycin is linked with its ability to suppress the VEGF-VEGFR2-mediated PI3K/Akt signaling pathway, FAK phosphorylation, paxillin relocation into focal adhesions and actin stress fiber formation.

Azithromycin inhibits lung tumor growth and tumor angiogenesis in xenograft mice

It was then investigated whether azithromycin exerts inhibitory effects on tumor angiogenesis in vivo and tumor growth using a A549 lung tumor xenograft mouse model. The mice tolerated 20 mg/kg azithromycin well, as no significant body weight loss was observed (data not shown). Azithromycin was revealed to significantly suppressed lung tumor growth (Fig. 5A).

To examine whether the inhibitory effect of azithromycin on lung tumor growth is attributable to decreased tumor angiogenesis, blood vessel density and morphology of tumor tissue sections was examined using anti-CD31 antibody, which specifically stains tumor ECs (26). A significant decrease in density of blood vessels was observed in azithromycin-treated tumors compared with the control (Fig. 5B and C). Notably, the morphology of the majority of blood vessels was non-lumen structures (cell clusters and spots) in azithromycin-treated tumors, whereas blood vessels in control tumors were all luminal structures (Fig. 5B). Consistently, decreased protein levels of VEGF and VEGFR2, but not HIFα, were observed in azithromycin-treated tumors (Fig. 5D). These data demonstrated that azithromycin inhibited lung tumor growth through suppressing tumor angiogenesis.

Discussion

Combination therapies using angiogenesis inhibitors and cytotoxic chemotherapeutic agents may achieve additional anti-tumor efficacy as it targets two aspects of tumor development: Tumor cell growth and vascularization. Angiogenesis has been the target of major drug discovery programs, and angiogenesis inhibitors with different mechanisms of actions have been evaluated in clinical trials for cancer treatment over the last decade (2,3). To the best of our knowledge, the present study demonstrated for the first time that azithromycin acts as a potential angiogenesis inhibitor and may be repositioned from its traditional use in infection disease to the therapy of lung cancer.

The primary endothelial cells, HLT-ECs (isolated from human lung tumor), were selected to demonstrate the biological effects of azithromycin on a lung tumor angiogenesis model. Azithromycin was revealed to inhibit capillary network formation of HLT-ECs in a dose-dependent manner (Fig. 1A and B), suggesting that azithromycin inhibits lung tumor angiogenesis in vitro. VEGF is the most important angiogenic factor that stimulates endothelial cell proliferation, migration, tube formation and survival- all necessary components of an angiogenesis response (6). With the exception of migration, azithromycin inhibited all aspects of VEGF-induced angiogenesis, as shown by decreased proliferation and spreading, and increased apoptosis in HLT-ECs (Fig. 2). In accordance with in vitro data, azithromycin potently inhibited VEGF/bFGF induced angiogenesis in vivo (Fig. 1C and D). These results demonstrate that azithromycin is an angiogenesis inhibitor, and blocks multiple steps of angiogenesis.

In addition, azithromycin significantly suppressed VEGF-induced activation of VEGFR2, followed by the decreased phosphorylation of PI3K/Akt and FAK, and disruption of focal adhesion assembly and actin stress fiber formation in HLT-ECs (Fig. 4). Inhibitors targeting the PI3K/Akt were shown to decrease VEGF secretion and angiogenesis (27,28). The present study supports previous studies (27,28) and additionally demonstrates that PI3K/Akt may perform essential roles for anti-angiogenic activities of azithromycin. Bloomstein et al (2) reported that VEGF and its main receptor VEGFR2 are two validated molecular targets for patients with NSCLC. The ability of azithromycin to target VEGF and VEGFR2 makes it an attractive addition to the armamentarium in the treatment of NSCLC.

Notably, azithromycin has a potent ability to inhibit human lung tumor growth, an effect achieved to a great extent by angiogenesis suppression. In xenograft lung tumor mice, azithromycin significantly inhibited tumor growth throughout the treatment (P=0.023; Fig. 5A). Vascularization analysis revealed that the generation of new blood vessels and the expression of VEGF and VEGFR2 in azithromycin-treated mice tumors were significantly decreased compared with the control (Fig. 5B-E), demonstrating that azithromycin inhibits angiogenesis in lung tumors. This finding supports the previous studies that angiogenesis inhibitors alone can inhibit tumor growth (29). Notably, azithromycin has been reported to exhibit inhibitory effects on tumor cell proliferation (16). It is possible that the inhibition of lung tumor growth occurs through the effects of azithromycin on the biological function of endothelial cells and/or tumor cells. The present data demonstrated that HLT-ECs were more susceptible compared with lung cancer cells to azithromycin treatment (Fig. 3), suggesting that endothelial cells were possibly the primary target of azithromycin in lung tumor growth inhibition.

In conclusion, to the best of our knowledge, the present study reports for the first time that azithromycin is a novel angiogenesis inhibitor. It potently inhibits angiogenesis in vitro and in vivo through suppressing multiple aspects of VEGF-induced angiogenic response. The molecular mechanisms of the action of azithromycin on HLT-ECs are attributed to its inhibition of VEGFR2-mediated downstream signaling pathways. Notably, azithromycin effectively inhibits lung tumor growth via suppressing angiogenesis. These findings suggest that azithromycin may be translated into clinical trials for lung cancer treatment and also emphasize the therapeutic value of angiogenesis inhibition in lung cancer.

Acknowledgements

The present study was supported by a research grant provided by Wuhan No. 6 Hospital, Affiliated Hospital to Jianghan University (grant no. WH6201206006) and the National Foundation Research Grant of China (grant no. 81300413).

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July-2017
Volume 14 Issue 1

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Spandidos Publications style
Li F, Huang J, Ji D, Meng Q, Wang C, Chen S, Wang X, Zhu Z, Jiang C, Shi Y, Shi Y, et al: Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2‑mediated signaling pathways in lung cancer. Oncol Lett 14: 89-96, 2017
APA
Li, F., Huang, J., Ji, D., Meng, Q., Wang, C., Chen, S. ... Li, C. (2017). Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2‑mediated signaling pathways in lung cancer. Oncology Letters, 14, 89-96. https://doi.org/10.3892/ol.2017.6103
MLA
Li, F., Huang, J., Ji, D., Meng, Q., Wang, C., Chen, S., Wang, X., Zhu, Z., Jiang, C., Shi, Y., Liu, S., Li, C."Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2‑mediated signaling pathways in lung cancer". Oncology Letters 14.1 (2017): 89-96.
Chicago
Li, F., Huang, J., Ji, D., Meng, Q., Wang, C., Chen, S., Wang, X., Zhu, Z., Jiang, C., Shi, Y., Liu, S., Li, C."Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2‑mediated signaling pathways in lung cancer". Oncology Letters 14, no. 1 (2017): 89-96. https://doi.org/10.3892/ol.2017.6103