β-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells—Possible Role of VEGF Downregulation

2.2. BCP Inhibits Hypoxia-Induced VEGF Expression in A549 Cells

To find a possible mediator for the antiangiogenic effect of BCP, the regulation of various proangiogenic factors in hypoxic A549 cells was analyzed using the LEGENDplex™ multiplex assay (Figure 2). Here, hypoxia caused only upregulation of interleukin (IL)-6 and VEGF, whereas platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiopoietin-1 were suppressed. No substantial change in secretion was found for angiopoietin-2, epidermal growth factor (EGF), fibroblast growth factor-basic (FGF-basic), IL-8, and tumor necrosis factor (TNF) α. In the case of the two upregulated factors, a downregulation of secretion could only be measured for VEGF. Furthermore, BCP led to a decrease in PECAM-1 levels, which, in contrast to VEGF, however, were not increased in the corresponding vehicle control compared with normoxia.

Further analysis of hypoxia-induced VEGF expression was performed at the protein level with ELISA and at the mRNA level using RT-qPCR. Thereby, a strong induction of VEGF release from hypoxic A549 cells compared with normoxic conditions was shown at the protein level (Figure 3A). BCP, in turn, caused a concentration-dependent inhibition of hypoxia-induced VEGF protein levels in cell culture supernatant, which was significantly evident from as low as 0.01 µM (Figure 3A). Again, co-incubation with the CB₂ receptor antagonist AM630 significantly abolished the BCP effect on VEGF formation (Figure 3B). Inhibition of VEGF expression by BCP was also registered at the mRNA level in A549 cells (Figure 3C). Interestingly, a significant effect was already observed at a BCP concentration of 0.01 µM, whereas a further increase in the concentration did not show any increase in the inhibitory effect.

2.3. BCP Decreases Hypoxia-Induced HIF-1α Protein Levels in A549 Cells

In a recently published work of our group, siRNA experiments demonstrated that hypoxia-induced VEGF protein formation in A549 cells proceeds independently of activation of the transcription factor HIF-1α [20]. In the case of BCP, however, we were interested in the regulation of this factor, which in addition to VEGF is responsible for the expression of other angiogenic factors (for review, see [25]) and genes involved in virtually all aspects of tumor biology, including invasion and metastasis, but also in the tumor′s response to radiation and chemotherapy (for review see [26,27,28,29]).

In the Western blot analyses we performed, BCP caused a reduction in HIF-1α in hypoxic A549 cells by approximately 30% and 50% after incubation with concentrations of 3 and 10 µM, respectively (Figure 4A, left). However, statistical significance could not be determined due to the high variability of the measured protein levels. Moreover, this inhibitory pattern did not correspond to VEGF regulation by BCP, where the latter elicited a highly significant VEGF decrease already at a concentration of 0.01 µM (Figure 3A). With respect to the transcription factor HIF-2α, virtually no interference by BCP was shown in the same experimental approaches (Figure 4A, right). In another experiment, the HIF-1α decrease detected in the presence of 10 µM BCP in hypoxic A549 cells proved to be reversible after preincubation with the CB₂ receptor antagonist AM630 (Figure 4B).

2.4. BCP Also Inhibits VEGF Expression in Hypoxic H358 Lung Cancer Cells and Suppresses Endothelial Tube Formation via Modulation of the H358 Cell Secretome

To exclude the possibility that the demonstrated antiangiogenic and VEGF-lowering effects of BCP were limited to A549 cells, key experiments were repeated with H358 cells, another NSCLC cell line. As shown in Figure 5A,B, BCP in H358 exhibited a similar pattern of effects as shown in A549 cells. Thus, concentration-dependent inhibition of HUVEC tube formation was also observed after incubation with CM of hypoxic H358 cells previously treated with BCP, which became statistically significant starting at a BCP concentration of 1 µM (Figure 5A). Similarly, BCP also caused a concentration-dependent inhibition of VEGF protein formation in hypoxic H358 cells, which was statistically significant at BCP concentrations between 0.1 and 10 µM (Figure 5B). In the case of the determined VEGF mRNA levels, again, a plateau effect in inhibition was noted starting at BCP concentrations of 0.01 µM (Figure 5C). On the other hand, the HIF-1α protein decrease previously registered in hypoxic A549 cells for higher BCP concentrations could not be confirmed in hypoxic H358 cells (Figure 5D). Interestingly, BCP, rather, led to an increase in HIF-1α protein levels here.

4.2. Cell Culture

Human NSCLC cells A549 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; #ACC-107; RRID: CVCL_0023). The NSCLC cell line NCl-H358 was obtained from ATCC (Manassas, VA, USA; #CRL-5807TM; RRID:CVCL_1559). Both lung cancer cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L glucose and with UltraGlutamine^TM I from Lonza Cologne (Cologne, Germany) supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell as single-donor cryogenic vials (Heidelberg, Germany; #C-12200; RRID: CVCL_2959). HUVECs were cultured in Endothelial Cell Growth Medium Kit (#C-22110, assigned as HUVEC complete medium) from PromoCell supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. All cell lines were cultivated in a humidified incubator at 37 °C and 5% CO₂. HUVECs were used between passages 3 and 7. The lung cancer cell lines were discarded after passage 20.

For incubation with test compounds, cells were washed with DPBS and cultured in serum-free DMEM. In the case of viability studies with HUVECs, experiments were performed in serum-free DMEM with 1% (v/v) FBS. Test compounds were first dissolved in ethanol (BCP), DMSO (AM630), or DPBS (rVEGF) and then diluted in DPBS. The final concentration of solvents in the treated cell media was 0.1% (v/v) ethanol and/or DMSO and 0.000025% (w/v) rHSA. In all experiments, the incubation media contained the same amount of solvent. Experiments under hypoxic conditions were performed in the humidified Baker Ruskinn InvivO₂^® 400 chamber (I&L Biosystems, Königswinter, Germany; hereafter referred to as hypoxia chamber) at 1% O₂, 5% CO₂, and 37 °C.

4.3. Generation of Conditioned Media from A549 and H358 Cells

To generate conditioned media (CM), A549 and H358 cells were seeded in 48-well plates at a density of 0.5 × 10⁵ cells per well and incubated for 16–24 h in a humidified incubator at 37 °C and 5% CO₂ (normoxic conditions). Subsequently, cells were washed, and serum-free DMEM was added. For equilibration, cells were incubated in the hypoxic chamber for 15 min. After equilibration, incubation with BCP was performed in a final volume of 300 µL per well for 48 h in the hypoxia chamber. Similarly, unconditioned media (UCM) containing serum-free DMEM with the respective vehicle control without cells were prepared. The same procedure was used in the experiments with AM630, except that a 30-min preincubation with this compound was performed under normoxic conditions before hypoxia equilibration. Finally, UCM and CM were collected, centrifuged at 1300× g and 4 °C for 5 min, and used for HUVEC tube formation (Section 4.4), migration (Section 4.5), and viability (Section 4.6) analysis or LEGENDplex™ multiplex assay (Section 4.7) and VEGF analysis (Section 4.8). For some experiments, the media of a treatment group were pooled to obtain a larger volume.

4.4. Tube Formation Assay

The tube formation assay as a method to analyze the angiogenic potential of HUVECs was performed to determine a corresponding effect of CM of previously BCP- or vehicle-treated hypoxic A549 and H358 cells or of BCP and vehicle without prior tumor cell interaction. To this end, a previously described method [18,51] was applied with slight modifications. First, 48-well plates were coated with 30 µL of ice-cooled Corning^® Matrigel^® matrix (BD Biosciences, Heidelberg, Germany; #356234, hereafter referred to as Matrigel) per well, which polymerized at 37 °C for 30 min. Meanwhile, HUVECs were harvested and resuspended in serum-free DMEM. Then, the HUVECs suspension was seeded at a density of 0.5 × 10⁵ cells per well into a coated 48-well plate containing reoxygenated UCM/CM or serum-free DMEM with vehicle or BCP. After an incubation period of 6 h (in the case of UCM and CM) or 3 h (in the case of serum-free DMEM with vehicle or BCP) in a humidified incubator at 37 °C and 5% CO₂, cells were fixed with 2% (v/v) PFA. Microscopic images were obtained using the Primovert inverted microscope (Carl Zeiss AG, Jena, Germany). To quantify tube formation, the number of tube-like structures that formed closed intersections was counted in four microscopic fields.

4.5. Migration Assay

The effect of CM obtained from A549 cells on HUVEC migration was determined with a Boyden chamber assay using Falcon^® cell culture inserts (#353097; Corning, Corning, NY, USA) as previously described [18,51]. HUVECs were harvested and resuspended in reoxygenated UCM or CM from A549 cells (Section 4.3). Subsequently, 300 µL of HUVEC suspension containing 1 × 10⁵ cells were seeded onto inserts containing a polyethylene terephthalate membrane with 8 µm pores. DMEM containing 10% (v/v) FBS served as a chemoattractant in the lower companion plate. Non-migrating HUVECs on the upper membrane surface of the insert were removed with a cotton swab after an incubation period of 24 h in a humidified incubator at 37 °C and 5% CO₂. Finally, the WST-1 assay was used to quantify the migrated cells on the membrane bottom.

4.6. WST-1 Assay

The WST-1 assay is a colorimetric test for the determination of cellular viability of HUVECs. In this assay, the water-soluble tetrazolium salt WST-1 (Roche Diagnostics, Mannheim, Germany) is cleaved by metabolically active cells to form a soluble formazan dye. In the corresponding experiments, after harvesting and resuspension in reoxygenated CM, 5 × 10³ HUVECs were seeded in 100 μL CM containing 1% (v/v) FBS per well of a 96-well plate. After an incubation period of 24 h, the metabolic activity of the cells was quantified by measuring the absorbance at 450 nm (wavelength correction at 690 nm) using a microplate reader (Infinite F200 Pro Tecan, Tecan Group, Männedorf, Switzerland).

4.7. LEGENDplex™ Multiplex Assay

CM of A549 cells were obtained as described in Section 4.3 and assayed for 10 key targets involved in angiogenesis using the LEGENDplex™ multiplex assay (BioLegend, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The specific kit was the Human Angiogenesis Panel 1 (10-plex) with V-bottom plate (#740698). For the experiments, samples were incubated with capture beads with fluorescent barcodes coated with monoclonal antibodies against the following analytes: angiopoietin-1, angiopoietin-2, EGF, FGF-basic, IL-6, IL-8, PECAM-1, PlGF, VEGF, and TNFα. After washing, the beads were incubated with phycoerythrin (PE)-conjugated monoclonal antibodies specific for a different epitope of each analyte tested. After further washing, samples were detected by flow cytometry (CytoFLEX LX; Beckman-Coulter, Krefeld, Germany). The PE intensities thereby informed absolute analyte amounts by interpolation from 5-log analyte standards measured in parallel.

4.8. Determination of VEGF-A Protein

CM of A549 and H358 cells were obtained as described in Section 4.3. Subsequently, the VEGF Quantikine^® ELISA Kit (#DVE00) from R&D Systems (Wiesbaden, Germany) was used to quantify VEGF-A according to the manufacturer’s instructions.

4.9. Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR)

NSCLC cells were seeded in 6-well plates at a density of 2.0 × 10⁵ per well and maintained in DMEM containing 10% (v/v) FBS in a humidified incubator at 37 °C and 5% CO₂ for 16–24 h. The cells were then washed, and serum-free DMEM was added under normoxic conditions. After equilibration in the hypoxia chamber for 15 min, cells were treated with BCP or its vehicle under hypoxic conditions. Following a 6 h incubation under hypoxic conditions, cells were lysed, and total RNA was isolated using the RNeasy Mini Kit from Qiagen (Hilden, Germany). Total RNA concentrations were measured using the NanoDrop™ OneC Microvolume UV-Vis spectrophotometer from Thermo Fisher Scientific to ensure the use of equal amounts of RNA for further RT-qPCR. The mRNA was quantified by RT-qPCR using the Applied Biosystems^® Taq-Man^® RNA-to-CT™ 1-Step Kit and primers for VEGF-A (Assay ID: Hs00900055_m1; FAM-MGB) and peptidylprolyl isomerase A (PPIA; Assay ID: Hs999904_m1; VIC-MGB) from Thermo Fisher Scientific according to the manufacturer’s instructions. PPIA served as a housekeeping gene to normalize VEGF mRNA levels before comparison with vehicle controls.

4.10. Isolation of Total Cellular Protein

NSCLC cells were seeded in 6-well plates at a density of 2.0 × 10⁵ per well and maintained in DMEM containing 10% (v/v) FBS in a humidified incubator at 37 °C and 5% CO₂ for 16–24 h. The cells were then washed, and serum-free DMEM was added under normoxic conditions. For equilibration, cells were incubated in the hypoxic chamber for 15 min. After equilibration, cells were incubated with BCP or its vehicle in a final volume of 1 mL per well for 3 h in the hypoxic chamber. Similarly, normoxia control was prepared in a separate 6-well plate in a humidified incubator at 37 °C and 5% CO₂. Cells were then washed and scraped in lysis buffer (2% [w/v] SDS, 40% [v/v] H₂O, 10% [v/v] glycerol, 50% [v/v] 125 mM Tris-HCl [pH 6.8]). Proteins were immediately denatured at 95 °C with continuous shaking for 10 min. Subsequently, the lysed cells were centrifuged at 20,817× g and 4 °C for 5 min, and the supernatants were collected.

For analysis of p-VEGFR2 and VEGFR2, HUVECs were seeded in 6-well plates at a density of 3.0 × 10⁵ cells per well and cultured for 16–24 h in HUVEC complete medium. After washing and cultivation in serum-free DMEM containing 10 µM BCP and, if necessary, 1 µM AM630, HUVECs were treated with 10 ng/mL rVEGF for 5 min. Protein isolation was then performed as described above.

The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) was used to quantify protein concentrations. Before protein samples were stored at −20 °C, 4% (v/v) β-mercaptoethanol was added.

4.11. Western Blot Analysis

Equal amounts of protein samples were separated on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were next blocked for 1 h in 5% (w/v) NFM in Tris-buffered saline containing 0.1% (v/v) Tween^® 20 (TBS-T buffer), washed in TBS-T buffer and incubated with primary antibodies in 5% (w/v) NFM in TBS-T buffer overnight at 4 °C. The primary antibodies used against VEGFR2 (#2479; RRID:AB_2212507) and p-VEGFR2 (Tyr1175) (#3770; RRID:AB_1642326) were purchased from Cell Signaling Technology (Frankfurt/Main, Germany). Antibodies against HIF-1α (#MA1-516; RRID:AB_325431; Thermo Fisher Scientific), HIF-2α (#NB100-122; RRID:AB_10000872; Novus Biologicals, Wiesbaden Nordenstadt, Germany), and β-actin (#A5441; RRID:AB_476744; Sigma-Aldrich) were used. Subsequently, membranes were washed with TBS-T buffer and incubated with secondary antibodies coupled to horseradish peroxidase (anti-rabbit antibody #7074, RRID:AB_2099233 or anti-mouse antibody: #7076; RRID:AB_330924) from Cell Signaling Technology in 5% (w/v) NFM in TBS-T buffer for 1 h at room temperature. A substrate chemiluminescence solution (100 mM Tris-HCl [pH 8.5], 1.25 mM luminol, 200 µM p-coumaric acid, 0.09% [v/v] H₂O₂) was then added, and signal detection was performed with the ChemiDoc XRS gel documentation system from Bio-Rad Laboratories (Munich, Germany). The Precision Plus Protein^TM Dual Colour Standard from Bio-Rad Laboratories was applied to identify the approximate molecular weight of the protein bands. The Quantity One 1-D analysis software (Bio-Rad Laboratories) was used to quantify the signal intensity of the protein bands. The specific protein bands were normalized to the signal of β-actin.