Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro

Abstract

Background: Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro.

Methods: Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay.

Results: Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation.

Conclusion: Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases.

Figures

Figure 1

Effect of papain on cell viability (panel A) and lactate dehydrogenase release (panel B). To test cell viability, HUVEC were seeded into fibronectin coated microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM and cells were cultured for 10 hours in EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and 10 μg/mL papain. Cells were stained using life cell staining calcein-AM. Cells were photographed at 4 fold magnification using a Nikon Eclipse Ti, the FITC filter set of the instrument and a Nikon Digital Sight DS-Fi1C CCD camera. Integrity of the monolayer and viability of cells was assessed visually. To investigate lactate dehydrogenase release, HUVEC were seeded into fibronectin coated 96-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain at concentrations as indicated. After 48 hours, released lactate dehydrogenase was determined using a 96-Cytotoxassay kit. Data are shown as mean ± standard error of measurement. Monolayers of papain treated cells are intact and cells are viable as demonstrated by calcein uptake, metabolization and retention (panel A). No increase of LDH in the supernatant was apparent at enzyme concentrations of up to 25 μg/mL (panel B).

Figure 2

Effect of papain on the expression of VEGF-receptor 2. HUVEC were seeded into fibronectin coated 8-well chamber slides and cultured in EGM. After 24 hours, medium was changed and 10 ng/mL VEGF or a combination of 10 ng/mL VEGF and 10 μg/mL papain. After 1 hour, cells were fixed with 2% paraformaldehyde and stained with primary mouse antibody against VEGFR2 and FITC labeled goat anti mouse secondary antibody (Panel B). Cell nuclei were counter stained using DAPI. Unspecific mouse IgG served as isotype control (Panel A). Although there is slight unspecific binding of secondary antibody visible on the isotype control, VEGFR2 was clearly present on cells after treatment with 10 μg/mL papain.

Figure 3

Effect of pre-treatment of papain on the phosphorylation status of key-regulatory proteins. HUVEC were seeded into fibronectin coated 25 cm2 tissue culture flasks and cultured in EGM. After 24 hours medium was changed to EBM containing 10 μg/mL Papain and cells were incubated for further four hours. Cells were washed with PBS to remove papain and treated for 15 minutes with EBM containing 10 ng/mL VEGF. Untreated cells served as control. Protein was isolated and phosphorylation was measured using a multipathway ELISA kit. Data are shown as mean ± standard error of measurement with the numbers indicating p-values, black bars indicating VEGF treatment alone and open bars indicating pre-treatment with papain. Pretreatment with papain profoundly interferes with the ability of cells to respond to angiogenic stimuli. Phosphorylation of MEK1 and p38-MAPK were significantly downregulated, whereas ERK1/2 was significantly upregulated. Phosphorylation of Akt1 and SAPK/JNK was also downregulated but with p-values between 0.1 and 0.15.

Figure 4

Effect of papain on cell proliferation of HUVEC. HUVEC were seeded into fibronectin coated 96-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain at concentrations as indicated. After 72 hours, cell growth was assayed using an MTT-assay kit. Data are shown as optical density at 450 nm with a reference wavelength of 620 nm, mean ± standard error of measurement. Papain exerts a significant inhibition of proliferation at concentrations between 6.25 μg/mL and 100 μg/mL.

Figure 5

Effect of proteolytic enzymes on cell migration and tube formation in HUVEC. HUVEC were seeded into fibronectin coated 12-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM. Cells were stained using calcein-AM, the monolayer was scratched with a 200 μL pipette tip and photographed at 10 fold magnification using a Nikon Eclipse Ti as described above. After 14 hours culture in EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain and photographed. covered area was measured using the TScratch software package, results were calculated as percent open area. Data are shown as mean ± SEM. Panel A shows photomicrographs at the beginning of the assay (column start) and after 14 hours incubation (column end) for cells treated with either 10 ng/mL VEGF or 10 ng/mL VEGF and 10 μg/mL papain. Migration fronts are marked by white lines. Panel B shows the percentage open area. Papain inhibited cell migration almost completely at a concentration of 10 μg/mL. Angiogenesis slides were coated with 10 μL Matrigel per well and incubated for 30 min at 37°C. Endothelial cells were and seeded into the wells at a density of 5000 cells per well in EBM containing 10 ng/mL VEGF. After 4 hours preincubation, papain was added at concentrations as indicated. After a further 20 hour incubation period, cells were labelled with 2 μM Calcein-AM. Micrographs of fluorescent cells were taken at 4 fold magnification using a Nikon Eclipse Ti as described above. Tube formation was quantified by the angiogenesis analyzer plugin for ImageJ. Panel C shows photomicrographs for the control, treatment with 10 ng/mL VEGF and with 10 ng/mL VEGF in combination with 1 and 10 μg/mL papain. Panel D shows the measured network length as percentage untreated control. Tube length decreased significantly to control levels after treatment with 1 μg/mL papain. At concentrations of 10 μg/mL tube formation was almost completely abrogated.

Figure 6

Effect of other plant derived proteolytic enzymes on cell growth and tube formation of HUVEC. HUVEC were seeded into fibronectin coated 96-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and bromelain respectively ficin at concentrations as indicated. After 24 hours, cell growth was assayed as described above. Data are shown as mean ± standard error of measurement. Treatment with bromelain as well as ficin resulted in a significant inhibition of proliferation at concentrations between 10 μg/mL and 25 μg/mL (Panel A). Panel B shows the effect of bromelain and ficin on tube formation, assayed as described above. Briefly, after 4 hours preincubation bromelain respectively ficin was added at concentrations as indicated. Data were measured as network length and shown as mean percent control ± standard error of measurement. Treatment with 1μg/mL bromelain inhibited tube formation significantly to control levels. At concentrations of 10 μg/mL bromelain or ficin tube formation was almost completely abrogated.