Plant derived cysteine proteases, especially bromelain have been used for decades in complimentary medicine as a remedy for inflammatory diseases. However, the mode of action by which plant cysteine proteases act is not well understood. Several mechanisms have been proposed: Cleavage of proteins from the cell surface
], Interference with key components of pathways
] and direct cytotoxicity via induction of apoptosis
To address these questions we investigated direct cytotoxicity of papain towards endothelial cells. Prolonged incubation of endothelial cells with concentrations of up to 25 μg/mL papain did not result in increased release of lactate dehydrogenase which would be indicative for cell lysis. Additionally MTT assays showed that papain at concentrations of up to 100 μg/mL did suppress cell growth only to slightly below untreated control. These results are in line with other studies were protease concentrations of up to 400 μg/mL have been used
]. Next we investigated the possibility of proteolytic removal of the main receptor for VEGF mediated angiogenesis, VEGFR2. Indeed, immunofluorescent staining showed that VEGFR2 remains on the cell surface even after prolonged incubation with papain.
We then investigated the phosphorylation status of AKT1, ERK1/2, MEK1/2, p38-MAPK and SAPK/JNK after pre-treatment with papain and found a modulation of phosphorylation levels of AKT1, MEK1/2, p38-MAPK and SAPK/JNK (down-regulated) and ERK1/2 (up-regulated). The PKB/AKT pathway is one of the main signaling pathways activated by VEGFR2
]. Down-regulation of AKT phosphorylation by cysteine proteases has been observed in other cells models in context with blocking NF-κB activation and subsequent induction of apoptosis
]. However, in context of the VEGF receptor 2 signaling cascade, AKT phosphorylates BAD and caspase9 thus inhibiting their apoptotic activity. Additionally AKT phosphorylates the FOXO family of proteins which leads to their inactivation and a decrease in transcription of proteins promoting apoptosis
]. In this context AKT acts as an inhibitor of apoptosis and is crucially required for VEGF mediated angiogenic activation
]. Targeting AKT inhibits angiogenesis in vivo
VEGF additionally signals via Ras → c-RAF → MEK → ERK
]. An effect of cysteine proteases on this pathway has first been reported by Mynott et al. in a T-cell model
]. In this model, treatment with bromelain down-regulated ERK-2 phosphorylation, and decreased IL-2, IFN-γ, and IL-4 mRNA accumulation. Since trypsin had no effect, Mynott could exclude unspecific proteolysis. Our data suggest that down-regulation of this signaling pathway includes down-regulation of MEK1/2 activity. MEK1/2 are protein kinases that mediate the phosphorylation of ERK1 or ERK2. This activates ERK1/2, which are protein-serine/threonine kinases with a broad spectrum of cytosolic and nuclear substrates. Interestingly we observed an up-regulation of ERK1/2 phosphorylation. This differs from the situation reported in other cell models and in vivo
where a marked inhibition of ERK1/2 has been observed
]. The reason for this observation may be the down-regulation of p38-MAPK which serves negative regulator for ERK1/2
Both p38-MAPK and JNK are mainly involved in cellular response to many types of stresses, but they also control proliferation, differentiation, survival and migration of specific cell types. Aside cellular stress JNK as well as p38 MAPK pathways can be activated by growth factors
]. The role of SAPK/JNK in angiogenesis is not fully understood yet. Boyd et al. found that JNK inhibition lead to inhibition of tube formation
] leading to the conclusion of JNK being a positive regulator of angiogenesis. This is underlined by the fact that inhibition of JNK significantly decreased endothelial proliferation and migration
]. We observed a down-regulation of SAPK/JNK which cold in part explain the reduced migration and tube formation we observed.
Activation of p38-MAPK induces endothelial cell migration
] and serves as a negative regulator for ERK1/2 and AKT in VEGF mediated angiogenesis
]. On the other hand, in endothelial cells exposed to chronic inflammatory activation p38-MAPK acquires a pro-angiogenic role
]. We observed a down-regulation of p38-MAPK which is in line with published observations in other models
Finally we investigated whether these data translate into altered endothelial cell function (cell growth, cell migration and capability to form tubes). Papain inhibited cell growth in a concentration response dependent manner with an IC50 of 7 μg/mL. Cell migration was almost completely abrogated at a concentration of 10 μg/mL and tube formation was significantly inhibited at a concentration of 1 mg/mL. At a concentration of 10 μg/mL, tube formation was almost completely abrogated. Inhibition of cell growth and tube formation could also be seen in bromelain and ficin treated endothelial cells, pointing towards antiangiogenic properties of plant derived cysteine proteases in general.