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

Thomas Mohr,Lucia Desser

doi:10.1186/1472-6882-13-231

Abstract

BackgroundVascular 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.MethodsCell 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.ResultsPapain 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.ConclusionPapain 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.

Highlights

Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy
These findings are confirmed by the lactate dehydrogenase (LDH) release assay which would be indicative for cell lysis (Figure 1B)
Proteases bromelain and ficin we investigated the capability of other plant derived cysteine proteases to inhibit endothelial cell growth (Figure 6A) and tube formation (Figure 6B)

Summary

Introduction

Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. AKT, ERK1/2 and p-38 MAPK constitute important regulatory proteins in angiogenesis, regulating growth, migration and survival of endothelial cells [8] These data suggest that plant derived cysteine proteases might display anti-inflammatory but an anti-angiogenic properties as well. In this study we present to our knowledge for the first time data on the phosphorylation state of AKT, MEK1/2, ERK1/2, STAT3, SAPK/JNK and on key endothelial functions such as cell growth, migration and the capability to form tubes after treatment with papain To put these data into a more general context, we tested whether other plant derived cysteine proteases display a similar effect on endothelial cell growth and tube formation

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