Elsevier

Fitoterapia

Volume 110, April 2016, Pages 181-188
Fitoterapia

Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice

https://doi.org/10.1016/j.fitote.2016.03.016Get rights and content

Abstract

Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and in the activation of protein C. Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of diketopiperazine on EPCR shedding. We investigated this issue by monitoring the effects of diketopiperazine on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice and underlying mechanism. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and 1-3 induced potent inhibition of PMA-, TNF-α-, IL-1β (in HUVECs), and CLP-induced EPCR shedding (in mice) via inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. 13 also inhibited the expression and activity of PMA-induced TACE in HUVECs suggesting that p38, ERK1/2, and JNK could be molecular targets of 1-3. These results demonstrate the potential of 1-3 as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding.

Introduction

A soluble form of endothelial cell protein C receptor (sEPCR) can be generated in vitro through proteolytic cleavage by metalloprotease activity, which is inducible by thrombin and other inflammatory mediators [1], [2]. This process is known as EPCR shedding. sEPCR circulates in plasma, retaining its ability to bind both protein C (PC) and activated PC (APC), but does not enhance PC activation [3]. It inhibits the anticoagulant activity of APC through the formation of a complex that does not bind to phospholipid membranes [4]. Owing to the shedding of membrane EPCR, sEPCR can be detected in plasma at a concentration of approximately 100 ng/mL, and high levels of sEPCR have been reported in systemic inflammatory diseases [5]. Previous in vitro studies have reported a dramatic increase in EPCR shedding from the endothelium induced by a wide variety of inflammatory mediators (interleukin (IL)-1β, hydrogen peroxide (H2O2), and phorbol myristate acetate), and thrombin, and this EPCR shedding is potentiated by the microtubule disrupting agent, nocodazole [1], [2], [6]. In addition, phosphorylation of p38 MAPK, ERK1/2, and JNK was increased by stimulation with PMA [7], [8], [9], and activation of TACE occurs upon activation of ERK or p38 [10], [11].

Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae [12]. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity [13]. In the course of screening for anti-EPCR shedding from natural products, the ethyl acetate extracts of two independently fermented bacterial strains derived from marine sources showed potent inhibition on EPCR shedding in human umbilical vein endothelial cells (HUVECs). Repeated chromatographic separation gave three of diketopiperazines, cyclo (l-Pro-d-Val) (1), cyclo (l-Pro-l-Tyr) (2) and cyclo (l-Pro-d-Leu) (3), and these proline-containing compounds have been reported from the fermented bacterial strains [14], fungal strains [15], and the other macroorganisms [16]. Although several of diketopiperazines have been reported to have diverse biological activities including antibiotic and anti-inflammatory activities [13], the effects of 1–3 on EPCR shedding have not yet been studied. Therefore, in the current study, we investigated the effect of 1–3 on PMA-induced EPCR shedding in human endothelial cells and in a cecal ligation and puncture (CLP) model of septicemia in mice.

Section snippets

Reagents

Anisomycin and IL-1βwere purchased from Sigma-Aldrich (St. Louis, MO). sEPCR and TNF-α were purchased from Abnova (Taiwan). FVa, FXa, prothrombin, and thrombin were obtained from Haematologic Technologies (Essex Junction, VT).

Microbial isolation and identification

Bacillus sp. HC001 was obtained from marine sediments from Jeju Island, South Korea, in 2013. The collected sediments were dried under air flow for 1 day on a clean bench. The dried sediments were plated on the SYP SW agar media (soluble starch, 10 g/L; yeast extract, 4 g/L;

Isolation and characterization of compounds 1–3

Compounds 1 and 2 were isolated as white amorphous solid from the extract of Bacillus sp. HC001 culture media by ethyl acetate extraction. Compound 3 was isolated as white amorphous solid from the ethyl acetate extract of the cultured Piscicoccus sp. 12L081.

Compound 1 (Fig. 1) was identified to be cyclo (l-Pro-d-Val) by the analyses of spectroscopic data including 1H and 13C NMR data, LC-ESI-MS data and specific rotation value. The protonated molecule of compound 1 was observed at m/z [M + H]+

Conflict-of-interest disclosure

The authors declare no competing financial interest.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2012R1A5A2A42671316 and 2014R1A2A1A11049526).

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    These authors contributed equally to this work.

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