Peptides against Low Density Lipoprotein (LDL) Aggregation Inhibit Intracellular Cholesteryl Ester Loading and Proliferation of Pancreatic Tumor Cells.

Aleyda Benitez-Amaro,Pilar Navarro,Marta Soler,Lia Ros-Blanco,Noemí Manero-Rupérez,Neus Martínez-Bosch,Vicenta Llorente-Cortés,Lene Claudi,Maria La Chica Lhoëst

doi:10.3390/cancers14040890

Abstract

Simple SummaryDyslipidemia is a modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers. A key component of dyslipidemia is a high level of small and dense low-density lipoproteins (LDLs). These LDLs have a high probability to be entrapped and modified (aggregated) in the extracellular matrix (ECM), becoming a source of cholesterol for tumor cells. However, the effect of aggregated LDLs on tumor progression has been unexplored. The aim of this work was to determine the effect of modified LDLs on intracellular cholesteryl ester/free cholesterol ratio (CE/FC) and cancer cell growth, and the efficacy of peptides designed to inhibit LDL aggregation on these processes. Our results show that aggregated LDL upregulates the intracellular CE/FC ratio and cell growth in pancreatic cancer and that these upregulatory effects were blocked by peptides against LDL aggregation. We propose that anti-LDL aggregation peptides deserve to be further investigated as anti-tumoral strategies.Dyslipidemia, metabolic disorders and/or obesity are postulated as risk factors for pancreatic ductal adenocarcinoma (PDAC). The majority of patients with these metabolic alterations have low density lipoproteins (LDLs) with increased susceptibility to become aggregated in the extracellular matrix (ECM). LDL aggregation can be efficiently inhibited by low-density lipoprotein receptor-related protein 1 (LRP1)-based peptides. The objectives of this work were: (i) to determine if aggregated LDLs affect the intracellular cholesteryl ester (CE)/free cholesterol (FC) ratio and/or the tumor pancreatic cell proliferation, using sphingomyelinase-modified LDL particles (Aggregated LDL, AgLDL); and (ii) to test whether LRP1-based peptides, highly efficient against LDL aggregation, can interfere in these processes. For this, we exposed human pancreatic cancer cell lines (PANC-1, RWP-1 and Capan-1) to native (nLDL) or AgLDLs in the absence or presence of LRP1-based peptides (DP3) or irrelevant peptides (IP321). Results of thin-layer chromatography (TLC) following lipid extraction indicate that AgLDLs induce a higher intracellular CE/FC ratio than nLDL, and that DP3 but not IP321 counteracts this effect. AgLDLs also increase PANC-1 cell proliferation, which is inhibited by the DP3 peptide. Our results indicate that AgLDL-induced intracellular CE accumulation plays a crucial role in the proliferation of pancreatic tumor cell lines. Peptides with anti-LDL aggregation properties may thus exhibit anti-tumor effects.

Highlights

Pancreatic ductal adenocarcinoma (PDAC) presents one of the toughest challenges in oncology
In agarose gels (Figure 1C), where low density lipoproteins (LDLs) run according to its negative charge, aggregated LDLs (AgLDLs) appeared as a diffuse band compared to the well-defined LDL band
We found a slight reduction of total cell number from 48 to 72 h in PANC-1 exposed to AgLDL (Figure 6C) concomitantly with an increase of dead cells (Figure S4), likely due to cell stress caused by a mitogen stimulus (AgLDLs) in the absence of serum for a long time

Summary

Introduction

Pancreatic ductal adenocarcinoma (PDAC) presents one of the toughest challenges in oncology. Low-density lipoproteins (LDLs) play a crucial role as a source of exogenous cholesterol for cells, especially when LDLs become entrapped, as shown in the intimal [10] and proposed in the stromal [11] ECM of atherosclerotic plaques and tumors, respectively. In the ECM of human atherosclerotic lesions, sphingomyelinase (SMase) is one of the main secretory phospholipases contributing to aggregation of intimal retained LDL [15]. These events can contribute to cancer progression, like in metastatic melanoma, where SMase has been reported to be activated by acidic extracellular pH [16], or in PDAC, where there is an initial and progressive alteration of the ECM [17] that may render PGs more interactive with lipoproteins

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