cancers Article Peptides against Low Density Lipoprotein (LDL) Aggregation Inhibit Intracellular Cholesteryl Ester Loading and Proliferation of Pancreatic Tumor Cells Aleyda Benitez-Amaro 1,2,† , Neus Martínez-Bosch 3,† , Noemí Manero-Rupérez 3 , Lene Claudi 1,2 , Maria Teresa La Chica Lhoëst 1,2 , Marta Soler 2 , Lia Ros-Blanco 2 , Pilar Navarro 1,3,4, * and Vicenta Llorente-Cortés 1,2,5, * 1 Institute of Biomedical Research of Barcelona (IIBB), Spanish National Research Council (CSIC), 08036 Barcelona, Spain; abenitez@santpau.cat (A.B.-A.); lclaudi@santpau.cat (L.C.); mlachica@santpau.cat (M.T.L.C.L.) 2 Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025 Barcelona, Spain; msoler@santpau.cat (M.S.); lros@santpau.cat (L.R.-B.) 3 Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Unidad Asociada IIBB-CSIC, 08003 Barcelona, Spain; nmartinez@imim.es (N.M.-B.); nmanero@imim.es (N.M.-R.) 4 August Pi Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain 5 CIBERCV, Institute of Health Carlos III, 28029 Madrid, Spain * Correspondence: pilar.navarro@iibb.csic.es (P.N.); vicenta.llorente@iibb.csic.es (V.L.-C.) † These authors contributed equally to this work. Simple Summary: Dyslipidemia 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 Citation: Benitez-Amaro, A.; tumor cells. However, the effect of aggregated LDLs on tumor progression has been unexplored. The Martínez-Bosch, N.; Manero-Rupérez, N.; Claudi, L.; La Chica Lhoëst, M.T.; aim of this work was to determine the effect of modified LDLs on intracellular cholesteryl ester/free Soler, M.; Ros-Blanco, L.; Navarro, P.; cholesterol ratio (CE/FC) and cancer cell growth, and the efficacy of peptides designed to inhibit LDL Llorente-Cortés, V. Peptides against aggregation on these processes. Our results show that aggregated LDL upregulates the intracellular Low Density Lipoprotein (LDL) CE/FC ratio and cell growth in pancreatic cancer and that these upregulatory effects were blocked by Aggregation Inhibit Intracellular peptides against LDL aggregation. We propose that anti-LDL aggregation peptides deserve to be Cholesteryl Ester Loading and further investigated as anti-tumoral strategies. Proliferation of Pancreatic Tumor Cells. Cancers 2022, 14, 890. https:// Abstract: Dyslipidemia, metabolic disorders and/or obesity are postulated as risk factors for pancreatic doi.org/10.3390/cancers14040890 ductal adenocarcinoma (PDAC). The majority of patients with these metabolic alterations have low Received: 24 January 2022 density lipoproteins (LDLs) with increased susceptibility to become aggregated in the extracellular Accepted: 8 February 2022 matrix (ECM). LDL aggregation can be efficiently inhibited by low-density lipoprotein receptor-related Published: 11 February 2022 protein 1 (LRP1)-based peptides. The objectives of this work were: (i) to determine if aggregated LDLs Publisher’s Note: MDPI stays neutral affect the intracellular cholesteryl ester (CE)/free cholesterol (FC) ratio and/or the tumor pancreatic cell with regard to jurisdictional claims in proliferation, using sphingomyelinase-modified LDL particles (Aggregated LDL, AgLDL); and (ii) to test published maps and institutional affil- whether LRP1-based peptides, highly efficient against LDL aggregation, can interfere in these processes. iations. 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 Copyright: © 2022 by the authors. also increase PANC-1 cell proliferation, which is inhibited by the DP3 peptide. Our results indicate Licensee MDPI, Basel, Switzerland. that AgLDL-induced intracellular CE accumulation plays a crucial role in the proliferation of pancreatic This article is an open access article tumor cell lines. Peptides with anti-LDL aggregation properties may thus exhibit anti-tumor effects. distributed under the terms and conditions of the Creative Commons Keywords: anti-LDL aggregation peptides; LDL atherogenicity; cholesteryl esters; pancreatic Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ ductal adenocarcinoma 4.0/). Cancers 2022, 14, 890. https://doi.org/10.3390/cancers14040890 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 890 2 of 15 1. Introduction Pancreatic ductal adenocarcinoma (PDAC) presents one of the toughest challenges in oncology. Patients diagnosed with local and regional disease show a five-year relative survival rate of about 9% [1], which places PDAC as the third leading cause of cancer-related death in developed countries. Many advances in PDAC biology have been made [2,3]; however, to change the dismal prognosis of PDAC, improvements must be made by addressing early diagnosis and improving the ineffective therapies that are currently available. Dyslipidemia, metabolic disorders and/or obesity have been postulated to be modifi- able risk factors for PDAC [4–6]. People with metabolic disorders including obesity and type 2 diabetes have increased numbers of small dense lipoproteins that, in addition to high triglycerides and low high-density lipoprotein (HDL), conform the characteristic lipid profile of atherogenic dyslipemia [7]. Small dense lipoproteins are prone to binding to the extracellular matrix (ECM)-proteoglycans (PGs) [8,9]. 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. Modulation of stroma-tumor crosstalk is essential for targeting PDAC progression [12,13]. In particular, pancreatic tumor stroma contains abundant ECM proteins, endothelial cells, immune cells and cancer-associated fibroblasts (CAFs) that represent up to 80% of the pancreatic tumor. In the atherosclerosis arena, the proatherogenic nature of LDLs is determined by the tendency of LDLs to be entrapped in the network of intimal PGs [8,9] and become aggregated [10,14]. 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 also 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. In the vasculature, PGs promote the formation of aggregated LDLs (AgLDLs), which are taken up through the low-density lipoprotein receptor-related protein 1 (LRP1), a receptor that is upregulated by cholesterol and hypoxia in ECM-adjacent cells. Overall, this generates a vicious cycle that efficiently produces intracellular lipid droplets (LDs) and prothrombotic cell phenotypes in the vasculature [18–20]. In addition, increased cholesterol loading and changes in lipid metabolism due to vascular cells from the stroma can also modulate tumor angiogenesis [21,22], which in turn can contribute to the strong immunosuppressive microenvironment of PDAC [23]. In pancreatic cancer, lipid droplets (which closely correlate with tumor aggressiveness) are mainly formed by cholesteryl esters (CEs) from exogenous sources [24,25]. Moreover, PDAC tumors are enriched in activated pathways associated with lipoprotein-related metabolism [26], and an aberrant accumulation of CEs has been observed in human pan- creatic cancer tissues and linked to cancer metastasis [27]. The sequence (Gly1127 –Cys1140 ) (P3) located in the CR9 domain of cluster II of the LRP1 α-chain interacts with the apolipoprotein B100 (ApoB100) of modified lipoproteins [28]. Peptides based on this P3 sequence electrostatically interact with ApoB100 and maintain ApoB100 conformation, preserving LDL against aggregation induced by phospholipolytic enzymes present in the ECM [29,30]. In the vascular scenario, innovative therapeutic attempts, such as peptides anti-LDL aggregation [29–32] and their antibodies [33–35], have successfully inhibited the formation of lipid droplet cell phenotypes and atheroscle- rosis. The retro-enantio (e.g., reversed d-amino acid order) peptide of P3, termed DP3 (Ac-dN·dE·dE·dD·dS·dN·dD·dE·dS·dD·dN·dD·G-NH2 ) and an ApoB-100 based peptide, termed IP321 (H-RLTRKRGLK-NH2 ), have been previously validated by our group as positive and negative controls, respectively, in biochemical and cell assays developed to test the anti-LDL aggregation and anti-cholesteryl ester loading properties of a new family of optimized peptides [30]. Cancers 2022, 14, 890 3 of 15 Currently, it is unknown whether LDL aggregation determines the intracellular CE content and growth of pancreatic tumor cells. Of note, the ECM of the stroma in tumors, like that of the intima in atherosclerosis, contains PGs and lipolytic enzymes that may strongly facilitate LDL retention and aggregation. Our hypothesis is that aggregated LDLs provide a relatively inexhaustible source of cholesterol for PDAC cells and are a contributing factor for cell growth. Our objectives were: (1) to analyze the impact of aggregated LDLs on intracellular CE accumulation and proliferation of human pancreatic tumor cells; (2) to analyze the effect of peptides against LDL aggregation on intracellular CE accumulation and tumor cell proliferation, and (3) to know whether the effect of aggregated LDLs on cell growth depends on their capacity to transfer CEs to tumor cells. 2. Materials and Methods 2.1. Peptide Sequence and Synthesis Peptide sequences were written from N to C, H- at the end terminus indicating amine ending, whereas -NH2 at the C terminus indicates amide. The amino acid sequence of LP3 peptide (parent) is H-GDNDSEDNSDEENC-NH2 , DP3 is the retro-enantio version of LP3 without cysteine: H-needsndesdndh-NH2 , where the amino acid letter code in lowercase stands for amino acids with D-chirality. As a negative control we used an ApoB-100- based peptide coded as IP321 with the sequence H-RLTRKRGLK-NH2 . All peptides were synthesized by Iproteos (Barcelona Knowledge Campus, Barcelona, Spain) by standard 9- fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) solid-phase peptide synthesis. Syntheses were performed manually on a 100 µmol scale/each using Rink amide ChemMatrix® resin. Peptide elongation and other solid-phase manipulations were done manually in polypropylene syringes, each fitted with a polyethylene porous disk. Solvents and soluble reagents were removed by suction. Washings between synthetic steps were done with dimethylformamide and dichloromethane using 5 mL of solvents/g resin each time. Nα- Fmoc-protected amino acids (four equivalents) were coupled using 2-(1H-benzotriazole-1- yl)-1,1,3,3-tetramethyluronium (TBTU, four equivalents), and N,N-diisopropylethylamine (eight equivalents). The extent of the reaction was monitored using the Kaiser test (primary amines). During couplings, the mixture was allowed to react with intermittent manual stirring. The Fmoc group was removed by treating the resin with 20% piperidine in (N,N- dimetilformamide) DMF (3 mL/g resin). The peptides were cleaved from the resin using a mixture of trifluoroacetic acid, triisopropylsilane and water (95:2.5:2.5). The crude products obtained were purified in reverse phase using a semiprep HPLC instrument equipped with a C18 column. The purity and identity of the synthesized peptides was assessed by HPLC, HPLC-MS and MALDI-TOF analysis. 2.2. LDL Isolation and Purification Human LDL (d1.019–d1.063 g/mL) was obtained from pooled normolipemic human plasma by sequential ultracentrifugation in a KBr density gradient. Briefly, very low density lipoproteins (VLDLs) were first discarded after spinning plasma at 36,000 rpm for 18 h at 4 ◦ C using a fixed-angle rotor (50.2 Ti, Beckman Coulter, Brea, CA, USA) mounted on an Optima L-100 XP ultracentrifuge (Beckman Coulter). Subsequently, VLDL-free plasma was layered with 1.063 g/mL KBr solution and centrifuged at 36,000 rpm for 18 h at 4 ◦ C. LDLs were dialyzed against 0.02 M Trizma, 0.15 M NaCl, 1 mM EDTA, pH 7.5 for 18 h, and then against normal saline for 2 h. Finally, isolated LDLs were filter-sterilized (0.22 µm Millex-GV filter unit, Merck KGaA, Darmstadt, Germany,). Protein concentration was determined using the BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA) and the cholesterol concentration with a commercial kit (IL test Cholesterol, Izasa Scientific, Alcobendas, Spain). 2.3. LDL Modification by Sphingomyelinase (SMase) in the Presence and Absence of Peptides To generate aggregated LDL (AgLDL), LDL (1.44 mg/mL) were incubated with 40 U/L of Bacillus cereus SMase (Sigma-Aldrich, Schnelldorf, Germany) in 20 mM Tris buffer Cancers 2022, 14, 890 4 of 15 (pH 7.0) containing 150 mM NaCl, 2 mM CaCl2 , and 2 mM MgCl2 at 37 ◦ C. From the methodological point of view, the in vitro modification of LDL by SMase has the main advantage versus other methods such as mechanical aggregation, in that the enzymatic modification is easily monitored and controlled. Therefore, the results using this kind of LDL modification are highly reproducible in terms of LDL aggregation degree (monitored by turbidimetry). LDL incubation with SMase was performed at several peptide concentra- tions ranging from 0 µM to 10 µM (0, 2.5, 5, 7.7, 8.8 and 10 µM). LDL lipolysis was stopped by the addition of EDTA (final concentration 0.5 mM). 2.4. Characterization of SMase-Induced LDL Aggregation by Turbidimetry The LDL composition was measured by commercial methods adapted to a Cobas 6000/c501 autoanalyzer (Roche Diagnostics, Indianapolis, IN, USA). Total cholesterol, triglycerides and ApoB reagents were obtained from Roche Diagnostics. PLs, NEFAs, and free cholesterol reagents were obtained from Wako Pure Chemicals (Tokyo, Japan). Sample turbidity (LDL aggregation monitorization) was determined by measuring absorbance of 100 µL of LDLs (1.44 mg/mL ApoB) in a 96-well microplates at 405 nm. 2.5. Cell Culture of Pancreatic Tumor Cell Lines Human pancreatic cancer cell lines PANC-1 [36], RWP-1 [37], and Capan-1 [38] were obtained from the cancer cell repository at Hospital del Mar Medical Research Institute (IMIM), Barcelona. Cells were cultured at 37 ◦ C in a cell incubator at 5% CO2 using DMEM (Invitrogen, Waltham, MA, USA) with 10% FBS. 2.6. Monitorization of the Efficacy of Peptides to Inhibit LDL-Induced Intracellular Cholesteryl Ester Accumulation Peptide inhibitory efficacy was monitored by determination of intracellular cholesteryl ester/free cholesterol (CE/FC) ratio. PANC-1, RWP-1 and Capan-1 were seeded in 12- well plates (20,000 cells). After 24 h, cell quiescence was induced for 24 h with 0.2% fetal calf serum. Quiescent cells were exposed for different times to LDL previously treated with SMase for 18 h in the presence or absence of peptides at a concentration of 10 µM (ratio peptide/apoB100 5:1). The degree of LDL aggregation was assessed by turbidimetry measurements before the incubation with the cells. Following the lipoprotein incubation period, all cell types were washed exhaustively: twice with phosphate buffered saline (PBS), twice with PBS supplemented with 1% bovine serum albumin (BSA), and once with PBS supplemented with both 1% BSA and 100 U/mL heparin. Cells were then harvested into 1 mL of 0.15 M NaOH for lipid extraction followed by neutral intracellular lipid partitioning through thin layer chromatography and lipid band analysis and quantification. 2.7. Determination of Intracellular Cholesteryl Ester/Free Cholesterol Ratio Lipids were extracted using the Bligh and Dyer method with minor modifications [24]. The lipid extract was dissolved in dichloromethane, applied to silica gel plates, and sep- arated by thin layer chromatography. Cholesterol and cholesterol palmitate were run as standards of free and cholesteryl ester, respectively. A primary solvent combination of heptane/diethyl ether/acetic acid (74:21:4, v/v/v) was used as a chromatographic mobile phase followed by heptane alone. After lipid separation, the plates were dried and stained as previously reported [24]. Finally, the spots corresponding to CE and FC were mea- sured by densitometry against the standard curve using a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA). Intracellular CEs detected in these cells upon exposure to LDL derives exclusively from CE supplied by LDL, as cells unexposed to LDL did not have intracellular CEs. The inhibitory effect of the peptide on the intracellular cholesterol accumulation was analyzed in terms of the decrease in the ratio of intracellular CE and FC content. Cancers 2022, 14, 890 5 of 15 2.8. Confocal Microscopy Analysis PANC-1, RWP-1, Capan-1 were seeded at 50,000 cells over glass coverslips in 24-well plates. Cells were left with 0.2% FBS for 24 h and exposed for 4 h to nLDL or AgLDL (100 µg/mL), in the absence or presence of DP3 or IP321 peptides (10 µM). Cells were fixed with PFA 4 % and permeabilized with 0.25 % Triton X-100. After blocking with 1% BSA, cells were incubated with BODIPY® 493/503 (stock 5 mg/mL, dilution 1/100, ThermoFisher Scientific, Waltham, MA, USA). Hoechst 33342 (stock 10 mg/mL, dilution 1/2000) staining was used to counter visualize nucleus. Confocal images were acquired with Axio-Observer Z1 (ZEISS, Oberkochen, Germany) laser confocal microscope adapted to for sequential image acquisition. To assure a correct normalization of fluorescence, we have adjusted exposure times to eliminate the autofluorescence of the negative control (PANC-1 cells unexposed to LDL). 2.9. Flow Cytometry Analysis Cells were grown in media supplemented with 10 % FBS for 24 h and arrested for 24 h in media supplemented with 0.2% FBS before LDL addition (time 0). At time 0, cells were exposed to LDL (added to the media without serum) for 24, 48 or 72 h. The experimental setting was designed to avoid serum interferences and to assure that cholesterol-induced cell growth was a direct response to cholesterol from LDL. Quiescent human pancreatic cancer cells, PANC-1, were exposed to nLDL or AgLDL (1.44 mg/mL) untreated or treated with peptide DP3 or IP321 (10 µM). Cells were then washed twice with PBS and removed from the culture dish with Trypsin-EDTA 0.25% (Invitrogen, Waltham, MA, USA). PBS supplemented with 1% FBS was used to inactivate the enzyme and harvest the cells. The cell suspension was centrifuged at 1200 rpm for 10 min. The resulting pellet was resuspended in PBS supplemented with propidium iodide (stock 1 mg/mL, dilution 1/1000) to detect dead cells. Live, dead and total cells were counted using a MACSQuant® Analyzer with MACSQuantify™ Software version 2.13 (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.10. Statistical Analysis Data were described as the mean ± SEM. Experiments were performed three times and included three technical replicates unless otherwise indicated. Comparisons among groups were performed by parametric student t-test (one-factor analysis of variance). Statistical significance was considered when p was <0.05. The statistical software package SPSS (V25, IBM, Armonk, NY, USA) was used for statistical analyses. 3. Results 3.1. Effect of SMase on Human LDL Aggregation Turbidimetry assays (absorbance at 405 nm) showed that SMase strongly increased human LDL aggregation after 24 h (from 0.123 ± 0.26 to 1.21 ± 0.12, p < 0.001) (Figure 1A). Gradient gel electrophoresis (GGE) (Figure 1B) and agarose gels (Figure 1C) were used to visualize the modification of LDLs caused by SMase. In native GGE gels, the position of the LDL particle was determined by particle size. SMase caused a partial loss of the band corresponding to LDLs (Figure 1B). In Figure 1B, small LDL aggregates are marked with a red arrow while the lack of biggest LDL bands of AgLDL can be attributed to the fact that the pore size of GGE gels precludes the entrance of the largest LDL aggregates. In agarose gels (Figure 1C), where LDLs run according to its negative charge, AgLDL appeared as a diffuse band compared to the well-defined LDL band. Cancers 2022, Cancers 2022, 14, 14, xx 66 of of 16 16 that the that the pore pore size size of of GGE GGE gels gels precludes precludes the the entrance entrance of of the the largest largest LDL LDL aggregates. aggregates. In In Cancers 2022, 14, 890 agarose gels (Figure 1C), where LDLs run according to its negative charge, AgLDL agarose gels (Figure 1C), where LDLs run according to its negative charge, AgLDL ap- 6 ofap- 15 peared as peared as aa diffuse diffuse band band compared compared toto the the well-defined well-defined LDL LDL band. band. Figure1.1. Figure Figure 1.Effects Effectsofof Effects ofSMase SMaseon SMase onhuman on human human LDL LDL LDL aggregation. aggregation. aggregation. (A) (A)(A) Bar BarBar graph graph graph showing showing showing the thethe effect effect effect of SMase of SMase of SMase on on LDL on LDL aggregation aggregation (turbidimetry (turbidimetry at 405 at 405 nm) (B) Gradient gel electrophoresis (GGE) and (C) Aga- LDL aggregation (turbidimetry at 405 nm)nm) (B) Gradient (B) Gradient gel electrophoresis gel electrophoresis (GGE)(GGE) and and (C) Aga- (C) Agarose rose gel rose gel electrophoresis electrophoresis showing showing LDLLDL electrophoretic electrophoretic mobility mobility ofof AgLDL AgLDL in in comparison comparison to to nLDL. gel electrophoresis showing LDL electrophoretic mobility of AgLDL in comparison to nLDL.nLDL. Data Data are Data are shown shown as as mean mean ±± SEM SEM ofof three three experiments experiments performed performed in in triplicate. triplicate. Statistical Statistical significance significance are shown as was determined mean determined by ± SEM of by Student’s three Student’s t-test.experiments t-test. O/N, performed O/N, overnight; overnight; LDL, in triplicate. LDL, low-density Statistical low-density lipoprotein; significance lipoprotein; nLDL, was nLDL, native native was determined LDL; AgLDL, AgLDL, Student’s t-test. by aggregated LDL.O/N, overnight;blots Full uncropped uncropped LDL,canlow-density be found found inlipoprotein; in Figure S5.nLDL, native LDL; Figure S5. LDL; aggregated LDL. Full blots can be AgLDL, aggregated LDL. Full uncropped blots can be found in Figure S5. 3.2. Effect 3.2. Effect of nLDLs nLDLs and and AgLDLs AgLDLs on on Intracellular Intracellular CE/FC CE/FC Ratio in in PANC-1, PANC-1, RWP-1 RWP-1 and 3.2. Effect ofofnLDLs and AgLDLs on Intracellular CE/FC RatioRatio in PANC-1, RWP-1 and and Capan-1Cells Capan-1 Capan-1 Cells Cells Wecompared We We comparedthe compared theeffect the effectofof effect ofnLDLs nLDLsand nLDLs andAgLDLs and AgLDLs(100 AgLDLs (100µg/mL, (100 μg/mL,222h) μg/mL, h)on h) onthe on theintracellular the intracellular intracellular cholesteryl cholesteryl ester ester (CE)/free (CE)/free cholesteryl ester (CE)/free cholesterol cholesterol (FC) (FC) ratio ratio in in the human pancreatic in the human pancreatic cancer cell the human pancreatic cancer cancer celllines cell lines lines PANC-1 PANC-1 (Figure PANC-1(Figure 2A), (Figure2A), RWP-1 2A),RWP-1 (Figure RWP-1(Figure (Figure 2B) 2B) 2B) andand Capan-1 Capan-1 and Capan-1 (Figure (Figure (Figure 2C) 2C)2C) by by by thin thinthin layer layer chroma- chromatog- layer chroma- tography raphy tography (TLC). (TLC). In the (TLC). Inabsence In the absence the absence of LDL,of LDL, of LDL, PANC-1 PANC-1 and Capan-1 and Capan-1 PANC-1 and Capan-1 cells cells did did not cells did not contain contain not contain CE, whileCE, CE, while RWP-1 RWP-1 has a has a moderate while RWP-1 moderate has a moderate CE CE content. content. At 2 h, CE content. At 2 h, AtnLDLs nLDLs did not 2 h, nLDLs did not didornot or only only slightly slightly or only increased increased slightly the increased thecontent CE the CE content CE content of these of these of these cells cellscells lines; lines; however, however, lines; however, AgLDLAgLDL strongly strongly AgLDL increased increased strongly the CE/FC the CE/FC increased the CE/FC ratio ratioratio in in all in all pancreatic pancreatic cancer cancer cells cells (Figure (Figure 2). 2). These These results results were were corroborated corroborated all pancreatic cancer cells (Figure 2). These results were corroborated by BODIPY staining by by BODIPY BODIPY staining staining andconfocal and and confocalmicroscopy, confocal microscopy,showing microscopy, showing showing higher higher higher upregulatory upregulatory upregulatory effects effects of AgLDLs of of effects AgLDLs AgLDLs as compared compared as compared as to to nLDLs nLDLs on on both both sizesize andand abundance abundance ofof lipid lipid droplet droplet (green (green dots) dots) in in to nLDLs on both size and abundance of lipid droplet (green dots) in PANC-1 (Figure 3A), PANC-1 PANC-1 (Figure (Figure 3A), 3A), RWP-1(Figure RWP-1 RWP-1 (Figure3B) (Figure 3B)and 3B) andCapan-1 and Capan-1(Figure Capan-1 (Figure3C) (Figure 3C)cell 3C) celllines. cell lines. lines. Figure Figure 2.Effects Figure2.2. Effectsof Effects ofnLDLs of nLDLsand nLDLs andAgLDLs and AgLDLson AgLDLs onthe on theintracellular the intracellularCE/FC intracellular CE/FC ratio CE/FC ratio in ratio in PANC-1, in PANC-1,RWP-1 PANC-1, RWP-1and RWP-1 and and Capan-1 Capan-1 pancreatic pancreatic tumor tumor cells. cells.Cells Cellswere were untreated untreated orortreated treated with withnLDL nLDL or AgLDL or AgLDL for 2 h. for 2 Repre- h. Rep- Capan-1 pancreatic tumor cells. Cells were untreated or treated with nLDL or AgLDL for 2 h. Rep- resentative sentative resentativethinthin thin layer layer layer chromatography chromatography chromatography (TLC) (TLC) images images (TLC) show images show cholesteryl cholesteryl show ester esterester cholesteryl (CE) (CE)(CE) and and and free choles- free free choles- cholesterol terolbands; (FC) terol (FC) bands; (FC) bands; the barthethe bar graphs graphs bar graphs show the show the intracellular intracellular intracellular show the CE/FC ratioCE/FC CE/FC ratio in in PANC-1 ratio in PANC-1 PANC-1 (A), RWP-1 (A),(B) (A), RWP-1 (B) and and Capan-1 RWP-1 (B) and Capan-1 (C) (C) cell cell types. Capan-1 (C) cell Datatypes. Data areData types. shown areas are shown as the the mean shown as the±mean mean SEM ±±ofSEM SEM threeofexperiments of three experiments three experiments performed performed in trip- trip- in triplicate. performed in licate. Statistical licate. Statistical Statistical significance significance significance was determined was was determineddetermined by Student’s by by Student’s Student’s t-test. t-test. t-test. CE, CE, cholesteryl CE, cholesteryl cholesteryl ester; ester; ester; FC, FC, freeFC, free cho- free cho- cholesterol; lesterol; lesterol; LDL, low-density LDL, low-density lipoprotein; lipoprotein; nLDLs, nLDLs, native native LDLs; AgLDLs, LDLs; aggregated aggregated AgLDLs, aggregated LDL. Uncropped LDL. Uncropped LDL, low-density lipoprotein; nLDLs, native LDLs; AgLDLs, LDL. Uncropped blots can blots can blots can be be found found in in Figure Figure S6. S6. be found in Figure S6. Cancers Cancers 2022, 2022, 14,14, 890x 77ofof1516 Figure3.3.Effects Figure EffectsofofnLDLs nLDLsand andAgLDLs AgLDLson onlipid lipiddroplet dropletformation formationininpancreatic pancreatictumor tumorcell celllines. lines. Confocal laser microscopy images show BODIPY-stained lipid droplets (green dots) and nuclei Confocal laser microscopy images show BODIPY-stained lipid droplets (green dots) and nuclei (blue) in the (A) PANC-1, (B) RWP-1 and (C) Capan-1 cell lines. Scale bar, 25 μm. LDL, low-density (blue) in the (A) PANC-1, (B) RWP-1 and (C) Capan-1 cell lines. Scale bar, 25 µm. LDL, low-density lipoprotein; nLDL, native LDLs; AgLDL, aggregated LDLs. lipoprotein; nLDL, native LDLs; AgLDL, aggregated LDLs. 3.3.Effect 3.3. Effectof of Peptide Peptide DP3 DP3 on SMase-Induced on SMase-Induced LDL LDL Modification Modification ToTostudy studythetheeffects effectsofofthe thepeptides peptidesononSMase-induced SMase-inducedLDL LDLaggregation, aggregation,LDL LDLwere were incubated with SMase in the absence or presence of DP3 (anti-LDL incubated with SMase in the absence or presence of DP3 (anti-LDL aggregation peptide) aggregation peptide) ororIP321 IP321(negative (negativecontrol controlpeptide), peptide),asaspreviously previouslydetailed detailed[29]. [29].The Theeffect effectofofpeptides peptidesonon SMase-inducedLDL SMase-induced LDL aggregation aggregation waswas tested tested at several at several peptide peptide concentrations concentrations (0,5,2.5, (0, 2.5, 7.7,5, 7.7, 8.8 8.810 and and 10 Turbidimetry µM). μM). Turbidimetry studies studies showed showed thatsignificantly that DP3 DP3 significantly reducedreduced SMase- SMase-LDL LDL aggregation aggregation at 18 hatin18a hdose-dependent in a dose-dependentmanner manner (Figure (Figure 4A).expected, 4A). As As expected, the nega- the negative IP321 control peptide did not inhibit SMase-LDL aggregation, at any dose tested. InIn tive IP321 control peptide did not inhibit SMase-LDL aggregation, at any dose tested. addition,agarose addition, agarosegelsgels showed showed that the AgLDLAgLDL bandbandwaswasmore morediffuse diffusethan thanthethewell-de- well- fined LDL defined LDLbandband (Figure (Figure 4B). 4B). AgLDLs in the the presence presence of ofDP3 DP3(at (atconcentrations concentrationshigher higher than5 5µM) than μM)exhibited exhibiteda asimilar similarband bandpattern patternasasnLDLs. nLDLs.InIncontrast, contrast,IP321 IP321diddidnot notchange change therunning the runningpattern patternofofAgLDLs. AgLDLs.We Weselected selectedthetheconcentration concentrationofof1010µM μMtotoperform performfurther further comparative comparativestudies studieswith withthese thesepeptides. peptides. 2022, 14, x Cancers 2022, 14, 890 8 of 16 8 of 15 Figure 4. Effects of peptides DP3 and IP321 on SMase-induced LDL aggregation. LDLs were used Figure 4. Effects of peptides DP3 and IP321 on SMase-induced LDL aggregation. LDLs were used at 1.44 mg/mL ApoB. The peptides DP3 and IP321 were tested at increasing concentrations (0, 2.5, at 1.44 mg/mL ApoB. The peptides DP3 and IP321 were tested at increasing concentrations (0, 2.5, 5, 7.7, 8.8 and 10 µM). The effects of peptides on LDL aggregation were monitored by measuring 5, 7.7, 8.8 and 10 μM). The effects of peptides on LDL aggregation were monitored by measuring turbidimetry (A)turbidimetry and agarose(A) gel and agarose gel mobility electrophoretic electrophoretic mobility (B). Results are(B). Results shown ±SD ofas mean ± SD of are shown as mean three independent three independent experiments (n experiments = 5 biological(nreplicates). = 5 biological * p