The roles of fatty acid modification in the activity of anticancer peptide

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The roles of fatty acid modification in the activity of anticancer peptide of R-lycosin-I. Cui Jian, Peng Zhang, Jing Ma, Shandong Jian, Qianqian Zhang, Bobo Liu, Songping Liang, Meiyan Liu, Youlin Zeng, and Zhonghua Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00605 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Molecular Pharmaceutics

The roles of fatty acid modification in the activity of anticancer peptide of R-lycosin-I Cui Jian†a, Peng Zhang†b, Jing Maa, Shandong Jianb, Qianqian Zhangb, Bobo Liua, Songping Liangbc, Meiyan Liua, Youlin Zeng*a and Zhonghua Liu*bc

a

The National and Local Joint Engineering Laboratory for New Petrochemical Materials and Fine Utilization of Resources, Hunan Normal University, Changsha Hunan 410081, China. E-mail: [email protected] b

The National &Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha Hunan 410081, China. E-mail: [email protected] c

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha Hunan 410081, China *Corresponding author †These authors contribute equally to this paper.

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Abstract We previously reported that R-lycosin-I, modified by amino acid substitution from Lycosin-I, was a peptide with anticancer activity and a linear amphipathic alpha-helix conformation, and it can induce cancer cell apoptosis and inhibit cell proliferation. However, the anticancer activity of R-lycosin-I was not highly improved. In order to further improve the anticancer activity of R-lycosin-I, fatty acids with different chain length from 12 to 20 carbons were introduced to the N-terminal of R-lycosin-I to yield five lipopeptides (R-C12, R-C14, R-C16, R-C18, R-C20). The physicochemical properties of the five lipopeptides were determined by hydrodynamic size, ζ-potential and circular dichroism spectroscopy, respectively. Then, the cytotoxic activity of these lipopeptides in A549 cells was evaluated with serum-containing and serum-free media, respectively, showing their anticancer activities were all increased through fatty acids modification. This may be owed to the increased hydrophobicity and the enhanced interaction with cancer cell membrane. the cytotoxic activity of R-C16 was 3~4-fold higher than that of original R-lycosin-I, and also was the strongest among them of all the five lipopeptide, whether in serum or serum free. Compared with R-lycosin-I, the lactate dehydrogenase (LDH) leakage assay and scanning electron microscopy (SEM) indicated that R-C16 had weakly destructive effect on cancer cell membrane, but it, might cause apoptosis to exert anticancer activity. Finally, the impacts of fatty acid length on the physicochemical properties and the anticancer potential of peptide were discussed. Our data consolidate work on fatty acid modified anticancer peptides.

Keywords: R-lycosin-I, Fatty acids, lipopeptides, Hydrophobicity, Anticancer activity


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1. Introduction Globally, due to cancer cells continue to spread and accumulate in the body to form tumor masses, it has already been one of the most serious diseases that threaten human health1-3. However, a wide variety of cancer treatment drugs have a common feature: the emergence of resistance against multiple drugs and their consequent undesirable side effects for the patients4-6. Thus, it is urgent to exploit novel anti-tumor drugs which have more powerful activity and circumvention of multiple drug resistance. Anticancer peptides (ACPs) have become the research focus mainly due to most of the ACPs are alpha-helical amphipathic and have consistent features of high activity, low immunogenicity, good biocompatibility, sequence multiplicity and a large number of functional molecules modification sites7-10. For their cationic and amphipathic features, ACPs may bind to cancer cells through electrostatic and hydrophobic interactions, and hence lead to cancer cells necrosis or apoptosis11-16. Although ACPs have effective anticancer activity, there still exist several disadvantages, such as low permeability to the membrane, poor stability, and sensitivity to proteolytic digestion17, which limit their further applications. Therefore, researchers have also concentrated efforts on the design and modification of peptide in recent years and many peptide-based modifications were reported. For example, the addition of histidine to cell-penetrating peptide PepFec enhances its pH sensitivity18 and we previously reported that the replacement of lysine by arginine in the sequence of Lycosin-I improves its activity19. Pegylation of novel GLP-1 receptor agonists improves their stability and prolongs their half-life20. The modification of TP10 by stearic acid increases the peptide-membrane interaction and the ability to transport oligonucleotide cargo21. In addition, stearic acid has been widely applied to improve the cell penetration of cell-penetrating peptide (CPP)22. So, the purpose of modification can be achieved by changing some physicochemical parameters of the peptide. Hydrophobicity has been reported as a critical parameter which can induce the hydrophobic core of the antimicrobial peptide inserted into themembrane23, 24. And it has been found that hydrophobicity affects many features of pores such as the formation rate, size, and stability25. R-lycosin-I is an amphiphilic α-helix anticancer active peptide, which is obtained by amino acid substitution (all lysine in Lycosin-I is replaced by arginine) and shows good anticancer activity and cell permeability19. Thus, like most antimicrobial peptides, the structural parameters such as charge, hydrophobicity, amphipathicity, and conformation23,24 also influence the activity of R-lycsion-I. Fatty acid-modified peptides have great development prospects, and they also have significant tumor inhibition while increasing their cell permeability26. Therefore, the hydrophobicity and anticancer activity of R-lycosin-I might be enhanced by fatty acid modification. In this research, fatty acids were covalently coupled to the N-terminus of R-lycosin-I. In order to systematically understand the relationship between chains length of fatty acids and anti-cancer activities, five R-lycosin-I analogs were designed and synthesized by fatty acids modification. Physicochemical characteristics of five lipopeptides were determined by DLS and CD. The anti-cancer



activity of five lipopeptides with and without serum were assayed by the CCK-8 method. The LDH leakage assays determined that R-C16 capable to induce cancer cell death via membrane disruption.

2. Materials and Methods 2.1 Reagents All Fmoc amino acids, HATU and HOBT were purchased from Advanced Automated Peptide Protein Technologies (AAPP TEC, Louisville, KY, USA). Rink amide-AM resin was obtained from GL Biochem (Shanghai, China). The Cell Counting Kit-8 (CCK-8) was order from Sigma. The LDH Cytotoxicity assay Kit purchased from Beyotime (Shanghai, China).

2.2 Cell cultures, animals and human blood Human lung carcinoma cells (A549), Human Umbilical Vein Endothelial Cells (HUVEC) and Human embryonic kidney 293T cells (HEK293T) were cultured in Dulbecco’s modified Eagles medium (DMEM, Invitrogen) involving 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 100 mg/ml streptomycin at 37°C containing 5% CO2. 5000 cells/well were housed in 96-well Plates for 24 h before the experiments. Healthy male 4 to 6-week-old Kunming mice were purchased from Slac & Jingda Corporation of laboratory animals, Changsha, China. Human blood was provided by the author and collected at the Hunan Normal University Hospital.

2.3 Synthesis of Fatty Acid Modified Peptides Peptides were synthesized using the Fmoc solid-phase peptide synthesis (SPPS) methods27 and purified by reversed-phase high-performance liquid chromatography. Upon completion of the peptide chain, the N-terminal Fmoc group was removed, and fatty acids (0.5 mmol) were added and coupled using HATU (0.5 mmol) and HOBT (0.5 mmol) in DMF as activators and NMM as base for 1 h. Peptides were cleaved using trifluoroacetic acid/1,2-Ethanedithiol/Thioanisole/Anisole (90%/5%/3%/2%(v/v)) and after 2 h precipitated in cold diethyl ether. RP-HPLC (C18, 4.6×250 mm) was used to purify the lipopeptides and R-lycosin-I by using 0.1% TFA/acetonitrile gradient from 0-80% in 48 minutes. The purified products were assessed using MALDI–TOF MS (AB, SCIEX).

2.4 Hydrodynamic size and zeta potential of peptides The dynamic light scattering (DLS) of Zeta sizer Nano ZSP (Malvern Instruments, UK) apparatus was used to determine the hydrodynamic size and zeta potential of R-lycosin-I and lipopeptides. R-lycosin-I and lipopeptides concentrations were diluted to 100 µM with MQ in a final volume of 1 mL. Measurements were carried out at 25oC, with two measurements in each group and one measurement set to three.

2.5 CD spectroscopy The secondary structure of all the peptides were confirmed in accordance with the


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protocol previously published by our group19. CD spectra was recorded between 180 nm and 280 nm on a Jasco J-815 CD spectrometer. The temperature was adjusted to 25oC and peptides concentrations were diluted to 100 µM with phosphate buffer solution and 50% TFE, respectively.

2.6 Transmission electron microscopy (TEM) The morphologies of the peptides were investigated using TEM (Tecnai G20 F20, American). 5 µL of each sample was dropped to the copper grid coated with a Formavar/carbon support film, after 1 min, the excess solution was blotted away with filter paper. Then, a drop of 2% uranyl acetate dihydrate solution was covered on the above grid. The samples were observed after air-dried.

2.7 Cytotoxicity The cells (3×103 cells/well) were seeded onto 96-well plates in DMEM containing 10% (v/v) FBS. Aliquots (90 µL) of this suspension were seeded in sterile 96-well plates and containing 10 µL of R-lycosin-I or lipopeptides at different concentrations. According to our previously described28, after 24 h incubation at 37oC, the CCK-8 assay was used to determine the cell viability.

2.8 Flow cytometry assay We used Annexin V-FITC / PI apoptosis detection kit (Bio-uniqure, China) detection of apoptotic cells. We cultured the cells using the previous method and then treated with 6µM peptides for 24 hours and then treated with 500 µL of anti-Annexin V antibody conjugated with 5 µL propidium iodide (PI) and 5 µL FITC as previously described Buffer staining19. A Flow cytometry was used to analyze the apoptosis of stained cells (BD, USA).

2.9 Scanning electron microscopy The SEM was performed to investigate cell membrane morphological changes in A549 cells after treatment with R-C16 and R-Lycosin-I. A549 cells were seeded at coverslips that are placed into a 6-well plate for SEM and incubated for 24 hours. After incubating the cells with R-C16 and R-Lycosin-I at a concentration of 15 µM for 1 h, cells were washed with PBS gently and fixed with 1 mL of 2.5% glutaraldehyde solution for 4 hours. Then the fixed cells were dehydrated with 30%, 50%, 70%, 75%, 80%, 90%, 100% ethanol, respectively, and dried by a freeze-drying device and coated with gold plating before observation. The sample were examined by using a scanning electron microscope (Hitachi Su8010, Japan).

2.10 LDH leakage assays The lactate dehydrogenase (LDH) release assay was used to determine the membrane integrity by the cytotoxicity Detection Kit (Beyotime, China)29,30. LDH assay was completed according to the manufacturer’s instructions. Briefly, A549 cells seeded in 96-well plate for 24 h. Then, 100 µL of serum-free medium containing various concentrations of R-lycosin-I and lipopeptides were added and incubated for 24 h. After



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treatment with R-lycosin-I and lipopeptides, reaction mixture (50 µL) was added to each well and reacted for 30 min at 25oC, followed by 50 µL of stop solution. Neither R-lycosin-I nor lipopetides was added as a control which take as no leakage. The fluorescence was detected by microplate reader at 490 nm. The cell treated with 1% TritionX-100 represented 100% leakage (total LDH release).

2.11 Determination of Hemolytic activity The hemolytic activity of R-lycosin-I and five lipopeptides were confirmed according to a literature procedure31, 32. Red blood cells were washed with PBS buffer and centrifuged for 5 minutes, 3000 rpm, and repeated three times. The serum was removed from the blood by centrifugation and suction, and cells were resuspended in PBS to a final concentration of 1%. The diluted RBC suspension (50 µL) was then mixed with: 50 µL of PBS as a negative control, 50 µL of 1% Triton-X 100 as a positive control, and 50 µL of lipopeptides at concentrations ranging from 0.63 to 40 µM. The mixtures were incubated for 1 h at 37°C, then centrifuged for 5 min at 12,000 rpm. The supernatant was added to 96-well plates to measure the absorbance at 450 nm by using a microplate reader.

2.12 Serum stability assay The serum stability of R-lycosin-I and lipopeptides were determined according to previously described19. R-lycosin-I and lipopeptides were incubated with 10% serum or PBS at 37oC for 24 h and 48 h, respectively. Then, R-lycosin-I and lipopeptides at a concentration of 40 µmol·L-1 were added to A549 cells and incubated at 37oC, in 5% CO2. After 24 h, the cell viability of R-lycosin-I and lipopeptides were detected by microplate reader by using the CCK-8 assay.

3. Results and discussion 3.1 Design and Synthesis of R-lycosin-I Conjugates To determine the effect of fatty acids on the anticancer peptide, we synthesized a series of R-lycisin-I derivatives with different hydrophobic chain lengths by fatty acid modification strategy. The corresponding lipopeptides were named as R-C12, R-C14, R-C16, R-C18, R-C20 based on their original peptide backbone and the length of hydrophobic chain (Table 1). C18 RP-HPLC was used to purify the lipopeptides and R-lycosin-I (Fig. 1A-E). Then MALDI-TOF MS was used to determine the relative molecular mass of lipopeptides (Fig. 1F-J). To investigate the hydrophobicity of lipopeptides, 50 µM of R-lycosin-I and five lipopeptides were mixed and then elution with a gradient of 0-80% /0.1% TFA acetonitrile. From (Fig. 2A), it is obvious that the hydrophobicity of lipopeptides is significantly higher than that of R-lycosin-I. The retention times of R-lycosin-I and lipopeptides (Table 1) also indicated the fatty acid chain length was positively correlated with the hydrophobicity of the lipopeptides. Table 1 Sequences, molecular masses and retention times of peptides. Named

Sequence


Mw

a

TR (min)

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a

The R-lycosin-I NH2-RGWFRAMRSIARFIARERLREHL-CONH2 2928.29 retention R-C12 CH3-(CH2)10-CONHRGWFRAMRSIARFIARERLREHL-CONH2 3110.61 time (TR) of R-C14 CH3-(CH2)12-CONHRGWFRAMRSIARFIARERLREHL-CONH2 3138.66 each R-C16 CH3-(CH2)14-CONHRGWFRAMRSIARFIARERLREHL-CONH2 3166.71 peptide was determined R-C18 CH3-(CH2)16-CONHRGWFRAMRSIARFIARERLREHL-CONH2 3194.77 by the R-C20 CH3-(CH2)18-CONHRGWFRAMRSIARFIARERLREHL-CONH2 3222.82 maximum height of the peaks from RP-HPLC, which indicates the hydrophobicity of the peptides.


15.50 22.66 24.10 25.88 27.63 30.66


Fig.1 Characterization of the lipopeptides by RP-HPLC and MALDL-TOF MS. The purification of (A) R-C12, (B) R-C14, (C) R-C16, (D) R-C18, (E) R-C20 were performed using RP-HPLC (column, Vydac, C18, 300Å, 4.6×250 mm). The lipopeptides were separated at a flow rate of 1.0 mL/min under a gradient of 0-80% acetonitrile/0.1% TFA. The elution of lipopeptides were monitored at 280 nm. MALDI-TOF MS of (F) R-C12, (G) R-C14, (H) R-C16, (I) R-C18, and (J) R-C20.


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3.2 Characterization of R-lycosin-I and Lipopeptides Physicochemical properties of lipopeptides such as size and zeta potential, are very important characteristics that can influence the anticancer activities. The sizes and surface potentials of 100 µM of R-lycosin-I and lipopeptides were evaluated by using DLS, the results were listed in Table 2 and Fig. S1. The results confirmed that most of the lipopeptides showed a decrease in average size relative to R-lycosin-I, and positive charges. The polydispersity indexes of lipopeptides were approximately 0.3-0.5 and two mainly size populations were presented (Fig. S1). These results confirmed that at least two conformations were formed in the buffer solution which was consistent with the helicity from CD spectrum (Table S1). Although the hydrophobicity of the lipopeptides was positively correlated with the fatty acid chain length (Table 1), the irregular Zeta potential and average size of lipopeptides indicated that the length of fatty acid chain of lipopeptide was not key factor for the decrease in average size and positive charges, it may be comprehensively caused by their hydrophobic environment, aggregate degree and conformation formed in buffer solution. Therefore, lipopeptides except for R-C20 have a moderate positive charge which will facilitate the electrostatic binding to the cancer cell membrane generally carrying more negatively charged molecules such as phosphatidylserine, negative glycoproteins and glycosaminoglycans,33-35 and have a smaller size which may facilitate peptide uptake by cancer cells according to the size effect19, 36. Table 2 The Zeta potential, average sizes and PDI of R-lycosin-I19 and lipopeptides Named

Zeta potential (mv)

Average sizes (d.nm)

PDI

R-lycosin-I

19.7±7.5

273.5±7.6

0.549±0.017

R-C12

6.6±2.2

138.4±5.0

0.476±0.064

R-C14

15.9±3.2

168.6±27.5

0.380±0.014

R-C16

9.6±0.2

117.6±5.5

0.318±0.006

R-C18

4.0±1.2

152.7±2.0

0.402±0.023

R-C20

33.8±3.9

254.5±26.0

0.391±0.055

Our previous work displayed that R-lycosin-I forms α-helix conformation19. The CD spectrum (Fig. 2B-C) revealed the secondary structures of R-lycosin-I and lipopeptides in phosphate buffer solution and in 50% TFE. Under the condition of 50% TFE, R-lycosin-I and lipopeptides exhibited an α-helical, but the helical content of R-lycosin-I (35.5% helix, table S1) was significantly lower than that of lipopeptides (from 62.99% to 100.00% helix), indicating the hydrophobicity play an important role in maintaining the α-helical structure. The peptide with long acyl groups (R-C18 and R-C20) took an alpha helix conformation in a buffer solution. The CD results coincided well with previous findings that the highly hydrophobic acyl group induces more α-helix structure of the peptides in buffer solution and the moderate chain length of acyl group was important to formation of aggregate, which is facilitate for



incorporation into the cells37. The morphology of lipopeptides was further characterized by negative staining TEM at 2 mM concentration in MQ. As shown in (Fig. 3), all of the peptides displayed homogeneous spherical nanoparticles with a diameter of ~50-140 nm.

Fig.2 The hydrophobicity and secondary structure of R-lycosin-I and lipopeptides. (A) is the peak retention time of the mixture of these six peptides eluting with the same acetonitrile gradient. (B) The secondary structure was measured in PBS (50 mM KH2PO4/K2HPO4 containing 100 mM KCl) at 25oC, and (C) 50% TFE at 25oC.


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Fig. 3 Negative staining TEM images of R-lycosin-I and lipopeptides. The R-lycosin-I and lipopeptides dissolved in MQ at 2 mM concentration were observed by negative staining TEM. Images of (A) R-lycosin-I, (B) R-C12, (C) R-C14, (D) R-C16, (E) R-C18 and (F) R-C20 peptides. Scale bars are 200 nm.

3.3 Cellular bioactivity and toxicity It is reported that fatty acids can increase the stability of peptides38 and increase peptide permeability to inhibit cancer cell proliferation26. Moreover, studies have shown that peptide (PepFect14) can improve cell transfection efficacy depending on complexes hydrophobicity31 via direct covalent conjugation with saturated fatty acid. Based on these finding, the anticancer activity of R-lycosin-I, R-C12, R-C14, R-C16, R-C18 and R-C20 against A549 cells and cytotoxic activity of two non-cancer cells (HUVEC cells and HEK293T cells) were investigated by using the CCK-8 assay. In serum-free conditions (Fig. 4A), compared to R-lycosin-I, the data showed that all lipopeptides had potent anticancer activity against A549 cells. This may be related to its superior physicochemical properties, such as smaller size and higher hydrophobicity. In serum-containing media, generally speaking, the anticancer activity of lipopeptides showed declining tendency, as shown in (Fig. 4B). Interestingly, the shorter and longer lipopeptides (especially R-C20) are less toxic than mid-length carbon chain lipopeptides. Surprisingly, in serum-free and serum conditions, the IC50 values of R-C16 was approximately 3~4-fold lower than R-lycosin-I. However, although the lipopeptides exhibited stronger anticancer activity than R-lycosin-I, they also show toxicity against HUVEC cells and HEK293T cells (Fig. S2). These results indicated that lipopeptides were non-selective for non-cancer cells in in serum and serum-free conditions. Next, flow cytometry was also run to detect peptides-induced A549 cell apoptosis (Fig. 4C). The result of the flow cytometry supported that the result of the cytotoxicity test in serum conditions. The percentage of apoptotic cells increased from 6.79% of control R-lycosin-I to 39.07% in the 6 µM R-C16 treated A549 cells. However, when treated with 6 µM R-lycosin-I, there are not apoptosis in A549 cells. In addition, at a dosage of 50 mg·kg-1, R-C16 did not cause any apparent toxic symptoms (such as Spin, jump up and down, lack of energy and shortness of breath) in mice within 48 h (n = 3, data not show). Therefore, R-C16 was selected for further investigation. Taken together, these results determined that fatty acids modification of R-lycosin-I demonstrated the boosted anticancer activity.



Fig.4 The peptides inhibition A549 cells growth and apoptosis. IC50 represents the concentration of lipopeptides in which cell viability is inhibited by 50%. (A) shows serum-free conditions and (B) represents serum conditions. (C) Flow cytometric analysis of peptides-induced apoptosis under serum conditions. A549 cells were treated with 6 µM peptides. (D) Corresponding bar graph of apoptosis data.

3.4 Hemolytic activity of R-lycosin-I conjugates The specificity of peptides or its analogs against cancer cells can be evaluated by therapeutic index values which are calculated as MHC (Hemolytic Activity)/IC50 (Anticancer activity); thus, the therapeutic index is proportional to the anticancer specificity39, 40. Furthermore, the hemolytic activity as lysis of blood cells is used to assess membrane activity which will be the first sign of toxicity of the lipopeptide complex after systemic administration31. As show in Fig. 5, the MHC values of R-lycosin-I, R-C12, R-C14, R-C16, R-C18 and R-C20 are around 40, 17, 22, 38, more than 40 µM, 42, respectively. The results showed both R-lycosin-I and five lipopeptides displayed dose-dependent hemolytic effects, R-C12 and R-C14 showed stronger hemolytic activity than R-lycosin-I, R-C16 and R-C20 showed equivalent hemolytic activity to R-lycosin-I. Interesting, R-C18 only induced about 25% hemolysis at 40 µM. The hemolytic effects of the lipopeptides roughly reflected the membrane activity which could be confirmed by the IC50 the value of lipopeptides in serum. In the other respect,


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the therapeutic index of R-C16 which is equal to about 8 was higher than that of other lipopeptides and R-lycosin-I indicating that the R-C16 has a greater specificity against cancer cells than that of other lipopeptide and R-lycosin-I.

Fig.5 Hemolytic activity of lipopeptides against human erythrocytes. R-lycosin-I and lipopeptides concentrations ranged from 0.63 to 40 µΜ. The absorbance of the supernatant was detected by microplate reader at 450 nm.

3.5 Serum stability of lipopeptides The serum stability of R-lycosin-I and lipopeptides were performed in fetal bull serum (FBS) to confirm whether the serum would affect the inhibitory activity of lipopeptides on cell growth. R-lycosin-I and lipopeptides were inculcated with 10% FBS or PBS for different times. The serum stability of R-lycosin-I was much lower than that of lipopeptides (Fig. 6). Within 24 h pretreatment (Fig. 6A), the cell viability of all lipopeptides on A549 cells was approximately 20%. The result was similar to the cell viability of untreated lipopeptides. Although the inhibitory activity of lipopeptides decreased slightly, it remained retained about 70% at 48 h (Fig. 6B). In contrast, the cell viability of R-lycosin-I on A549 cells was approximately 60% within 24 h of pretreatment and was approximately 90% after 48 h. Taken together, these results demonstrated that fatty acids modification conferred R-lycosin-I may have a greater serum stability.



Fig.6 The serum stability of lipopeptides. (A) The lipopeptides were incubated PBS containing 10% FBS for (A) 24 h and (B) 48 h at 37°C, respectively. At the specified time points, the pre-incubated R-lycosin-I and lipopeptides were added to corresponding wells and incubated in 24 h. The cell viability was detected by microplate reader using CCK-8 assay.

3.6 Membrane-lytic activity of R-lycosin-I and R-C16 It has been reported that several cationic antibacterial peptides also have anticancer activity9, 41-45. In addition, cationic antibacterial peptides not only bind to the surface of cancer cells but can also be inserted into the lipid portion of the bilayer, which ultimately destroys the integrity of the membrane and leads to cell death15. The extent of membrane permeability can be reflected by LDH release assays29. To determine the membrane-lytic activity of R-C16, the LDH leakage assay was performed after the A549 cells were treated with the R-lycosin-I or R-C16 for 24 h in serum. As shown in (Fig. 7D), the A549 cells was treated with R-C16 and R-lycosin-I at a series of concentrations as same as in the CCK-8 assay. However, the result form LDH leakage assay was different from that in the CCK-8 assay, indicating that R-C16 exhibited effective anticancer activity not entirely by destroying cell membrane. It has been previous reported that apoptosis is the atrophy of cells that maintains the integrity of the cell membrane and the production of apoptotic bodies46. In addition, the cell membrane integrity was directly visualized by scanning electron microscopy after the cells treatment with R-lycosin-I or R-C16. As shown in (Fig. 7A-C), untreated A549 cells had intact membrane with smooth surface. The cells treaded with R-lycosin-I or R-C16 appeared to have sponge-like morphology and porous membrane surface, indicating membrane disruption. Compared with R-lycosin-I, R-C16 showed cell atrophy and produced some apoptotic bodies. These data indicated that R-C16 could either induce the disruption of cell membrane or promote apoptosis to exert anticancer activity.


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Fig.7 The cell membrane integrity was observed using SEM and LDH leakage in A549 cells after treatment with R-lycosin-I and R-C16. (A) The morphology of the intact cell membrane, and after treatment of cells with (B) R-lycosin-I and (C) R-C16 at 15 µM concentration. (D) R-C16and R-lycosin-I effect on cell membrane leakage. Apoptotic bodies (right arrow) and pore formation (left arrow). Scale bar are 10 µm or 20 µm for SEM.

4. Conclusion In order to enhance the anticancer activity of R-lycosin-I, five lipopeptides were successfully synthesized by fatty acids modification method. Compared to R-lycosin-I, in chemical and physical respect, five lipopeptides show higher hydrophobicity and smaller hydrodynamic size. In biological activity respect, all lipopeptides have similar potent anticancer activity against cancer cells. However, all lipopeptides also have toxicity in non-cancer cells, which indicated those lipopeptide had non-selectivity for noncancer cells. Unexpectedly, at a dosage of 50 mg·kg-1, R-C16 did not cause any deadly or obvious poisoned symptoms in mice within 48 h. Moreover, the LDH assay and SEM demonstrated that lipopeptides not only can disrupt the cell membrane, and also may promote apoptosis to exert anticancer activity. In summary, although the exact mechanism of action of lipopeptides requires further investigation, our work has confirmed that the fatty acid conjugate is effective to enhance the anticancer activity of



R-lycosin-I and lay a ground for anticancer peptide modification.

Acknowledgments This research was financially supported by the National Nature Sciences Foundation of China (General Program: 2127206, 31670783), the Science Fund for Distinguished Young Scholars of Hunan Province (no. 14JJ1018), the Opening Fund of The National &Local Joint Engineering Laboratory of Animal Peptide Drug Development (Hunan Normal University), National Development and Reform Commission (2017KF003), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (no. 20134486).

Abbreviations LDH, lactate dehydrogenase; SEM, scanning electron microscopy; ACPs, Anticancer peptides; CPP, cell-penetrating peptide; A549, Human lung carcinoma cells; HUVEC, Human Umbilical Vein Endothelial Cells; DMEM, Dulbecco’s modified Eagles medium; FBS, fetal bovine serum; SPPS, solid-phase peptide synthesis; PI, propidium iodide; HEK293T, Human embryonic kidney 293T cells; MHC, Measurement of Hemolytic Activity.

Supporting Information

Size and zeta potential distribution graphs; biophysical data of R-lycosin-I conjugates; and cytotoxicity assay data. Conflicts of Interest The authors report no conflict of interest.

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Fig.1 Characterization of the lipopeptides by RP-HPLC and MALDL-TOF MS. 150x205mm (254 x 254 DPI)


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Fig.2 The secondary structure of the peptides and characterization of the hydrophobicity of the peptides by RP-HPLC. 150x123mm (254 x 254 DPI)



Fig. 3 Negative staining TEM images of R-lycosin-I and lipopeptides. 105x70mm (203 x 203 DPI)


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Fig.4 The peptides inhibition A549 cells growth and apoptosis. 152x125mm (220 x 220 DPI)



Fig.5 Hemolytic activity of lipopeptides against human erythrocytes. 82x65mm (220 x 220 DPI)


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Fig.6 The serum stability of lipopeptides. 152x69mm (220 x 220 DPI)



Fig.7 The cell membrane integrity was observed using SEM and LDH leakage in A549 cells after treatment with R-lycosin-I and R-C16. 153x125mm (178 x 178 DPI)


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Graphical Abstract 104x42mm (300 x 300 DPI)


The roles of fatty acid modification in the activity of anticancer peptide

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