Research Article Received: 25 February 2014
Revised: 23 May 2014
Accepted: 28 May 2014
Published online in Wiley Online Library: 23 June 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2667
Functional properties of a novel hybrid antimicrobial peptide NS: potent antitumor activity and efficient plasmid delivery Yun Zhang,a,b† Jingjing Song,a† Wei Zhang,a Ranran Liang,a Yinyun Ma,b Li Zhang,b Xiaojin Wei,b Jingman Nia,b* and Rui Wanga* Antimicrobial peptides have been widely recognized as potential candidates for treating tumor, especially for defending against multidrug-resistant cells. Previously, based on the structure of substance P, we have designed a novel class of hybrid antimicrobial peptide NS, which possesses potent antimicrobial activity against a broad spectrum of bacterial pathogens. In this study, we evaluated its cytotoxicity to tumor cells and studied the possible mechanism of action. We showed that NS could efficiently kill tumor cells by rapidly disrupting the tumor cell membrane and inhibiting the DNA synthesis. In addition, we also found that NS could efficiently deliver plasmids into cells and exhibit high transfection efficiency after the introduction of a stearyl moiety to its N-terminus, like many reported cell-penetrating peptides. Taken together, this study revealed the potential multiple functions of NS, providing fundamental support for further therapeutic application as potential antitumor agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: NS; antitumor activity; membrane disruption; DNA interaction; plasmid transfection
Introduction
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* Correspondence to: Rui Wang and Jingman Ni, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences and School of Pharmacy, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected]; [email protected] †
These authors contributed equally to this work
a Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China b School of Pharmacy, Lanzhou University, Lanzhou 730000, China Abbreviations: AMPs, antimicrobial peptides; SP, substance P; CPPs, cell-penetrating peptides; NLS, nuclear location sequence; NBS, neonatal bovine serum; FBS, fetal bovine serum; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; PI, propidium iodide; BSA, bovine serum albumin; EtBr, Ethidium bromide; GFP, green fluorescent protein.
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Although the cancer therapy has experienced tremendous progress, the constant use of conventional chemotherapeutical agents easily results in the frequent appearance of multidrug-resistant tumors, threatening human health and quality of life. Based on this, many researches concentrated on the development of novel antitumor drugs with a different mechanism of action that disfavors drug resistance. Antimicrobial peptides (AMPs) have been considered as a new class of alternative chemotherapeutics that overcome limitations of current anticancer drugs because of high antitumor activity, tumor selectivity, and low susceptibility to common resistant mechanism [1,2]. Antimicrobial peptides were widespread in most of species, including microorganisms, plants, insects, amphibians, and mammals [3,4]. They not only exhibit broad antimicrobial activity against bacteria, fungi and virus [5–7] but also play an essential role in the innate immune response [8,9]. Recently, AMPs also attract more and more attention in antitumor treatment. The cationic and amphiphilic properties drive the rapid binding of AMPs to tumor cell membrane subsequently leading to the membrane disruption and the leakage of cytoplasmic components via a nonreceptor-mediated pathway [1,2,10,11]. In addition, some AMPs have been shown to translocate into tumor cells and subsequently interact with the cellular targets, resulting in cell death [12,13]. Furthermore, the structural similarities between AMPs and cell-penetrating peptide (CPPs) provide a possibility that AMPs may act like CPPs [14,15]. Thus, these desirable features prompt us to exploit the multiple functions of potential AMPs in tumor therapy, leading to further advance in the development of more effective treatment strategies.
Substance P (SP) is an 11-residue neuropeptide member of the tachykinin family, which plays important roles in most of biological activities, including cancer, wound healing, exocrine gland secretion, neuroendocrine, and immune regulation [16]. Notably, SP has also been reported to exhibit direct antimicrobial activity [17,18]. To improve its potential as novel therapeutic agent, we designed a series of new hybrid antimicrobial peptide analogs, being made up of two fragments: One is derived from nuclear location sequence (PKKKRKV), providing the cationic cluster for analogs; and the other is derived from SP, providing the hydrophobic cluster for analogs. NS, one of the analogs, showed significant antimicrobial activities (data were not shown). Because many antimicrobial peptides are reported to exhibit antitumor activity [1,19], we are interested if NS can remarkably inhibit the proliferation of tumor cells. Therefore, we studied the antitumor activity of NS as well as its action mechanism in this work.
ZHANG ET AL. the purified plasmids were dissolved in distilled water and stored at 20 °C.
Materials and Methods Materials Rink amide MBHA resin, protected amino acids, and other reagents and solvents for the peptide synthesis were purchased from GL Biochem Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and ethidium bromide (EtBr) were obtained from Sigma (St. Louis, MO, USA). Caspase inhibitor Z-VAD-FMK was purchased from Beyotime Biotechnology (Jiangsu, China). Peptide Synthesis NS and its related peptides shown in Table 1 were synthesized on an MBHA resin (0.54 mmol/g) using the standard Fmoc-based solidphase method, as described previously [20,21]. Stearic acid was coupled as Fmoc amino acids; the fluorescein moiety (FITC) was conjugated to the N-terminus via an aminohexanoic acid spacer by treating a resin bound peptide (0.1 mmol) with FITC (0.1 mmol) and diisopropyl ethyl amine (0.5 mmol) in N,N-dimethylformamide (DMF) for 12 h. The final cleavage was performed using a mixture of trifluoroacetic acid, thioanisole, water, phenol, and 1,2ethanedithiol (82, 5, 5, 5, 5, 2.5, v/v) for 3 h at room temperature. All crude peptides were purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) (Waters) using μ-BONDAPAKTM C18 column (19 mm × 300 mm) with gradient elution of 10–90% acetonitrile/water in the presence of 0.1% trifluoroacetic acid. The final purity of the peptides (>95%) was assessed by analytical RP-HPLC with sunfireTM column (3.9 mm × 150 mm, Waters). The molecular mass of synthetic peptides was conformed by electrospray ionization mass spectrometry. The sequence and characterization of peptides are shown in Table 1. Cell Cultures and Amplification of Plasmid DNA HeLa cells were maintained in RPMI1640 medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% heat-inactivated neonatal bovine serum (Sijiqing Biotech, Hangzhou, China). U251-MG, MDA-MB-231, and Cos-7 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). All cell lines were cultured in a 5% CO2 humidified atmosphere at 37 °C. pGL3 or pEGFP-C1 plasmid (4.7 kb), expressing luciferase or enhanced green fluorescent protein (EGFP) respectively, was transformed in Escherichia coli DH5α and amplified in LB medium at 180 rpm at 37 °C overnight. The plasmids were purified according to an EndoFree Plasmid Kit (Tiangen, Beijing, China). Thereafter,
Cytotoxicity Assays The antiproliferative effects of all peptides were evaluated using MTT assay. Cells (5 × 103 cells/well) were seeded in 96-well plates 24 h before treatment. After incubation with various concentrations of peptide for indicated times, 20 μl of 5 mg/ml MTT solution was added to each well and incubated for another 4 h at 37 °C. The absorbance was measured using microplate reader (Model 680, Bio-Rad, Hercules, CA, USA) at 570 nm. The effect of caspase inhibitor Z-VAD-FMK on the cytotoxicity of NS was also determined by MTT assay, as described previously [22]. HeLa cells (5 × 103 cells/well) were cultured in 96-well plates. After 24 h, cells were preincubated for 1 h with or without 20 μM Z-VAD-FMK. Thereafter, the cells were treated with NS at different concentrations for 24 h. Finally, cell viability was examined as described previously. Lactate Dehydrogenase (LDH) Leakage Assay Membrane integrity was assessed using the CytoTox-ONETM assay (Promega, Madison, WI, USA). Cells (5 × 103 cells/well) were seeded in 96-well plate 24 h before experiment. After 1 h of incubation with different concentrations of peptide in serum-free medium, 40 μl of medium was transferred to 96-well plate and incubated with 40 μl of reaction mixture for 15 min followed by addition of 20 μl stop solution. Fluorescence was detected at 560/590 nm. Untreated cells were defined as no leakage, and 100% leakage was defined as total LDH release by lysing cells in 0.2% Triton X-100. PI Uptake Assay To observe the cell membrane disruption after NS treatment, HeLa cells (2 × 105 cells/well) were seeded in 24-well plates and attached overnight at 37 °C. After incubation with NS at different concentrations for 1 h, the cells were washed with cold phosphate buffered saline (PBS), and then, PI (50 μg/ml) was added to each sample for 10 min staining. The positive rates of PI in cells after NS treatment were analyzed using FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). A total of 104 events were recorded for each sample, and the ratio of PI positive cells to the total cell population was defined as cell membrane disruption rate. Scanning Electron Microscopy and Transmission Electron Microscopy The morphological changes of HeLa cells after peptide treatment were observed by scanning electron microscopy (SEM) and
Table 1. Amino acid sequences, calculated and observed molecular masses, net charge, and RP-HPLC retention times of NS, stearyl-NS, and FITClabeled NS Molecular mass Peptides NS Stearyl-NS FITC-NS
Amino acid sequences PKKKRKVWKLLQQFFGLM-NH2 Stearyl-PKKKRKVWKLLQQFFGLM-NH2 FITC-AHX-PKKKRKVWKLLQQFFGLM-NH2c
Net charge
RP-HPLC retention times (min)b
+7 +6 +6
18.2 28.6 20.6
a
Calculated
Measured
2274.4 2540.9 2275.8
2274.3 2540.6 2776.5
a
Molecular masses were determined by electrospray ionization mass spectrometry. TM RP-HPLC retention time was measured by analytical HPLC with sunfire column (3.9 mm × 150 mm, Waters). c The fluorescein moiety (FITC) was attached to the N-terminus via an aminohexanoic acid (AHX) spacer. b
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NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY transmission electron microscopy (TEM). HeLa cells were seeded onto coverslips under the bottom of the six-well microplate for 24 h. The cells were treated with NS at a concentration of 10 μM for 1 h. Then, samples were prepared according to a published report [23] and finally observed by using SEM (JSM-6380LV, JEOL, Tokyo, Japan) and TEM (JSM-1230, JEOL, Tokyo, Japan). Cell Cycle Analysis The cellular DNA content and cell cycle perturbation was analyzed by PI DNA staining and monitoring its fluorescence via flow cytometry. Cell cycle analysis was performed using the previous method with a minor modification [24]. HeLa cells were seeded in six-well plates for 24 h at a density of 4 × 105 cells/well, followed by addition of NS with various concentrations. After 24-h treatment, the cells were harvested, washed twice with PBS, and fixed with 70% cold ethanol at 4 °C overnight. Before analysis, the fixed cells were further washed twice with cold PBS containing 1% bovine serum albumin and then incubated with PI solution (50 μg/ml PI, 0.1% sodium citrate, 0.1 mg/ml ribonuclease A, and 0.1% Triton X-100) for 30 min at 4 °C. Finally, cells were subjected to DNA content analysis by monitoring PI fluorescence with an FACScalibur flow cytometer. Data from at least 20 000 cells were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) to calculate cell cycle distributions. Cellular Uptake Assays HeLa cells (2 × 105 cells/well) were seeded in 24-well microplates 24 h before treatment. After 1 h of incubation with serum-free medium containing 2-μM FITC-labeled NS, the cells were harvested and washed twice with cold PBS and subsequently resuspended in 500 μl of PBS. Finally, the fluorescent intensity of 10 000 cells was analyzed using an FACScalibur flow cytometer with the 488-nm laser excitation. To gain a direct insight into the distribution of FITC-labeled peptide in HeLa cells, cells were plated in a glass-bottom culture dish for 24 h and then incubated with FITC-labeled NS using conditions and concentrations described earlier. After 1-h incubation at 37 °C, the cells were washed with cold PBS three times and imaged using an inverted Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Complexes Formation The complexes at various N/P ratios (peptides amino groups: DNA phosphate) were prepared by mixing 0.5 μg of plasmids with appropriate concentrations of peptides in 50 μl medium (one tenth of the final treatment volume). According to the positive charges of the peptide and negative charges of the plasmid, N/P ratios were calculated theoretically. For instance, final concentration of stearyl-NS was 0.5 μM at N/P ratio of 1. After incubation for 30 min at 37 °C, the formed complexes were diluted to a final volume of 500 μl with medium for subsequent use. Gel Retardation Assay
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Particle Size and ζ -Potential Measurements The peptide/pGL3 plasmid complexes were prepared at N/P ratios ranging from 1 to 4. The complexes were diluted in water to 1-ml volume, and then the size and ζ -potential were measured by Nano-ZS ZEN3600 (Malvern Instruments, Malvern, UK) at 25 °C, respectively. Vitro Transfection Assays To evaluate the ability of peptides to deliver plasmid DNA to cells, Cos-7 cells were seeded in a glass-bottom culture dish and cultured for 24 h. Cy5-labeled pGL3 plasmid was prepared using the method described by a Label IT Tracker Kit (Mirus, Madison, WI, USA). The peptide/Cy5-labeled pGL3 plasmid complexes were added into the dish at N/P ratio of 3. After incubation for 4 h, the cells were washed three times with PBS. Then, the fluorescence was observed with a confocal laser scanning microscope. To further determine the transfection efficiency of peptide/plasmid complexes, 1 × 105 Cos-7 cells were seeded in 24-well plate 24 h before experiment. Cells were treated with peptide/pGL3 plasmid complexes at different N/P ratios in serum-free DMEM for 4 h followed by addition of fresh DMEM containing 10% FBS and further incubated for 20 h. Thereafter, cells were washed and lysed using 100 μl reporter lysis buffer (Pierce, Rockford, IL, USA) for 15 min at 37 °C. Luciferase activity was detected using luciferase detection assay (Promega, Nacka, Sweden) and normalized to protein content. Protein concentrations in the cell lysate were examined using a BCA protein assay kit (Pierce, Rockford, IL, USA). EGFP Expression Assay To analyze the EGFP gene expression, Cos-7 cells were seeded in a glass-bottom culture dish 24 h before treatment; 0.5 μg of pEGFP plasmid was mixed with NS or stearyl-NS at N/P ratio of 3 as described in the Complexes Formation Section. Cells were treated with peptide/EGFP plasmid complexes at N/P ratio of 3 for 4 h in serum-free DMEM followed by addition of fresh DMEM containing 10% FBS and further incubated for 20 h. Thereafter, images were captured using an inverted Zeiss LSM 710 confocal microscope.
Results Cytotoxity of Peptide To evaluate the antitumor activity of NS, we initially determined its ability to inhibit cell proliferation. The cervical carcinoma cell line (HeLa), glioblastoma cell (U251-MG), and breast cancer cell line (MDA-MB-231) were chosen and treated with various concentrations of the tested peptide for 24 h; subsequently, cell viability was determined by the MTT assay (Figure 1A). The results showed that NS could kill tumor cells in a dose-dependent manner. Viability of the cells was reduced more than 90% after 24-h treatment at a concentration of 20 μM, exhibiting potent antitumor activity. In order to investigate the characteristics of the NS action, we examined its cytotoxicity to HeLa cells for the indicated term of incubation. As shown in Figure 1B, the proliferation of cells was inhibited in a time-dependent manner after NS
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DNA condensation was evaluated by the gel retardation assay [15]. Briefly, the peptide/pGL3 plasmid complexes at various N/P ratios were prepared by mixing appropriate volumes of peptide solution with 0.5 μl of pGL3 plasmid (200 ng/μl in water). The complexes were diluted to a total volume of 8 μl with NaCl solution (150 mM) and then incubated for 30 min
at 37 °C. Subsequently, the complexes were loaded on the 0.8% (w/v) agarose gel and imaged by staining the gel with EtBr (0.5 μg/ml).
ZHANG ET AL.
Figure 1. The cytotoxicity of NS to tumor cells. (A) The inhibition effect of NS on different tumor cells. Cells were treated with various concentrations of NS for 24 h and then detected by MTT assay. (B) Cell survival rate of HeLa cells treated with various concentrations of NS for 1, 4, 8, and 24 h. (C) The effect of Z-VAD-FMK on cellular viability during NS treatment. HeLa cells were preincubated with or without 20 μM Z-VAD-FMK for 1 h and further incubated with NS (1, 2, and 5 μM) for 24 h. Cell viability was determined using the MTT method. Data are mean ± SEM of three repeated experiments.
treatment; however, we found that pan-caspase inhibitor Z-VADFMK did not recover the viability of cells by treating HeLa cells with Z-VAD-FMK for 1 h before NS treatment (Figure 1C), demonstrating that NS induced caspase-independent cell death. Notably, rapid cell death was detected after 1 h of treatment with NS at high concentration, implying NS might exhibit cytotoxicity by disrupting the tumor cell membrane, just like many naturally occurring antimicrobial peptides [1,19]. Effect of NS on the Cell Membrane In order to verify the membrane disrupting activity of NS, we determined the effect of NS on the integrity of cell membrane. LDH leakage assay was employed to measure acute membrane disturbance. LDH release was detected in HeLa cells after NS treatment
and revealed the membrane-lytic activity of NS (Figure 2A). In addition, the PI uptake assay also was used to study the change of cell membrane integrity after peptide treatment. PI can permeate unhealthy/damaged membranes [2], so positive PI fluorescence indicates damaged cell membranes. Figure 2B showed the uptake of PI in HeLa cells after 1 h of treatment with NS at indicated concentration (5, 10, and 20 μM). As expected, the positive rates of PI in HeLa cells after NS treatment could increase with an increase in the peptide concentration, whereas the uptake of PI in untreated cells was negligible, supporting the results indicated by the LDH leakage assay. These results suggested that NS could disrupt and damage the cell membrane, resulting in death of the target cells. To better understand the death mode in HeLa cells treated with NS, we performed morphological investigation by TEM and
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Figure 2. LDH leakage (A) and PI uptake (B) in HeLa cells treated with various concentrations of NS for 1 h. Data are mean ± SEM of three repeated experiments.
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NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY SEM with the results presented in Figure 3. The untreated control cells presented plenty of microvilli and adherent smooth surface, whereas the membranes of HeLa cells treated with NS were shriveled, invaginated, disrupted, and characterized with significant loss of microvilli and pore formation. Furthermore, TEM was used to examine the ultrastructural changes of HeLa cells after NS treatment. Normal HeLa cells displayed microvilli protruding from the entire surface and a smoothly outlined nucleus with chromatin in the form of heterochromatin. In contrast, following the NS treatment, HeLa cells showed loss of plasma membrane integrity and electron-lucent cytoplasm with intact nuclear membrane, which were consistent with the characteristics of necrosis. According to the results derived from the LDH leakage assay, PI uptake assay, SEM, and TEM, we concluded that NS could disrupt cell membrane and lead to cell necrosis. Effect of NS on the Cell Cycle Distribution Although NS killed tumor cells by rapid membrane disruption, we could not conclude that the membrane was the only target against which NS exerted its antitumor activity. Therefore, we assessed the impact of NS on cell cycle distribution to attempt to look for some intracellular target. For this purpose, we performed flow cytometric DNA cell cycle analysis on NS-treated HeLa cells (Figure 4). This method relies on fluorescent probes that bind DNA, thus enabling the entire DNA content per nucleus to be monitored with precision [25]. Relevant differences in cell cycle distribution were observed in cells treated with 5-μM NS. The 39.5% G0/G1 value in the NS-treated cells was significantly reduced compared with 61.4% for the untreated cells. The percentage of cells in S phase for NS-treated cells was 52.8%, which was higher when compared with the untreated controls, 26.2%. Differences in the frequency of cells in the G2/M phase were imperceptible. Our results indicated that NS could induce S-phase arrest in HeLa cells. Deriving from the association of S-phase arrest and DNA synthesis inhibition [26–28], we conclude that NS could perturb DNA replication and inhibit its synthesis, consequently arresting the growth of HeLa cells in the S phase.
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Cellular Uptake of Peptide The cell penetration ability of NS was assessed by FACS and confocal microscopy. Figure 5A revealed that the fluorescence intensity of NS in HeLa cells at 2 μM was significantly high, supporting its strong cellular uptake. Furthermore, to gain a direct insight into the internalization of NS, the images of cells treated with 2μM FITC-labeled NS for 1 h were taken using a confocal laser scanning microscopy (Figure 5B). It was evident that distribution of potent fluorescence signal was almost in cellular nuclei, indicating that NS can translocate into cell and accumulate in the nuclei. Plasmid Transfection Ability of NS Based on the cell penetration ability of NS, we presumed that NS may act as an efficient vector for gene transfection like many reported CPPs [29,30]. Deriving from the success of stearylation of CPPs for plasmid delivery [20,31–33], we conjugated stearic acid to the N-terminus of NS and evaluated the efficiencies of NS and stearyl-NS for plasmid transfection.
Figure 5. Cellular uptake of NS in HeLa cells. Cells were incubated with 2-μM FITC-labeled NS for 1 h in serum-free medium. (A) Flow cytometry histogram of peptide internalized into cells. (B) Confocal images of cellular uptake of peptide. DIC represents the differential interference contrast images of experimental groups taken by confocal microscopy under natural light.
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Figure 3. Morphological changes of HeLa cells after NS treatment. Cells were incubated with NS (10 μM) for 1 h and then observed using SEM and TEM.
Figure 4. Cell cycle distribution of HeLa cells after NS treatment for 24 h. All the results were obtained from three independent determinations.
ZHANG ET AL. On the basis of noncovalent nanoparticle formation strategy [34], positively charged peptides and negatively charged pDNA are mixed together to form steady complexes. The nanoparticle formation efficiency was assessed by gel retardation assay. As shown in Figure 6A, the pDNA migration was completely retarded by both NS and stearyl-NS at N/P ratio of 2, indicating that they could efficiently condense pDNA into the nanoparticles. Stearyl-NS had an enhanced DNA condensable ability as more pDNA could not migrate into the gel at N/P ratio of 1. The ζ -potential and particle size stearyl-NS/plasmid complex was characterized as a function of increasing N/P ratios. Figure 6B showed that the ζ -potential of complex was increased with increasing N/P ratios. Additionally, the particle size of stearylNS/plasmid complex was in range of 177–278 nm (Figure 6C). After the characterization of complex, we evaluated the ability of complex to transfect plasmids into cells. We first assessed plasmid delivery ability of peptides by confocal microscopy. On the basis of the confocal images (Figure 7A), Cy5-labeled plasmid signals were detected in almost all cells after incubation with stearyl-NS/Cy5-labeled plasmid complex, whereas NS could deliver little Cy5-labeled plasmid complex into cells, indicating that NS could exhibit enhanced plasmid delivery ability when associated with stearic acid. Furthermore, to further evaluate the transfection efficiencies of NS and stearyl-NS, Cos-7 cells were transfected with complexes of luciferase-encoding pGL3 plasmid and different peptides. As seen in Figure 7B, NS showed slightly higher transfection efficiency than pGL3 plasmid alone. Intriguingly, luciferase expression levels of stearyl-NS were significantly increased compared with unmodified peptide at various N/P ratios, displaying remarkably enhanced transfection efficiency. Additionally, in accordance with the result of luciferase expression, stearyl-NS/EGFP plasmid complex at N/P ratio of 3 mediated significant EGFP expression, whereas no EGFP expression in Cos-7 cells was observed for NS/plasmid complex (Figure 7C). These results suggested that NS could efficiently mediate plasmid delivery after the N-terminal stearylation.
Discussion Although there is established effectiveness in a wide range of tumors, the currently used antitumor drugs cause several side effects and induce drug resistance, resulting in narrow therapeutic indexes. Nowadays, antimicrobial peptides can provide a potential in cancer therapy as a result of more effective and specific treatment than standard therapies [1,2]. Thus, researchers pay more attention to the development of novel antimicrobial peptide for better tumor treatment. In this study, we found that the novel hybrid antimicrobial peptide NS exhibited a significant cytotoxic effect against tumor cells via membrane disruption and inhibition of DNA synthesis. In addition, NS was found to show cell-penetrating ability and efficiently deliver plasmid DNA into cells when combined with stearyl moiety. Recently, membrane binding/translocation have been reported to play a crucial role in the cytotoxicity of AMPs to tumor cell [35,36], as it facilitates the membrane permeabilization and cellular accumulation of AMPs, subsequently producing morphological and biochemical hallmarks of cell death. In this study, we clearly showed that NS displayed a rapid cell killing activity. The remarkable LDH release and PI uptake seen when treating the cells with NS indicated that NS triggered acute membrane disruption, just like many antimicrobial peptides [1,19]. This conclusion is consistent with the dramatic morphological changes observed by SEM and TEM. Thus, our results revealed that necrotic mechanism was directly involved in the antitumor action of NS. As we know, resistance is one major cause resulting in the failure of tumor therapy. It is often associated with the transporters that efficiently pump out the chemical drugs from the cells [37]. Notably, rapid cell death via membrane disruption is an irreversible receptor-independent process. Therefore, it is difficult for tumor cell to develop resistance to NS. Furthermore, the membrane-lytic activity endows NS a remarkable superiority that it may exhibit better cytotoxicity to multidrug-resistant tumor cells like many antimicrobial peptides [2], complementing common drawbacks of classical chemotherapy.
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Figure 6. Characterization of stearyl-NS/plasmid complex. (A) Ability of peptides to form complexes with pDNA at different N/P ratios (0.25–3) was evaluated by gel retardation assay. (B) ζ -potential of stearyl-NS/plasmid complex at N/P ratios. (C) Particle size of stearyl-NS/plasmid complex at N/P ratios.
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J. Pept. Sci. 2014; 20: 785–793
NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY
Figure 7. Effect of stearyl-NS on plasmid transfection. (A) Cellular uptake of peptide/Cy5-labeled plasmid complex at N/P ratio of 3 in Cos-7 cells after 4h incubation. (B) Luciferase expression mediated by stearyl-NS/pGL-3 plasmid complex at N/P ratios. (C) EGFP expression mediated by stearyl-NS/EGFP plasmid complex at N/P ratio of 3. All the results were obtained from three independent determinations. DIC represents the differential interference contrast images of experimental groups taken by confocal microscopy under natural light.
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endosomal entrapment of both CPP and respective cargo, limiting the relative efficient of unmodified CPPs in promoting plasmid delivery [29,30]. Stearylation of CPPs has been shown to be a successful strategy for efficient plasmid delivery, as a lone modification on polyarginine [31,32], TP10 [43], or if incorporated into multifunctional envelope-type nano device [44]. The significant role played by stearylated CPPs in plasmid transfection has consequently resulted in growing attention to design of new modified CPPs. Recently, some groups have shown that antimicrobial peptides can translocate into cells like CPPs [15,45,46]. The possibility to utilize AMPs for cellular plasmid transduction has gained wide interest. We have demonstrated previously that N-terminal stearylation of [D]-K6L9, a membrane-disruptive antimicrobial peptide, was successful in promoting efficient plasmid delivery [15]. In the current work, we studied whether NS could also exhibit delivery potential for the cellular transduction of plasmid. In concordance with the previous reports on stearylated CPPsmediated plasmid delivery [31–33], the introduction of N-terminal stearyl moiety has a substantial impact on NS peptide’s ability to deliver plasmids into cells. The level of transfection was remarkably high when using stearyl-NS, whereas unmodified NS had poor effect on the transfections of plasmids. The high transfection efficiency of the stearyl-NS/DNA complex could attribute to the increased cellular uptake. The hydrophobic stearyl moiety renders the complex more lipophilic, presumably facilitating its penetration through the cell membrane. In addition, the peptides consisting of NLS are well adapted to nuclear localization and facilitate efficient plasmid transfection [33]; therefore, the NLS linked to the N-terminus of NS may play an important role in the transfection efficiency of the stearyl-NS. Furthermore, we believe that
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Moreover, we found that NS could penetrate into cells and accumulate inside cellular nuclei, so the antitumor mechanism of NS maybe involved in the intracellular action. In this study, we observed that the caspase inhibitor Z-VAD-FMK did not prevent NS-induced cell death, demonstrating NS exerted its antitumor activity through caspase-independent pathway. Some reports have shown that antimicrobial peptides can target the cellular DNA and interrupt DNA functions (such as the DNA synthesis/replication), thereby contributing to the cell death [38,39]. Nuclear localization signal (NLS) has been proven to facilitate nuclear delivery [33,40]; the introduction of NLS indeed resulted in the nuclear accumulation of NS, implying that the observed antitumor effect is likely associated with DNA interaction of NS after internalization. A possible contribution of DNA interaction in the efficient antiproliferation effect of NS was evaluation by cell cycle analysis. The significant increase in cell number of S phase when treating cells with NS indicated that NS caused apparent S arrest in tumor cells. Earlier studies have demonstrated that the S-phase arrest caused in tumor cells correlated with efficient inhibition of DNA synthesis [27,28]. Therefore, another plausible explanation for the remarkably potent antitumor activity may be that the DNA binding affinity of NS facilitates its perturbation in DNA replication, consequently leading to cell cycle arrest in S phase and finally growth inhibition. Naked therapeutic nucleic acid-based molecules used in gene expression modulation have a strong negative charge and high molecular weight, preventing them to enter cells freely. CPPs are one group of such nonviral vectors, which are increasingly utilized for gene delivery because of their high internalization efficiency, low cytotoxicity, and flexible structural design [41,42]. The main barrier when utilizing CPPs for delivery is the
ZHANG ET AL. endosomal escape is probably the critical determinant for efficient plasmid transfection obtained with the stearyl-NS. The N-terminal stearylation [31,32,43,44] and membrane-lytic activity [15] of peptides have a substantial effect on improving endosomal escape of the nanoparticles. With the aid of both the stearyl moiety and the membrane-lytic ability, the stearyl-NS/DNA complexes would escape from endosomal compartments, thus facilitating the efficient transfection. The remarkable transfection efficiency observed with stearyl-NS illustrates that stearylated antimicrobial peptides would represent another alternative for the development of an efficient transfection system and can be also used as an efficient vector for tumor gene therapy. In conclusion, our results demonstrated that the novel antimicrobial peptide NS could kill tumor cells by rapidly disrupting cell membrane and arresting DNA synthesis, which are not affected by the known resistance mechanism. Gratifyingly, NS modified with stearyl moiety could result in the efficient plasmid delivery, implying that it might act as a potential delivery vector for further tumor gene therapy. Taken together, the multiple functions of NS provide direct evidence that it is a highly interesting candidate for future tumor treatment. Acknowledgements We are grateful for the grants from the National Natural Science Foundation of China (Nos. 81273440, 91213302, 20932003, and 21272102) and the Key National S&T Program ‘Major New Drug Development’ of the Ministry of Science and Technology (2012ZX09504-001-003).
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NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY 36 Liu S, Yang H, Wan L, Cai HW, Li SF, Li YP, Cheng JQ, Lu XF. Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery. Acta Pharmacol. Sin. 2011; 32: 79–88. 37 Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2002; 2: 48–58. 38 Boman HG, Agerberth B, Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect. Immun. 1993; 61: 2978–2984. 39 Park CB, Kim HS, Kim SC. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem. Biophys. Res. Commun. 1998; 244: 253–257. 40 Pouton CW, Wagstaff KM, Roth DM, Moseley GW, Jans DA. Targeted delivery to the nucleus. Adv. Drug Deliv. Rev. 2007; 59: 698–717. 41 Bolhassani A. Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochim. Biophys. Acta 2011; 1816: 232–246.
42 Hoyer J, Neundorf I. Peptide vectors for the nonviral delivery of nucleic acids. Acc. Chem. Res. 2012; 45: 1048–1056. 43 Lehto T, Simonson OE, Mäger I, Ezzat K, Sork H, Copolovici DM, Viola JR, Zaghloul EM, Lundin P, Moreno PM, Mäe M, Oskolkov N, Suhorutšenko J, Smith CI, Andaloussi SE. A peptide-based vector for efficient gene transfer in vitro and in vivo. Mol. Ther. 2011; 19: 1457–1467. 44 EI-Sayed A, Masuda T, Akita H, Harashima H. Stearylated INF7 peptide enhances endosomal escape and gene expression of PEGylated nanoparticles both in vitro and in vivo. J. Pharm. Sci. 2012; 101: 879–882. 45 Jones S, Howl J. Enantiomer-specific bioactivities of peptidomimetic analogues of mastoparan and mitoparan: characterization of inverso mastoparan as a highly efficient cell penetration peptide. Bioconjug. Chem. 2012; 23: 47–56. 46 Luque-Ortega JR, van’t Hof W, Veerman EC, Saugar JM, Rivas L. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in leishmania. FASEB J. 2008; 22: 1817–1828.
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Revised: 23 May 2014
Accepted: 28 May 2014
Published online in Wiley Online Library: 23 June 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2667
Functional properties of a novel hybrid antimicrobial peptide NS: potent antitumor activity and efficient plasmid delivery Yun Zhang,a,b† Jingjing Song,a† Wei Zhang,a Ranran Liang,a Yinyun Ma,b Li Zhang,b Xiaojin Wei,b Jingman Nia,b* and Rui Wanga* Antimicrobial peptides have been widely recognized as potential candidates for treating tumor, especially for defending against multidrug-resistant cells. Previously, based on the structure of substance P, we have designed a novel class of hybrid antimicrobial peptide NS, which possesses potent antimicrobial activity against a broad spectrum of bacterial pathogens. In this study, we evaluated its cytotoxicity to tumor cells and studied the possible mechanism of action. We showed that NS could efficiently kill tumor cells by rapidly disrupting the tumor cell membrane and inhibiting the DNA synthesis. In addition, we also found that NS could efficiently deliver plasmids into cells and exhibit high transfection efficiency after the introduction of a stearyl moiety to its N-terminus, like many reported cell-penetrating peptides. Taken together, this study revealed the potential multiple functions of NS, providing fundamental support for further therapeutic application as potential antitumor agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: NS; antitumor activity; membrane disruption; DNA interaction; plasmid transfection
Introduction
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* Correspondence to: Rui Wang and Jingman Ni, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences and School of Pharmacy, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected]; [email protected] †
These authors contributed equally to this work
a Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China b School of Pharmacy, Lanzhou University, Lanzhou 730000, China Abbreviations: AMPs, antimicrobial peptides; SP, substance P; CPPs, cell-penetrating peptides; NLS, nuclear location sequence; NBS, neonatal bovine serum; FBS, fetal bovine serum; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; PI, propidium iodide; BSA, bovine serum albumin; EtBr, Ethidium bromide; GFP, green fluorescent protein.
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Although the cancer therapy has experienced tremendous progress, the constant use of conventional chemotherapeutical agents easily results in the frequent appearance of multidrug-resistant tumors, threatening human health and quality of life. Based on this, many researches concentrated on the development of novel antitumor drugs with a different mechanism of action that disfavors drug resistance. Antimicrobial peptides (AMPs) have been considered as a new class of alternative chemotherapeutics that overcome limitations of current anticancer drugs because of high antitumor activity, tumor selectivity, and low susceptibility to common resistant mechanism [1,2]. Antimicrobial peptides were widespread in most of species, including microorganisms, plants, insects, amphibians, and mammals [3,4]. They not only exhibit broad antimicrobial activity against bacteria, fungi and virus [5–7] but also play an essential role in the innate immune response [8,9]. Recently, AMPs also attract more and more attention in antitumor treatment. The cationic and amphiphilic properties drive the rapid binding of AMPs to tumor cell membrane subsequently leading to the membrane disruption and the leakage of cytoplasmic components via a nonreceptor-mediated pathway [1,2,10,11]. In addition, some AMPs have been shown to translocate into tumor cells and subsequently interact with the cellular targets, resulting in cell death [12,13]. Furthermore, the structural similarities between AMPs and cell-penetrating peptide (CPPs) provide a possibility that AMPs may act like CPPs [14,15]. Thus, these desirable features prompt us to exploit the multiple functions of potential AMPs in tumor therapy, leading to further advance in the development of more effective treatment strategies.
Substance P (SP) is an 11-residue neuropeptide member of the tachykinin family, which plays important roles in most of biological activities, including cancer, wound healing, exocrine gland secretion, neuroendocrine, and immune regulation [16]. Notably, SP has also been reported to exhibit direct antimicrobial activity [17,18]. To improve its potential as novel therapeutic agent, we designed a series of new hybrid antimicrobial peptide analogs, being made up of two fragments: One is derived from nuclear location sequence (PKKKRKV), providing the cationic cluster for analogs; and the other is derived from SP, providing the hydrophobic cluster for analogs. NS, one of the analogs, showed significant antimicrobial activities (data were not shown). Because many antimicrobial peptides are reported to exhibit antitumor activity [1,19], we are interested if NS can remarkably inhibit the proliferation of tumor cells. Therefore, we studied the antitumor activity of NS as well as its action mechanism in this work.
ZHANG ET AL. the purified plasmids were dissolved in distilled water and stored at 20 °C.
Materials and Methods Materials Rink amide MBHA resin, protected amino acids, and other reagents and solvents for the peptide synthesis were purchased from GL Biochem Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and ethidium bromide (EtBr) were obtained from Sigma (St. Louis, MO, USA). Caspase inhibitor Z-VAD-FMK was purchased from Beyotime Biotechnology (Jiangsu, China). Peptide Synthesis NS and its related peptides shown in Table 1 were synthesized on an MBHA resin (0.54 mmol/g) using the standard Fmoc-based solidphase method, as described previously [20,21]. Stearic acid was coupled as Fmoc amino acids; the fluorescein moiety (FITC) was conjugated to the N-terminus via an aminohexanoic acid spacer by treating a resin bound peptide (0.1 mmol) with FITC (0.1 mmol) and diisopropyl ethyl amine (0.5 mmol) in N,N-dimethylformamide (DMF) for 12 h. The final cleavage was performed using a mixture of trifluoroacetic acid, thioanisole, water, phenol, and 1,2ethanedithiol (82, 5, 5, 5, 5, 2.5, v/v) for 3 h at room temperature. All crude peptides were purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) (Waters) using μ-BONDAPAKTM C18 column (19 mm × 300 mm) with gradient elution of 10–90% acetonitrile/water in the presence of 0.1% trifluoroacetic acid. The final purity of the peptides (>95%) was assessed by analytical RP-HPLC with sunfireTM column (3.9 mm × 150 mm, Waters). The molecular mass of synthetic peptides was conformed by electrospray ionization mass spectrometry. The sequence and characterization of peptides are shown in Table 1. Cell Cultures and Amplification of Plasmid DNA HeLa cells were maintained in RPMI1640 medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% heat-inactivated neonatal bovine serum (Sijiqing Biotech, Hangzhou, China). U251-MG, MDA-MB-231, and Cos-7 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). All cell lines were cultured in a 5% CO2 humidified atmosphere at 37 °C. pGL3 or pEGFP-C1 plasmid (4.7 kb), expressing luciferase or enhanced green fluorescent protein (EGFP) respectively, was transformed in Escherichia coli DH5α and amplified in LB medium at 180 rpm at 37 °C overnight. The plasmids were purified according to an EndoFree Plasmid Kit (Tiangen, Beijing, China). Thereafter,
Cytotoxicity Assays The antiproliferative effects of all peptides were evaluated using MTT assay. Cells (5 × 103 cells/well) were seeded in 96-well plates 24 h before treatment. After incubation with various concentrations of peptide for indicated times, 20 μl of 5 mg/ml MTT solution was added to each well and incubated for another 4 h at 37 °C. The absorbance was measured using microplate reader (Model 680, Bio-Rad, Hercules, CA, USA) at 570 nm. The effect of caspase inhibitor Z-VAD-FMK on the cytotoxicity of NS was also determined by MTT assay, as described previously [22]. HeLa cells (5 × 103 cells/well) were cultured in 96-well plates. After 24 h, cells were preincubated for 1 h with or without 20 μM Z-VAD-FMK. Thereafter, the cells were treated with NS at different concentrations for 24 h. Finally, cell viability was examined as described previously. Lactate Dehydrogenase (LDH) Leakage Assay Membrane integrity was assessed using the CytoTox-ONETM assay (Promega, Madison, WI, USA). Cells (5 × 103 cells/well) were seeded in 96-well plate 24 h before experiment. After 1 h of incubation with different concentrations of peptide in serum-free medium, 40 μl of medium was transferred to 96-well plate and incubated with 40 μl of reaction mixture for 15 min followed by addition of 20 μl stop solution. Fluorescence was detected at 560/590 nm. Untreated cells were defined as no leakage, and 100% leakage was defined as total LDH release by lysing cells in 0.2% Triton X-100. PI Uptake Assay To observe the cell membrane disruption after NS treatment, HeLa cells (2 × 105 cells/well) were seeded in 24-well plates and attached overnight at 37 °C. After incubation with NS at different concentrations for 1 h, the cells were washed with cold phosphate buffered saline (PBS), and then, PI (50 μg/ml) was added to each sample for 10 min staining. The positive rates of PI in cells after NS treatment were analyzed using FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). A total of 104 events were recorded for each sample, and the ratio of PI positive cells to the total cell population was defined as cell membrane disruption rate. Scanning Electron Microscopy and Transmission Electron Microscopy The morphological changes of HeLa cells after peptide treatment were observed by scanning electron microscopy (SEM) and
Table 1. Amino acid sequences, calculated and observed molecular masses, net charge, and RP-HPLC retention times of NS, stearyl-NS, and FITClabeled NS Molecular mass Peptides NS Stearyl-NS FITC-NS
Amino acid sequences PKKKRKVWKLLQQFFGLM-NH2 Stearyl-PKKKRKVWKLLQQFFGLM-NH2 FITC-AHX-PKKKRKVWKLLQQFFGLM-NH2c
Net charge
RP-HPLC retention times (min)b
+7 +6 +6
18.2 28.6 20.6
a
Calculated
Measured
2274.4 2540.9 2275.8
2274.3 2540.6 2776.5
a
Molecular masses were determined by electrospray ionization mass spectrometry. TM RP-HPLC retention time was measured by analytical HPLC with sunfire column (3.9 mm × 150 mm, Waters). c The fluorescein moiety (FITC) was attached to the N-terminus via an aminohexanoic acid (AHX) spacer. b
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NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY transmission electron microscopy (TEM). HeLa cells were seeded onto coverslips under the bottom of the six-well microplate for 24 h. The cells were treated with NS at a concentration of 10 μM for 1 h. Then, samples were prepared according to a published report [23] and finally observed by using SEM (JSM-6380LV, JEOL, Tokyo, Japan) and TEM (JSM-1230, JEOL, Tokyo, Japan). Cell Cycle Analysis The cellular DNA content and cell cycle perturbation was analyzed by PI DNA staining and monitoring its fluorescence via flow cytometry. Cell cycle analysis was performed using the previous method with a minor modification [24]. HeLa cells were seeded in six-well plates for 24 h at a density of 4 × 105 cells/well, followed by addition of NS with various concentrations. After 24-h treatment, the cells were harvested, washed twice with PBS, and fixed with 70% cold ethanol at 4 °C overnight. Before analysis, the fixed cells were further washed twice with cold PBS containing 1% bovine serum albumin and then incubated with PI solution (50 μg/ml PI, 0.1% sodium citrate, 0.1 mg/ml ribonuclease A, and 0.1% Triton X-100) for 30 min at 4 °C. Finally, cells were subjected to DNA content analysis by monitoring PI fluorescence with an FACScalibur flow cytometer. Data from at least 20 000 cells were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) to calculate cell cycle distributions. Cellular Uptake Assays HeLa cells (2 × 105 cells/well) were seeded in 24-well microplates 24 h before treatment. After 1 h of incubation with serum-free medium containing 2-μM FITC-labeled NS, the cells were harvested and washed twice with cold PBS and subsequently resuspended in 500 μl of PBS. Finally, the fluorescent intensity of 10 000 cells was analyzed using an FACScalibur flow cytometer with the 488-nm laser excitation. To gain a direct insight into the distribution of FITC-labeled peptide in HeLa cells, cells were plated in a glass-bottom culture dish for 24 h and then incubated with FITC-labeled NS using conditions and concentrations described earlier. After 1-h incubation at 37 °C, the cells were washed with cold PBS three times and imaged using an inverted Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Complexes Formation The complexes at various N/P ratios (peptides amino groups: DNA phosphate) were prepared by mixing 0.5 μg of plasmids with appropriate concentrations of peptides in 50 μl medium (one tenth of the final treatment volume). According to the positive charges of the peptide and negative charges of the plasmid, N/P ratios were calculated theoretically. For instance, final concentration of stearyl-NS was 0.5 μM at N/P ratio of 1. After incubation for 30 min at 37 °C, the formed complexes were diluted to a final volume of 500 μl with medium for subsequent use. Gel Retardation Assay
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Particle Size and ζ -Potential Measurements The peptide/pGL3 plasmid complexes were prepared at N/P ratios ranging from 1 to 4. The complexes were diluted in water to 1-ml volume, and then the size and ζ -potential were measured by Nano-ZS ZEN3600 (Malvern Instruments, Malvern, UK) at 25 °C, respectively. Vitro Transfection Assays To evaluate the ability of peptides to deliver plasmid DNA to cells, Cos-7 cells were seeded in a glass-bottom culture dish and cultured for 24 h. Cy5-labeled pGL3 plasmid was prepared using the method described by a Label IT Tracker Kit (Mirus, Madison, WI, USA). The peptide/Cy5-labeled pGL3 plasmid complexes were added into the dish at N/P ratio of 3. After incubation for 4 h, the cells were washed three times with PBS. Then, the fluorescence was observed with a confocal laser scanning microscope. To further determine the transfection efficiency of peptide/plasmid complexes, 1 × 105 Cos-7 cells were seeded in 24-well plate 24 h before experiment. Cells were treated with peptide/pGL3 plasmid complexes at different N/P ratios in serum-free DMEM for 4 h followed by addition of fresh DMEM containing 10% FBS and further incubated for 20 h. Thereafter, cells were washed and lysed using 100 μl reporter lysis buffer (Pierce, Rockford, IL, USA) for 15 min at 37 °C. Luciferase activity was detected using luciferase detection assay (Promega, Nacka, Sweden) and normalized to protein content. Protein concentrations in the cell lysate were examined using a BCA protein assay kit (Pierce, Rockford, IL, USA). EGFP Expression Assay To analyze the EGFP gene expression, Cos-7 cells were seeded in a glass-bottom culture dish 24 h before treatment; 0.5 μg of pEGFP plasmid was mixed with NS or stearyl-NS at N/P ratio of 3 as described in the Complexes Formation Section. Cells were treated with peptide/EGFP plasmid complexes at N/P ratio of 3 for 4 h in serum-free DMEM followed by addition of fresh DMEM containing 10% FBS and further incubated for 20 h. Thereafter, images were captured using an inverted Zeiss LSM 710 confocal microscope.
Results Cytotoxity of Peptide To evaluate the antitumor activity of NS, we initially determined its ability to inhibit cell proliferation. The cervical carcinoma cell line (HeLa), glioblastoma cell (U251-MG), and breast cancer cell line (MDA-MB-231) were chosen and treated with various concentrations of the tested peptide for 24 h; subsequently, cell viability was determined by the MTT assay (Figure 1A). The results showed that NS could kill tumor cells in a dose-dependent manner. Viability of the cells was reduced more than 90% after 24-h treatment at a concentration of 20 μM, exhibiting potent antitumor activity. In order to investigate the characteristics of the NS action, we examined its cytotoxicity to HeLa cells for the indicated term of incubation. As shown in Figure 1B, the proliferation of cells was inhibited in a time-dependent manner after NS
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DNA condensation was evaluated by the gel retardation assay [15]. Briefly, the peptide/pGL3 plasmid complexes at various N/P ratios were prepared by mixing appropriate volumes of peptide solution with 0.5 μl of pGL3 plasmid (200 ng/μl in water). The complexes were diluted to a total volume of 8 μl with NaCl solution (150 mM) and then incubated for 30 min
at 37 °C. Subsequently, the complexes were loaded on the 0.8% (w/v) agarose gel and imaged by staining the gel with EtBr (0.5 μg/ml).
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Figure 1. The cytotoxicity of NS to tumor cells. (A) The inhibition effect of NS on different tumor cells. Cells were treated with various concentrations of NS for 24 h and then detected by MTT assay. (B) Cell survival rate of HeLa cells treated with various concentrations of NS for 1, 4, 8, and 24 h. (C) The effect of Z-VAD-FMK on cellular viability during NS treatment. HeLa cells were preincubated with or without 20 μM Z-VAD-FMK for 1 h and further incubated with NS (1, 2, and 5 μM) for 24 h. Cell viability was determined using the MTT method. Data are mean ± SEM of three repeated experiments.
treatment; however, we found that pan-caspase inhibitor Z-VADFMK did not recover the viability of cells by treating HeLa cells with Z-VAD-FMK for 1 h before NS treatment (Figure 1C), demonstrating that NS induced caspase-independent cell death. Notably, rapid cell death was detected after 1 h of treatment with NS at high concentration, implying NS might exhibit cytotoxicity by disrupting the tumor cell membrane, just like many naturally occurring antimicrobial peptides [1,19]. Effect of NS on the Cell Membrane In order to verify the membrane disrupting activity of NS, we determined the effect of NS on the integrity of cell membrane. LDH leakage assay was employed to measure acute membrane disturbance. LDH release was detected in HeLa cells after NS treatment
and revealed the membrane-lytic activity of NS (Figure 2A). In addition, the PI uptake assay also was used to study the change of cell membrane integrity after peptide treatment. PI can permeate unhealthy/damaged membranes [2], so positive PI fluorescence indicates damaged cell membranes. Figure 2B showed the uptake of PI in HeLa cells after 1 h of treatment with NS at indicated concentration (5, 10, and 20 μM). As expected, the positive rates of PI in HeLa cells after NS treatment could increase with an increase in the peptide concentration, whereas the uptake of PI in untreated cells was negligible, supporting the results indicated by the LDH leakage assay. These results suggested that NS could disrupt and damage the cell membrane, resulting in death of the target cells. To better understand the death mode in HeLa cells treated with NS, we performed morphological investigation by TEM and
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Figure 2. LDH leakage (A) and PI uptake (B) in HeLa cells treated with various concentrations of NS for 1 h. Data are mean ± SEM of three repeated experiments.
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NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY SEM with the results presented in Figure 3. The untreated control cells presented plenty of microvilli and adherent smooth surface, whereas the membranes of HeLa cells treated with NS were shriveled, invaginated, disrupted, and characterized with significant loss of microvilli and pore formation. Furthermore, TEM was used to examine the ultrastructural changes of HeLa cells after NS treatment. Normal HeLa cells displayed microvilli protruding from the entire surface and a smoothly outlined nucleus with chromatin in the form of heterochromatin. In contrast, following the NS treatment, HeLa cells showed loss of plasma membrane integrity and electron-lucent cytoplasm with intact nuclear membrane, which were consistent with the characteristics of necrosis. According to the results derived from the LDH leakage assay, PI uptake assay, SEM, and TEM, we concluded that NS could disrupt cell membrane and lead to cell necrosis. Effect of NS on the Cell Cycle Distribution Although NS killed tumor cells by rapid membrane disruption, we could not conclude that the membrane was the only target against which NS exerted its antitumor activity. Therefore, we assessed the impact of NS on cell cycle distribution to attempt to look for some intracellular target. For this purpose, we performed flow cytometric DNA cell cycle analysis on NS-treated HeLa cells (Figure 4). This method relies on fluorescent probes that bind DNA, thus enabling the entire DNA content per nucleus to be monitored with precision [25]. Relevant differences in cell cycle distribution were observed in cells treated with 5-μM NS. The 39.5% G0/G1 value in the NS-treated cells was significantly reduced compared with 61.4% for the untreated cells. The percentage of cells in S phase for NS-treated cells was 52.8%, which was higher when compared with the untreated controls, 26.2%. Differences in the frequency of cells in the G2/M phase were imperceptible. Our results indicated that NS could induce S-phase arrest in HeLa cells. Deriving from the association of S-phase arrest and DNA synthesis inhibition [26–28], we conclude that NS could perturb DNA replication and inhibit its synthesis, consequently arresting the growth of HeLa cells in the S phase.
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Cellular Uptake of Peptide The cell penetration ability of NS was assessed by FACS and confocal microscopy. Figure 5A revealed that the fluorescence intensity of NS in HeLa cells at 2 μM was significantly high, supporting its strong cellular uptake. Furthermore, to gain a direct insight into the internalization of NS, the images of cells treated with 2μM FITC-labeled NS for 1 h were taken using a confocal laser scanning microscopy (Figure 5B). It was evident that distribution of potent fluorescence signal was almost in cellular nuclei, indicating that NS can translocate into cell and accumulate in the nuclei. Plasmid Transfection Ability of NS Based on the cell penetration ability of NS, we presumed that NS may act as an efficient vector for gene transfection like many reported CPPs [29,30]. Deriving from the success of stearylation of CPPs for plasmid delivery [20,31–33], we conjugated stearic acid to the N-terminus of NS and evaluated the efficiencies of NS and stearyl-NS for plasmid transfection.
Figure 5. Cellular uptake of NS in HeLa cells. Cells were incubated with 2-μM FITC-labeled NS for 1 h in serum-free medium. (A) Flow cytometry histogram of peptide internalized into cells. (B) Confocal images of cellular uptake of peptide. DIC represents the differential interference contrast images of experimental groups taken by confocal microscopy under natural light.
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Figure 3. Morphological changes of HeLa cells after NS treatment. Cells were incubated with NS (10 μM) for 1 h and then observed using SEM and TEM.
Figure 4. Cell cycle distribution of HeLa cells after NS treatment for 24 h. All the results were obtained from three independent determinations.
ZHANG ET AL. On the basis of noncovalent nanoparticle formation strategy [34], positively charged peptides and negatively charged pDNA are mixed together to form steady complexes. The nanoparticle formation efficiency was assessed by gel retardation assay. As shown in Figure 6A, the pDNA migration was completely retarded by both NS and stearyl-NS at N/P ratio of 2, indicating that they could efficiently condense pDNA into the nanoparticles. Stearyl-NS had an enhanced DNA condensable ability as more pDNA could not migrate into the gel at N/P ratio of 1. The ζ -potential and particle size stearyl-NS/plasmid complex was characterized as a function of increasing N/P ratios. Figure 6B showed that the ζ -potential of complex was increased with increasing N/P ratios. Additionally, the particle size of stearylNS/plasmid complex was in range of 177–278 nm (Figure 6C). After the characterization of complex, we evaluated the ability of complex to transfect plasmids into cells. We first assessed plasmid delivery ability of peptides by confocal microscopy. On the basis of the confocal images (Figure 7A), Cy5-labeled plasmid signals were detected in almost all cells after incubation with stearyl-NS/Cy5-labeled plasmid complex, whereas NS could deliver little Cy5-labeled plasmid complex into cells, indicating that NS could exhibit enhanced plasmid delivery ability when associated with stearic acid. Furthermore, to further evaluate the transfection efficiencies of NS and stearyl-NS, Cos-7 cells were transfected with complexes of luciferase-encoding pGL3 plasmid and different peptides. As seen in Figure 7B, NS showed slightly higher transfection efficiency than pGL3 plasmid alone. Intriguingly, luciferase expression levels of stearyl-NS were significantly increased compared with unmodified peptide at various N/P ratios, displaying remarkably enhanced transfection efficiency. Additionally, in accordance with the result of luciferase expression, stearyl-NS/EGFP plasmid complex at N/P ratio of 3 mediated significant EGFP expression, whereas no EGFP expression in Cos-7 cells was observed for NS/plasmid complex (Figure 7C). These results suggested that NS could efficiently mediate plasmid delivery after the N-terminal stearylation.
Discussion Although there is established effectiveness in a wide range of tumors, the currently used antitumor drugs cause several side effects and induce drug resistance, resulting in narrow therapeutic indexes. Nowadays, antimicrobial peptides can provide a potential in cancer therapy as a result of more effective and specific treatment than standard therapies [1,2]. Thus, researchers pay more attention to the development of novel antimicrobial peptide for better tumor treatment. In this study, we found that the novel hybrid antimicrobial peptide NS exhibited a significant cytotoxic effect against tumor cells via membrane disruption and inhibition of DNA synthesis. In addition, NS was found to show cell-penetrating ability and efficiently deliver plasmid DNA into cells when combined with stearyl moiety. Recently, membrane binding/translocation have been reported to play a crucial role in the cytotoxicity of AMPs to tumor cell [35,36], as it facilitates the membrane permeabilization and cellular accumulation of AMPs, subsequently producing morphological and biochemical hallmarks of cell death. In this study, we clearly showed that NS displayed a rapid cell killing activity. The remarkable LDH release and PI uptake seen when treating the cells with NS indicated that NS triggered acute membrane disruption, just like many antimicrobial peptides [1,19]. This conclusion is consistent with the dramatic morphological changes observed by SEM and TEM. Thus, our results revealed that necrotic mechanism was directly involved in the antitumor action of NS. As we know, resistance is one major cause resulting in the failure of tumor therapy. It is often associated with the transporters that efficiently pump out the chemical drugs from the cells [37]. Notably, rapid cell death via membrane disruption is an irreversible receptor-independent process. Therefore, it is difficult for tumor cell to develop resistance to NS. Furthermore, the membrane-lytic activity endows NS a remarkable superiority that it may exhibit better cytotoxicity to multidrug-resistant tumor cells like many antimicrobial peptides [2], complementing common drawbacks of classical chemotherapy.
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Figure 6. Characterization of stearyl-NS/plasmid complex. (A) Ability of peptides to form complexes with pDNA at different N/P ratios (0.25–3) was evaluated by gel retardation assay. (B) ζ -potential of stearyl-NS/plasmid complex at N/P ratios. (C) Particle size of stearyl-NS/plasmid complex at N/P ratios.
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Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2014; 20: 785–793
NS: POTENT ANTITUMOR ACTIVITY AND EFFICIENT PLASMID DELIVERY
Figure 7. Effect of stearyl-NS on plasmid transfection. (A) Cellular uptake of peptide/Cy5-labeled plasmid complex at N/P ratio of 3 in Cos-7 cells after 4h incubation. (B) Luciferase expression mediated by stearyl-NS/pGL-3 plasmid complex at N/P ratios. (C) EGFP expression mediated by stearyl-NS/EGFP plasmid complex at N/P ratio of 3. All the results were obtained from three independent determinations. DIC represents the differential interference contrast images of experimental groups taken by confocal microscopy under natural light.
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endosomal entrapment of both CPP and respective cargo, limiting the relative efficient of unmodified CPPs in promoting plasmid delivery [29,30]. Stearylation of CPPs has been shown to be a successful strategy for efficient plasmid delivery, as a lone modification on polyarginine [31,32], TP10 [43], or if incorporated into multifunctional envelope-type nano device [44]. The significant role played by stearylated CPPs in plasmid transfection has consequently resulted in growing attention to design of new modified CPPs. Recently, some groups have shown that antimicrobial peptides can translocate into cells like CPPs [15,45,46]. The possibility to utilize AMPs for cellular plasmid transduction has gained wide interest. We have demonstrated previously that N-terminal stearylation of [D]-K6L9, a membrane-disruptive antimicrobial peptide, was successful in promoting efficient plasmid delivery [15]. In the current work, we studied whether NS could also exhibit delivery potential for the cellular transduction of plasmid. In concordance with the previous reports on stearylated CPPsmediated plasmid delivery [31–33], the introduction of N-terminal stearyl moiety has a substantial impact on NS peptide’s ability to deliver plasmids into cells. The level of transfection was remarkably high when using stearyl-NS, whereas unmodified NS had poor effect on the transfections of plasmids. The high transfection efficiency of the stearyl-NS/DNA complex could attribute to the increased cellular uptake. The hydrophobic stearyl moiety renders the complex more lipophilic, presumably facilitating its penetration through the cell membrane. In addition, the peptides consisting of NLS are well adapted to nuclear localization and facilitate efficient plasmid transfection [33]; therefore, the NLS linked to the N-terminus of NS may play an important role in the transfection efficiency of the stearyl-NS. Furthermore, we believe that
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Moreover, we found that NS could penetrate into cells and accumulate inside cellular nuclei, so the antitumor mechanism of NS maybe involved in the intracellular action. In this study, we observed that the caspase inhibitor Z-VAD-FMK did not prevent NS-induced cell death, demonstrating NS exerted its antitumor activity through caspase-independent pathway. Some reports have shown that antimicrobial peptides can target the cellular DNA and interrupt DNA functions (such as the DNA synthesis/replication), thereby contributing to the cell death [38,39]. Nuclear localization signal (NLS) has been proven to facilitate nuclear delivery [33,40]; the introduction of NLS indeed resulted in the nuclear accumulation of NS, implying that the observed antitumor effect is likely associated with DNA interaction of NS after internalization. A possible contribution of DNA interaction in the efficient antiproliferation effect of NS was evaluation by cell cycle analysis. The significant increase in cell number of S phase when treating cells with NS indicated that NS caused apparent S arrest in tumor cells. Earlier studies have demonstrated that the S-phase arrest caused in tumor cells correlated with efficient inhibition of DNA synthesis [27,28]. Therefore, another plausible explanation for the remarkably potent antitumor activity may be that the DNA binding affinity of NS facilitates its perturbation in DNA replication, consequently leading to cell cycle arrest in S phase and finally growth inhibition. Naked therapeutic nucleic acid-based molecules used in gene expression modulation have a strong negative charge and high molecular weight, preventing them to enter cells freely. CPPs are one group of such nonviral vectors, which are increasingly utilized for gene delivery because of their high internalization efficiency, low cytotoxicity, and flexible structural design [41,42]. The main barrier when utilizing CPPs for delivery is the
ZHANG ET AL. endosomal escape is probably the critical determinant for efficient plasmid transfection obtained with the stearyl-NS. The N-terminal stearylation [31,32,43,44] and membrane-lytic activity [15] of peptides have a substantial effect on improving endosomal escape of the nanoparticles. With the aid of both the stearyl moiety and the membrane-lytic ability, the stearyl-NS/DNA complexes would escape from endosomal compartments, thus facilitating the efficient transfection. The remarkable transfection efficiency observed with stearyl-NS illustrates that stearylated antimicrobial peptides would represent another alternative for the development of an efficient transfection system and can be also used as an efficient vector for tumor gene therapy. In conclusion, our results demonstrated that the novel antimicrobial peptide NS could kill tumor cells by rapidly disrupting cell membrane and arresting DNA synthesis, which are not affected by the known resistance mechanism. Gratifyingly, NS modified with stearyl moiety could result in the efficient plasmid delivery, implying that it might act as a potential delivery vector for further tumor gene therapy. Taken together, the multiple functions of NS provide direct evidence that it is a highly interesting candidate for future tumor treatment. Acknowledgements We are grateful for the grants from the National Natural Science Foundation of China (Nos. 81273440, 91213302, 20932003, and 21272102) and the Key National S&T Program ‘Major New Drug Development’ of the Ministry of Science and Technology (2012ZX09504-001-003).
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