Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine

19 May 2016 - School of Pharmacy, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, ... Cationic amphipathic peptides (CAPs) are known to be able to c...
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Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine- or Arginine-Rich Cationic Hydrophobic Peptides in Cancer Cells Xiaoli Liu,†,‡,∥ Rui Cao,†,∥ Sha Wang,†,§ Junli Jia,† and Hao Fei*,† †

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou, Jiangsu 215123, P. R. China ‡ School of Life Science, Shanghai University, 99 Shangda Road, Baoshan District, Shanghai 200444, P. R. China § School of Pharmacy, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China S Supporting Information *

ABSTRACT: Cationic amphipathic peptides (CAPs) are known to be able to cause membrane destabilization and induce cell death, yet how the hydrophobicity, amphipathicity, and lysine (K)/arginine (R) composition synergistically affect the peptide activity remains incompletely understood. Here, we designed a panel of peptides based on the well-known anticancer peptide KLA. Increasing hydrophobicity enhanced the cytotoxicities of both the K- and R-rich peptides. Peptides with an intact amphipathic helical interface can cause instant cell death through a membrane lysis mechanism. Interestingly, rearranging the residue positions to minimize amphipathicity caused a great decrease of cytotoxicity to the K-rich peptides but not to the R-rich peptides. The amphipathicity-minimized R-rich peptide 6 (RL2) (RLLRLLRLRRLLRL-NH2) penetrated the cell membrane and induced caspase-3-dependent apoptotic cell death. We found that the modulation of hydrophobicity, amphipathicity, and K/R residues leads to distinct mechanisms of action of cationic hydrophobic peptides. Amphipathicityreduced, arginine-rich cationic hydrophobic peptides (CHPs) may represent a new class of peptide therapeutics.

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INTRODUCTION Cationic amphipathic peptides are derived from antimicrobial peptides, a class of host defense peptides expressed in many species.1−3 Most antimicrobial peptides are rich in cationic and hydrophobic amino acids, which can arrange into amphipathic α-helix or β-structure and display potent cell-killing effect. It was later discovered that some antimicrobial peptides have anticancer activity.4−8 The cell membranes of both bacteria and cancer cells contain net negative charges in elevated proportions.5 The electrostatic and hydrophobic interactions between the peptides and biomembrane surface form the basis of these peptides’ cytotoxicity. Most CAPs cause cell death through membranolytic mechanisms,9,10 in which the plasma or the mitochondrial membrane is selectively disrupted.5,11 Some CAPs can cause cell death through nonmembranolytic mechanisms, for example, through interactions with intracellular enzymes or receptors.5,12−14 Many naturally occurring α-helical CAPs (e.g., cecropins or melittin) and β-structure CAPs (e.g., defensins or lactoferricin) have strong membranolytic ability. On the basis of the structures of naturally occurring CAPs, various synthetic peptides have been designed to achieve the membrane disruption of cancer cells.4,15,16 A well-known example of an amphipathic peptide, KLA (1 in Figure 1A), was originally designed as an α-helical antimicrobial peptide.17 On its own, however, 1 has a low mammalian cell toxicity because of its © XXXX American Chemical Society

poor cell uptake efficiency. Researchers have concentrated on increasing its uptake efficiency using various modifications. Fusion to protein transduction domains,18,19 connection to a specific antibody20 or targeting sequence,21,22 and combination with nanoparticles23,24 have successfully helped 1 penetrate into cells and induce apoptosis. Naturally occurring antimicrobial peptides contain two main types of cationic amino acids, lysine and arginine. Most artificially designed membranolytic peptides are lysine (K) rich peptides.4,15−17,25,26 Fewer examples have been reported of artificially designed arginine (R) rich peptides, with one prominent class being the RW-rich peptides.27−29 Lysine and arginine have distinct modes of action when they encounter the lipid bilayer. Recent studies revealed that the guanidinium group allows arginine to interact with both the phosphate and glycerol groups of the membrane and form multiple hydrogen bonds, producing a less polar membrane-soluble ion pair complex30 and facilitating the penetration of argininecontaining hydrophobic sequences deep into the membrane.31 R-rich peptides have become the design basis used to create cell-penetrating peptides with high cell uptake efficiency. Replacing K in 1 with R (RLA, 4 in Figure 1A) can significantly Received: December 27, 2015

A

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with positively charged groups on one side and hydrophobic groups on the other (Figure 1B). To design peptides with a decreased hydrophobic moment, we took a minimal approach by removing the two C-terminal amino acids of 2 (LK) or of 5 (LR) and reinserted them before the location where the second heptad repeat begins (the underlined residues shown in Figure 1A). Such a two amino acid insertion rotated the second heptad by nearly 200° in an αhelix. This yielded peptides 3 (KL2) and 6 with identical amino acid compositions to 2 and 5 but with diminished hydrophobic moments because of the even distribution of the cationic and hydrophobic residues (Figure 1B). This design also maintained the intermittent distribution of the hydrophilic and hydrophobic residues in the peptide primary structure, avoiding the formation of amphipathic regions that could lead to selfaggregation. Secondary Structures and Self-Aggregation Behaviors of the Designed Peptides. The secondary structures of the peptides in water and in the presence of model lipid membranes were examined using CD spectroscopy. All designed peptides were unstructured random coils in water, displaying a minimum at 195 nm in the CD spectra (Figure 2A). When mixed with negatively charged large unilamellar lipid vesicles (LUVs) made from a 1:1 mixture of DMPC/ DMPG, 2 and 5 formed obvious α-helices, with the appearance of double negative peaks at 208 and 222 nm and a single positive peak at 195 nm; 3 and 6 appeared as partial random coiled structures mixed with partially folded helical structures, as the double negative peaks were not obvious (Figure 2B). 3Drendered helical peptide modeling showed a distinct difference between the two series of peptides (Figure 2C). In the model of 5 (and similarly 2), a continuous hydrophobic face allowed amphipathic helix formation along the entire length of the peptide, whereas in the model of 6 (and similarly 3), only disintegrated segments of the hydrophobic regions could be found in the neighboring turns, which was unfavorable for the formation of a fully folded helical structure at the lipid−solution interface. Amphipathic molecules tend to minimize unfavorable interactions with the aqueous environment via an aggregation process; in this process, hydrophilic regions are exposed, and hydrophobic domains are shielded by nucleation. Thus, we investigated whether the designed peptides with or without a predicted hydrophobic interface would aggregate in water. To confirm their tendencies of forming aggregates in an aqueous solution, the fluorescent dye 8-anilino-1-naphthalene sulfonate (ANS) was used to probe the hydrophobic microdomains in solution. ANS binds preferentially to clustered hydrophobic residues, and when it does, its fluorescence quantum yield significantly increases.38,39 The data show that 2 and 5 aggregated into micelles, as the enhanced fluorescence became apparent when the peptide concentrations reached thresholds, indicative of their respective critical micelle concentrations. On the contrary, 3 and 6 did not show any fluorescence enhancement, indicating their lack of aggregation in an aqueous environment (Figure 2D, E). These self-aggregation data, together with the CD spectra and 3D modeling data, consistently suggest that the two series of designed peptides display distinct folding and assembly behaviors based on the large differences of their predicted helical amphipathicity. Cytotoxic and Membranolytic Activities of the Designed Peptides. The cytotoxicities of these peptides were next investigated. HeLa cells were incubated with various

Figure 1. Peptide designs and their helical wheel projections. (A) Compound numbers (CN), sequences, hydrophobicities (H), and hydrophobic moments (μH) of the designed peptides. All peptides were N-terminally appended with an NH2-HGG sequence, of which histidine was introduced for luminescent iridium complex labeling and imaging purposes and GG was introduced for space separation from the subsequent helical domain. H and μH were calculated using HeliQuest. (B) Helical wheel presentations of the peptides showing the relative positions of the residues in a hypothetical α-helix structure. Blue circles refer to the cationic residues, and yellow circles refer to the Leu residues. Arrow direction and length indicate the predicted hydrophobic moment and strength. Graphics were generated using Heliquest.com.

improve its membrane permeability and increase its mitochondrial accumulation while maintaining low cytotoxicity.32 The gross hydrophobicity of a CAP also affects its activity. Introducing more hydrophobic residues can often enhance the cytotoxicity of a peptide.33 In addition to hydrophobicity, another important parameter known as amphipathicity (as measured by hydrophobic moment) has also been reported to be a strong modulator of membranolytic activity. Peptides with a higher hydrophobic moment have enhanced antibacterial and hemolytic activities.34,35 Some studies intentionally disrupted the hydrophobic−hydrophilic interface by introducing D-amino acids to replace L-amino acids or by introducing β structure forming isoleucine to reduce the helicity of the peptides and have successfully increased the therapeutic indices.4,15,36,37 Despite the current knowledge, close comparisons of the correlated effects of the hydrophobicity, amphipathicity, and K/ R composition on the anticancer cell activity of CAPs have been rarely reported. In this study, on the basis of the structure of 1, we designed a panel of cationic peptides by modulations of the hydrophobicity by alanine/leucine substitution, the helical amphipathicity by reducing the hydrophobic moment, and the internalization efficiency by lysine/arginine substitution. This systematic investigation allowed us to find an arginine-rich peptide, 6 (RL2), with minimum amphipathicity that could selectively induce stress-triggered apoptosis in cancer cells.

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RESULTS Peptide Designs. Peptides were designed based on the framework structure of 1, which contains two repeats of the heptad KLAKLAK (Figure 1). In the first series, two peptides, 2 (KL1) (KLLKLLKKLLKLLK-NH2) and 5 (RL1) (RLLRLLRRLLRLLR-NH2), were designed by replacing alanine with leucine and substituting lysine with arginine. With the increased number of leucines, they exhibited increased hydrophobicity compared with 1 and 4 (Figure 1A). A helical wheel projection shows the obvious amphiphilic α-helical structures of 2 and 5, B

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Figure 2. Circular dichroism (CD) spectra of 100 μM peptides in the presence of (A) H2O or (B) 5 mM negatively charged DMPC/DMPG (1:1) LUVs. LUV-only background was subtracted. (C) 3D modeling of the designed peptides; only peptides 5 and 6 are shown as the examples. Arginines are in blue and hydrophobic regions in gray with leucine side chains omitted for clarity. Structures were modeled using the 3D-HM Web site,40 and graphics were rendered using PyMOL. (D, E) Peptide self-aggregation and micelle formation in solution. Plots show 1,8-ANS fluorescence intensity responding to peptide concentration.

concentrations of the peptides for 24 h, and the cell viabilities were determined using the MTT assay. Of the peptides with strong amphipathicity, 2 and 5 exhibited strong cytotoxicity, with IC50 values of 3.3 ± 0.4 μM and 3.7 ± 0.1 μM, respectively. In the case of 3, when the helical amphipathicity was minimized, its cytotoxicity significantly decreased, with an IC50 value of 34.5 ± 1.6 μM. Interestingly, however, in the case of 6, the peptide’s cytotoxicity was not dramatically affected, as it still had an IC50 value of 4.3 ± 0.2 μM (Figure 3A,C). Most cationic amphipathic peptides cause cell death through membrane disruption or pore formation mechanisms. The MTT results led us to ask whether the cytotoxicity of the designed peptides was due to compromised plasma membrane integrity. To this end, we used a fluorescence leakage assay employing a HeLa cell line with cytosol-expressed green fluorescent protein (HeLa-GFP). In this assay, the HeLa-GFP

cells were incubated with various concentrations of peptides for a short time, and the fluorescence of the GFP released into the culture media corresponded to the extent of cell membrane leakage caused by the peptides. The half-leakage concentration (LC50) was calculated for quantitative comparisons. Compared to in vitro liposome-based leakage assays, this assay has the advantage of allowing direct observation of the actual cell membrane response to peptide action. The results show that both 2 and 5 had a strong and instant membrane lysis ability with very low LC50 values of 3.6 ± 0.1 μM and 3.9 ± 0.2 μM, respectively; 3 and 6 exhibited much higher LC50 values of 38.8 ± 0.5 μM and 25.9 ± 0.7 μM, respectively (Figure 3B,C). For 2 and 5, the close values of their respective LC50 and IC50 values suggest that the membrane disruption or the pore formation mechanism accounts for the majority of the lethality to the cancer cells. C

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Figure 3. Comparison of the cytotoxicity and membranolytic activity of the peptides. (A) Lethality of the HeLa cells after treatment using peptides of various concentrations. (B) Fluorescence of the GFP leakage from HeLa-GFP cells showing the membranolytic ability of the peptides. (C) Comparison of the IC50 and the LC50 of peptides. Data are presented as the mean values ± standard deviations of three independent experiments.

Figure 4. Different cell interactive behaviors of the R-rich peptides. (A) Different HeLa cell morphologies after co-incubation with 5 or 6 (5 μM) for 30 min. Scale bar: 50 μm. (B) PI/Hoechst co-staining shows cell membrane compromised differently in 5 μM peptide treated cells. Scale bar: 100 μm. (C) Time-lapse MTT assay of R-rich peptide-treated HeLa cells. (D) Time-lapse imaging of luminescently labeled peptides (5 μM) in HeLa cells. Scale bar: 50 μm.

In the case of 3, the reduced amphipathicity reduced both its cell lethality and its membranolytic activity by an order of magnitude. These results confirmed that amphipathicity contributes greatly to the cytotoxicity of KL-rich peptides. It was then interesting to see that despite its strong cytotoxicity, peptide 6 exhibited weak membranolytic ability, suggesting an alternative cell death mechanism to the instant membrane disruption of 2 and 5. Different Cell Interactive Behaviors of Peptides 5 and 6. The previous results showed that 5 and 6 displayed similar IC50 values but very different membranolytic behavior toward HeLa cells. We next investigated in greater detail this pair of Rrich peptides, studying the differences in peptide-treated cell morphologies, rates of cell death, and peptide localizations in the cells. After incubation with 5 μM 5 or 6 for 30 min, HeLa cells exhibited very different morphological characteristics under the microscope (Figure 4A). In the 5-treated sample, the cells exhibited drastic membrane leakage, with many

membrane fragments dispersed in the culture medium. In the 6-treated sample, the cells shrank and became round, but their morphology stayed intact. The difference of the membrane integrity of the cells after peptide treatments could be more clearly demonstrated by fluorescent imaging using nuclear dyes. Hoechst 33342 stains the nuclei of live or dead cells, and propidium iodide (PI) stains only dead cells. As depicted in Figure 4B, 5-treated cells showed PI/Hoechst co-staining within 5 min; 6-treated cells were initially stained only by Hoechst but became co-stained after 30 min. We observed that, unlike 5, which killed cells instantly, 6 caused cell death over a longer time window. To more accurately profile the rates of death in 5- and 6-treated cells, we performed a “time-lapse” MTT assay in which the cells were incubated with peptides for different amounts of time, and the lethality was subsequently assessed. The results show that the lethality of 5 treated cells readily reached a plateau after a 5 min D

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Figure 5. Peptide 6 induced selective stress-triggered apoptosis in cancer cells. (A) Analysis of cell apoptosis using flow cytometry, with panels showing PI/annexin V staining of the control and 6-treated cells; FL1-A represents the fluorescence of annexin V, and FL2-A represents the fluorescence of PI. (B) Caspase-Glo assay showing increased caspase-3 activity after cell exposure to the peptides at indicated times. (C) Western blot of cleaved caspase-3 in HeLa cells after exposure to 6 (5 μM) for the indicated periods of time. Actin was used as a loading control. (D) Decline of caspase-3 activity and cell lethality of 6-treated (5 μM, 30 min) HeLa cells after introducing caspase-3 inhibitor. (E) Cytotoxicity of peptides 5 and 6 in several cancer and noncancer cell lines. (F) Effect of 6 on the intracellular ROS production of several cancer and noncancer cell lines after coincubation with the cells for 30 min. Data are shown as the fold changes of the peptide-incubated group compared with the nontreated control group (P < 0.01). (G) Lethality in HeLa cells after co-incubation with various concentrations of 5 or 6 at 0 or 37 °C for 30 min. (H, I) Hemolysis assay of 5 and 6 using mouse red blood cells.

Like most of the designed CAPs, 5, with its distinct hydrophobic−hydrophilic helical interface, could accumulate on the cell membrane and perhaps self-assemble into ordered transmembrane porelike structures that cause drastic membrane lysis and induce instant cell death. On the other hand, 6 lacks the ability to form an amphipathic secondary structure for intermolecular assembly or ordered peptide−lipid assembly; thus, 6 would likely penetrate into the cell without destroying the cell membrane, causing cell death, instead, through an intracellular mechanism.

incubation, while 6 induced cell death lagged behind, with the lethality gradually rising over 6 h (Figure 4C). In order to visualize peptide localizations in the cells, we labeled the N-terminal histidine residue of each peptide with a lipophilic organometallic iridium dye to obtain phosphorescence.41 Time-lapse images were captured using a confocal microscope after the cells were treated with 5 μM iridiumlabeled peptides. The results showed that 5 appeared to be concentrated at the plasma membrane, while 6 penetrated and dispersed into the cytosol within minutes (Figure 4D). E

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lines using a redox-sensitive fluorescent probe, 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) (Figure 5F). The results showed that the 5 μM-6-treated cancer cells all exhibited significantly increased ROS levels, whereas the same treated noncancer cells did not show any increase; these results suggest that the intracellular accumulation of 6 specifically elevates the level of ROS in cancer cells, which might be the causal factor that triggers downstream apoptotic cell death. To further confirm the cell killing effects of 6 involved cell metabolic pathways, we proposed that altering the treatment temperature would change the peptide-induced lethality to cells. We found that low-temprature (0 °C) incubation with the peptides greatly decreased the cytotoxicity of 6 while barely affecting the cytotoxicity of 5 in HeLa cells (Figure 5G). This indicates that the cytotoxicity mechanism of 6 is energydependent. Many cationic peptides with anticancer activity can also induce hemolysis, which prevents their clinical translation. To evaluate the safety profile of the designed peptides, we examined their hemolytic activity using red blood cells. Amphipathic peptide 5 showed very strong hemolytic properties, with a low HC50 value of 2.9 ± 0.3 μM, while its counterpart, 6, with minimized amphipathicity, exhibited much reduced hemolytic activity, with an HC50 value at over 200 μM (Figure 5H,I; similar results also observed for 2 and 3, shown in Figure S8). This drastic decrease in the hemolytic activity of 6 is likely due to its inability to form ordered helical amphipathic structures. These results are similar to the previously reported D,L-diastereomers of KL-rich peptides, in which intermittent D to L amino acid replacement effectively reduced the peptide helicity as well as the hemolytic activity.45 To further explore its therapeutic value, we investigated the cytotoxicity of the all D amino acid peptide 6 (D-6) against HeLa cells. Similar to 6, D-6 showed an IC50 of 4.1 ± 0.5 μM in HeLa cells; it was internalized by the cells and induced caspase-3 activation (Figure S9), which is consistent with a chirality-independent membrane-centric mechanism. Its resistance to enzyme degradation and lower immunogenicity could be an advantage for therapeutic development.

6 Induces Selective Stress-Triggered Apoptosis in Cancer Cells. On the basis of the relatively slow onset of death of 6-treated cells and the intracellular localization of 6, we postulated that an apoptotic cell death mechanism could be involved. Using flow cytometry analysis, we found that 6treated cells retained their integrity and a similar scatter distribution to the control cells, which allowed for further labeling analysis (Figure S7). Co-staining the cells with PI and annexin V demonstrated that a large proportion of the 6-treated cells became stained with annexin V, which indicated early apoptotic cell death; some of the cells showed dual staining with both PI and annexin V, indicating late-stage apoptotic cell death (Figure 5A). The activation of caspase-3 is a hallmark of apoptosis. Using an enzyme-specific fluorogenic substrate, caspase-3 activity was monitored in 6-treated cells. The results showed that high caspase-3 activity was detected within 5 min after adding the peptide and soon began to decline over time, falling quickly within 1 h and reaching baseline at 4 h (Figure 5B). This was consistent with the time-lapse MTT results (Figure 4C), in which the cell lethality increased markedly within the first hour and then reached a plateau over the next several hours. Similar to the control sample, no incremental changes of the caspase-3 activity was detected in 5-treated cells. In addition, a Western blot showed an increased level of cleaved caspase-3 in the 6treated sample after 5 min (Figure 5C). Furthermore, the lethality to 6-treated HeLa cells could be partially mitigated using the caspase-3 inhibitor Ac-DEVD-CHO, which caused an obvious decrease in cell death from 56 ± 5% to 20 ± 3% (Figure 5D). These data indicated that, indeed, 6 caused cell death through apoptosis. We next examined how 6, compared to 5, would manifest its cytotoxicity and selectivity in cancer cells compared to noncancer cells. A panel of human cancer cells, including A549, MCF-7, and HeLa, and several human noncancer cell lines, including HLF, 293T, and HUVEC, were used for comparisons. MTT results showed that 5 displays strong cytotoxicity in all the cell lines, and noncancer cells were slightly more resistant to 5-treatment, which might be due to the greater anionic membrane surface charge of cancer cells that led to stronger cationic peptide association (Figure 5E). On the contrary, 6 showed better selectivity of cytotoxicity against cancer cells, with a 4- to 9-fold difference of IC50 values between cancer and noncancer cells. Similar assays were performed using KL-rich peptides; the results showed that 2 displayed strong cytotoxicity across all the cell lines, while 3 had relatively weak cytotoxicity (Table S1). For each individual noncancer cell line, 6 appeared to be better tolerated than 5. The improved cell line selectivity of 6 suggests that, in addition to the cell surface charge difference, 6 may also exploit other mechanistic differences of cancer and noncancer cells. It is known that tumor cells are more metabolically active than normal tissue cells, leading to altered mechanisms in dealing with the intracellular oxidative stress caused by reactive oxygen species (ROS). Although the absolute ROS amount of a particular cell line can be affected by a number of factors, such as the cell volume and growth rate and how the ROS levels evolve during the cancer cell lineage development, which remains controversial,42,43 it is commonly accepted that cancer cells are more sensitive to ROS-promoting agent treatment because of their stronger reliance on the ROS stress−response pathway than normal cells.43,44 Therefore, we next investigated the effect of 6 on the ROS levels in cancer and noncancer cell

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DISCUSSION In this study, four novel peptides were designed for systematic comparisons of how peptide hydrophobicity, amphipathicity, and type of cationic residues (i.e., K or R) contribute to the cytotoxicity in cancer cells. An arginine-rich, hydrophobicityenhanced, amphipathicity-minimized peptide 6 with apoptosisinducing ability was uncovered based on close comparisons on these parameters. We found that replacing alanine with leucine of the previously reported 1 and 4 resulted in 2 and 5 with increased hydrophobicity and potent cytotoxicity. In an earlier report, an amphipathic peptide RL9 (RRLLRRLRR-NH2) was shown to bind to the cell membrane surface without entering the cell.46 This peptide contains six arginines and forms an amphipathic helical structure in the presence of negatively charged lipids similar to 5, but it has fewer hydrophobic leucines than 5 and exhibits low cytotoxicity in CHO cells. We next found that rearranging the residue positions of the second heptad of 2 and 5 resulted in peptides 3 and 6, with minimum helical amphipathicity. Both peptides exhibited reduced helicity when mixed with an LUV preparation and also showed a lack of self-aggregation in a water solution. While F

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discriminating normal and tumor cells may be developed for pharmaceutical applications.

3 showed significantly reduced membranolytic and cytotoxic activities, 6 showed strong cytotoxicity, despite its reduced membranolytic activity. The difference between 3 and 6 lies in the different physicochemical features of the Lys and Arg residues. It is known that Lys could be deprotonated in the membrane, whereas Arg maintains its charge.47 The guanidinium group of arginine has special affinity for the polar head groups of phospholipids; thus, the arginine residues of the peptide would facilitate its penetration into the cytosol. Our imaging result confirmed that 6 could cross the cell membrane and enter the cytoplasm. On the other hand, although 5 and 6 both have six arginines, 5 could not be internalized by the cell. Peptide 5 has a distinct distribution of hydrophilic and hydrophobic residues in its helical conformation, which would cause stronger retention and aggregation at the solution−membrane interface and override arginine’s ability of membrane penetration. In contrast, the low helical polarity of 6, together with the guanidinium groups of the arginine residues, facilitates the transport of 6 across the cell membrane. The difference of the cell interactive behaviors of 5 and 6 determined their different modes of action in killing cancer cells. Our study provides evidence that 5 kills cancer cells in an instant membranolytic way, whereas 6 used a progressive apoptotic cell death mechanism. The apoptotic cell death may be a consequence of general membrane stress caused by the insertion of the cationic−hydrophobic peptides into the cytoplasmic membrane systems, such as the endoplasmic reticulum or the mitochondria, impairing the cell metabolism machinery. Because of their rapid growth, frequent cell divisions, and active preparation for metastasis, cancer cells are subject to metabolically challenged conditions and are significantly reliant on ROS-response pathways, which can lead to the increased sensitivity of cancer cells to stress-inducing treatments, compared to normal cells. Many existing first-line chemotherapeutics exploit the stress-induced death mechanism of cancer cells,48 although there is often limited selectivity between cancer and noncancer cells.49,50 Our recent study also showed that a cationic hydrophobic cyclometalated iridium complex can induce endoplasmic reticulum stress and apoptosis in cancer cells.51 In this study, we demonstrated that 6 can induce a significantly higher level of ROS in cancer cells; thus, 6 displays a higher selectivity in killing cancer cells than normal cells. The increased metabolic vulnerability of cancer cells may be exploited as an opportunity for drug designs with new mechanisms of action. Properly designed cationic−hydrophobic peptides (CHPs) lacking strong amphipathicity, such as 6, could take advantage of two mechanisms for more selective killing of cancer cells: (i) preferential association and penetration of the more anionic membrane of cancer cells (but avoiding direct membrane disruption) and (ii) causing intracellular membrane stress and progressive cell death to the more metabolically challenged cancer cells. The sequential and synergistic combination of the two mechanisms may give CHPs an extra advantage in selectivity over the more broadly studied CAPs or the nontargeted chemotherapeutics in killing cancer cells. In conclusion, our study closely compared a panel of designed peptides to demonstrate that the factors of charge/ hydrophobicity balance, amphipathicity, and K/R composition synergistically contribute to their killing kinetics and mechanisms. When combined with designs of stability and targeting motifs, novel peptides with even better selectivity in

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EXPERIMENTAL SECTION

Materials and General Methods. All chemical reagents (analytical reagent grade) were used as received without further purification. All buffer components were biological grade and were used as received. The peptides used in this study were purchased from Shanghai BOTAI Bioscience & Technology Co., Ltd. (China) (>95% purity as assessed and purified using HPLC and characterized using MS; experimental details are listed in the Supporting Information). The quality control data are as follows: 1 (purity, 97.3%; MW calculated, 1774.27, found, 1773.9), 2 (purity, 99.5%; MW calculated, 1942.6, found, 1943.3), 3 (purity, 96.3%; MW calculated, 1942.6, found, 1941.8), 4 (purity,95.8%; MW cculated,1942.35, found, 1942.0), 5 (purity, 98.1%; MW calculated, 2110.68, found, 2110.0), 6 (purity. 96.3%; MW calculated, 2110.68, found, 2110.0). The HPLC chromatogram and electrospray-ionization mass spectra of the peptides are included in the Supporting Information (Figures S1− S6). The lyophilized peptides were stored at −20 °C. Before use, peptides were quantitated based on the peptide bond absorbance at 205 nm (Scopes method). Peptides dissolved in Milli-Q water or solutions were stored for a short term at 4 °C. [Ir(ppy)2(H2O)2]OTf was received from SunaTech, Inc. (Suzhou, China). MTT (Sigma) and 1,8-ANS (Aladdin) were used as indicated below. PI, Hoechst 33342 (Beyotime), Ac-DEVD-CHO (Beyotime), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime) were used according to reference guides. Fetal bovine serum, penicillin−streptomycin solution (SV30010), and culture media were obtained from Hyclone. Cell Lines and Culture. The human cancer cell lines HeLa (human cervical carcinoma cell) and HeLa-GFP, A549 (human lung adenocarcinoma cell line), MCF-7 (human breast cancer cell line), HUVEC (human umbilical vein endothelial cells), 293T (human embryonic kidney cells), and HLF (human lung fibroblasts) were purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). HUVEC, HLF, 293T, HeLa, MCF-7, and HeLa-GFP cell lines were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. A549 cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were incubated at 37 °C under 5% CO2. Circular Dichroism (CD) Spectroscopy. Peptides were dissolved in deionized (DI) water or 5 mM negatively charged large unilamellar vesicles (LUV) at a concentration of 100 μM. LUVs were used as a model membrane system for the CD studies. LUVs of DMPC/DMPG (1:1) were prepared using extrusion techniques. Briefly, DMPC and DMPG solutions in chloroform were dried under a gentle stream of nitrogen. Then, the samples were placed under vacuum for 16 h to remove the residual solvent. The lipid film was hydrated using PBS solution and vortexed for 10 min. Then, the lipid solutions were extruded using 100 nm pore size filters to obtain LUVs. The CD spectra of the peptide solutions were recorded at room temperature using a quartz cuvette (0.05 cm path length) and Chirascan PLUS (Applied Photophysics, Ltd.). The spectra were obtained from 190 to 260 nm with the solvent subtracted at a 5 nm/min scanning speed and averaged of three runs of each sample. The acquired CD signal spectra were then converted to the mean residue ellipticity ([θ]mr). [θ]mr was calculated using the equation [θ]mr = θobs/(10Icn), where θobs is the measured ellipticity in mdeg, I is the path length in cm, c is the concentration of peptide in M, and n is the number of amino acid residues of the constructs (n = 14 for the designed peptides). Peptide Self-Aggregation Assay. A series of peptide solutions containing 40 μM 1,8-ANS were prepared using the following procedure. Briefly, 2.5 μL of ANS solution (8 mM in ethanol) was added to a series of 1.5 mL centrifuge tubes, and the solvent was evaporated; then, the appropriate volumes of peptide solutions (1 mg/ mL in H2O) and H2O were added to obtain peptide concentrations ranging from 1.56 to 300 μg/mL with a total final volume of 500 μL. The mixed solutions were agitated using an oscillator at 250 rpm and room temperature for 10 min; then, the fluorescence emission G

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intensity (excitation 330 nm, emission 492 nm) was measured using a Hitachi F4600 fluorescence spectrophotometer. Cell Viability Assay. We used an MTT assay to examine the cytotoxic effect of the compounds in the cell lines. Cells were seeded in a complete growth medium in 96-well plates (Costar Corning, NY) at a density of 1 × 104 cells/well and were grown for 24 h before treatment. The growth medium was then substituted with fresh medium containing the peptides to be tested at the appropriate concentrations close to the IC50 values. After incubation for 24 h, 20 μL of aqueous MTT solution (5 mg/mL) was added to each cell; the cells were incubated for another 4 h, and the medium and MTT mixtures were removed. Next, 150 μL of DMSO was added to each well and incubated at 37 °C for 10 min. The absorbance of each sample at 490 nm was measured using a microplate reader (PerkinElmer, Victor X4). For time-lapse MTT, the cells were treated with the peptides for a shorter time series before adding the MTT solution. HeLa-GFP Fluorescence Leakage Assay. HeLa-GFP cells were seeded in 96-well plates at a density of 1 × 104 cells/well and grown for 24 h before treatment. The growth medium was then substituted with PBS containing the peptides to be tested at various concentrations. After incubation for 5 min, the culture solution was collected and centrifuged at 1000g. The supernatant was then transferred to a black 96-well plate for fluorescence determination. The fluorescence intensity of each sample at 485/535 nm was measured using a microplate reader (PerkinElmer, Victor X4). Triton X-100 (1%) was used to prepare a positive control of 100% leakage. Peptide Luminescence Labeling Using the Iridium Complex [Ir(ppy)2(H2O)2](OTf). Peptides were dissolved in Milli-Q water at a concentration of 1 mM, and the peptides and the iridium complex were diluted to an equivalent concentration (200 μM) with PBS and mixed at a 1:1 volume to allow a coordination reaction for 2 h at 37 °C in the dark. The mixture exhibited green fluorescence (excitation 328 nm) after the complete reaction. Confocal Cell Imaging. HeLa cells were seeded at a density of 1 × 105 cells/35 mm in a Fluorodish and were incubated at 37 °C under 5% CO2 for 24 h. Using a confocal microscope (Nikon A1R), the locations of the Ir-labeled peptides in the live HeLa cells were imaged after the cells were exposed to the peptides for different amounts of time (0, 5, and 15 min). Luminescence images were captured using an emission window of 500−560 nm (excitation 403 nm). Flow Cytometry. Cells were seeded in a 6-well plate and incubated at 37 °C under 5% CO2 for 24 h. The cells were then incubated with 5 μM 5 or 6 for 15 min and were later processed according to the manufacturer’s instructions of the annexin V-FITC apoptosis kit. Briefly, the cells were collected, centrifuged, and washed using PBS solution twice. Aliquots of annexin V-FITC binding buffer, annexin V-FITC, and PI were then added to the cells. After incubation in the dark for 20 min at room temperature, each sample was examined using a BD Accuri C6 flow cytometer (BD Corp. USA). Caspase-3 Activity and Inhibition Assay. To detect caspase-3 activity, the cells were cultured in 96-well plates and treated with 5 μM 5 or 6 for 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, or 4 h and analyzed using the Caspase-Glo assay kit (G8091, Promega, Madison, WI, USA) according to the manufacturer’s instructions. 100 μL of caspase-Glo 3/7 reagent was added to each well, and the plate was gently agitated using a plate shaker at 300 rpm for 30 s, followed by incubation at room temperature for 2 h. The samples were then transferred to a white 96-well plate, and the luminescence of each sample was measured using a microplate reader (PerkinElmer, Victor X4). For the inhibitor assay, the cells were first treated for 30 min with 50 μM Ac-DEVD-CHO, a specific irreversible inhibitor of caspase-3; then, they were incubated with or without 5 μM 6 for 30 min. Cell survival was determined using the MTT assay. Western Blot. HeLa cells were plated at a density of 8 × 105 cells/ 100 mm tissue culture dish in 8 mL of culture medium for 24 h. The cells were then treated with 8 mL of medium containing 5 μM 6 for 5, 10, or 15 min. Then, the drug-containing medium was removed, and the cells were washed using cold PBS; 400 μL of the cell lysis buffer (Beyotime) (containing 1 mM PMSF) was added to the samples,

which were incubated for 3 min on ice. Samples of the cell lysate were boiled in loading buffer for 10 min. All of the samples were subjected to electrophoresis (15% SDS−PAGE), followed by transfer to a PVDF membrane. The blots were blocked using 5% nonfat dry milk for 1 h at room temperature and incubated with rabbit monoclonal antibody against cleaved caspase 3 (Beyotime) at 4 °C overnight. After incubation, the PVDF membranes were washed using TBST plus 0.5% Tween-20 for 30 min, followed by incubation for 2 h at room temperature with HRP-labeled goat anti-rabbit secondary antibody (Beyotime). The blots were washed again using TBST plus 0.5% Tween-20 for 30 at 5 min intervals. The protein bands were revealed using the BeyoECL Plus system and a luminescent image analyzer (LAS 4000 EPUV mini, Fujifilm). Determination of ROS Generation. Changes of the intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) using a microplate reader. Cells in 96-well plates were incubated with DCFH-DA at 37 °C for 20 min and treated with or without 6 (5 μM) for 30 min. The cells were washed using PBS; then, the fluorescence intensity of each sample at 485/535 nm was measured using a microplate reader (PerkinElmer, Victor X4). Hemolysis Assay. Fresh mouse blood was collected in anticoagulant tubes containing heparin. The red blood cells were washed three times with 0.9% saline and purified and diluted to 4.0 × 108 cells/mL for the hemolysis assays. Various concentrations of the peptides were incubated with the RBCs at 37 °C for 1 h. Then, the test groups were centrifuged at 1000 rpm for 10 min, and the supernatants from each group were measured using a microplate reader (PerkinElmer, Victor X4) at 540 nm. RBCs treated with 1% Triton X-100 were used as a positive control, and the release rate of hemoglobin of this group was set as 100%. The data represent the mean ± standard deviation of three independent experiments.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b02016. HPLC chromatograms and electrospray ionization mass spectra of peptides; flow cytometry analysis of cell morphology or integrity; effects of D-6 on HeLa cells; hemolysis assay of 2 and 3; cytotoxicity of 2, 3 against several cancer and noncancer cell lines (PDF)

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AUTHOR INFORMATION

Corresponding Author

*H.F.: phone, (+86) 512 62872584; fax, (+86) 512 62872181; e-mail, [email protected]. Author Contributions ∥

X.L. and R.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to group members for detailed discussions and valuable technical advice. This work was supported by the National Natural Science Foundation of China (Grants 81573339, 31170777, 21302213).

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ABBREVIATIONS USED CAP, cationic amphipathic peptide; CHP, cationic−hydrophobic peptide; LUV, large unilamellar lipid vesicle; CD spectroscopy, circular dichroism spectroscopy; ANS, 8-anilino1-naphthalene sulfonate; PI, propidium iodide; ROS, reactive H

DOI: 10.1021/acs.jmedchem.5b02016 J. Med. Chem. XXXX, XXX, XXX−XXX

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oxygen species; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; DMSO, dimethylsulfoxide

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