OCI-AML-2 (AML-2) cell line was obtained from the Ontario Cancer Institute (Toronto, Canada), and Jurkart and HL-60 cell lines from the American Type Culture Collection (ATCC, USA). These three cell lines were cultured at 37°C in a 5% CO₂ atmosphere using α-MEM medium (GibcoBRL, Gland Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Sigma, ST. Louis, MO, USA). MCF-7 and NIH/3T3 cell lines from ATCC and SNU-601 cell line from the Korean Cancer Cell Bank (Seoul, Korea) were cultured under the same conditions but using RPMI-1640 medium (GibcoBRL, Gland Island, NY, USA) containing 10% heat-inactivated FBS. Cells were maintained in suspension or as monolayer cultures, and subcultured.

Peptides were synthesized by using a solid phase method using (based on) Fmoc (9-fluorenyl-methoxycarbonyl)-chemistry 8. Rink amide 4-methyl benzhydrylamine (MBHA) resin (0.55 mmol/g) was used as the support. Coupling of Fmoc-amino acids was performed by DCC (dicyclohexyl-carbodiimide/HOBt (1-hydroxybenzotriazole). After completion of peptide chain elongation, the protected final peptide resins were treated with the reagent K. The crude peptide obtained was then washed repeatedly with diethylether, dried in vacuum, and purified by reverse-phase (RP) HPLC (LC10A, Shimadzu, Tokyo, Japan) on a Waters 15-m Deltapak C18 column. The purity of the peptide was checked by analytical RP-HPLC using an Ultrasphere C18 column (4.625 cm, Beckman, Miami, FL, USA). Purified peptides were hydrolyzed with 6 N HCl at 110°C for 22 h, and then dried in vacuum. The residues were dissolved in 0.02 N HCl and subjected to amino acid analysis (8500 A, Hitachi, Tokyo, Japan) to determine peptide concentration. The molecular weights of the synthetic peptides were determined by MALDI (matrix-assisted laser desorption ionization) mass spectra, respectively (data not shown).

The in vitro cytotoxicity of the Pep27 analogues produced was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma, ST. Louis, USA] assay, as described by 9. IC₅₀ values were determined directly from semilogarithmic dose-response curves. Experiments were carried out at least in duplicate.

Peptide hemolytic activities were measured as described by 10, by determining hemoglobin release by 4% suspensions of fresh human erythrocytes at 414 nm. Briefly, human erythrocyte cells were centrifuged and washed three times with phosphate-buffered saline (PBS: 35 mM phosphate buffer/0.15 M NaCl, pH 7.0). 100 ml of human red blood cells suspended 8% (v/v) in PBS were the inoculated into 96-well plates. 100 ml of the peptide solution, Pep27 or its analogues, was then added to each well. The plates were incubated for 1 h at 37°C, and centrifuged at 150 g for 5 min. 100 ml aliquots of the supernatant obtained were transferred to flat-bottom 96-microtiter plates. Hemolysis was determined by measuring absorbance at 414 nm with an ELISA plate reader (Molecular Devices Emax, Sunnyvale, CA, USA). Hemolysis rates of 0% or 100% were determined as the same way with PBS and 0.1% Triton-100, respectively. Hemolysis percent was calculated using the following equation: % hemolysis = [(Abs414nm in the peptide solution - Abs414nm in PBS)/(Abs414nm in 0.1% Triton-100 - Abs414nm in PBS)] × 100.

FITC was freshly dissolved in DMSO to 10 mg/ml, and added to 1 mg/ml of peptide in 100 mM sodium bicarbonate, pH 9.3 to a final concentration of 1 mg/ml FITC. After incubation for 4 hr in the dark at room temperature, 1 M ethanolamine was added to inactivate the residual FITC. The solution was then left in the dark for an additional 2 hr, and chromatographed sequentially on a gel-filtration column followed by a 120 cm Sephadex G-50 column to remove unconjugated dye. The FITC-labeled peptide (FITC-Pep27anal2) was obtained by reverse phase HPLC using a C18 column. Conjugation between FITC and the peptide of interest was confirmed by SDS-PAGE gel as intense peptide fluorescence under UV light.

Jurkat cells grown on glass coverslips overnight were treated with FITC-labeled Pep27anal2 at a concentration of 5 μg/ml. The cells were washed with PBS at 30, 60 and 120 min being treated of Pep27anal2, and then fixed using 3.7% formaldehyde for 15 min. Coverslips were mounted on glass slides, FITC was excited using a 488-nm laser, and images were manipulated using the manufacture's software.

Annexin V binding was assessed by flow cytometry, and cell staining was evaluated using FITC-labeled annexin V (green fluorescence), and propidium iodide (PI) (negative for red fluorescence) simultaneously. This test was able to discriminate between intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+) (Vermes et al., 1995). Jurkat cells (10⁵/ml) were exposed to etoposide (100 μM), mellitin (12.5 μM) and Pep27anal2 (12.5 μM and 62.5 μM) for 4 hr. Cells were washed with PBS, resuspended in 400 μl of binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5.mM CaCl2), and incubated with 10 μl of annexin V-FITC (MedSystems Diagnostics, GmbH, Australia) for 10 min at room temperature. After incubation, cells were washed with binding buffer and exposed to 1 μg of PI in a final volume of 400 μl. FITC and PI fluorescences were determined using a flow cytometer (Becton Dickinson, Franklin Lakes, FL, USA)

Jurkat cells were harvested and immediately fixed in phosphate-buffered glutaraldehyde solution (pH 7.2) for 2 hrs and then fixed in 1% buffered osmium tetroxide for 2 hrs at 4°C. After processing through a graded series of ethanol and propylene oxide, the cells were embedded in Epon. A semi-thin (1 μm) section was cut and stained with toluidine blue for the ultrastructural examination. Ultra-thin (60–90 nm) sections were also prepared from the Epon block above using an ultramicrotome (LKB-V, Sweden), stained with uranyl acetate and lead citrate, and examined under a Hitachi H-7600 electron microscope (Hitachi, Kyoto, Japan). Apoptosis detection was done by taking photographs (×1,000) of the entire field of each ultra-thin sectioned sample. The degree of apoptosis (apoptosis index) was defined as the number of apoptotic cells expressed as a percentage of the total cell count.

Cytochrome c immunoblotting was performed using a slight modification of a previously described method 11. Jurkat cells were collected by centrifugation at 200 g for 5 min at 4°C. The cells were then washed twice with ice-cold PBS, pH 7.4, and centrifuged at 200 g for 5 min. The cell pellet obtained was then resuspended in 600 μl of extraction buffer, containing 20 mM Hepes-KOH (pH 7.5), 10 mM KCl, 250 mM sucrose, 1 mM EGTA, 1 mM EDTA, 1.5 mM MgCl₂, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride.

After incubation for 30 min on ice, the cells were homogenized (20 strokes), spun at 14,000 g for 15 min, supernatants were separated from the mitochondrial fraction, and both were stored at -70°C, until required for gel electrophoresis. The mitosol fraction was obtained by adding 0.1% SDS. Fifty μg of cytosolic protein extracts and the mitosol extracts were loaded into the lanes of an 15% SDS-polyacrylamide gel, separated and blotted onto a nitrocellulose membrane (Amersham, Piscataway, NJ, USA) at 150 mA overnight in transfer buffer (20 mM Tris-base, 150 mM glycine, 20% methanol). Non-specific binding was blocked by incubating the membrane in 5% non-fat milk in TBS-T (Tris-buffered saline and Tween-20; 10 mM Tris-HCl pH 8.0, 50 mM NaCl, and 0.05% Tween-20) for 3 h at RT. The membrane was incubated in anti-cytochrome c monoclonal antibody (7H8.2Cl2) solution (1:2000 dilution, BD PharMingen, San Diego, CA, USA) containing 0.5% non-fat milk in TBS-T. After overnight incubation with agitation at 4°C, the membrane was washed three times with 0.5% non-fat milk in TBS-T. The secondary antibody, a horseradish peroxidase-coupled anti-mouse antibody (Sigma, ST. Louis, MO, USA), was incubated at a 1:1000 dilution for 2 h at RT. The membrane was then washed four times (5 min) with 5% non-fat milk in TBS-T.

The peptide samples used for NMR experiments contained 1.5 mM peptide in water /2, 2, 2-trifluoro-(d₃)-ethanol (TFE) mixture (50:50; v/v) at pH 7.2, which was prepared after lyophilizing samples in an aqueous solution. NMR spectra were recorded at 15°C on a Bruker DRX-500 spectrometer equipped with a triple-resonance probe and an x, y, z-shielded pulsed-field gradient coil. Two-dimensional (2D) NMR spectra were recorded in the phase-sensitive mode using time-proportional phase incrementation 12 for quadrature detection in the t₁ domain. The 2D experiments such as TOCSY was performed using a MLEV-17 spin-lock pulse sequence with a mixing time of 69.7 ms and 2D-NOESY with mixing time of 300 ms. Solvent suppression during TOCSY and NOESY experiments was achieved using a WATERGATE pulse sequence 13 in combination with a pulsed-field gradient pulse. All NMR spectra were acquired with 2048 complex the t₂ data points and 256 increments in the t₁ dimension, with 32 scans per increment. Slowly exchanging amide protons were identified by lyophilization of a fully protonated sample in 50% H₂O/ 50% TFE, redissolution in 50% D₂O/ 50% TFE solution, and the immediate acquisition of a series of one-dimensional NOESY and 2D-NOESY spectra. ₃J_HNα coupling constants were measured from the 1D spectrum. NMR data were processed on a Silicon Graphics Indigo II workstation using XWINNMR (Bruker Instruments, Karlsruhe, Germany) software. Proton chemical shifts were expressed relative to the methyl resonance of sodium 2, 2-dimethyl-2-silapentane-5-sulfonic acid (DSS), used as an internal standard.

Data are expressed as means ± SEM. One-way ANOVA and the Student's t-test were performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 7.5 (SPSS, Korea). Results were considered significant when p was <0.05.

The amphipathic features of α-helical antimicrobial peptides plays an important role against target cells. Furthermore, a number of parameters, including a net positive charge, α-helicity, and overall hydrophobicity have been shown to modulate the antibiotic activity of the α-helical amphipathic antimicrobial peptides. Recent investigations using amphipathic α-helical model peptides have revealed that the modulation of the hydrophilic-hydrophobic balance of α-helical antimicrobial peptides is a crucial factor in the design of peptides with potent antibiotic activity 14. In a preliminary experiment, we could not find that Pep27 has anticancer activity. Therefore, we designed Pep27 analogue peptides and evaluated their anticancer and chemosensitizing effects. In order to elucidate the relationship between the hydrophobic and the hydrophilic regions of antibiotic peptides with respect to antibiotic activity and cytotoxicity, we designed and synthesized certain Pep27 analogues, on the basis of the α-helical wheel diagram of Pep27. In particular, Pep27 hydrophobicity was increased by substituting tryptophan. The amino acid sequences used in the present study are summarized in Table 1. Synthesized peptides were purified by RP-HPLC and their molecular weights were confirmed by MALDI-MS.

RP-HPLC has often been used to experimentally determine the quantitative hydrophobic-hydrophilic balance of amphipathic peptides. Peptides are separated by RP-HPLC according to their hydrophobic interactions with C-18 of the stationary phase. Such hydrophobic interactions can be considered comparable to the interaction between amphipathic antimicrobial peptides and the lipid bilayer of plasma membranes. In order to investigate the possibility of a correlation between peptide hydrophobicity and the biological activities on cancer cells, the hydrophobic characteristics of the peptides were investigated by comparing their retention times on RP-HPLC (Table 1). As expected, all Pep27 analogues eluted later than Pep27, and the analogue Pep27anal2 had the longest retention time.

To evaluate the anticancer activities of the Pep27 analogues, different concentrations of these peptides were administered to various cancer cell lines, and cytotoxicities were then determined by MTT assay. In this study, we used both anchorage-dependent and independent cancer cell lines. These included AML-2 an acute myelogenous leukemia cell line, HL-60 an acute promyelocytic leukemia cell line, Jurkat a T cell leukemia cell line, SNU-601 a gastric cancer cell line and MCF-7 a breast cancer cell line. Pep27 analogue peptides showed similar anticancer activities in five cancer cell lines tested. Of the analogues tested, Pep27anal2 showed most anticancer activity (Fig. 1). The IC₅₀ and IC₉₀ values of Pep27anal2 in five cancer cell lines tested were 10 – 28 μM and 35 – 55 μM, respectively (Fig. 1 and Table 2). Since the substitution of (²R→W) and (⁴E→W) to form Pep27anal1 showed less activity than Pep27anal2, the additional substitution of (¹¹S→W) and (¹³Q→W) in Pep27anal2 was viewed as being important for anticancer activity. On the other hand, other Pep27 analogues substituted with more than two tryptophans at different sites did not show more anticancer activity than Pep27anal2 in 5 cell lines tested. Moreover, of the Pep27 analogues produced, Pep27anal2 had the greatest hydrophobicity, based on its RP-HPLC retention time (Table 1).

In order to determine whether or not Pep27anal2 can cause membrane damage, an RBC hemolysis test was performed (Table 3). Of the Pep27 analogues, only Pep27anal 2 at 12.5 μM for 1 h at 37°C, hemolysed 18% of RBCs whereas mellitin at the same concentration induced 100% hemolysis, suggesting that Pep27anal2 may cause membrane damage like mellitin. Since the mechanism of Pep27anal 2 peptides is believed to involve disruption of the cell membrane, confocal microscopy used to determine whether Pep27anal2 can enter cells and act as a membrane toxin. Jurkat cells were treated with Pep27anal2-FITC at a concentration of 12.5 μM for 30, 60 and 120 min. After incubation cells were fixed with 3.7% formalin for 30 min and then observed under a confocal microscope. Confocal microscopy showed ring-shaped cells, suggesting that Pep27anal2-FITC is localized to the cell membrane without disturbing its integrity, and then moves from the membrane intracellularly with time (Fig. 2). However, Jurkat cells treated with 12.5 μM Pep27anal2 for more than 60 min showed cell shrinkage and membrane blebbing, suggesting apoptosis, and indicating that Pep27anal2 can enter cells by an unknown mechanism and induce apoptosis whilst maintaining membrane integrity, within 2 hr of treatment.

One of the earliest events to occur during apoptosis is the externalization of phosphatidylserine, a phospholipid that is normally restricted the inner leaflet of the plasma membrane. Importantly, this precedes membrane damage. Moreover, the externalization of phosphatidylserine can be monitored using annexin V, a phosphatidylserine-specific binding protein 15. Another important secondary event during apoptosis is the loss of plasma membrane integrity. Intact plasma membranes exclude certain dyes, like PI, the most commonly used marker of membrane integrity 16.

In the present study, Jurkat cells were treated with 100 μM of etoposide, 12.5 μM of mellitin or 12.5 μM or 63 μM of Pep27anal2 for 4 hr. Apoptosis and membrane integrity were then determined using annexin V and propidium iodide, respectively, as described in "Materials and methods". In Fig. 4. living cells are confined to the lower left quadrant. Dead cells appear first in the lower right quadrant (apoptosis), where death was due to the externalization of PS (detected with annexin V-FITC). The upper right quadrant shows cell death resulting from the loss of cell membrane integrity (PI incorporation). Fig. 3 shows that Pep27anal2 caused a higher level of annexin V-FITC staining with a little increase of PI staining than etoposide, whereas mellitin increased the distribution of PI staining without an increase of annexin V-FITC staining.

An electron microscopic examination revealed that Pep27anal2 induced apoptosis in Jurkat cells, as characterized by condensation of the cytoplasm, cell shrinkage, loss of plasma membrane microvilli, condensed or fragmented nuclei, and the formation of membrane vesicles, as in etoposide (Fig. 4). Cells treated with Pep27anal2 increased apoptosis significantly (P < 0.05) in a time-dependent manner. Moreover, 62.5 μM Pep27anal2 induced more apoptosis than 100 μM of etoposide (Fig. 4).

Cytochrome c is a well-characterized mitochondrial protein and is involved in cellular energy metabolism. The precursor of cytochrome c, apocytochrome c, is synthesized in the cytoplasm. Upon translocation to mitochoindria, cytochrome c is refolded and acquires a heme moiety, which is required for functionality in the mitochondrial respiration chain. The heme-bound form of cytochrome c is called holocytochrome c, which can activate the caspases responsible for apoptosis by interacting with protease activating factors when released into the cytosol 17. Jurkat cells (5 × 10⁵/ml) were treated with Pep27anal2 at a concentration of 12.5 μM for 1 h, 2 h, and 4 h and at 62.5 μM for 4 h. After these incubations, cytochrome c in the cytosol fraction, obtained by 14,000 g centrifugation for 15 min, and in the mitosol, obtained by adding 0.1% SDS to the mitochondrial fraction, was determined by Western blotting. Pep27anal2 at 62.5 μM did not induce cytochrome c release from mitochondria into the cytosol within 4 hrs (Fig. 5).

Complete proton resonance assignments for Pep27 and Pep27anal2 were obtained using the standard sequential resonance assignment procedure. Once the individual spin systems had been classified, backbone sequential resonance assignments were completed by d_αN (i,i+1) NOE connectivities in the 2D-NOESY spectra. Figure 6 summarizes the sequential and short-range NOE connectivities observed for Pep27 and Pep27anal2 in TFE/H₂O (1v/1v). The observations of continuous d_NN(i,i+1) contacts, together with the characteristic d_αN(i,i+3) and d_αβ (i,i+3) NOEs strongly support the existence of α-helices (Fig. 6). The amide proton temperature coefficient also supports the hypothesis that intramolecular hydrogen bonds stabilize the structures of the Pep27 and Pep27anal2 helices. This result is further supported by the backbone amide proton exchange data of both peptides 18, and the small ³J_HNα coupling constants of Pep27anal2 (that of Pep27 could not be measured). The NMR structures of Pep27 and Pep27anal2 were calculated using the experimental restraints derived from the 2D-NOESY spectra. A total of 50 distance geometry geometric structures served as starting structures for the dynamic simulated-annealing calculations for the peptides in TFE/H₂O solution. The 20 lowest-energy structures (<SA>_k) of 50 simulated annealing structures were selected for detailed structural analysis. A best-fit superposition of 20 <SA>_k structures and an energy-minimized average structure () are shown in Fig. 7. The main secondary-structural feature of Pep27 is one α-helix spanning residues, His6 -Leu15 in TFE/H₂O (1v/1v), whereas Pep27anal2 has a more stable α-helix spanning Trp 4-Leu15 (Fig. 7).