Cells from the HCT116 human colon carcinoma-derived cell line were provided by Dr Bert Vogelstein (The Johns Hopkins University, Baltimore, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) and 5% antibiotic/antimycotic mixed stock solution (Nacalai Tesque) at 37°C and 5% CO₂. Cell passage and maintenance were performed as previously described (12).

The primary amino acid structure of the original LL-37 is LLGD FFR KSK EKI GKE FKR IVQ RIK DFL RNL VPR TES. hCAP is a 27-mer peptide that lacks the first and last 5 amino acids of LL-37. FF/CAP18 (FRK SKE KIG KFF KRI VQR IFD FLR NLV) was designed by replacing a glutamic acid residue and lysine residue with phenylalanine of hCAP18 to enhance its antimicrobial and anticancer properties (11).

HCT116 cells were seeded at a density of 1.2×10⁶ cells/ml in a 100-mm dish and incubated for 2 days. The cells were washed with phosphate-buffered saline and cultivated in DMEM containing 10% exosome-depleted fetal bovine serum Media Supplement (System Biosciences, Palo Alto, CA, USA) and 5% antibiotic/antimycotic mixed stock solution (Nacalai Tesque). After 48 h, the medium was harvested to collect exosomes. Exosomes were isolated by ultrafiltration using a Vivaspin 20-100K (GE Healthcare, Little Chalfont, UK) and miRCURY™ Exosome Isolation Kit-Cells, Urine and CSF (Exiqon, Vedbaek, Denmark). The culture medium was centrifuged at 300 x g for 5 min and at 1,200 x g for 20 min to remove cells and debris. The supernatant (20 ml) was ultrafiltered and condensed to 1 ml using the Vivaspin 20-100K to concentrate the exosomes. Exosome isolation was performed using a miRCURY™ Exosome Isolation Kit according to the manufacturer’s instructions (Exiqon). The protein levels of exosomes resuspended in 100 µl of resuspension buffer (Exiqon) were quantified using the DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After confirmation by transmission electron microscopy (TEM), a combination method was used to isolate exosomes from FF/CAP18-treated cells.

To examine the quality of isolated exosomes, the size, size distribution and morphology of non-treated HCT116 cells were evaluated. A total of 5 µl of each exosome sample (n=3) was applied to a 400-mesh copper grid for 10 min prior to negative staining with 10 µl of 2% phosphotungstic acid for 10 min. The grid was washed with 10 µl of distilled water, dried, and viewed by TEM using a model H-7650 microscope (Hitachi, Tokyo, Japan).

Cells and exosomes were homogenized in lysis buffer [1 M Tris-HCl (pH 7.4), 3 M NaCl, 1% Triton X-100, 6 mM sodium deoxycholate, and 0.5% protease inhibitor; Nacalai Tesque] by ultrasonic fragmentation as previously described (12). Protein concentration was measured using the DC protein assay kit. Cell lysates (20 µg) and exosome lysates (10 µg) were electrophoresed on 5-20% polyacrylamide gels (ATTO, Tokyo, Japan) and transferred to Immobilon-P membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with phosphate-buffered saline containing 3% skimmed milk and 0.05% Tween-20, and then incubated with primary mouse monoclonal antibodies against CD63 (1:1,000; 10628D; Invitrogen; Thermo Fisher Scientific, Inc.), CD81 (1:1,000; 10630D; Invitrogen; Thermo Fisher Scientific, Inc.) and α-tubulin (1:1,000; sc-32293; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing, the membranes were incubated with sheep anti-mouse IgG conjugated to horseradish peroxidase (1:2,000; GE Healthcare) as the secondary antibody. Signals were detected using a SuperSignal™ West Femto Maximum Sensitivity Substrate Trial Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and images were obtained with the LumiCube device (Liponics, Inc., Tokyo, Japan) and analyzed with Just TLC software (Sweday, Sodra Sandby, Sweden).

Synthetic FF/CAP18 was conjugated with 5-carboxyfluorescein (FAM; Scrum Co. Ltd., Tokyo, Japan). HCT116 cells were examined at 1 and 6 h after treatment with FAM-FF/CAP18 (40 µg/ml). The nuclei were visualized by staining with 1 µg/ml 4′,6-diamidino-2-phenyl-indole (Dojindo Laboratories, Kumamoto, Japan) 6 h after treatment with FAM-FF/CAP18. The mitochondria were also visualized by staining with 200 nM MitoRed (Dojindo Laboratories). The staining was visualized using a BZ-8100 fluorescence microscope (Keyence, Tokyo, Japan). Each experiment was conducted in triplicate.

The rate of cell apoptosis following treatment with FF/CAP18 was measured using the Muse^® Annexin V and Dead Cell assay kit (Merck Millipore). This assay is based on the appearance of phosphatidylserine on the surface of cells early during apoptosis, which can bind Annexin V. In the late phase of apoptosis or necrosis, phosphatidylserine on the cell surface binds to Annexin V and DNA binds to 7-AAD, which is a fluorescent intercalator of GC regions. The combination of Annexin V and 7-AAD is used to detect apoptotic cells and distinguish between apoptotic phases. HCT116 cells were treated with FF/CAP18 (20, 30 or 40 µg/ml). After 48 h, the cells were treated with trypsin and collected in 1.5-ml micro-tubes. These tubes were centrifuged at 800 x g for 5 min. The supernatant was aspirated and the cell pellets were individually resuspended in 100 µl fresh medium and added to 100 µl of Muse Annexin V and Dead Cell assay kit reagent. The cellular mixture was incubated for 20 min at room temperature and the cells were examined using a Muse Cell Analyzer (Merck Millipore). These experiments were repeated three times (n=3).

Cell viability was measured by the WST-8 assay (Dojindo Laboratories). HCT116 cells were seeded (2×10³ cells/well) in a 96-well plate with complete culture medium and incubated at 37°C in 5% CO₂. After 24 h, exosomes released by FF/CAP18-treated or untreated HCT116 cells were added to the corresponding cells and co-incubated for another 48 h. WST-8 reagent was added to each well and the samples were incubated for 4 h. Absorbance was measured at a wavelength of 450 nm using a Synergy™ HT (BioTek, Winooski, VT, USA). These experiments were repeated three times (n=3). Cell viabilities (%) were calculated as relative values based on the absorbance of non-treated cells (100%).

Exosomes were collected according to the exosome isolation protocol. Total RNA, including miRNAs, was extracted using the miRCURY™ RNA Isolation Kit-Cell and Plant (Exiqon) according to the appendix protocol of the miRCURY™ Exosome Isolation Kit-Cells, Urine and CSF (Exiqon). RNA quality [concentration, optical density (OD) 260/280, 260/230 nm] was determined using a BioSpecnano device (Shimadzu, Kyoto, Japan). The 3D-Gene™ human microRNA chips for miRNA expression analysis (Toray Industries, Inc., Tokyo, Japan) were used to analyze the effect of FF/CAP18 treatment on miRNA expression in exosomes. Total RNAs labeled with 3D-Gene miRNA labeling kit (Toray Industries, Inc.) were hybridized onto the 3D-Gene Human miRNA Oligo chip (Toray Industries, Inc.). Fluorescent signals were scanned with the 3D-Gene Scanner (Toray Industries, Inc.) and analyzed using 3D-Gene Extraction software (Toray Industries, Inc.). The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The data were globally normal-ized per array, such that the median of the signal intensity was adjusted to 25. Cellular RNA extraction and miRNA microarray were performed as previously described (12).

Data are presented as the mean ± standard deviation. Statistical significance between two groups was determined by the Student’s t-test of variance with least difference post hoc test. P<0.05 was considered to indicate statistically significant differences.

To ensure successful isolation of exosomes, the collected exosomes from non-treated HCT116 cells were observed as microvesicle clusters (Fig. 1A). For size distribution, 78% of exosomes were 40-100 nm (Fig. 1B). The amount of exosomes was 3.8×10⁻⁸ and 4.9×10⁻⁸ mg/cell in untreated controls and FF/CAP18-treated cells, respectively.

The characterization of exosomes, as determined by western blotting, is shown in Fig. 1C. The investigated CD63 and CD81 proteins were previously reported as being exosome-specific markers (21,22). CD63 was detected at 30-60 kDa, as expected from the datasheet for the CD63 antibody. CD81 was detected at 25 kDa. These two proteins were detected in HCT116 cells and exosomes before and after treatment with FF/CAP18. In HCT116 cells, the levels of CD63 and CD81 were increased after FF/CAP18 treatment. The expression levels of exosomes from FF/CAP18-treated cells were similar to those in controls, although both proteins were detected.

At 1 h after treatment with FAM-FF/CAP18, FF/CAP18 interacted with the cell membranes (Fig. 2A). Some FAM-FF/CAP18 was observed in the cells. At 6 h after treatment, FF/CAP18 was incorporated in the cytoplasm (Fig. 2B). No FF/CAP18 was detected on the cell membrane. As shown in Fig. 2B, the peptide localized in the cytoplasm, but not in the mitochondria or nucleus.

Cells were classified into four groups based on the reactivity of Annexin V and 7-AAD combined, as previously described (23). An increased concentration of FF/CAP18 was accompanied by an increased affinity for Annexin V (Fig. 2C). Treatment with FF/CAP18 at 20 and 30 µg/ml decreased the number of live cells (Annexin V^- and 7-AAD⁻) (Fig. 2C, upper right panel and lower left panel). Treatment with FF/CAP18 at 40 µg/ml increased the number of late apoptotic cells and apoptotic cell death (Annexin V⁺ and 7-AAD⁺). To determine the density of FF/CAP18 treatment, HCT116 cells were exposed to FF/CAP18 at a concentration of 33.8 µg/ml; this concentration was selected for the experiment as this dose caused the death of ~50% of HCT116 cells (Fig. 2D).

Exosomes were quantified with the DC protein assay to evaluate the effect of FF/CAP18 on the exosome secretion volume. The amount of pooled exosomes released from cells during FF/CAP18 exposure was 5.0×10^-8 mg/cell. The amount of exosomes released from non-treated control cells was 3.8×10⁻⁸ mg per cell. To determine whether exosomes affect the viability of HCT116 cells, exosomes released from control HCT116 cells (Exo CTRL) and from cells treated with FF/CAP18 (Exo FF) were harvested and added to HCT116 cells in the WST-8 assay. The effect on cell viability was evaluated at exosome protein concentrations of 15.63, 31.25, 62.5, 125 and 250 µg/ml. The cell viability (Exo FF group) was significantly decreased at 125 and 250 µg/ml (Fig. 3, P<0.01). The effect of Exo CTRL was less prominent compared with that of Exo FF.

An attempt was made to analyze the alterations in the miRNAs in exosomes to reveal the cause of cell viability suppression. A miRNA microarray revealed that 137 miRNAs were increased by 2-fold or more in exosomes released by FF/CAP18-treated HCT116 cells compared with exosomes released by non-treated cells (Table I). By contrast, 17 miRNAs were increased by 2-fold or more in HCT116 cells following FF/CAP18 treatment compared with non-treated cells (Table II). Three miRNAs (miR-584-5p, miR-1202 and miR-3162-5p) were upregulated in both HCT116 cells and exosomes following treatment with FF/CAP18.