N. fowleri trophozoites (ATCC No. 30215) were cultured under axenic conditions in Nelson's medium at 37℃ [16]. Before using N. fowleri trophozoites, their pathogenicity and cytotoxicity were tested in mice or target cells, respectively [13]. N. fowleri lysate was prepared according to the method of a previous paper [12]. Non-pathogenic N. gruberi (ATCC No. 30960) was cultured at 27℃ in modified PYNFH medium [17]. U87MG cells were grown in a monolayer in 75-cm² flasks containing 12 ml of Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, Missouri, USA) with 10% fetal bovine serum in a fully-humidified incubator containing 5% CO₂ at 37℃.

The U87MG cells were seeded on 6-well tissue culture plate (Nunc, Roskilde, Denmark). For non-contact culture system, a tissue-culture insert (Nunc) with a membrane pore size of 0.2 µm was placed in a 6-well tissue culture plate containing 2 ml of DMEM without fetal bovine serum. Then, N. fowleri trophozoites were put onto a tissue-culture insert and co-cultured with U87MG cells for 30 min, 2 hr, and 4 hr in 5% CO₂ incubator at 37℃. For a control group, 3 × 10⁵/well of U87MG cells or N. fowleri trophozoites were seeded in a 6-well tissue culture plate. For other control experiments, N. fowleri trophozoites were cultured with Nelson's medium, and U87MG cells were cultured with DMEM. After 30 min, 2 hr, and 4 hr, the conditioned media were collected and used for LDH release assay, and U87MG cells for FACS analysis. The cultures were assessed under an inverted microscope (Nikon, Tokyo, Japan) and photographed for documentation during culture.

Flow cytometry experiments were performed to identify early apoptotic cells and to discriminate late apoptotic/necrotic and living cells. Apoptotic cells were detected by flow cytometry using a FACS Vantage (Becton Dickinson, Franklin Lakes, New Jersey, USA), after staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide (PI) using the Annexin V-FITC kit™ (Trevigen Inc., Gaithersbug, Maryland, USA). The U87MG cells and N. fowleri trophozoites were incubated at density of 3 × 10⁵ in a 6-well tissue culture plate for 30 min, 2 hr, and 4 hr. The U87MG cells were harvested by washing with PBS followed by treatment with trypsin-EDTA solution (Sigma). The cell pellet was resuspended in the binding buffer at a concentration of 1 × 10⁵ cells/ml. After adding annexin V fluorescein isothiocyanate and PI and incubating for 15 min at room temperature in the dark, the cells were run on a FACS Vantage. In this study, we used tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or N. fowleri lyaste for the control. TRAIL is known as the death ligand, which induces preferential apoptosis of transformed tumor cells, and N. fowleri lysate shows the cytotoxic effect on target cells. For each experiment, 1 × 10⁴ cells were analyzed using the CellQuest software (Beckton Dickinson). In FACS analysis, the lower right quadrant represents early apoptotic cells positive for annexin V and negative for PI, and the upper right quadrant represents apoptotic or necrotic cells positive for both annexin V and PI. The lower left quadrant represents healthy cells negative for annexin V and PI staining as a control.

To evaluate the cytotoxicity of N. fowleri on target cells under a non-contact culture system, lactate dehydrogenase (LDH) release assay was performed according to the previous study [13]. Briefly, 3 × 10⁵ of N. fowleri trophozoites as effector cells and 3 × 10⁵ of U87MG cells as target cells were co-cultured in a non-contact condition which was previously described above. The supernatant containing LDH released from target cells was collected at 30 min, 2 hr, and 4 hr. As control groups, U87MG cells were directly treated with N. fowleri lysate or N. gruberi lysate (1 mg/ml) for 4 hr. For LDH release assay, 50 µl of reacted supernatant in each well was transferred onto 96-well assay plates (Nunc). After 50 µl of the reconstituted assay buffer in CytoTox96® Non-radioactive Cytotoxicity Assay Kit (Promega, Madison, Wisconsin, USA) was added, the plate was incubated for 30 min at room temperature, and then 50 µl of stop solution was added. The reactions were read at 490 nm with ELISA reader. Values were means ± standard errors of 3 experiments in triplicate. The formula of in vitro cytotoxicity was as follows: Cytotoxicity (%)=Sample release-Spontaneous release/Maximum release-Spontaneous release×100

Statistical differences between groups or samples were determined with Student's 2-sample t-test. The difference was considered significant when P was < 0.05.

The U87MG cells co-cultured with N. fowleri trophozoites at 30 min, 2 hr, and 4 hr in a non-contact system showed serious morphologic changes such as the cell membrane destruction and a reduction in cell number during the culture periods (Fig. 1). However, U87MG cells treated with 1 mg/ml of N. fowleri lysate revealed only a little morphologic change similar to an untreated control group (Fig. 1).

To detect the externalization of phosphatidylserine in early phase of apoptosis, we performed annexin V-FITC binding assay. N. fowleri trophozoites induced the apoptosis of U87MG cells in a non-contact culture system, as which showed the distinguishment between viable, apoptotic, or necrotic cells by staining of annexin V in combination with PI (Fig. 2). In FACS dot plots, apoptotic cells shifted the lower right quadrants, annexin V-positive and PI-negative staining populations. The U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system significantly increased the percentage of apoptotic cells in comparison with the control or N. fowleri lysate-treated group (P < 0.01). When U87MG cells were co-cultured with N. fowleri for 30 min, 2 hr, and 4 hr in a non-contact system, the populations of annexin V-positive and PI-negative cells were 16.1, 16.6, and 16.7%, respectively (Fig. 2). The U87MG cells treated with N. fowleri lysate for 4 hr showed the decreasing populations of the necrotic and apoptotic cells (2.5 and 2.7%, respectively). Otherwise, when U87MG cells were treated with 100 ng/ml of TRAIL (a positive control) for 4 hr, the population of annexin V-positive and PI-negative cells was 13.3% (Fig. 2).

To further confirm the in vitro cytotoxicity of N. fowleri trophozoites, we performed LDH release assay. When U87MG cells were co-cultured with N. fowleri trophozoites for 30 min, 2 hr, and 4 hr in a non-contact system, N. fowleri trophozoites showed 40.5, 44.2, and 45.6% cytotoxicity in a time-dependent manner, respectively (Fig. 3). When the U87MG cells were treated with 1 mg/ml of N. fowleri lysate, the cytotoxicity of amebae on target cells ranged from 10.2 to 13.6% (Fig. 3). In other words, N. fowleri trophozoites co-cultured with target U87MG cells for 4 hr in a non-contact system showed maximum 45.6% cytotoxicity, whereas N. fowleri lysate showed maximum 13.6% cytotoxicity (P < 0.01). Under the same conditions, non-pathogenic N. gruberi trophozoites showed 10.2, 12.4, and 13.2% of cytotoxicity, whereas the cytotoxicity of N. gruberi lysate ranged from 6.1 to 6.4% (Fig. 3).