CLNAs used in the study, which include α-eleostearic acid, β-eleostearic acid, β-calendic and jacaric acid, with an estimated purity >97%, were purchased from Larodan Fine Chemicals AB, Sweden. The stock solution (0.2 M) was prepared by dissolving the powder in sterile, cell culture-tested ethanol (Sigma-Aldrich Co., USA). All other chemicals were purchased from Sigma-Aldrich unless otherwise stated.

The human eosinophilic leukemia EoL-1 cell line obtained from the Riken Cell Bank (Tsukuba Science City, Japan) was originally established from the peripheral blood of a 33-year-old male with eosinophilic leukemia (15). The cells were maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin sulfate and 0.25 μg/ml amphotericin B in 0.85% saline) in 5% humidified air at 37°C.

Cell proliferation was measured using both the colorimetric MTT assay and fluorometric cell proliferation assay. Briefly, EoL-1 cells (1×10⁵/ml) seeded in 96-well microtiter plates were incubated at 37°C with different concentrations of jacaric acid for various periods of time. The anti-proliferative response was measured by a standard MTT colorimetric assay (16) and recorded by a Benchmark microplate reader (Bio-Rad Laboratories, USA). The result was also confirmed by using the CyQuant^® NF Cell Proliferation Assay kit (Molecular Probes; Invitrogen Corp., USA) (17) and recorded using a fluorescence plate reader (Tecan Polarion, USA).

EoL-1 cells (1×10⁵/ml) were synchronized with 0.5% heat-inactivated FBS (HI-FBS) overnight and incubated at 37°C with different concentrations of jacaric acid. Afterwards, the cells were stained by propidium iodide (PI) (Sigma-Aldrich), and the cell cycle profile was analyzed by flow cytometry (FACSCanto™ flow cytometer; BD BioSciences, USA) using the software ModFit LT V3.0 (Verity Software House).

The measurement was performed according to the manufacturer’s instructions in the Cell Death Detection ELISA^PLUS kit (Roche Applied Science, USA). Briefly, EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for various periods of time. The supernatant was transferred to the well of a streptavidin-coated 96-well microtiter plate, and the absorbance was measured at 405 nm. The degree of apoptosis was expressed as the enrichment factor, which was calculated as follows: Enrichment factor = DNA fragments in treated sample/DNA fragments in the control.

Induction of apoptosis can also be measured using Annexin V-GFP/PI dual staining method. Briefly, EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for 24 h. The cells were then resuspended in Annexin V binding buffer (BD Biosciences) supplemented with Annexin V-GFP fusion protein and PI. The samples were incubated for 30 min at room temperature and were analyzed for PerCP-Cy5.5 with an emission wavelength of 695 nm versus FITC fluorescence with an emission wavelength of 525 nm by the FACSCanto flow cytometer. The percentages of cells in the four quadrants were calculated by WinMDI (version 2.9) software.

EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for 24 h. Afterwards, the cells were resuspended in PBS supplemented with JC-1 dye (Molecular Probes; Invitrogen). The samples were incubated for 30 min at 37°C and then analyzed for red fluorescence (FL-2) with an emission wavelength of 575 nm vs. green fluorescence (FL-1) with an emission wavelength of 525 nm by the FACSCanto flow cytometer. The percentages of cells with membrane depolarization were calculated by WinMDI (version 2.9) software.

Protein expression was determined by western blot technique using a panel of specific antibodies. EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for 72 h. Cell pellets were collected and total proteins were extracted using cell lysis buffer. The protein concentration was measured using Bradford reagent (Sigma-Aldrich), and the protein samples were resolved on 12% polyacrylamide gels and transferred to PVDF membranes. The membranes were first incubated with the following primary antibodies: mouse anti-human Bax, Bcl-2, caspase-3, CDK2 and rabbit anti-human cyclin E antibodies (all from Santa Cruz Biotechnology, USA), rabbit anti-human pp53 antibody (Cell Signaling) and mouse anti-human β-actin antibody (Sigma-Aldrich), followed by incubation with HRP-conjugated secondary antibodies (GE Healthcare Ltd., UK) and finally developed with ECL reagent (Santa Cruz).

The procedures were similar as previously described with slight modifications (18). In brief, EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for 9 days. At day 4 and day 7 of the treatment, treated cells were washed with plain medium, and the cells (1×10⁵/ml) were further incubated with fresh medium supplemented with jacaric acid. Cell morphology was then determined by the preparation of cytospin smears. The EoL-1 cells (5×10⁴) were fixed onto a microscopic slide by cytocentrifugation at 500 rpm for 5 min using the Shandon Cytospin 3 centrifuge (Shandon Scientific Ltd., UK). The cells were air-dried and then stained with Hemacolor staining solutions (Diagnostica Merck, USA) for 15 sec and destained under de-ionized water. Finally, the air-dried cells on the slides were mounted with neutral mounting medium, Canada balsam (Sigma-Aldrich), and morphology was examined under a light microscope.

The procedures of Lung et al (18) were followed with slight modifications. EoL-1 cells (1×10⁵/ml) were incubated with different concentrations of jacaric acid at 37°C for 9 days. At day 4 and day 7 of the treatment, treated cells were washed with plain RPMI medium, and the cells (1×10⁵/ml) were further incubated with fresh medium supplemented with jacaric acid. The cells were harvested and fixed with 100% ice-cold methanol for 10 min at 4°C and washed once with PBS. Afterwards, the cells were incubated with normal mouse IgG (1 mg/ml; Sigma-Aldrich) for 30 min before staining with mouse anti-human EPO or mouse anti-human MBP monoclonal antibodies (0.5 mg/ml; Pharmingen Inc., USA) for 2 h. Lastly, the cells were incubated with FITC-labeled goat anti-mouse IgG (0.5 mg/ml; Sigma-Aldrich) for 1 h. The stained cells were analyzed for FITC fluorescence with an emission wavelength of 525 nm by the FACSCanto flow cytometer.

Each experiment was repeated at least three times and only the results of the most representative experiments are shown. The data are expressed as the arithmetic mean ± standard error (SE). One-way analysis of variance (ANOVA) or unpaired Student’s t-test was used for statistical analysis and the differences were considered as statistically significant at P<0.05.

To determine the anti-proliferative effect of CLNA isomers on the EoL-1 cells, the MTT reduction assay was performed. As shown in Fig. 2A, all four CLNA isomers were found to exhibit an inhibitory effect on the proliferation of EoL-1 cells in a concentration-dependent manner. In contrast, the solvent control (up to 0.1% v/v ethanol) did not exert any significant inhibitory effect on EoL-1 cells (<5% inhibition, data not shown). Jacaric acid was found to be the most potent isomer among all the CLNA isomers tested and therefore it was chosen as the specific target for further investigation in the present study. Fig. 2B shows that jacaric acid inhibited the growth of EoL-1 cells in a time- and concentration-dependent manner, and the estimated 50% inhibitory concentration (IC₅₀) after a 48-h treatment was found to be 5.33±0.86 μM. The growth-inhibitory effect of jacaric acid on EoL-1 cells was confirmed by the CyQuant^® NF Cell Proliferation Assay kit (Fig. 2C). Notably, jacaric acid exhibited no direct cytotoxicity on normal primary myeloid cells such as murine peritoneal macrophages, as the percentage of cell viability of the macrophages remained >90% even when the cells were incubated with 100 μM jacaric acid for 48 h (data not shown).

To determine the possible mechanisms of the anti-proliferative effect of jacaric acid on EoL-1 cells, cells were stained with propidium iodide after incubation with jacaric acid for 72 h, and the cell cycle profile was analyzed by flow cytometry. As shown in Fig. 3A, jacaric acid triggered cell cycle arrest at the G₀/G₁ phase, accompanied by a decrease in the percentage of cells at the S phase. To further elucidate the underlying mechanisms, western blotting was carried out in order to examine the protein expression levels of cyclin E, CDK2 and pp53 (Fig. 3B), which are known to be involved in the transition of G₀/G₁ to S phase (19,20). Our results showed that the protein expression levels of cyclin E and CDK2 decreased in the jacaric acid-treated EoL-1 cells, whereas there was an increase in the expression level of the pp53 protein (Fig. 3C–E). Collectively, the results indicate that jacaric acid treatment of EoL-1 cells leads to cell cycle arrest at the G₀/G₁ phase, and modulates the expression of certain cell cycle-regulatory proteins such as cyclin E, CDK2 and pp53.

Apart from triggering cell cycle arrest, another possible mechanism for the growth-inhibitory effect of jacaric acid is the induction of apoptosis. To examine whether jacaric acid induces apoptosis in the EoL-1 cells, the Cell Death Detection ELISA^PLUS kit was used according to the manufacturer’s instructions. It was found that jacaric acid induced DNA fragmentation in the EoL-1 cells, as revealed by the increase in the enrichment factor in the jacaric acid-treated EoL-1 cells in a time- and concentration-dependent manner (Fig. 4A), suggesting that jacaric acid induced apoptosis in the EoL-1 cells. To gain further insight into the molecular mechanism underlying the induction of apoptosis, the effects of jacaric acid on altering the expression levels of different apoptosis-regulatory proteins were examined by western blot analysis (Fig. 4B). The expression level of the anti-apoptotic protein Bcl-2 was decreased whereas the expression levels of Bax and caspase-3 proteins were increased (Fig. 4C–E). Apart from the Cell Death Detection ELISA, the Annexin V assay was used to detect the externalization of phosphatidylserine (PS) which is a hallmark of early apoptosis (21). In addition, JC-1 dye staining was used to measure the mitochondrial membrane potential which is known to decrease prior to PS externalization and DNA fragmentation (22,23). Our results showed that jacaric acid induced PS externalization in the EoL-1 cells at 24 h of incubation in a concentration-dependent manner as demonstrated by the Annexin V assay (Fig. 5), and reduced the mitochondrial membrane potential of the EoL-1 cells after staining with JC-1 dye (Fig. 6). Together these results suggest that jacaric acid inhibits the growth of EoL-1 cells by inducing apoptosis.

Induction of cellular differentiation can also lead to suppression of cell growth (24). As shown in Fig. 7B–D, after treatment with different concentrations of jacaric acid for 9 days, there were more refractive vacuoles in the treated-EoL-1 cells than those in the control (Fig. 7A). This suggests that the granule matrix was altered leading to the presence of small refractive vacuoles when observed under a light microscope. In order to confirm the induction of eosinophilic differentiation of EoL-1 cells by jacaric acid, the expression levels of two eosinophil-specific proteins, eosinophil peroxidase (EPO) and the major basic protein (MBP), were measured by flow cytometry. As shown in Fig. 7E and F, a significant increase in the expression levels of EPO and MBP proteins was noted after 9 days of treatment with 2 μM jacaric acid. Collectively, our results indicate that prolonged exposure (9 days) of EoL-1 cells to a low concentration (1–2 μM) of jacaric acid induces eosinophilic differentiation of the cells as represented by morphological changes and an elevation in EPO and MBP proteins.