Identification of a Bioactive Compound against Adult T-cell Leukaemia from Bitter Gourd Seeds

In our previous report, an 80% ethanol bitter gourd seed extract (BGSE) was found to suppress proliferation of adult T-cell leukemia (ATL) cell lines. The present study aimed to identify the bioactive compounds from BGSE specific against ATL. From the result of an HPLC-MS analysis, α-eleostearic acid (α-ESA) was present in BGSE at 0.68% ± 0.0022% (±SD, n = 5). In the cell proliferation test, α-ESA potently suppressed proliferation of two ATL cell lines (ED and Su9T01; IC50 = 8.9 and 29.3 µM, respectively) more than several other octadecanoic acids. However, α-ESA moderately inhibited phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMC; IC50 = 31.0 µM). These results suggest that BGSE-derived α-ESA has potential as a functional food constituent because of its activity against ATL, particularly against ED cells. Moreover, α-ESA might be effective for the prevention of moderate adverse effects of ATL on normal T cells.


Introduction
Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type I (HTLV-I) infected individuals. After transmission of HTLV-I, 2%-5% of carriers are likely to develop ATL after a long latency period (30-50 years) [1]. These patients have been frequently identified as being from a restricted area of tropical regions [2]. It is currently very difficult to effectively treat patients with ATL using existing therapeutic methods, and most clinical trials focus on chemotherapeutic treatment and allogeneic hematopoietic stem cell transplantation. Therefore, it is important to find appropriate therapeutic methods to prevent the development of ATL or to prolong survival after its occurrence.
In our previous report, we screened 52 agricultural plant samples for their ability to inhibit proliferation in seven kinds of ATL related cell lines to start structure of a study for finding potential drug candidates with the prevention of ATL. We found that an 80% ethanol bitter gourd (Momordica charantia L.) seed extract (BGSE) showed an inhibitory effect on the proliferation of ATL-related human leukemia cells [3].
Bitter gourd belongs to the Cucurbitaceae family and is cultivated worldwide as a vegetable crop. The fruit is not only used as a food, but also for its medicinal properties, such as anti-microbial, anti-diabetic, anti-HIV and anti-tumor activities, which were described in a recent review [4]. BGSE has also been reported to have anti-leukemic potential on human acute myelogenous leukemia cells (HL-60) [5]. However, HL-60 and ATL cell lines comprise different types of leukemia cells, and as there have been no reports about bioactive compounds from BGSE active against ATL, the effect of BGSE on ATL cell proliferation requires elucidation. The aim of the present study was to identify bioactive compounds in BGSE exhibiting activity against ATL. Figure 1a,b show the HPLC-DAD chromatogram (a) and HPLC-MS-total ion chromatogram (TIC) (b) of BGSE. As shown in Figure 1a, a major peak (peak 1: retention time (RT) = 8.570 min, λ = 270 nm) was detected for BGSE. The TIC showed several peaks (Figure 1b). Figure 1c shows the MS spectrum of the peak with the RT: 8.570 min. in Figure 1b, which shows a deprotonated molecular ion signal at m/z 277. This compound was estimated to be C 18 H 29 O 2 and was proposed to be α-ESA on the basis of reference data [6]. α-ESA was analyzed using the same method.  Figure 1c). From these results, peak 1 in Figure 1 was determined to be α-ESA, and α-ESA is the main compound in BGSE.

Identification of Active Compounds in BGSE
We also attempted to quantitatively determine α-ESA in BGSE. Figure 2 shows the α-ESA calibration curve. The calibration curve showed good linearity (R 2 = 0.9977). α-ESA was contained in the concentration range of 34.0 and 34.2 µg/5 mg of freeze-dried bitter gourd seeds (0.68% ± 0.0022%, ±SD, n = 5). In our previous report, BGSE suppressed the proliferation of ATL cell lines [3]. The current study shows the presence of α-ESA in BGSE. Tsuzuki et al. reported that α-ESA accounted for about 60% of the total fatty acid composition of bitter gourd seed oil [7]. Therefore, α-ESA might have greatly contributed to suppressing the proliferation of ATL cell lines.

The Inhibitory Effects of Octadecanoic Acid Analogs on ATL Cell Lines
Of the octadecanoic acids, conjugated linoleic acids (CLA), which includes α-ESA, has been acknowledged to have numerous biological activities, such as anti-obesity, anti-diabetes, anti-cancer, anti-arthritis, anti-asthma, and anti-cardiovascular disease effects [8]. However, no study has yet reported the anti-leukemic effects of α-ESA on ATL cell lines. We examined the inhibitory effects of related compounds, seven octadecanoic acid groups, on the proliferation of two types of ATL cell lines (ED and Su9T01). As shown in Table 1, α-ESA (C18:3, n-5), γ-linolenic acid (C18:3, n-6), and α-linolenic acid (C18:3, n-3), which belong to the triunsaturated fatty acid group, substantially inhibited ED cell growth (IC 50 values of 8.9, 61.3 and 129.9 µM, respectively) and Su9T01 cell growth (IC 50 values of 29.3, 174.3 and 167.4 µM, respectively). Linoleic acid (C18:2, n-6), which belongs to the diunsaturated fatty acid group, inhibited ED and Su9T01 cell growth (IC 50 values of 100.7 and 180.1 µM, respectively). In ED cells, these compounds exhibited higher activity than EGCG, which was used as the positive control [9] (IC 50 = 152.7 µM). On the other hand, in Su9T01 cells, only α-ESA exhibited higher inhibitory activity than EGCG (IC 50 = 166.0 µM). We cannot calculate the exactly IC 50 values of monounsaturated fatty acid group (oleic acid (C18:1, n-9) and elaidic acid (C18:1, n-9)) and saturated fatty acid (stearic acid (C18:0)) in both cell lines. This assay was employed to compare the effects of octadecanoic acids on ED and Su9T01 cell growth; α-ESA showed the highest inhibitory activity. IC 50 values decreased roughly in proportion to the number of double bonds; therefore, the number of double bonds was an important determinant of anti-proliferation activity. Indeed, the triunsaturated fatty acid group was more potent than EGCG.

The Effect of α-ESA on ATL Cell Line and Phytohemagglutinin-Activated Human Peripheral Blood Mononuclear Cell (PBMC) Proliferation
As shown in Figure 3, we compared the suppressive effect of α-ESA on ED, Su9T01 cells and PBMCs. PBMCs are commonly used as the healthy/normal cell model in comparison to cancer cell lines. Significant differences were observed between ED cells and PBMCs treated at 6, 19 and 56 µM α-ESA (p < 0.05). Su9T01 cells and PBMCs treated with between 1 and 500 µM α-ESA were not significantly different. These results confirmed that the α-ESA strongly and selectively inhibited ED cells and moderately inhibited PBMCs, which are healthy normal T cells. α-ESA at 166 and 500 µM significantly decreased proliferation of all cell types. α-ESA showed inhibitory effects on ED, Su9T01 cells and PBMCs (IC 50 of 8.9, 29.3 and 31.0 µM, respectively).  [11]. α-ESA also may have similar potential with nutritional function for cancer prevention. The present study is the first to show the inhibitory effects of α-ESA on ATL cells in vitro. Specifically, α-ESA showed an inhibitory effect in the rank order: ED cells > Su9T01 cells ≥ PBMCs. Sasaki et al. reported that tumor suppressor in lung cancer 1 (TSLC1) gene expression was different between ED and Su9T01 cells [12,13]. Therefore, using gene expression analysis, future studies should investigate the regulatory mechanism of TSLC1 and its DNA methylation, as well as the possible role of α-ESA in the inhibition of ED and Su9T01 proliferation and TSLC1 expression.

Identification of Compounds in BGSE
The freeze-dried powder of bitter gourd seeds (5 mg) was extracted with 80% EtOH (0.5 mL) by vortexing for 30 s, followed by centrifugation at 1,500 rpm for 3 min. The supernatant was used for high performance liquid chromatography-diode array detector (HPLC-DAD) and mass spectrometry (MS) analysis. The α-ESA HPLC analysis method was a modification of the methods of Amakura et al. and Řezanka et al. [6,14].

α-ESA Calibration Curve
The α-ESA standard was dissolved in 80% ethanol and serial dilutions were analyzed by HPLC-DAD. α-ESA content was calculated using the following linear equation based on the calibration curve: Y = 215772X + 493606, R 2 = 0.9977. Y is the area detected by DAD (270 nm), and X is the α-ESA content in μg/mL.

ATL Cell Proliferation Assay
We used two ATL cell lines (ED and Su9T01) that are highly sensitive to inhibition of cell proliferation, as determined in our previous study [3]. ED cells were kindly provided by Dr. M. Maeda (Kyoto University, Kyoto, Japan) and Su9T01 cells were kindly provided by Dr. N. Arima (Kagoshima University, Kagoshima, Japan). The test compounds were dissolved in dimethyl sulfoxide and subjected to assay screening. The method of ATL assay is described in a previous report [3]. IC 50 calculation was some curve fitted onto the determined proliferation inhibition points.

Isolation and Culture of PBMCs
The method of isolation and culture of PBMCs is as follows. Heparinised blood (5 mL) was diluted by adding 5 mL of PBS. The diluted blood samples were divided into four equal parts, loaded on 4 mL of Ficoll and centrifuged at 400 × g for 30 min. The PBMC layer was located within the interphase between the Ficoll and plasma. The Ficoll contained the erythrocytes and most of the granulocytes. The plasma was removed using a pipette until ~5 mL above the PBMC interphase. The cells were washed three times with PBS (centrifuged at 200 × g for 15 min) and resuspended in RPMI 1640 medium supplemented with 10% foetal bovine serum containing 100 U/mL penicillin G, 100 μg/mL streptomycin, 2 ng/mL IL-2 and 128-fold dilution of phytohemagglutinin to a final cell density of 1 × 10 6 cells/mL. The PBMC proliferation assay was conducted using the same method as for the ATL proliferation assay [3].

Statistics
Each experiment was conducted at least three times. All data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistically significant differences were calculated by Student's t-test.

Conclusions
α-ESA was shown to be the main bioactive compound in BGSE, and contributes to the inhibition of ED cell differentiation and proliferation without damaging normal cells, leading to the disruption of ATL pathogenesis.