Adlay Testa (Coix lachryma-jobi L. var. Ma-yuen Stapf.) Ethanolic Extract and Its Active Components Exert Anti-Proliferative Effects on Endometrial Cancer Cells via Cell Cycle Arrest

Cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to verify the effect of ethanol extracts of four parts of the adlay seed in human endometrial cancer cells (HEC-1A and RL95-2). Ethanol extracts of the hull, testa, bran, and polish adlay of the seed are referred to as AHE, ATE, ABE, and PAE, respectively. The cells were treated with AHE, ATE, ABE, and PAE (200 μg/mL) for 48 h. As shown in Figure 1A,B, ATE treatment significantly reduced the viability of HEC-1A and RL95-2 cells at 48 h. PAE only slightly reduced HEC-1A cell viability at 48 h. AHE, ABE, and PAE showed no effect on RL95-2 cell viability at 48 h. These results suggest that ATE exerted potential anti-cancer effects compared to the other components. Therefore, ATE was subjected to further investigation. This study used paclitaxel, a standard initial therapy for advanced endometrial cancer, as a positive control.

The potential anticancer effect of different ATE fractions on the proliferation of human endometrial cancer cells (HEC-1A, and RL95-2) were screened using MTT. The cells were treated with ATE-Hex, ATE-EA, ATE-Bu, and ATE-H₂O at 200 μg/mL for 48 h. The results show that the ATE-EA fraction significantly reduced the viability of RL95-2 cells (Figure 1D), but. in HEC-1A (Figure 1C), a statistical trend was found at 48 h. ATE-Hex and ATE-Bu fractions only slightly reduced HEC-1A (Figure 1C) and RL95-2 cell (Figure 1D) viability at 48 h. Finally, the cell viability assay showed no significant difference between the untreated control and ATE-H₂O groups at 48 h on both cell lines. Collectively, these results indicate that major anti-cancer components possibly exist in the lower-polarity fraction of ATE, especially ATE-EA. Therefore, the ATE-EA fraction was selected for further investigation.

The MTT cell proliferation assay was then performed to further assess the effects of different concentrations of ATE-EA on human endometrial cancer cell (HEC-1A and RL95-2) proliferation. We treated cells with ATE-EA ranging from 25 to 200 μg/mL for 48 h. As shown in Figure 1E,F, ATE-EA significantly inhibited the viability of HEC-1A cells (Figure 1E) and RL95-2 cells (Figure 1F) at 200 μg/mL at 48 h, compared to the untreated control. These data suggest a selective targeting of human endometrial cancer cells by ATE-EA. Later, chromatographic subfractions of the ATE ethyl acetate fraction (ATE-EA), nine samples in total, were screened for cytotoxic activity on human endometrial cancer cells (HEC-1A and RL95-2) using the MTT assay, and the results are presented in Figure 1G, H. ATE-EA-A–H chromatographic subfractions showed potent growth inhibition effects on both HEC-1A (Figure 1G) and RL95-2 (Figure 1H) cells at 48 h. These data suggest that these chromatographic subfractions exert anti-proliferation activity against human endometrial cancer cells.

The growth inhibitory effect of 11 phenolic compounds (identified in ATE-EA as follows: protocatechnic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, syringic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, and ferulic acid) were explored in HEC-1A and RL95-2 cells using the MTT assay. Phenolic compounds in ATE-EA were identified by HPLC analysis based on the reference [23]. The results, as shown in Figure 2A,C, display growth inhibitory effects of the phenolic compounds mixture at serial concentrations (100, 200, 400, and 800 μg/mL) on both HEC-1A and RL95-2 cells at 48 h, compared to ATE-EA (100 and 200 μg/mL). This result suggests that the ATE-EA at the 200 ug/mL dose has a better inhibitory effect than that phenolic mixtures. Moreover, the individual growth inhibitory effect of these 11 phenolic compounds were explored (200 μg/mL each) compared to the untreated control to identify their relative anti-cancer effects on both HEC-1A and RL95-2 cells using the MTT assay. The results suggest that protocatechnic acid, caffeic acid, and p-hydroxybenzaldehyde inhibited the proliferation of HEC-1A and RL95-2 cells (Figure 2B, D). HEC-1A and RL95-2 cells were more sensitive to ATE-EA than the mixed phenolic compounds at same concentrations (200 μg/mL).

We evaluated the potential anti-cancer effects of five flavonoid compounds (liquiritigenin, chrysoeriol, quercetin, naringenin, and quercetin-3,5,7,3’,4-pentamethylether) individually as well as combined at different four concentrations in HEC-1A and RL95-2 cells (Figure 3A–D). The flavonoid contents were analyzed by HPLC based on the reference [24]. The results confirm the previous findings that ATE-EA reduced cell growth when the dosage was 200 μg/mL. The mixed flavonoid compounds suppressed cell growth in vitro and showed only a decreasing trend. Our results indicate that liquiritigenin, chrysoeriol, quercetin, naringenin, and quercetin-3,5,7,3’,4-pentamethylether produced anti-proliferative effects at different doses (12.5, 25, 50, and 100 μg/mL) in the HEC-1A cell line. The red boxes represent the individual flavonoid compounds with series concentrations.

The potential growth inhibitory effect of the mixture of steroid compounds as well as four steroids in ATE-EA, including campesterol, β-sitosterol, stigmasterol, and stigmastanol, were explored on HEC-1A and RL95-2 cells using the MTT assay (Figure 4A–D) and compared to ATE-EA at the doses of 100 and 200 μg/mL over 48 h. The results suggest that steroids partly governed the inhibitory ability of ATE-EA. These results are consistent with ATE-EA at the optimal dose of 200 μg/mL. The results demonstrate that HEC-1A and RL95-2 cells were more sensitive to ATE-EA than the four mixed steroids. The red boxes represent the individual steroid compounds with the serial concentrations.

We evaluated the proliferation suppressive efficiency of the fatty acid mixture as well as four individual fatty acids, including palmitic acid, stearic acid, oleic acid, and linoleic acid, of ATE-EA on both HEC-1A and RL95-2 cells using the MTT assay (Figure 5A–D). The results show that treatment with ATE-EA (200 μg/mL) and the mix of fatty acid compounds at both 400 and 800 ug/mL for 48 h significantly reduced HEC-1A and RL95-2 cell growth. Both palmitic acid and linoleic acid at the optimal dose showed a considerably strong inhibitory effect on HEA-1A and RL95-2 cell lines. A similar trend was observed for ATE-EA, which compared better to the mixed compounds in these cell assays.

To better understand the relationship between the inhibitory effects and the mixtures of phenolics, flavonoids, steroids, and fatty acids in the ATE-EA, the above four mixtures of compounds were compared to the ATE-EA. The results suggest that the inhibitory effect of ATE-EA on cell growth was concentration-dependent. In addition, the strongest cell growth inhibition was observed and increased with the dosage of the four mixed compounds at concentrations of 400 and 800 μg/mL in both HEC-1A and RL95-2 cell lines (Figure 6A,B). Therefore, the involved phenolic components—flavonoids, steroids, and fatty acids of ATE-EA—contributed to the anti-inhibitory activities on endometrial cancer cells.

To identify whether the cytotoxic effect of ATE-EA is associated with induction of cell cycle arrest, HEC-1A and RL95-2 cells were treated with ATE-EA at different concentrations (100 and 200 μg/mL) for 8, 12, 24, and 48 h and changes in cell cycle distribution were measured using flow cytometry (Figure 7A–H). ATE-EA (100 μg/mL) treatment for 8, 12, 24, and 48 h increased the percentage of HEC-1A and RL95-2 cells in the G0/G1 phase. Doubling the concentration of ATE-EA to 200 μg/mL increased the sub-G1 phase at 12, 24, and 48 h. Collectively, ATE-EA showed dose-dependent cell cycle arrest of HEC-1A and RL95-2 cells at the sub G1 checkpoint and G2/M checkpoint.

The different phenolic compounds presented in ATE-EA were characterized using high performance liquid chromatography (HPLC) analysis. Figures S1 and S2 show the chromatograms of the phenolic compound analysis and the reference standards. Briefly, identifying the major phenolic compounds in ATE-EA was done by comparing the chromatograms of analyzed samples to those of the standards (protocatechnic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, p-hydroxybenzaldehyde, syringic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, and ferulic acid). This analysis allowed identification of the retention times of the phenolic compounds presented in ATE-EA as well as the retention times relative to the standard via HPLC analysis (see Figures S1 and S2 and Table S2 in Supplementary Materials). The major phenolic and flavonoid compounds in ATE-EA are shown in Table 1 and Table 2 (cf. Table S3), respectively. In addition, five major flavonoid compounds, namely chrysoeriol, liquiritigenin, naringenin, quercetin, and quercetin-3,5,7,3’,4-pentamethylether, were identified in ATE-EA by the curves of the five major flavonoid standards.

The different phytosterols in ATE-EA were identified using gas chromatography (GC)/flame ionization detection (FID). This chromatographic method proved efficient at detecting and separating steroids as it can be applied to different types of detectors with no loss of analytical sensitivity. Figure S3 shows the analysis of ATE-EA and the relevant reference standards. Individual phytosterols in ATE-EA were identified by comparing their retention time with those of the available standards (β-sitosterol, stigmasterol, stigmastanol, and campesterol) and the retention time relative to the internal standard cholesterol via GC/FID. The major phytosterols in ATE-EA are shown in Table 3. The phytosterols were eluted at time points ranging from 20.20 to 22.15 min. The internal standard used in quantifying the phytosterols was eluted in 18.36 min.

McCoy’s 5A medium, sodium bicarbonate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paclitaxel, one of the most effective and widely used anti-cancer drugs for treating various tumors, was purchased from LC Laboratories (MO, USA). Bovine serum albumin (BSA), sodium bicarbonate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other chemicals such as rhodamine-123, propidium iodide (PI), RNase A, sodium citrate, and standards for analysis were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents for cell culture (DMEM/F12 medium, fetal bovine serum (FBS), epidermal growth factor, fibroblast growth factor, insulin, penicillin, and streptomycin) were purchased from Gibco (Grand Island, NY, USA). Coomassie Brilliant Blue R-250 was purchased from Bio-Rad (Hercules, CA, USA). Analytical-grade solvents used during the purification procedures, including acetonitrile, n-butanol (Bu), ethanol, ethyl acetate (EtOAc), hexane, and methanol were obtained from Merck (Darmstadt, Germany).

The powder from all ground components was sieved through a 20-mesh sieve (aperture, 0.94 mm). The powder samples (100.0 g) were extracted with 10 volumes (w/v) of 95% ethanol (1.0 L), and filtration was used to separate samples at room temperature for 24 h. Then, dried ethanol extracts were obtained from the filtrate by concentration and stored at −20 °C. Ethanol extracts of four seed parts, namly the hull, testa, bran, and polish adlay of the adla seed, are referred to as AHE (ethanolic extracts of adlay hull), ATE (ethanolic extracts of adlay testa), ABE (ethanolic extracts of adlay bran), and PAE (ethanolic extracts of polished adlay), respectively.

As illustrated in Figure 8, the ethanolic extracts of the adlay testa were extracted and partitioned. Adlay testa powder (2.0 kg) was extracted with ethanol three consecutive times for 24 h and filtered. The filtrate, ethanol, and residues were separated in each 24-h extraction and concentrated to obtain 107.6 g (4.48%, based on the dry weight) of ethanolic extract under vacuum conditions. ATE, the ethanolic extracts of adlay testa, were suspended in water with 10% methanol and partitioned with hexane until the hexane fraction was colorless. Then, ATE-Hex (28.8 g, 1.2%) and ATE-EA (32.0 g, 1.33%) were obtained from the hexane fraction and dried under a vacuum or defatted by partitioning with ethyl acetate (EtOAc), respectively. Finally, ATE-Bu (10.0 g, 0.42%) from the residue in the butanoic fraction was obtained by partitioning with butanol and dried. ATE-Wa (24.4 g, 1.02%) was freeze-dried from the aliquoted fraction. Separation was done using silica gel and the mechanisms of isolation was based on the polarity of compounds. The different fractions were obtained from the EtOAc fraction by chromatographed successively on silica gel using eluting solutions with increasing polarity. ATE-EtOAc was subjected to column chromatography using silica gel and eluted with a Hex/EtOAc/MeOH gradient. Eight subfractions were obtained in the below combinations according to the similarity of each collection in thin layer chromatography (TLC): A (∼0–10% EA/Hex), B (∼10–20% EA/Hex), C (∼20–30% EA/hex), D (∼30–40% EA/Hex), E (∼40–50% EA/Hex), F (∼50–80% EA/Hex), G (∼80–100% EA/Hex), H (∼0–20% MeOH/EA), and I (∼20–100% MeOH/EA). The screening of the effective fractions was done through bioassay-guided fractionation procedures.

Human endometrial cancer cell lines (HEC-1A and RL95-2) were obtained from the Food Industry Research and Development Institute (FIRDI, Taiwan, ROC) and the Culture Collection and Research Center (CCRC, Taiwan, ROC). The HEC-1A cell line and RL95-2 cell line were cultured in McCoy’s 5A and DMEM/Ham’s F-12, respectively. All cells were maintained under the recommended conditions with 10% FBS and 1% antibiotics (10,000 units/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin with 8.5 g/L NaCl). The atmosphere was 5% CO₂ at 37 °C.

The MTT assay was used to detect the cell proliferation effects of extracts of adlay seeds or paclitaxel. MTT was added to each well after the cancer cells were incubated in the presence of the extracts and paclitaxel treatment. The cancer cells (HEC-1A and RL95-2 cells) were at a density of 2000 cells/well with 100 μL of complete medium/well prepared before the above processing. An aliquot of 100 μL DMSO was added, and the plates were shaken and measured until the crystals dissolved at which point the absorbance was read at 570 nm on an ELISA plate reader (Model 550, Bio-Rad Laboratories, Hercules, CA, USA). The procedures of the MTT assay were performed as previously described [17,42]. The relative cell viability data were reported as the percentage of cells treated with the vehicle in at least triplicate experiments.

The easiest and rapidest method for detecting apoptosis is DNA staining with a propidium iodide (PI) solution and measurement in CellQuest Software with a FACSVantage (Becton Dickinson, Franklin Lakes, NJ) flow cytometry system. Cells treated with extracts from adlay seeds or paclitaxel were fixed in 75% ethanol at −20 °C. Cells were resuspended in 100 μL of phosphate buffered saline (PBS) containing 10 μL of RNase A (10 mg/mL) and incubated for 1 h at 37 °C after washing twice with cold PBS. The cells were then stained with 400 μL of PI (50 mg/mL) for 30 min at room temperature in the dark. For details of the procedures of cell cycle analysis by flow cytometry, see the work in [27,43].

HPLC analysis was conducted on a Hitachi liquid chromatograph equipped with a L-6200 intelligent pump (Manasquan, NJ, USA) and L-7455 photodiode array detector (Manasquan, NJ, USA). Gradient elution was performed by varying the proportion of solvent A (water–acetic acid, 95:5 v/v) to solvent B (acetonitrile–acetic acid, 99.5:0.5 v/v), with a flow rate of 1 mL/min. A C18 guard column and a C18 reversed-phase packing column (250 × 4.6 mm 5 μM) were used (YMC Co., INC). Solvent B started at 10% for 10 min, then increased from 10% to 15% from 10 to 20 min, 15% to 16% from 20 to 35 min, 16% to 17% from 35 to 50 min, and finally 17% to 21% from 50 to 55 min. Phenolic compounds in the ATE-EA were identified based on the comparison of retention times and absorbance spectra at 280 nm to the reference standards using calibration curves.

GC/FID with a Varian 3400 GC (Palo Alto, CA) equipped with an EQUITY-5 column (30 m × 0.53 mm i.d., 0.15 μm film thickness) (Supelco, Saint Louis, MO, USA) was used for the composition analysis of steroids in the ATE-EA. Injector and FID temperatures were 320 and 330 °C, respectively. The carrier gas and makeup gas were hydrogens at a constant flow rate of 30.0 mL/min and nitrogen at flow rate of 28.0 mL/min, respectively. The temperature gradient had an initial temperature of 100 °C, then increased to 250 °C/min, held for 5 min, and then held at 300 °C for 30 min.

We prepared a standard stock solution in hexane that included campesterol, stigmasterol, β-sitosterol, and stigmastanol. Different concentrations of work solutions were prepared daily by dilution a standard stock and stored at 4 °C. A solution of 0.05 mL cholesterol (2.0 mg/mL) as the internal standard (IS) was added to the EA fraction of the ATE (250.0 mg) in 5.0 mL hexane.

The fatty acid content was detected by promulgation for confirmation of national standards [44]. As shown in Table S1, palmitic acid, stearic acid, oleic acid, and linoleic acid account for the major content of ATE-EA.

Data are presented as mean ± standard deviation. Determination of statistical significance was performed by one-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison test using SPSS (IBM Corporation, Armonk, NY, U.S.A). Differences with p < 0.05 were considered significant. Results are representative of three independent experiments.