MGDG extracted from spinach enhances the cytotoxicity of radiation in pancreatic cancer cells

The spinach (Spinacia oleracea) subspecies Anna was used in this study. Dried spinach was extracted with ethanol; the extract was diluted in 70% aqueous ethanol and subjected to Diaion HP-20 column chromatography (Sigma-Aldrich, St. Louis, MO, USA), then eluted with 95% aqueous ethanol. The eluate was evaporated to dryness; the residue was redissolved in chloroform and the resultant solution was subjected to PSQ60B silica gel column chromatography (Fuji Silysia Chemical, Tokyo, Japan). After washing with chloroform/ethyl acetate (1:1 v/v), the column was eluted with ethyl acetate and the eluate was purified using a Sep-Pak C₁₈ cartridge (Waters, Milford, MA, USA) that was then eluted with methanol. The MGDG fraction was evaporated, yielding pure MGDG (~98%). The purity was confirmed by normal-phase silica gel high-performance liquid chromatography (Shiseido, Tokyo, Japan) coupled to an evaporative light scattering detector (M&S Instruments, Osaka, Japan), with chloroform/methanol (1/1, v/v) used as the eluent.

MIAPaCa-2, PANC-1, BxPC-3 and AsPC-1 human pancreatic cancer cell lines as well as Raji and HL60 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml). HCT116 colon carcinoma cell lines with wild-type p53 (HCT116 p53^+/+) and their isogenic derivatives lacking p53 (HCT116 p53^−/−) were a gift from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA). The cells were maintained in McCoy’s 5A medium. Primary normal human dermal fibroblasts (NHDFs) were purchased from Cell Systems Corp. (Kirkland, WA, USA) and maintained according to the supplier’s instructions. The cytotoxicity of MGDG was evaluated with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cytotoxicity when combined with irradiation was assessed with the colony forming assay. For the MTT assay, cells were treated with 0, 2, 5, 10, 20, 50 or 100 μM MGDG or corresponding doses of dimethyl sulfoxide (DMSO) for 24, 48 or 72 h at 37 °C in a humidified atmosphere of 5% CO₂; 2 h after adding MTT solution (0.6 mg/ml in Milli-Q-purified water), cells were lysed in 200 μl of fresh DMSO. For the colony forming assay, MIAPaCa-2, BxPC-3 and AsPC-1 cells were treated with 40–60 μM MGDG or 0.8% DMSO for 24 h, and then exposed to 0, 2, 4 and 8 Gy of radiation. After 9–12 days, colonies were fixed with a solution of 10% methanol and 20% acetic acid, stained with Methylene Blue and counted under a light microscope.

X-ray irradiation was performed using a MBR-1505R2 instrument (Hitachi, Tokyo, Japan) at a voltage of 150 kV and a current of 5 mA with a 1-mm-thick aluminum filter (0.5 Gy/min at the target) for in vitro and in vivo studies. Mice were anesthetized by intraperitoneal administration of somnopentyl (0.1 mg/g body weight) and were then anesthetized and immobilized in a customized harness that exposed the implanted tumors while shielding the remainder of the body with lead during irradiation.

Apoptosis was evaluated based on DNA fragmentation using the APO-Direct Assay Staining kit (BD Biosciences, San Diego, CA, USA) as previously described [22]. In this assay, DNA breaks are labeled with fluorescein isothiocyanate (FITC)-2′-deoxyuridine-5′-triphosphate and cells are analyzed by flow cytometry. MIAPaCa-2 cells were treated with MGDG (25 μM) alone for 24 h, radiation (5 Gy) alone for 12 h, or with a combination of both. The cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and then fixed in 1% paraformaldehyde for 15min followed by 70% ethanol overnight at −20 °C. A DNA labeling solution containing FITC was added for 30min; the cells were the resuspended in PBS, and apoptosis was detected by flow cytometry on a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed with CellQuest software (Becton Dickinson), and apoptotic cells were quantified as a percentage of the total number of cells.

MIAPaCa-2 cells were seeded at a density of 2 × 10⁶/well in a 6-well plate for 24 h, then washed with PBS and incubated with various concentrations of MGDG for 24 h. Cells were washed twice with PBS and lysed in lysis buffer composed of 20 mM HEPES (pH 7.5), 10% glycerol, 1.5 mM MgCl₂, 1 mM TritonX-100, and protease inhibitor cocktail (Roche Life Science, Tokyo, Japan) followed by boiling for 5min. To isolate cytosolic and mitochondrial fractions, MIAPaCa-2 cells were harvested by scraping on ice, and resuspended in 500 μl buffer A composed of 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail. After incubation on ice for 1 h, cells were lysed by repeated passage (20–30 times) through a 27-gauge needle. The lysates were centrifuged at 750 × g for 5min at 4 °C and the supernatant was centrifuged at 10,000 × g for 15min at 4 °C. The mitochondrial pellet was washed once in buffer A and lysed in Laemmli sample buffer. The supernatant was centrifuged at 100,000 × g for 30min at 4 °C to obtain the cytosolic fraction. Protein concentrations were measured with the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes that were blocked with 5% non-fat milk in PBS and probed overnight at 4 °C with primary antibodies against the following proteins: actin (Santa Cruz Biotechnology, Dallas, TX, USA), poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Danvers, MA, USA), caspase-3 (Cell Signaling Technology), pro-caspase-3 (GeneTex, Irvine, CA, USA), B cell lymphoma (Bcl)-2 (Santa Cruz Biotechnology), and Bcl-2-associated X protein (Bax) (Santa Cruz Biotechnology). Immunoreactivity was detected with an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, UK) and protein bands were visualized using an Amersham Imager 600 (GE Healthcare). Signal intensity was quantified using Multi Gauge v.3.0 software (Fujifilm, Tokyo, Japan).

The effect of MGDG on the cell cycle was evaluated by flow cytometry as previously described [23]. Briefly, MIAPaCa-2 cells (3 × 10⁵ cells in a 25-ml flask) were treated with 40 μM MGDG, 8 Gy of radiation, or a combination of both for 24 h. Cells were irradiated within 12 h of adding MGDG and incubated for 12 h, then fixed on ice for 30min in PBS (pH 7.4) containing 2% formaldehyde and stored at −20 °C until analysis. Cells were washed and incubated for 15min in phosphate citric acid buffer composed of 20% Triton X and 5 mg/ml ribonuclease A in PBS, then resuspended in 50 mg/ml propidium iodide for at least 15min at room temperature in the dark; the DNA content of the samples was analyzed by flow cytometry using a FACScan instrument (Becton Dickinson) with a 488-nm laser run at 15 mW and a 585/420-nm bandpass filter. At least 20,000 events were acquired using CellQuest software (Becton Dickinson). The experiment was performed at least twice. The G1, S and G2 fractions were identified by selecting the areas consisting of living cells and excluding those containing dead cells.

Induction of DNA damage was investigated by detecting phosphorylated histone 2AX (γ-H2AX)-positive foci by immunocytochemistry [24]. Briefly, MIAPaCa-2 cells were subcultured in 35-mm dishes and treated with 40 μM MGDG for 1 h and/or 8 Gy of radiation. Cells were then fixed in 4% paraformaldehyde in PBS for 20min, permeabilized with 0.1% Triton X-100 in PBS for 5min, and blocked in 5% bovine serum albumin in PBS for 60min. The cells were incubated with rabbit anti γ-H2AX antibody (1:200; Cell Signaling Technology, Danvers, MA, US) overnight at 4 °C, followed by incubation with tetramethyl rhodamine isothiocyanate-conjugated anti-rabbit secondary antibody (1:20; Dako, Glostrup, Denmark) for 90min at room temperature. Nuclei were stained with 4′,6-diaidino-2- phenylindole, and cells were visualized with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). Nuclear γ-H2AX foci in 200 cells in each treatment group were manually counted, and data are presented as the mean ± standard deviation of three random fields.

The alkaline comet assay was performed using a kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Briefly, 250 ml of 0.65% normal agarose was prepared and a drop was placed on a frosted slide, covered with a coverslip and allowed to solidify. The cell suspension (100 ml) was mixed at 1:10 with 0.5% low-melting-point agarose and a 100 μl volume of the mixture was pipetted onto the slides and allowed to solidify. A final layer of 0.5% low-melting-point agarose was added, and slides were then immersed for 1 h at 4 °C in the dark in cold lysis solution composed of 2.5 M NaCl, 100 mM EDTA, 300 mM NaOH, 10 mM Tris, and 34 mM N-lauroylsarcosine (pH 10), with 10% DMSO and 1% Triton X-100 added just before use. Slides were placed in a submarine-type electrophoresis tank containing 300 mM NaOH and 1 mM EDTA (pH 13.5) for 15min. Electrophoresis was then carried out at 0.8 V/cm for 15min. Slides were rinsed three times with neutralization buffer (0.5 M Tris, pH 7.4) and stained with a solution of 1 μl SYBR Green Gold in 30 ml Tris/EDTA buffer.

Male BALB/cAJcl-nu/nu mice (4 weeks old; n = 10 per group) were used for the xenograft model. Experiments were carried out in strict accordance with institutional ethics guidelines. Each mouse received a subcutaneous intratumoral injection of 2 × 10⁷ MIAPaCa-2 cells resuspended in the Matrigel (BD Biosciences) and were then randomly divided into the following four groups: control, MGDG (injection with 2 mg MGDG solution twice on alternate days to ensure good penetration into the tumors), irradiation (5 Gy), and the combination of both. For this procedure, MGDG was used at a concentration of 50 mg/ml (62.5 mM) in solvent, and 4 mg MGDG solution (two injections of 2-mg MGDG solution) were used to prepare 50 μM MGDG. To assess the tumor growth-inhibitory effect of MGDG and/or radiation, tumor size was measured two or three times a week by calculating the volume using the formula L × W² × (π/6), where L and W are the longest and shortest diameters of the tumor, respectively. All animal experiments were approved by the Institutional Animal Care and Use Committee (permission no. 100605R1) and were performed according to Kobe University Animal Experimentation Regulations.

Data are presented as mean ± standard error. Differences between groups were evaluated with the Student’s t test. Data were considered statistically significant at P < 0.05.

Compared to DGDG or SQDG, MGDG showed distinct cytotoxic effects on human cancer cell growth (Table 1). MGDG showed dose- and time-dependent cytotoxicity in all four human pancreatic cancer cell lines, as determined with the MTT assay (Fig. 1a). The half-maximal inhibitory concentrations (IC₅₀) in MIAPaCa-2, BxPC-3, AsPC-1 and PANC-1 cells at 72 h were 18.5 ± 1.7, 26.9 ± 1.3, 22.7 ± 1.9, and 25.6 ± 2.5 μM, respectively. In contrast, MGDG showed almost no cytotoxicity in NHDFs, and combined MGDG treatment and irradiation did not have a synergistic effect in NHDFs as compared to irradiation alone (Fig. 1b). The colony forming assay revealed fewer MIAPaCa-2, BxPC-3 and AsPC-1 cell colonies upon treatment with the combination treatment as compared to irradiation alone (P < 0.05) (Fig. 2).

The single treatments increased the rate of apoptosis (5.2% for MGDG and 8.8% for radiation), while the combination of both yielded a higher proportion of apoptotic cells (21.5%), suggesting that they had a synergistic effect on apoptosis induction (Fig. 3).

Molecular events underlying apoptosis in MIAPaCa-2 cells treated with MGDG were investigated by the western blotting. Cytochrome c release from mitochondria to the cytosol increased in a concentration-dependent manner in the presence of 50 and 75 μM of MGDG; that is, cytochrome c level was reduced in mitochondria and increased in the cytosol relative to control cells (Fig. 4a, b). The activation of cleaved PARP and cleaved caspase-3 was also increased by MGDG treatment (Fig. 4c, d), with the latter possibly resulting from increased mitochondrial release of cytochrome c. Bax was also upregulated in a dose-dependent manner at 25 an 50 μM, whereas Bcl-2 level remained largely unaffected (P < 0.05) (Fig. 4e, f). A possible reason for slight decrease in Bax and Bcl-2 levels at 75 μM MGDG is cell loss caused by severe MGDG toxicity. Results from the western blot analysis were compared with the Student’s t test [22].

The effects of MGDG and radiation on the cellular DNA damage response was evaluated by quantifying γ-H2AX foci in MIAPaCa-2 cells. Compared to irradiation alone, combination treatment increased the number of γ-H2AX foci in MIAPaCa-2 cells (Fig. 5a, b). The results indicate that MGDG and irradiation induce DNA damage in a synergistic manner.

We performed the alkaline comet assay to detect defects in DNA repair. At 30min, the proportion of comet tails indicating DNA fragments was higher in the combination treatment (46.5%) than in the MGDG (3.2%) and irradiation (30.5%) groups (P < 0.05) (Fig. 5c). At later time points, most cells had completed DNA repair or had undergone apoptosis. These results indicate that MGDG potentiated the suppressive effects of radiation on DNA repair.

To determine whether cell cycle was affected by MGDG or irradiation, MIAPaCa-2 cell cycle distribution was analyzed by flow cytometry. MGDG induced a slight increase in the G2/M fraction (29.9–36.2%) (Fig. 6a, b), while irradiation caused G2/M arrest (29.9–65.3%) (Fig. 6a, c). The cell cycle distribution following combination treatment was similar to that of irradiation alone (Fig. 6c, d). These results indicate that MGDG does not act synergistically with radiation to induce cell cycle arrest.

The effects of MGDG and radiation were assessed in a mouse xenograft model using MIAPaCa-2 cells. After 23 days, the tumor volumes were 7475.3 ± 986.1 mm³ (control), 7598.8 ± 1532.0 mm³ (MGDG), 5892.7 ± 1313.3 mm³ (radiation), and 2539.8 ± 552.7 mm³ (MGDG and radiation) (Fig. 7a, b). The tumor growth inhibitory effect was greater in mice receiving intratumoral injection of MGDG combined with irradiation as compared to either of these approaches alone (P < 0.05) (Fig. 7b).