α-mangostin (chemical structure shown in Fig. 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. RPMI-1640 medium, penicillin-streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc. (Logan, UT, USA). 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were obtained from Sigma-Aldrich. Cell lysis buffer and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The fluorescein isothiocyanate (FITC)-conjugated Annexin V Apoptosis Detection kit was purchased from BD Biosciences (San Diego, CA, USA). Anti-β-actin (#4967), anti-Bax (#2772), anti-Bcl-2 (#2876), anti-caspase-3 (#9662), anti-cleaved caspase-3 (#9661), anti-caspase-9 (#9502), anti-poly(ADP-ribose) polymerase (PARP; #9542), anti-ERK1/2 (#9102), anti-phosphorylated (p)-ERK1/2 (#4376), anti-p38 (#9212), anti-p-p38 (#4631), anti-c-myc (#9027), anti-Ki-67 (#9027) and goat anti-rabbit horseradish peroxidase (HRP)-conjugated (#7074) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The DeadEnd™ fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit was purchased from Promega Corp. (Madison, WI, USA).

The human tongue mucoepidermoid carcinoma cell line, YD-15, was purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO₂ atmosphere. The culture medium was replaced every 2–3 days. For α-mangostin treatment, the YD-15 cells were seeded at a density of approximately 3×10⁴ cells/cm² in a 175-cm² flask and were allowed to adhere overnight.

The effects of α-mangostin on YD-15 cell survival were determined by MTT assay. The YD-15 cells were seeded in 96-well plates at a density of 2×10⁴ cells/ml in a volume of 200 µl/well. Following 24 h of incubation, the cells were treated with 10, 15, 20, 25 or 30 µM α-mangostin for 24 h in triplicate. Following treatment, the medium was discarded and 40 µl 5 mg/ml MTT solution were added followed by incubation for an additional 2 h. The medium was then aspirated, and the formazan product generated by the viable cells was solubilized by the addition of 100 µl DMSO. The absorbance of the solutions at 595 nm was determined using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The percentage of viable cells relative to the untreated (control) cells was estimated.

To assess apoptosis, the nuclei of the YD-15 cells were stained with DAPI. The cells were seeded onto 60-mm dishes at a density of 1×10⁵ cells/ml and incubated with 10 or 15 µM α-mangostin for 24 h. Following treatment, the cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 15 min in an incubator. Following fixation, the cells were washed twice with PBS and the cell nuclei were stained with DAPI in PBS. Fluorescence signals were visualized using a fluorescence microscope (BX41; Olympus Co., Tokyo, Japan) at ×200 magnification.

The Annexin V/propidium iodide (PI) assay was performed according to the manufacturer's instructions (BD Biosciences). Briefly, the YD-15 cells were treated with or without 10 or 15 µM α-mangostin for 24 h, washed twice with cold PBS, and incubated with fluorescein isothiocyanate (FITC)-conjugated Annexin V and phycoerythrin (PE)-conjugated PI in binding buffer at room temperature for 15 min in the dark. The samples were analyzed using a FACSCalibur™ flow cytometer (BD Biosciences).

Cell cycle progression was assayed by measuring DNA fragmentation with PI staining. The YD-15 cells were treated with or without 10 or 15 µM α-mangostin for 24 h, washed twice with PBS and fixed with 70% ethanol for 30 min. Following fixation, the DNA fragments were stained in PBS containing PI and RNase (Sigma-Aldrich) for 30 min at room temperature. After sorting out the viable cells, the fluorescence intensity was measured using a FACSCalibur™ flow cytometer (BD Biosciences).

The cells were grown in culture flasks under the same conditions as described above and treated with 10 or 15 µM α-mangostin for 24 h. The cells were washed with PBS and treated with trypsin-EDTA for 1 min. Cell pellets were obtained by centrifugation, lysed in lysis buffer (Invitrogen Life Technologies) and centrifuged at 13,000 rpm for 5 min at 4°C to obtain whole-cell lysates. Protein concentrations were determined using a Bradford Protein assay kit (Bio-Rad Laboratories Inc.). The samples were stored at −80°C. The proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto nitrocellulose membranes (Bio-Rad Laboratories Inc.). The membranes were blocked with Tris-buffered saline (TBS) containing 5% non-fat dry milk and 0.1% Tween-20 at 4°C for 2 h. After blocking, the membranes were incubated with anti-β-actin, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-9, anti-PARP, anti-ERK1/2, anti-p-ERK1/2, anti-p38, anti-pp38 and anti-c-myc antibodies overnight at 4°C, with gentle shaking. Following incubation with the primary antibodies, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies for 2 h at room temperature with gentle shaking. After washing the membranes 3 times for 10 min in TBS containing 0.1% Tween-20, bands were detected using enhanced chemiluminescence (ECL) western blotting detection reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. β-actin was used as a loading control. Band density was measured using the ImageJ software (NIH, Bethesda, MD, USA) program.

Five-week-old male BALB/c nude (nu/nu) mice were purchased from the animal production company of Orient Bio, Inc. (Gyeonggi-do, Korea) and maintained in a controlled environment at 23±5°C with 40±10% relative humidity with artificial lighting from 8:00 a.m. to 8:00 p.m. in facilities approved by the Companion and Laboratory Animal Science Department of Kongju National University (Chungnam, Korea). The animals were housed in cages and allowed access to sterilized water and commercial rodent chow (Biopia, Seoul, Korea) ad libitum. All animal experiments were performed following the approval of the Institutional Animal Care and Use Committee according to the guidelines of Kongju National University.

The YD-15 cells were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin at 3°C in a humidified 5% CO₂ atmosphere. The YD-15 cells were harvested by exposure to trypsin-EDTA. The cells were then washed twice and resuspended in RPMI-1640 medium. The YD-15 cells were then injected subcutaneously (1×10⁷ cells/0.2 ml medium/animal) into the left and right flanks of the mice using a 27-gauge needle. When the tumors were palpable, the mice were assigned randomly into 3 groups with 3 mice in each (the vehicle-treated controls, and the groups treated with 10 or 20 mg/kg body weight α-mangostin). The doses of α-mangostin (10 and 20 mg/kg) were selected for the in vivo experiments using mice based on the results of a previous study by Akao et al (8), in which mice administered >20 mg/kg α-mangostin exhibited a significant increase in natural killer (NK) cell activity. Therefore, we selected 20 mg/kg as the dose for use in the present study, as this is the dose that others have reported has no harmful effect. For administration, α-mangostin was dissolved in 0.1% DMSO and further diluted in PBS before injection. α-mangostin was administered intraperitoneally 5 times per week at a dose of 10 or 20 mg/kg body weight, while the control group mice were administered the vehicle only (DMSO in PBS). Tumor weight and size were monitored twice each week. Tumor size was measured using Vernier calipers (Mitutoyo, Kawasaki, Japan). The mice were sacrificed by ether inhalation at 22 days following treatment and the tumors were excised for the measurement of tumor weight. A portion of the tumor was embedded in paraffin and used for TUNEL assays and immunohistochemical analysis.

Apoptotic cell death was quantified using a Promega DeadEnd Colorimetric TUNEL system kit according to the manufacturer's instructions (Promega Corp.). Briefly, the tumor tissues were fixed in 10% formalin overnight and embedded in paraffin. The blocks were then cut into 5-µm-thick slices. The tissue sections attached to microscopic slides were then deparaffinized by immersion in xylene and the slides were then washed with 100% ethanol. The samples were rehydrated by sequential immersion in a graded ethanol series (95, 85, 70 and 50%). The tumor sections were visualized using 3′-diaminobenzidine tetrahydrochloride (DAB) solution, treated with mounting reagent, and observed under a microscope (BX41; Olympus Co.) at x200 magnification.

The tumor sections were deparaffinized with two changes of xylene for 10 min, rehydrated with two changes each of 100 and 95% ethanol for 1 min, and rinsed with tap water for 10 min. The sections were then incubated at 4°C with anti-cleaved caspase-3, anti-p-ERK1/2, anti-p-p38 and anti-Ki-67 antibodies overnight and incubated for 1 h at room temperature with a HRP-conjugated goat anti-rabbit antibody followed by incubation for 1 h. The tumor sections were visualized using DAB solution, treated with mounting reagent, and observed under a microscope (×200 magnification).

The results are all expressed as the means ± standard deviations (SD). Differences between mean values for the individual groups were assessed by one-way analysis of variance (ANOVA) with Dunnett's t-tests. A P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was conducted to examine the effects of α-mangostin on YD-15 cell viability. The YD-15 cells were treated with 0, 10, 15, 20, 25 and 30 µM α-mangostin for 24 h. Cell viability was inhibited at a concentration of 10 µM (Fig. 2A). Compared with the untreated control cells, the cells treated with 10 µM α-mangostin displayed approximately 15% inhibition, whereas those treated with 15 µM α-mangostin exhibited approximately 58% inhibition. Moreover, the number of viable cells decreased in a concentration-dependent manner. DAPI staining was performed to identify the morphological changes associated with nuclear and chromosomal condensation. The YD-15 cells were treated with 0, 10 and 15 µM α-mangostin for 24 h. DAPI staining was utilized to observe the cells by fluorescence microscopy. As a result, increased apoptosis (indicated by increased chromatin condensation) was observed in the cells treated with 10 and 15 µM α-mangostin compared to the control group (Fig. 2B). These results indicate that α-mangostin decreased the viability of the YD-15 cells, creating apoptotic bodies, thus leading to cellular apoptosis.

Apoptotic cells were analyzed quantitatively by Annexin-V FITC/PI staining to examine the effects of α-mangostin on YD-15 cells. The percentage of apoptotic cells in the untreated (control) group was 16.06% (early apoptotic cells, 4.30%; late apoptotic cells, 11.76%). However, the number of apoptotic cells increased significantly with the increasing concentration of α-mangostin. The percentages of apoptotic cells following treatment with 10 and 15 µM α-mangostin were 28.1 (early apoptotic cells, 10.79%; late apoptotic cells, 17.31%) and 33.65% (early apoptotic cells, 9.84%; late apoptotic cells, 23.81%), respectively (Fig. 3A). Flow cytometry was used to examine the effects of α-mangostin on the YD-15 cell cycle. The percentage of sub-G1 cells in the control group was 0.28% (Fig. 3B). The number of cells in the sub-G1 phase tended to increase in a concentration-dependent manner (10 µM, 1.38% and 15 µM, 6.08%; Fig. 3B). These results suggest that the inhibitory effects of α-mangostin on cell viability are caused by sub-G1 arrest related to apoptosis.

The expression of Bcl-2 family proteins, responsible for controlling apoptosis, was determined by western blot analysis in order to elucidate the mechanisms responsible for the apoptosis induced by α-mangostin in YD-15 cells. The α-mangostin-treated group exhibited an increased expression of the pro-apoptotic factor, Bax, and a decreased expression of the anti-apoptotic factor, Bcl-2, in a concentration-dependent manner (Fig. 4). These results imply that the apoptotic mechanism induced by α-mangostin is associated with Bcl-2 family proteins.

The expression levels of caspase-3, caspase-9 and PARP were measured by western blot analysis in order to determine the association between the induction of apoptosis by α-mangostin and caspase activation in YD-15 cells. The cells treated with α-mangostin exhibited increased caspase-3 and -9 activity in a concentration-dependent manner (Fig. 5A). The level of cleaved-PARP increased as well. Cleaved caspase-3 was visualized by immunohistochemical analysis in order to examine the effects of α-mangostin on caspase expression in tumor tissues collected from mice with tumor xenografts (Fig. 5B). The tumor tissue from mice treated with α-mangostin exhibited increased levels of cleaved caspase-3 in a concentration-dependent manner, suggesting that the mechanism of apoptosis induced by α-mangostin is related to caspase activation, which in turn induces PARP segmentation.

The levels of ERK1/2, p38 and c-myc were examined by western blot analysis in order to determine whether the induction of apoptosis by α-mangostin involves the MAPK pathway. The inhibition of ERK1/2 and p38 activation, and decreased c-myc expression were observed in the cells treated with α-mangostin in a concentration-dependent manner (Fig. 6A). The levels of p-ERK1/2 and p-p38 were determined by immunohistochemical analysis to determine whether the effects of α-mangostin involve the MAPK pathway in tumor tissue from mice with tumor xenografts (Fig. 6B). The tumor tissue of the mice treated with α-mangostin exhibited decreased levels of p-ERK1/2 and p-p38 in a concentration-dependent manner.

The effects of α-mangostin on tumor volume in mice with YD-15 tumor xenografts were investigated. α-mangostin was administered by intraperitoneal injection at the dose of 10 mg/kg (low-dose) and 20 mg/kg (high-dose). The mice in the control group received the vehicle (0.5% DMSO in PBS) with the same timing and dosing schedule used for the treatment group. A significant difference in tumor volume emerged in the control group from day 8 onwards following treatment. The mice treated with 20 mg/kg α-mangostin exhibited a tumor volume inhibition rate of 89.9% compared with the control group (Fig. 7A). The sizes of the final tumors in the control and α-mangostin-treated (10 and 20 mg/kg body weight) mice were 639, 105 and 43 mm³, respectively. Tumor weights in the experimental nude mice were also determined to be 0.25, 0.08 and 0.05 g in the control, and 10 and 20 mg/kg α-mangostin-treated groups, respectively, indicating a decreasing trend in tumor weight upon the administration of α-mangostin (Fig. 7B). A TUNEL assay was performed on the extracted tumor tissue to examine cell apoptosis. As a result, many apoptotic cells were confirmed in the mice treated with α-mangostin compared with the control group (Fig. 7C). Immunohistochemical analysis was also performed to examine the expression of Ki-67, the protein responsible for the rate of tumor growth (Fig. 8). The tumor samples from the mice treated with α-mangostin exhibited a decreased Ki-67 expression in a concentration-dependent manner, suggesting that α-mangostin induced the apoptosis of YD-15 cells (in the tumor xenografts), thus inhibiting tumor growth.