Mangosteen (Garcinia mangostana Linn) pericarps (Figure 1A) collected in Thailand were dried, ground, and successively extracted in water and 50% ethanol. After freeze-drying the 50% ethanol extract, the resultant dried powder was suspended in water partitioned with ethyl acetate. The ethyl acetate extract was then purified by chromatography on silica gel with the n-hexane-ethyl acetate system and recrystallized to give α-mangostin at > 98% purity. The chemical structure of α-mangostin is shown in Figure 1B. For in vitro use, crystallized α-mangostin was dissolved in dimethyl sulphoxide (DMSO), and aliquots of stock 20 mM solution were stored at -20°C.
Cell lines and animals
The murine BJMC3879 mammary adenocarcinoma cell line was derived from a metastatic focus within a lymph node of an inoculated BALB/c mouse in an earlier study; the cell line continues to show a high metastatic propensity, especially to lymph nodes and lungs [18–20], a trait retained through culture. The BJMC3879luc2 mammary carcinoma cell line used in our investigations was generated by stable transfection of the luc2 gene (an improved firefly luciferase gene) into the parent BJMC3879 cell line . BJMC3879luc2 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum with streptomycin/penicillin at 37°C under 5% CO2. MDA-MB231, a human mammary carcinoma cell line stably expressing the green fluorescence protein (GFP) was maintained in DMEM or RPMI-1640 medium containing 10% fetal bovine serum. Most of the in vitro analyses of caspase, cytochrome c release, Bid and cell cycle were conducted using the mouse BJMC3879luc2 cells, but Akt-phosphorylation analysis was performed in both human MDA-MB231 cells in vitro and mouse BJMC3879luc2 cells in vivo.
Thirty six-week-old female BALB/c mice were used in this study (Japan SLC, Hamamatsu, Japan). The animals were housed five per plastic cage on wood chip bedding with free access to water and food under controlled temperature (21 ± 2°C), humidity (50 ± 10%), and lighting (12-12 hours light-dark cycle) conditions. All animals were held for a one-week acclimatization period before study commencement. This animal experiment was approved by the Animal Experiment Committee of Osaka Medical College. Mice were treated in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals at Osaka Medical College, the Japanese Government Animal Protection and Management Law (No.105) and the Japanese Government Notification on Feeding and Safekeeping of Animals (No.6).
BJMC3879luc2 and MDA-MB231 cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 2 mM L-glutamine under an atmosphere of 95% air and 5% CO2 at 37°C. These cells were plated into 96-well plates (1 × 104 cells/well) one day before α-mangostin treatment. They were subsequently incubated for 24 hours with culture medium containing DMSO vehicle alone or with medium containing α-mangostin at various concentrations up to 20 μM. Cell viability was determined using a CellTiter-Bule Cell Viability Assay (Promega Co., Madison, WI, USA). The IC50 for each cell line under these conditions was found to be 12 μM α-mangostin in BJMC3879luc2 and 20 μM in MDA-MB231 cells; thus, all in vitro studies were performed using exposure to these respective concentrations of α-mangostin for 24 hours.
Caspase activity and TUNEL assay
BJMC3879luc2 cells were grown in two-well chambered slides and treated with 12 μM α-mangostin for 24 hours. The cells were then fixed in 4% formaldehyde solution in phosphate buffer and terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL) staining was performed according to the manufacturer's protocol (Wako Pure Chemical Industries, Osaka, Japan).
BJMC3879luc2 cells were plated into 96-well plates at a concentration of 1 × 104 cells/well one day before α-mangostin treatment. Cells were treated with 12 μM α-mangostin or DMSO alone for 48 or 72 hours; subsequent cell viability was measured using a CellTiter-Blue Cell Viability Assay (Promega). The activities of caspase-8, caspase-9 and caspase-3 were measured using a luminescent assay kit (Promega). Caspase activity was measured in terms of the luminescent signal produced by caspase cleavage of the corresponding substrate using a Luminoskan Ascent kit (Thermo Electron Co., Helsinki, Finland). Caspase activity levels were then adjusted to account for the corresponding cell viability data as previously reported .
Release of cytochrome c
After incubation with either DMSO alone or with 12 μM α-mangostin for 24 hours, both floating and attached BJMC3879luc2 cells were harvested, rinsed once in PBS, re-suspended in cell lysis buffer, incubated for one hour at room temperature, and centrifuged at 1000 × g for 15 minutes. The resultant supernatant was diluted at least five-fold. Supernatants containing the cytosolic fraction were collected separately and the protein concentrations were determined. To evaluate cytochrome c release into the cytosol, cytochrome c was measured using a Cytochrome c ELISA kit (R&D Systems, Inc, Minneapolis, MN, USA).
Caspase inhibitor experiment
BJMC3879luc2 cells were treated for 24 hours with either 10 μM or 100 μM of the following caspase inhibitors: z (N-benzyloxycarbonyl)-VAD-fmk (fluoromethyl ketone) against broad-spectrum caspases; Ac (acetyl)-DNLD-CHO (aldehyde) against caspase-3; z-IETD-fmk against caspase-8; and z-LEHD-fmk against caspase-9. With the exception of the caspase-3 inhibitor, which was purchased from Peptide Institute, Inc., Osaka, Japan, these caspase inhibitors were purchased from MBL, Inc. Nagoya, Japan. Although DEVD has generally been used as a caspase-3 inhibitor, it has recently been demonstrated as non-specific to caspase-3 [23, 24]; in the present experiment, we therefore decided to use Ac-DNLD-CHO to inhibit caspase-3. Two hours after inhibitor treatments, cells were exposed to 12 μM α-mangostin and cell viability was measured 24 hours later using a fluorescent assay kit (CellTiter-Blue Cell Viability Assay, Promega).
Flow cytometric analysis was conducted on trypsinized BJMC3879luc2 cell suspensions that were harvested after 24 hours exposure to 12 μM α-mangostin and fixed in cold 70% ethanol. The cells were stained with a 50 μg/ml propidium iodide solution containing 100 μg/ml RNase A for 30 minutes at 37°C and then placed on ice just prior to flow cytometric analysis (EPICS Elite ESP; Coulter Co., Miami, FL, USA). The percentage of cells in each phase of the cell cycle was determined using a Multicycle Cell Cycle Analysis program (Coulter).
Total protein was extracted from whole cell lysates of BJMC3879luc2 cells and MDA-MB231 cells treated with DMSO (control) or α-mangostin according to the IC50 data previously stated. Total protein (40 μg) was fractionated on 14% Tris-glycine gels under reducing conditions and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies for the following proteins: Bid, total Akt, phospho-Akt-Thr308, phospho-Akt-Ser473, and β-actin. Membranes were then incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase (HRP). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), with the exception of the antibodies for Bid (R&D Systems) and phospho-Akt-Ser473 (Cell Signaling Technology, Danvers, MA, USA). Antibody binding was subsequently visualized by exposure to an enhancing chemiluminescence reagent (Amersham ECL; GE Healthcare UK Ltd., Buckinghamshire, UK). Blots were visualized using a LAS-3000 image analyzer (Fujifilm, Co., Tokyo, Japan).
Measurement of Akt phosphorylation
MDA-MB231 cells were treated with 20 μM α-mangostin or DMSO (vehicle control) for up to six hours. Protein was extracted using cell lysis buffer containing protease and phosphatase inhibitor cocktail. Total Akt, Akt phosphorylated-threonine 308 (phospho-Akt-Thr308) and Akt phosphorylated-serine 473 (phospho-Akt-Ser473) were measured with phosphorylation assay kits (AlphaScreen SureFire for Akt signaling and GAPDH, Perkin Elmer, Waltham, MA, USA) using a multilabel plate reader (model EnSpire™ Alpha, Perkin Elmer). Data were corrected against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values and expressed as mean ± SD.
In vivostudy of α-mangostin in a metastatic mammary cancer model
Two dosages of α-mangostin - 10 and 20 mg/kg/day - were selected for the in vivo studies in mice based on the results of preliminary investigations. In brief, no differences in body or organ weights were found in mice on a four-week toxicity study of crude α-mangostin administered 0, 4, 20, 40 and 120 mg/kg by gavage. The study demonstrated that mice treated with more than 20 mg/kg/day showed significant increases in NK activity ; therefore, since 20 mg/kg/day appears to be the highest concentration that shows no harmful effect, we chose 20 mg/kg as the dose to use in the present study.
It was difficult and expensive to obtain large amounts of the pure α-mangostin. Rather than subject the mice to the stress of daily gavage as well as to minimize unwanted loss of an invaluable test agent, α-mangostin was continuously administered via subcutaneously implanted mini-osmotic pumps (Alzet model 2002, Durect Co., Cupertino, CA, USA) that were calibrated to release 0.5 μl of solution per hour. α-Mangostin solutions (15 mg/ml and 30 mg/ml) in a DMSO/100% ethanol (1:3, v/v) vehicle were prepared. Control mice received the DMSO/100% ethanol vehicle alone. Since the pumps were calibrated to release for 14 days, they were replaced every other week.
BJMC3879luc2 cells, at a concentration of 2.5 × 106 cells/0.3 ml in phosphate-buffered saline, were subcutaneously inoculated into the right inguinal region of 30 female BALB/c mice. Three weeks later, when tumors had reached approximately 0.4-0.6 cm in diameter, mice were exposed to 0, 10 or 20 mg/kg/day α-mangostin via the mini-osmotic pumps for six weeks. Individual body weights were recorded weekly. Each mammary tumor was also measured weekly using digital calipers, and tumor volumes were calculated using the formula of maximum diameter × (minimum diameter)2 × 0.4 . All surviving mice were euthanized with isoflurane anesthesia after week six. One hour prior to euthanasia, all animals were intraperitoneally injected with 50 mg/kg 5-bromo-2'-deoxyuridine (BrdU, Sigma Co., St. Lois, MO, USA) as a means to quantify the degree of tumor malignancy through DNA synthesis.
Bioluminescence imaging in vivo
At week six, five mice from each group were anesthetized by isoflurane inhalation administered via the SBH Scientific anesthesia system (SBH Designs, Inc., Windsor, Ontario, Canada). Each anesthetized mouse received an intraperitoneal injection of 3 mg of D-luciferin potassium salts (Wako Pure Chemical Industries). Bioluminescence imaging with a Photon Imager (Biospace Lab, Paris, France) was performed. The bioluminescent signals received during the six minute acquisition time were quantified using Photovision software (Biospace Lab).
At necropsy following euthanasia at week six, the tumors and lymph nodes were removed from each mouse, fixed in 10% phosphate buffered formaldehyde solution and processed through to paraffin embedding. The lymph nodes from the axillary and femoral regions were routinely removed, along with lymph nodes that appeared abnormal. Other organs that appeared abnormal were also excised and preserved in the fixative solution. Lungs were inflated with formaldehyde solution prior to excision and immersion in fixative; the fixed individual lobes were subsequently removed from the bronchial tree and examined for metastatic foci and similarly processed through to paraffin embedding. All paraffin-embedded tissues were cut at 4 μm and sequential sections were either stained with hematoxylin and eosin (H&E) for histopathological examination or left unstained for immunohistochemical analysis.
p53 and phospho-Akt immunohistochemistry
The labeled streptavidin-biotin (LSAB) method (Dako Co., Glostrup, Denmark) was used for p53 immunohistochemistry. Unstained sections were immersed in distilled water and heated for antigen retrieval prior to incubation with a p53 mouse monoclonal antibody (Clone Pab240, Santa Cruz Biotechnology) that reacts to the mutant protein in fixed specimens. Phosphorylated Akt expression in tissues was evaluated using phospho-Akt rabbit antibodies for Thr308 (Santa Cruz Biotechnology) and Ser473 (Cell Signaling Technology).
Apoptosis and active-caspases in mammary tumors
For quantitative analysis of cell death, sections from paraffin-embedded tumors were assayed using the TUNEL method in conjunction with an apoptosis in situ detection kit (Wako Pure Chemical Industries), with minor modifications to the manufacturer's protocol. TUNEL-positive cells - regarded mainly as apoptotic cells - were counted in viable regions peripheral to areas of necrosis in tumor sections. The slides were scanned at low-power (× 100) magnification to identify those areas having the highest density of TUNEL-positive cells. Four fields neighboring an area of high TUNEL positivity were then selected and counted at higher (× 200-400) magnification. The number of TUNEL-positive cells was expressed as number per cm2.
Active caspase expression in the mammary tumor tissues was immunohistochemically detected using anti-cleaved caspase-3 and anti-cleaved caspase-9 rabbit polyclonal antibodies (Cell Signaling Technology). Immunohistochemistry was conducted using the LSAB method, and CSA II amplification (Dako) was additionally applied to detect cleaved caspase-9.
DNA synthesis in mammary tumors
The tumors from five animals from each treatment group were subsequently evaluated for DNA synthesis rates as inferred by BrdU incorporation. DNA was denatured in situ by incubating unstained paraffin-embedded tissue sections in 4 N HCl solution for 20 minutes at 37°C. The incorporated BrdU was visualized after exposure to an anti-BrdU mouse monoclonal antibody (Clone Bu20a; Dako). The numbers of BrdU-positive S-phase cells per 250 mm2 were counted in four random high power (× 400) fields of viable tissue and the BrdU labeling index was expressed as number per cm2.
Blood microvascular density in mammary tumors
Immunohistochemistry based on the LSAB method (Dako) was performed to quantitatively assess blood microvessel density in primary mammary carcinomas; a rabbit polyclonal antibody against CD31 (Lab Vision Co., Fremont, CA, USA), a marker specific for blood vessel endothelium, was used. The numbers of CD31-positive blood microvessels were counted as previously described ; briefly, slides were scanned at low-power (× 100) magnification to identify those areas having the highest number of vessels and the five areas of highest microvascular density were selected and counted at higher (× 200-400) magnification.
Dilated lymphatic vessels with cancer cell invasion
Mammary tumor sections from paraffin-embedded tissues were immunohistochemically stained using the LSAB method (Dako). A hamster anti-podoplanin monoclonal antibody (AngioBio Co., Del Mar, CA, USA), against a lymphatic endothelium marker was used. To quantitatively assess the number of lymphatic vessels having intraluminal tumor cells in the whole periphery area of the primary mammary carcinomas, the slides were scanned at low-power (x100) magnification to identify podoplanin-positive lymphatic vessels. Whether the lymphatic vessel contained mammary cancer cells or not was then confirmed at higher (x200-400) magnification .
Significant differences in the quantitative data between groups were analyzed using the Student's t-test via the method of Welch, which provides for insufficient homogeneity of variance. The differences in metastatic incidence were examined by Fisher's exact probability test, with either P < 0.05 or P < 0.01 considered to represent a statistically significant difference. Survival rates were analyzed using the Holm-Sidak method.