Preparation of ginger extract
Ginger was obtained from the local farmer’s market and extracts were prepared by soaking grated ginger in methanol overnight for four consecutive days. The supernatant was collected daily and was finally concentrated in vacuo (Buchi Rotavap, Buchi, Switzerland), followed by freeze-drying using a lyophiliser to a solid powder form. GE stock solution was prepared by dissolving 100 mg/ml of dimethyl sulfoxide, and various concentrations were obtained by appropriate dilutions. The entire study was conducted using a single batch of GE to avoid batch-to-batch variation and maximise the product constancy.
Cell lines, media, antibody and reagents
Normal prostate epithelial cells (PrEC) and prostate cancer (LNCaP, C4-2, C4-2B, DU145 and PC-3), breast (MDA-MB-231 and MCF-7) and cervical (HeLa) cancer cell lines were used in the present study. The medium used to culture these cells was Roswell Park Memorial Institute-1640 (RPMI-1640) or Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and 1 % antibiotic (penicillin/streptomycin). Primary antibodies to p21, cyclin E and BAX and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cyclin D1, cdk4, p-Rb, Bcl2, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose)polymerase (PARP) were from Cell Signaling (Beverly, MA, USA), Ki67 was from Zymed (South San Francisco, CA, USA) and β-actin was from Sigma (St Louis, MO, USA).
In vitro proliferation and colony survival assay
Cells were plated in ninety-six-well plates and treated with gradient concentrations (1–1000 μg/ml) of GE the next day. After 72 h of incubation, cell proliferation was determined using the Alamar blue cell proliferation assay. The magnitude of the fluorescent signal is proportional to the number of live cells, and is monitored using 530–560 nm excitation wavelength and 590 nm emission(25)
wavelength. For the colony assay, PC-3 cells were treated with 250 μg/ml of GE for 48 h, washed and replaced with regular RPMI medium. After 10 d, colonies were fixed with 4 % formaldehyde, stained with crystal violet and counted.
Cell-cycle progression studies by flow cytometry
For cell-cycle analysis, PC-3 cells were treated with vehicle (dimethyl sulfoxide) or GE at various doses (50, 100, 250, 500 and 1000 μg/ml) for 24 h or at a fixed dose of 250 μg/ml for various time points (12, 24, 48 and 72 h). At the end of incubation, cells were fixed with 70 % ethanol overnight, stained with propidium iodide containing RNase A, followed by data acquisition on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyses using Flo-Jo software (Ashland, OR, USA).
Western blots were performed as described earlier(26)
. Briefly, proteins were resolved by polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline containing 0·05 % Tween-20 and 5 % fat-free dry milk and incubated first with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualised with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA).
Mitochondrial and cytosolic fractionation
To determine the release of cytochrome c
from the mitochondria to the cytosol by immunoblotting, control or GE-treated (250 μg/ml) PC-3 cells were incubated on ice for 5 min in 100 μl of ice-cold cell lysis and mitochondria intact buffer (250 mM-sucrose, 70 mM-KCl and 100 μg digitonin/ml in PBS). The cells were pelleted and the supernatant containing cytosolic protein was stored at −80°C. The pellets were incubated at 4°C for 10 min in immunoprecipitation buffer (50 mM-Tris-HCl (pH 7·4), 150 mM-NaCl, 2 mM-EDTA, 2 mM-ethylene glycol tetra-acetic acid, 0·2 % Triton X-100, 0·3 % Nonidet P-40, 1 × Complete protease inhibitor; Roche Diagnostics Corporation, Indianapolis, IN, USA). The samples were centrifuged at high speed for 10 min at 4°C, and the supernatant containing mitochondrial protein was stored at −80°C(27)
. Proteins were subjected to immunoblot analysis as described above.
After treatment with 250 μg/ml of GE, PC-3 cells taken on glass coverslips were fixed with ice-cold methanol, followed by blocking with 2 % bovine serum albumin in PBS. Ki67, cleaved caspase-3 and PARP antibodies (1:250 dilution) were incubated with coverslips for 2 h at 37°C. The cells were washed with 2 % bovine serum albumin/PBS for 10 min at room temperature before incubating with a 1:500 dilution of Alexa 488- or Alexa 555-conjugated secondary antibodies. Cells were mounted with Prolong Gold antifade reagent that contains 4,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, CA, USA).
JC-1 staining for mitochondrial transmembrane potential
Control and 250 μg/ml of GE-treated cells were labelled with JC-1 reagent for 15 min at 37°C. After washing, cell fluorescence was measured on a flow cytometer using orange–red emission filters.
Caspase-3/7 activity assay
Control or 250 μg/ml of GE-treated lysates were tested for caspase-3-like activity using Ac-DEVD-7-amino-4-trifluoromethyl-coumarin, which detects the activities of caspase-3 and caspase-7 according to the manufacturer’s protocol (Calbiochem, San Diego, CA, USA). The results were evaluated using a fluorescence microplate reader and are expressed as relative fluorescence units.
In vivo tumour growth and treatment
Male Balb/c nude mice (6 weeks old) were obtained from the NCI (Frederick, MD, USA), and 106 PC-3 cells in 100 μl PBS were injected subcutaneously in the right flank without any basement membrane extracts such as Matrigel. The animals were given autoclave-sterilised standard diet pellets and water ad libitum. When tumours were palpable, mice were randomly divided into two groups. From each group, six mice were housed individually in one cage. The control group received vehicle and the treatment group received 100 mg/kg body weight of GE daily by oral administration. Tumour growth was monitored weekly using a vernier caliper and body weight was also recorded. All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
Histopathological and immunohistochemical staining
After 8 weeks of vehicle or 100 mg/kg GE treatment, tumour, lung, spleen, adrenal, liver, gut, brain, kidney, heart, testes and bone marrow were formalin-fixed, paraffin-embedded and 5 μm thick sections were stained with Ki67, cleaved caspase-3, PARP and haematoxylin and eosin. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining of tumour tissue sections was performed using the DeadEnd Fluorometric TUNEL System (Promega Inc., Madison, WI, USA) according to the manufacturer’s instructions.
All the experiments were repeated at least three times. Results are expressed as mean values of at least three independent experiments and standard deviations, and P values (Student’s t test) were calculated in reference to control values using Excel software.