Cells and culture conditions
Androgen-independent human PC-3 prostate cancer cells were obtained from ATCC (Rockville, MD) and cultured in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT), gentamicin (50 mg/l), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM) under conditions indicated in the figure legends. Actual experiments were performed in RPMI 1640 medium.
Antibodies and Reagents
Antibody to actin was obtained from Sigma Corporation (St. Louis, MO). Antibodies to Bcl-XL
, Bcl-2 and XIAP were obtained from Cell Signaling Technology (Beverly, MA). Antibodies to RelA, p50, IκBα and TOPO I were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary horseradish peroxidase-conjugated donkey anti-rabbit antibodies were purchased from Amersham (Arlington Hts., IL). TRAIL was purchased from Biomol (Plymouth Meeting, PA). Etoposide was obtained from Calbiochem (San Diego, CA). The hZIP1 antibody and CMV-hZIP1 expression vector were kindly provided by Dr. R. Franklin (University of Maryland) and have been described previously (18
). FluoZin-3 AM was obtained from Molecular Probes (Eugene, OR).
Stable Transfection of PC-3 cells with hZIP1
To create the hZip1 C-end FLAG-tagged expression vector, hZip1 ORF was obtained by PCR with 5′-atcttg aagctt gcc acc atg gga ccg tgg gga gag cca gag ctc ctg gtg-3′ (forward) and 5′-atcttg tctaga tta att aat cta ctt atc gtc gtc atc ctt gta atc gat ttg gat gaa gag cag gcc-3′ (reverse) primers using pRC-CMV-hZip1vector as a template and then cloned into the HindIII/Xba1 restriction sites of the pRC-CMV plasmid. PC-3 cells were transfected with either the hZIP1 expression vector or the CMV control vector using the TransIT-Prostate transfection kit (Mirus Bio, Madison, WI). Selection was performed using G418 (1.5 mg/mL; Invitrogen/Life Technologies), and screening of clones was based on Western Blot analysis with anti-hZIP1 antibody to determine hZIP1 expression. Stable transfectants were maintained in medium containing G418 (500 μg/mL).
Cells were incubated with 1 μg/ml of zinc in the form of ZnSO4 for 1 hour, washed twice with PBS, loaded with 5 μM FluoZin-3 AM at room temperature for 30 min and analyzed by flow cytometry. Analysis was performed using FACScan (Becton Dickinson, Franklin Lakes, NJ). Individual fluorescent populations were determined through the use of acquisition and analysis software (Cell Quest, Becton Dickinson).
Atomic Absorption Spectroscopy
The total zinc concentration of cells, plasma and tumor tissue specimens was measured by flame mode using a Shimadzu AA-6300 atomic absorption spectrophotometer. Cells were incubated with 1.5 μg/ml of zinc in the form of ZnSO4 for 3 hours. Harvested cells were rinsed three times in PBS, digested in 2% SDS and boiled for 10 minutes. Protein concentrations were measured with BCA protein assay (Pierce, Rockford, IL). To examine accumulation of zinc in tumor tissue specimens, portions of the tumor specimens were weighted, homogenized, digested in 2% SDS and boiled for 10 minutes. Zinc levels were re-calculated according to wet tissue weight. The plasma samples were diluted with 0.1 N nitric acid before the determination of zinc concentration.
Luciferase reporter assay
Cells were transfected with pNF-κB-luc (Stratagene, La Jolla, CA), pRL-TK or pGL3-control-luc (Promega, Madison, WI) plasmids. Twenty-four hours after transfection, cells were treated with 1.5 μg/ml of zinc in the form of ZnSO4 for 3 hours in RPMI medium followed by incubation with 10 ng/ml of TNF-α for an additional 3 hours. Samples were assayed for firefly and renilla luciferase activities using the Dual-Glo Luciferase assay System (Promega) and normalized as instructed by the manufacturer.
Western Blot Analysis
Nuclear and cytoplasmic extracts and whole cell lysates were prepared as described previously (27
). Protein concentrations were measured with BCA protein assay reagents (Pierce, Rockford, IL). Equivalent amounts of proteins (20 μg) were mixed with an equal volume of 2X Laemmli sample buffer, boiled and resolved by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The proteins were transferred from the gel to a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad) (15 V, 3 mA/cm2
for 24 minutes). Membranes were then incubated in a blocking solution containing 5% nonfat dry milk overnight to inhibit nonspecific binding. The membranes were then incubated with specific antibody (1–3 μg/ml) for 2 hours. After washing in TRIS/0.1% Tween 20 for 30 minutes, membranes were incubated for another 30 minutes with horseradish peroxidase-conjugated secondary antibody. The membranes were then washed and developed with enhanced chemiluminescence (ECL Western Blotting Kit, Amersham, Arlington Heights, IL).
IL-6, IL-8 and VEGF levels in cell culture supernatants and tumor tissue extracts were determined by ELISA kits (R&D Systems, Minneapolis, MN). Tumor tissue specimens were homogenized in 1% Tween 20/PBS containing a proteinase inhibitor cocktail (Roche Applied Science) as previously described (28
) and then centrifuged. Protein concentrations were measured with BCA protein assay reagents (Pierce, Rockford).
Measurement of apoptosis
DNA fragmentation was detected using the APO-BRDU kit (The Phoenix Flow Systems, Inc., San Diego, CA) according to the protocols provided with the kit.
The gelatinolytic activity of MMP-9 was determined in the conditioned cell culture supernatants using zymogram gels (Bio-Rad, Hercules, CA) as suggested by the manufacturer.
Surface expression of ICAM-1 was determined by staining cells with FITC-conjugated anti-ICAM-1 antibodies (R&D Systems, Minneapolis, MN) for 30 min on ice. Stained cells were washed twice with PBS and analyzed by flow cytometry. Analysis was performed on the FACScan (Becton Dickinson, Franklin Lakes, NJ). Individual fluorescent populations were determined through the use of acquisition and analysis software (Cell Quest, Becton Dickinson).
Cells were stained with Calcein AM (2 μM), pre-incubated with 1.5 μg/ml of zinc in the form of ZnSO4 for 6 hours and plated in triplicates onto 96-well plates (2.5 × 103cells per well) pre-coated with fibronectin (50 μg/ml). Cells were allowed to attach at 37°C for 30 minutes. Wells were then washed with PBS twice and images were captured using a fluorescence microscope equipped with a digital camera.
Analysis of cell invasiveness
Invasiveness was determined using a BD Falcon™ HTS FluoroBlok system (BD Biosciences, Bedford, MA) in triplicate for each condition. Cells (2.5 × 103) were seeded in the upper compartment of the FluoroBlok chamber in the absence of serum with or without 1.5 μg/ml of zinc in the form of ZnSO4. Serum-containing medium in the lower compartment served as a chemoattractant. Cells were incubated at 37°C for 12 hours and then stained with 2μM of Calcein-AM. An intervening membrane was present to block fluorescence from labeled cells present in the top chamber of the insert system. Images have been captured using fluorescence microscope equipped with a digital camera.
Assessment of in vivo tumor growth
For in vivo studies, 1 × 106 PC-3-hZIP1 or PC-3-CMV cells were inoculated s.c. in the flank region of 6 week old male C.B17/Icr-scid mice using a 27-gauge needle. All animal procedures were done according to local guidelines on animal care and with appropriate institutional certification. Animals were fed an autoclaved AIN-93M diet (Harlan Teklad, Madison, WI) and water ad libitum. Dietary zinc supplementation (2000 ppm zinc) was provided by adding ZnSO4 to the drinking water and started one week prior to tumor cell implantation. Tumors were measured twice weekly and their volumes were calculated by the formula: [volume = 0.52 × (width)2 × length]. None of the mice showed signs of wasting or other visible indications of toxicity. After 23 days of xenograft implantation animals were sacrificed by CO2 asphyxiation. At the termination of the experiment, blood was collected from the retro-orbital plexus under anesthesia from both experimental and control groups. Tumor tissue specimens were collected to assess zinc levels, the status of NF-κB activity and VEGF and IL-8 contents. Zinc levels in plasma and tissue samples were examined by Atomic Absorption Spectroscopy.
EMSA and supershift analysis
Isolation of nuclear extracts and EMSA were performed as previously described (29
). For supershift assay anti-p50 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the binding reaction buffer. Incubation continued for 20min at 25°C. Complexes were separated by electrophoresis on a 5% polyacrylamide gel in 1xTAE for 4h at 140V, dried and exposed to HyBlot CL autoradiography film (Denville Scientific) at −80°C.
Statistical analysis was performed by ANOVA. Where only two groups are compared, a Student’s t-test was applied. Results are expressed as the mean ± SEM. A P-value of <0.05 was considered statistically significant.