Cell lines and culture
The human metastatic breast carcinoma cell lines MCF7 and MDA-MB-231 as well as ovarian cancer cell lines SK-OV-3 and SNU8 were used throughout the study. These cells were provided by the Korean Cell Line Bank, Seoul, Korea and were cultured in DMEM supplemented with 10% FBS, 100 u/ml penicillin and 100 μg/ml streptomycin. MCF-10A, a line of healthy epithelial cells from human mammary gland (CRL-10317) provided by the American Type Culture Collection (Manassas, VA, USA) was used as a control. This cell line was maintained in the DMEM/Ham's Nutrient Mixture F-12 (1:1) with the addition of epidermal growth factor (20 ng/ml), cholera enterotoxin (100 ng/ml), insulin (10 μg/ml) and hydrocortisone (500 ng/ml) in the presence of 5% horse serum.
Construction of the ANT2 siRNA expression vector
ANT2 siRNA-1, siRNA-2 and siRNA-3 were synthesized by Bioneer (Daejeon, Korea), and pSilencer™ 3.1-H1 puro plasmids for DNA vector-based siRNA synthesis were purchased from Ambion (Austin, TX, US). The oligonucleotide pairs of ANT2 siRNA-1, siRNA-2, and siRNA-3 are complementary to exon 2 or exon 4 (Genbank accession number NM001152), and the sequences of ANT2 siRNA-1, siRNA-2 and siRNA-3 are 5'-GCAGAUCACUGCAGAUAAGTT-3', 5'-CTGACATCATGTACACAGG-3' and 5'-GATTGCTCGTGATGAAGGA-3', respectively. The oligonucleotide pairs were designed to contain a terminal BamHI or HindIII restriction site for subcloning into the BamHI or HindIII site of pSilencer™ 3.1-H1 puro vector to generate pSilencer™ 3.1-H1 puro ANT2 siRNA vectors (shRNAs). These vectors produce a shRNA with a TTCAAGAGA linker sequence that forms looped structures. This linker is processed with Dicer to generate an ANT2-specific siRNA. A negative scrambled siRNA (Ambion) control with no significant homology to mouse or human gene sequences was designed to detect nonspecific effects.
For transfection, cells were plated on either six-well plates (2 × 105 cells per well) or 100 mm dishes (2 × 106 cells) and were allowed to adhere for 24 hours. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for the transfections. pSilencer™ 3.1-H1 puro ANT2 siRNA vectors or pSilencer™ 3.1-H1 puro scramble siRNA vector were transfected into the cells. Transfected cells were then cultured for 4 hours and the culture media were replaced with fresh media supplemented with 10% FBS. The cells were harvested at 24–48 hours after transfection.
Reverse transcription-polymerase chain reaction
After 48 hours of transfection, the cells were collected and total RNA was extracted using Trizol (Invitrogen) according to the manufacturer's instructions. For RT-PCR analysis, 5 μg total RNA was reverse-transcribed using RT-PCR kits (Promega, Madison, WI, USA). PCR was used to amplify target cDNA with the following conditions: 35 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes. The PCR products were analyzed using standard agarose gel electrophoresis.
The primers used for RT-PCR are ANT1 forward, 5'-ACAGATTGTGTGGTTT-3' and reverse, 5'-TTTTGTGCATTAAGTGGTCTTT-3' ; ANT2 forward, 5'-CCGCAGCGCCGGAGTCAAA-3' and reverse, 5'-AGTCTGTCAAGAATGCTCAA-3' ; ANT3 forward, 5'-AACCAAGAGAACCACGTAGAA-3' and reverse, 5'-CTTAGAACAGACTTGGCTC-3' ; TNF-receptor I forward, 5'-CTGCCTCAGCTGCTCCAAA-3' and reverse, 5'-CGGTCCACTGTGCAAGAAGAG-3' ; and β-actin forward, 5'-GGAAATCGTGCGTGACATTAAGG-3' and reverse, 5'-GGCTTTTAGGATGGCAAG GGA C-3'.
Anti-ANT, anti-Bcl-xL, anti-Bax, anti-α-tubulin as well as anti-caspase-3 antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA) and polyclonal anti-ANT3 antibody was donated by Dr HH Schmid (University of Minnesota, MN, USA).
For western blot analyses, cells were harvested after 48 hours of transfection and were lysed with lysis buffer (5 mM/l ethylenediamine tetraacetic acid; 300 mM/l NaCl; 0.1% NP-40; 0.5 mM/l NaF; 0.5 mM/l Na3VO4; 0.5 mM/l phenylmethylsulfonyl fluoride; and 10 μg/ml each of aprotinin, pepstatin and leupeptin; Sigma, St Louis, MO, USA). After centrifugation at 15,000 × g for 30 minutes, the concentrations of supernatant proteins were analyzed by Bradford reagent (Bio-Rad, Hercules, CA, USA).
For the analysis of protein contents, 50 μg total proteins was electrophoresed in 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) and were then incubated with the respective antibodies indicated above. Immunoblots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
Apoptosis and DNA fragmentation assays
Approximately 2 × 105/ml MDA-MB-231 cells were transfected with respective pSilencer™ 3.1-H1 puro ANT2 siRNA-1, siRNA-2 and siRNA-3 vectors as well as with pSilencer™ 3.1-H1 puro scramble siRNA vector for the indicated times. The transfected cells were harvested, washed twice with PBS and were then incubated for 15 minutes at room temperature with a solution of annexin V conjugated with fluorescence isothiocyanate (2.5 μg/ml) and propidium iodide (PI) (5 μg/ml) (BD Pharmingen, San Diego, CA, USA) for flow cytometry (Epics XL; Coulter, Marseille, France) to detect the levels of apoptosis. Genomic DNA was extracted using genomic DNA extraction kits (G-DEX™IIc; Invitrogen, Seoul, Korea) and was subjected to electrophoresis in 2% agarose gels for DNA fragmentation analysis.
ATP assays were conducted using CellTiter-Glo™ Luminescent Cell Viability assay kits (Promega) that quantify ATP levels in viable cells. This bioluminescence assay utilizes luciferase, which induces light emission during the interaction between ATP and luciferin. Lyophilized enzyme/substrate mixtures (250 μl) were transferred to opaque 96-well microplates containing cell lysates. The plates were incubated at room temperature for 10 minutes to stabilize luminescence signals and then the stabilized signals were quantified with an Orion Luminometer (Berthold Detection Systems, Oak Ridge, TN, USA).
Cell cycle analysis
The cells transfected with pSilencer™ 3.1-H1 puro ANT2 siRNA or pSilencer™ 3.1-H1 puro scramble siRNA vector were trypsinized, counted, centrifuged and fixed in ethanol for 3 hours. These cells were then washed twice in PBS and centrifuged. Pellets were resuspended with a solution containing RNase (0.02 mg/ml) (Sigma), incubated at 37°C for 30 minutes and were stained with PI (0.02 mg/ml) (Sigma). The cells were analyzed by flow cytometry (Epics XL; Coulter).
Measurement of mitochondrial membrane potentials
To measure mitochondrial membrane potential disruption, the cells transfected with pSilencer™ 3.1-H1 puro ANT2 siRNA or pSilencer™ 3.1-H1 puro scramble siRNA vector were harvested, washed twice with PBS and were incubated with 20 nM 3,3'-diethyloxacarbocyanine (Molecular Probes, Eugene, OR, USA) for 15 minutes at 37°C. Mitochondrial membrane potential values were determined by flow cytometry (Epix XL; Coulter).
In vitro bystander effect assays
MDA-MB-231 cells (1.5 × 103) were cultured with the culture media of pSilencer™ 3.1-H1 puro ANT2 siRNA-1/siRNA-2/siRNA-3-transfected cells for 24 hours and were then harvested. Cell death was determined by annexin V–fluorescence isothiocyanate/PI staining followed by flow cytometry analysis (Epics XL; Coulter).
Fluorescent-activated cell sorter analysis (intracellular and surface staining)
Respective pSilencer™ 3.1-H1 puro ANT2 siRNA-1, siRNA-2 and siRNA-3 vectors as well as pSilencer™ 3.1-H1 puro scramble siRNA vector were transfected into MDA-MB-231 cells for the indicated times. Six hours before harvesting, the cells were treated with brefeldin A (10 μg/ml), washed twice with PBS, fixed with 2% paraformaldehyde, permeabilized with buffer (1% BSA, 0.1% saponine, 0.1% sodium azide in PBS) and were then stained with phenylethylene-conjugated anti-TNFα, anti-IFNγ, anti-IL-12 as well as anti-mouse IgG antibodies for 1 hour at 4°C (BD Pharmingen). Surface staining was performed with phenylethylene-conjugated anti-TNF-receptor 1 as well as anti-mouse IgG antibodies and then analyzed by flow cytometry (Epics XL; Coulter).
Antitumor effect of ANT2 iRNA in vivo
For tumor challenges, we established tumor models in 6-week-old to 8-week-old Balb/c nude mice by subcutaneously injecting 5 × 106 MDA-MB-231 cells into the right flanks. Treatments were started from 2 weeks after tumor inoculation when tumor volumes were <100 mm3. Intratumoral injections of PBS, pSilencer™ 3.1-H1 puro scramble siRNA vector (100 μg) or respective pSilencer™ 3.1-H1 puro ANT2 siRNA-1, siRNA-2 and siRNA-3 supplemented with Lipofectamine 2000 (200 μl) were performed three times per day for 5 days. Tumor sizes were measured using a caliper every week until 56 days after tumor challenges, and tumor volumes calculated using the formula: m12 × m2 × 0.5236 (where m1 represents the shortest axis and m2 the longest axis)
In situ detection of apoptosis detection
In situ detection of apoptosis was performed using ApopTag Fluorescein (Intergenco, New York, USA). After the blocking of endogenous peroxidase with 3% hydrogen peroxide for 5 minutes, the sections were digested with proteinase K (20 μg/ml) for 15 minutes at room temperature and were then treated with bovine testicular hyaluronidase (0.5 mg/ml) for 30 minutes at 37°C. DNA was end-labeled with deoxynucleotidyl transferase-mediated dUTP nick end-labeling and was detected with peroxidase-conjugated antidigoxigenin antibody. The reactivity was visualized by the mixtures of diaminobenzidine and hydrogen peroxide.
Data were analyzed using the Student's t test. P < 0.05 was considered statistically significant.