Introduction
Apoptosis can occur via a death receptor-mediated pathway or a mitochondrial pathway, and mitochondria-mediated apoptosis is initiated by multiple stimuli such as TNF, CD95 and stresses
Adenine nucleotide translocase (ANT) is a nuclear-encoded protein abundantly located in the inner mitochondrial membrane, and the role of this protein is to catalyze the exchange of mitochondrial ATP with cytosolic ADP. ANT therefore plays an important role in cellular energy metabolism by influencing mitochondrial oxidative phosphorylation. In addition, ANT is the major component of mitochondrial permeability–transition pore complex (PTPC) that interacts with Bcl2 family proteins, thereby contributing to mitochondria-mediated apoptosis
Human ANT has four isoforms (ANT1, ANT2, ANT3 and ANT4) and the relative expressions of these isoforms are dependent on developmental stages, proliferation status as well as tissue types or cell types. ANT3 is ubiquitously expressed in all tissues and the degrees of ANT3 expression are correlated with the levels of oxidative metabolism. ANT1 is highly expressed in terminally differentiated tissues such as skeletal muscles, heart and brain, whereas ANT4 is a murine stem and germ cell-specific isoform whose DNA methylation plays a key role in its transcriptional silencing in somatic cells
iRNA is currently used for knockdown of a particular gene expression to identify the functions of a targeted gene and to examine the potential usage of a targeted gene as a therapeutic method. iRNA is an evolutionarily conserved phenomenon, and a multistep process produces active siRNA from RNase III endonuclease (Dicer). The consequent 21-nucleotide to 23-nucleotide siRNAs then mediate the degradation of complementary homologous RNA
In the present article we investigate the anticancer effects of ANT2 RNA interference in breast cancer models
Materials and methods
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
Transfection
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'.
Western blotting
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 ×
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 assay
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).
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
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:
Statistical analysis
Data were analyzed using the Student's
Results
ANT2 expression markedly upregulated in human breast cancer cell lines and efficiently suppressed by RNA interference
To explore the functions of ANT2 in cancer, we first examined its mRNA expression levels in human cancer cell lines obtained from various organs (Figure
Expression and silencing efficiency of adenine nucleotide translocator 2 in cancer cell lines
Expression and silencing efficiency of adenine nucleotide translocator 2 in cancer cell lines. Expression of adenine nucleotide translocator (ANT) 2 in cancer cell lines and the silencing efficiency of ANT2 siRNA and shRNA in human breast and ovarian cancer cell lines. (a) RT-PCR analysis for detecting ANT2 mRNA expression in various human cancer cell lines. To evaluate ANT2 expression levels in the human cancer cell lines of various origins (SNU668, SNU719, SNU638, NCI-H358, A549, NCI-H889, SK-HEP-1, SNU449, SNU423, SK-OV-3, SNU8, SNU840, MCF7, MDA-MB-231 and SK-BR-3), total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human ANT2 or β-actin (internal control). (b) RT-PCR analysis and western blotting to detect ANT isoform expressions in breast cancer cell lines as well as a non-neoplastic breast cell line. To compare ANT isoform mRNA levels in non-neoplastic breast epithelial cell line MCF10A with other breast cancer cell lines such as MCF7 and MDA-MB-231, total RNA was extracted from the respective cell lines and subjected to RT-PCR using specific primers for human ANT1/ANT2/ANT3 or β-actin. In addition, to detect ANT protein levels, total cell extracts were used for performing western blotting with anti-ANT, anti-ANT3 and anti-β-actin antibodies. (c) RT-PCR analysis and western blotting for detecting the level of ANT2 repression mediated by ANT2 siRNA and ANT2 shRNA. To assess the extinction of endogenous human ANT2 mRNA in MCF7 and MDA-MB-231 cells due to ANT2 RNA interference, respective cell lines were transfected with ANT2 siRNAs, ANT2 shRNAs, scramble siRNA as well as scramble shRNA for 48 hours. Total RNA was then extracted from respective samples and subjected to RT-PCR using specific primers for human ANT2 or β-actin. To indirectly detect the reduction of ANT2 protein by ANT2 RNA interference, MCF7 cells were transfected with ANT2 shRNA-1, shRNA-2 and shRNA-3 as well as scramble shRNA, and 48 hours later total cell extracts were collected for performing western blotting with anti-ANT and anti-β-actin antibodies.
To expand the knowledge of ANT mRNA expressions among breast cancer cell lines, the MCF7 and MDA-MB-231 cell lines, as well as the non-neoplastic mammary epithelial cell line MCF10A, the relative expressions of three ANT isoforms from these cell lines were investigated at mRNA and protein levels (Figure
Additional file
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ANT2 protein levels were also tested from the breast cell lines by western blotting (Figure
Owing to the unique feature of overt ANT2 overexpression in breast cancer and ovarian cancer cell lines, we hypothesized that the downregulation of ANT2 in these cell lines may induce serious physiological effects that result in cell growth inhibition or cell death. To evaluate the functions of ANT2 in cancer development or the possible utility of ANT2 downregulation for breast cancer therapy, therefore, ANT2-specific knockdown experiments were performed with human breast cancer cell lines. Initially, respective siRNA-1, siRNA-2 and siRNA-3 oligonucleotides directed at exon 2 or exon 4 of ANT2 mRNA were synthesized to determine whether ANT2 siRNA suppresses ANT2 expression. At the same time, DNA oligonucleotides representing the siRNA duplex were cloned into pSilencer™ 3.1H1 puro vector to produce a high-level silencing effect based on a DNA vector system. The synthesized shRNA derived from DNA templates was composed of two identical 21-nucleotide sequence motifs in an inverted orientation, separated by a 9 base pair nonhomologous spacer (shRNA).
To confirm the silencing efficacies of ANT2 siRNA-1, siRNA-2 and siRNA-3 as well as pSilencer™ 3.1H1-ANT2 siRNA (ANT2 shRNA-1, siRNA-2 and siRNA-3), the cell lines MCF7 and MDA-MB-231 were transfected with the above samples as well as scramble siRNA and pSilencer™ 3.1H1-scramble siRNA (scramble shRNA) as negative controls. After culturing for 48 hours, their efficacies in extinguishing ANT2 mRNA expression were evaluated by RT-PCR. As shown in Figure
Taken together, we have uniquely identified the higher expression of ANT2 mRNA from human breast cancer cell lines MCF7, MDA-231 and SK-BR-3 as well as ovarian cancer cell lines SK-OV-3 and SNU8. To investigate the possible utility of ANT2 downregulation as a cancer therapy, ANT2 shRNA systems were adopted and the actual downregulation of ANT2 mRNA as well as protein mediated by ANT2 shRNA was confirmed from human breast cancer cell lines. As a result, we established a shRNA system to investigate the therapeutic value of ANT2 downregulation in breast cancer
ANT2 depletion induces cell cycle arrest (G1 arrest) and apoptotic cell death
To explore the potential of ANT2 shRNA as a treatment for human breast cancer, the diverse phenotypic changes of respective cancer cell lines affected by ANT2 shRNA were investigated. Firstly, the cell survival and proliferation rate were investigated using MDA-MB-231. After transfecting MDA-MB-231 cells with ANT2 shRNA-1 for 24 hours, the transfected cells became less confluent as compared with the cells transfected with scramble shRNA. Many ANT2 shRNA transfected cells even became rounded and detached from culture plates, providing one line of evidence about cell death or cell cycle arrest (Figure
Apoptotic changes of human breast cancer cell lines transfected with adenine nucleotide translocator 2 shRNA
Apoptotic changes of human breast cancer cell lines transfected with adenine nucleotide translocator 2 shRNA. (a) Cell morphology analysis: MDA-MB-231 cells were transfected with scramble shRNA or adenine nucleotide translocator (ANT) 2 shRNA-1 and then examined under a phase contrast microscope after 24 hours of transfection. (b) Cell proliferation assay: after 2 or 4 days of transfection with ANT2 shRNA-1 as well as scramble shRNA, the numbers of viable cells were determined using a hemacytometer after staining dead cells with Trypan Blue. (c) ATP assay: cells were transfected with ANT2 shRNA-1 as well as scramble shRNA and then lysed to quantify total intracellular ATP levels after 24 hours of incubation. Results are tabulated in relative ATP production by normalizing luminescence units (RLU) with total protein levels. (d) Cell cycle analysis: after 24 or 48 hours of transfection with scramble shRNA and ANT2 shRNA-1, the cells were trypsinized, fixed in ethanol and stained with propidium iodide to determine DNA contents. Cell cycle distributions were analyzed by flow cytometry. (e) Apoptosis analysis: cells were transfected with specific siRNA or shRNA against ANT2, and then 48 hours later the transfected cells were stained with annexin V–fluorescence isothiocyanate (FITC) and propidium iodide (PI) for flow cytometry analysis. Data are representative of three independent experiments.
We finally tested whether the knockdown of ANT2 shRNA induces cell death in the breast cancer cell lines MCF7 and MDA-MB-231
Taken together, these results suggest that the downregulation of ANT2 efficiently inhibits cell growth and proliferation by reducing ATP levels as well as inducing G1 arrest, which ultimately leads to the apoptosis of human breast cancer cells
ANT2 shRNA induces apoptosis by reducing the membrane potential of mitochondria and activating caspase 3 in MDA-MB-231 cells
To confirm apoptosis was responsible for the cell death induced by ANT2 shRNA in breast cancer cell lines, we examined the presence of genomic DNA fragmentation. As we expected, DNA laddering – a characteristic of apoptotic cell death – was abundantly observed from ANT2 shRNA-1 transfected MDA-MB-231 cells (Figure
Adenine nucleotide translocator 2 shRNA regulated Bcl-2 family members and disrupted mitochondrial membrane potentials
Adenine nucleotide translocator 2 shRNA regulated Bcl-2 family members and disrupted mitochondrial membrane potentials. Adenine nucleotide translocator (ANT) 2 shRNA regulated the levels of Bcl-2 family members and disrupted mitochondrial membrane potentials to induce apoptosis in MDA-MB-231 cells. (a) DNA laddering assay: cells were transfected with ANT2 shRNA-1, and then 48 hours later total genomic DNA was extracted and subjected to electrophoresis in 2% agarose gel to examine DNA fragmentation patterns. (b) Western blotting for identifying apoptotic mechanisms: cells were transfected with ANT2 shRNA-1, shRNA-2 and shRNA-3. Cytoplasmic extracts (free of mitochondria) were prepared after 24 hours of incubation for performing western blotting with anti-Bax, anti-Bcl-xL, anti-cleaved caspase 3 and anti-α-tubulin antibodies. (c) Analysis for detecting mitochondrial membrane potential: cells were transfected with ANT2 shRNA-1, and then 24 or 48 hours later the transfected cells were stained with 3,3'-diethyloxacarbocyanine. Mitochondrial membrane potentials were quantified by flow cytometry. Data are representative of three independent experiments.
It has been reported that Bcl-2 family molecules can physically interact with ANT and that this interaction regulates ANT-mediated mitochondrial permeability transition pore formation
We assumed the apoptosis triggered by ANT2 shRNA must be involved in the mitochondria-mediated apoptotic pathway because ANT2 protein is abundant in mitochondrial membranes. The potentials of mitochondrial membranes were measured by 3,3'-diethyloxacarbocyanine staining to prove our hypothesis. As expected, the cells transfected with ANT2 shRNA-1 displayed about 10-fold shifted curves to the left after 24 and 48 hours of transfection, indicating that mitochondrial membrane potentials are disrupted in apoptotic cells transfected with ANT2 shRNA (Figure
Taken together, these results implicate that ANT2 shRNA changes the Bcl-2 family balance in mitochondrial membranes, favoring a proapoptotic pore-forming status, and thereby causes the disruption of mitochondrial membrane potentials resulting in cell apoptosis.
Cell death by ANT2 shRNA amplified by the cytotoxic bystander effect in MDA-MB-231 cells
Before transfecting MDA-MB-231 cells with ANT2 shRNA-1, shRNA-2 and shRNA-3, we also assessed transfection efficiencies using GFP (Green Fluorescence Protein)-expressing vector. Although the transfection efficiency was only about 30%, the proportion of cells killed by ANT2 shRNA treatment exceeded 60%, raising the possible existence of bystander effect. To confirm the presence of a bystander effect in MDA-MB-231 cells, we cultured nontransfected cells with the supernatants obtained from ANT2 shRNA-1, shRNA-2 and shRNA-3 transfected cells and then evaluated cell death by annexin V–PI staining.
As shown in Figure
Adenine nucleotide translocator 2 shRNA induced cytotoxic bystander effects on MDA-MB-231 cells
Adenine nucleotide translocator 2 shRNA induced cytotoxic bystander effects on MDA-MB-231 cells. To evaluate bystander effects, MDA-MB-231 cells were transfected with adenine nucleotide translocator (ANT) 2 shRNA-1, shRNA-2 and shRNA-3. After 48 hours of incubation, nontransfected cells were cultured with the media obtained from ANT2 shRNA-1, shRNA-2 or shRNA-3 transfected cells for the next 24 hours. These nontransfected cells were then stained with annexin V–fluorescence isothiocyanate (FITC) and propidium iodide (PI) for flow cytometric analysis. Data are representative of three independent experiments.
Bystander effect associated with TNFα production and increased TNF-receptor I expression in ANT2 shRNA transfected cells
To determine the cause of this bystander effect, we examined the levels of proinflammatory cytokines such as TNFα, IFNγ and IL-12 from MDA-MB-231 cells treated with ANT2 shRNA or scramble shRNA by intracellular fluorescent-activated cell sorter. TNFα expression was elevated eight-fold in the cells treated with ANT2 shRNA-1 but the levels of IFNγ and IL-12 were unchanged (Figure
Bystander effects generated in MDA-MB-231 cells associated with TNFα production and TNF-receptor I expression
Bystander effects generated in MDA-MB-231 cells associated with TNFα production and TNF-receptor I expression. Bystander effects generated by adenine nucleotide translocator (ANT) 2 shRNA in MDA-MB-231 cells were associated with TNFα production and TNF-receptor I (TNFRI) expression. (a) Intracellular staining of TNFα, IFNγ and IL-12 p40. Cells were transfected with ANT2 shRNA-1. After 48 hours of incubation, the transfected cells were treated with brefeldin A for the next 6 hours. The cells were harvested, fixed in paraformaldehyde and then stained with phenylethylene-conjugated anti-TNFα, IFNγ or IL-12 p40 as well as anti-mouse IgG antibodies (negative control). Intracellular levels of TNFα, IFNγ and IL-12 p40 were analyzed by flow cytometry. (b) RT-PCR and fluorescent-activated cell sorter analysis for detecting the expression of TNFRI at transcriptional and translational levels. Cells were transfected with ANT2 shRNA-1. After 24 hours of incubation, total RNA was extracted and subjected to RT-PCR using specific primers for human TNFRI and β-actin. The RT-PCR products were analyzed by 1% agarose gel electrophoresis. In addition, the surface expression of TNFRI was measured by flow cytometry after staining cells with phenylethylene-conjugated anti-TNFRI antibody. (c) Partial neutralization of bystander effect mediated by anti-TNFα antibody. Cells were transfected with ANT2 shRNA-1. After 48 hours of incubation, the supernatants were collected and then mixed with or without TNFα antibody before transferring into nontransfected cells. These nontransfected cells were cultured for the next 24 hours and stained with annexin V–fluorescence isothiocyanate (FITC) and propidium iodide (PI) for flow cytometric analysis.
In addition, we evaluated TNF family death receptor I expression by RT-PCR and flow cytometry. Both transcription and surface expression of TNF-receptor I were upregulated by the transfection with ANT2 shRNA (Figure
ANT2 shRNA inhibits tumor growth
We finally evaluated the antitumor effects of ANT2 shRNA
Adenine nucleotide translocator 2 shRNA inhibited tumor growth
Adenine nucleotide translocator 2 shRNA inhibited tumor growth
Discussion
Energy generation by cells in the form of ATP can be achieved in two main ways: oxidative phosphorylation in the mitochondria, and glycolysis in the cytoplasm. Most normal cells mainly utilize mitochondrial oxidative phosphorylation for ATP synthesis but switch to glycolysis at lower oxygen tension
ANT exchanges ADP and ATP across the mitochondrial inner membrane. Specifically, ANT1 and ANT3 export ATP produced by mitochondrial oxidative phosphorylation, whereas ANT2 imports ATP produced by glycolysis into mitochondria to supply the energy required for mitochondrial functions
In the present study, we used DNA vector-based ANT2 siRNA as a tool to inhibit ANT2 transcripts. It was found that DNA vector-based ANT2 siRNA (shRNA) reduced ATP levels, induced cell cycle G1 arrest and effectively induced apoptotic cell death. Moreover, this apoptosis was accompanied by mitochondrial membrane potential disruption, cytochrome c release (data not shown) and caspase-3 activation. These facts encouraged us to speculate that ANT2 repression may disable ADP/ATP exchanges in mitochondrial membranes and/or promote PTPC formation as well as apoptosis. Interestingly, we also observed that ANT2 shRNA treatment upregulated proapoptotic Bax expression and downregulated antiapoptotic Bcl-xL expression. We speculate that these changes of gene expressions may contribute to the apoptotic cell death induced by ANT2 shRNA. Bcl2 family members in the mitochondrial membranes modulate the activities of ANTs, and the Bcl2 family and ANT interactively participate in the formation of PTPC
In contrast to our results, a recent study using siRNA oligonucleotides to knockdown ANT2 showed that ANT2 suppression neither induced apoptosis nor had any discernible effect on the mitochondrial network as well as the cell cycle. This suppression, however, did result in chemosensitization and increased mitochondrial transmembrane potential as well as reactive oxygen species levels
Another important finding from the present study was the bystander effect induced by ANT2 shRNA treatment. We demonstrated that this phenomenon was due to TNFα secretion and TNF-receptor I upregulation from ANT2 shRNA-treated cells. In addition, the cell death induced by the bystander effect was mainly interpreted as necrosis. How the expressions of TNFα and TNF-receptor I were induced by ANT2 shRNA treatment, however, and why the bystander effect induced necrotic cell death, are not known. More experiments are needed to address these questions.
In a previous report, mitochondrial H+-ATPase inhibitor was found to increase mitochondrial membrane permeability, to decrease cellular ATP levels and to stimulate reactive oxygen species production, subsequently leading to the activation of stress-activated pathways and p21 induction
In summary, the knockdown of ANT2 using ANT2 shRNA systems in breast cancer models induces apoptotic cell death followed by ATP depletion, G1 stage arrest and the disruption of mitochondrial membrane potentials
Conclusion
This present study demonstrates that ANT2 silencing by DNA vector-based iRNA effectively induces apoptotic cell death and tumor growth inhibition in breast cancer models
Abbreviations
ANT = adenine nucleotide translocator; BSA = bovine serum albumin; DMEM = Dulbecco's modified Eagle's medium; dsRNA = double-stranded RNA; FBS = fetal bovine serum; IFN = interferon; IL = interleukin; iRNA = interfering RNA; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; PI = propidium iodide; PTPC = permeability–transition pore complex; RT = reverse transcription; shRNA = short-hairpin RNA; siRNA = small interfering RNA; TNF = tumor necrosis factor.
Competing interests
The authors have applied for the domestic patent and will apply for the international patent regarding the utilization of ANT2 siRNA technology for a therapeutic method for cancer treatment. Seoul National University College of Medicine will retain the patent. The authors declare that they have no other competing interests.
Authors' contributions
J-YJ performed most of the experiments and was responsible for producing the results, for the data analysis, and for preparing the manuscript. YC contributed to the conduction of additional experiments. Y-KJ was responsible for analysis of the data and for writing the paper. C-WK contributed to the design of the project, to data analysis and to writing of the paper. All authors reviewed and agreed the final manuscript.