Introduction
Bcl-2 and Bcl-xL proteins are inhibitors of the mitochondrial apoptosis pathway; they exert their action by blocking their proapoptotic counterparts, including Bid and Bax, thereby preventing the release of cytochrome
c and the activation of caspase [
1,
2]. Bcl-xL shows remarkable homology to Bcl-2 and inhibits apoptosis as effectively as Bcl-2 in some cells. Furthermore, Bcl-xL is capable of preventing cell death when Bcl-2 fails to do so, suggesting that these proteins exert independent effects on the mitochondrial apoptotic pathway [
3]. Given that Bcl-2 and Bcl-xL are capable of inhibiting anticancer drug-induced apoptosis, which is mediated by the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, overexpression of Bcl-2 and Bcl-xL might confer resistance to chemotherapy [
4]. In fact, overexpression of Bcl-2 and Bcl-xL is observed in several cancers, including hematologic malignancies, as well as a range of solid tumors, including nasopharyngeal, colorectal, prostate, and breast cancer [
5‐
7].
Antisense oligodeoxynucleotides (AS ODNs) are short, synthetic stretches of DNA that hybridize with specific mRNA strands corresponding to target genes. By binding to mRNA, AS ODNs prevent the translation of target proteins, thereby blocking gene expression. Phosphorothioate ODNs, in which the oxygen atom of the phosphodiester moiety of the DNA backbone is replaced by sulphur, are the most commonly used first-generation AS ODNs because they have acceptable physical and chemical properties while showing resistance to nucleases [
8]. Several studies indicate that overexpression of Bcl-2 inhibits apoptosis induced by anticancer drugs, radiation, and other DNA-damaging agents [
9,
10]. In addition, increased sensitivity to anticancer drugs after treatment with AS Bcl-2 is observed in solid tumors, such as breast, prostate, lung, and gastric cancer [
11‐
14]. Similarly, downregulation of Bcl-xL protein expression by AS ODNs in various tumor cell lines resulted in activation of apoptosis, as well as decreased cellular proliferation and increased sensitivity to cytotoxic chemotherapeutic agents [
15,
16].
The two CpG motifs of AS
Bcl-2, which are unmethylated dinucleotide sequences of cytosine followed by guanine, are associated with potent immune stimulation [
17]. CpG ODNs administered in the vicinity of various animal tumors show marked antitumor activity [
18,
19]. However, it remains unclear whether immune stimulation is responsible for the antitumor effects of AS
Bcl-2 ODNs.
In the present study we examined the effects of downregulation of Bcl-2 and Bcl-xL on the chemosensitivity of breast cancer cells in vitro and in vivo with the aim of using this approach as a specific targeting therapy. The possibility of using growth inhibition as a mechanism by which AS Bcl-2 ODNs enhance chemosensitivity was also explored. Furthermore, we studied the effect of Bcl-2 gene transfection on the chemosensitivity of a breast cancer cell line that normally expresses a low basal level of Bcl-2. Finally, we attempted to evaluate the effects of the two AS Bcl-2 CpG motifs on immunostimulatory function and antitumor activity in athymic mice.
Materials and methods
Materials
The human breast cancer cell lines BT-474, ZR-75-1, MDA-MB-231, and MDA-MB-453 were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 (Gibco BRL, New York, USA) containing 10% heat-inactivated fetal bovine serum and antibiotics. Cultures were maintained in a humidified incubator at 5% CO2 and 37°C.
ODNs and anticancer drugs
Phosphorothioate ODNs, purified by reverse-phase high-performance liquid chromatography, were purchased from Biologica (Tokyo, Japan). The following AS
Bcl-2 oligonucleotide sequence, corresponding to the first six codons of the human Bcl-2 open reading frame, was used: AS 5'-TCTCCCAGCGTGCGCCAT-3' [
20]. The following Bcl-2 oligonucleotide sequences were used as controls: 5'-TCTCCCAGCATGTGCCAT-3' as a two-base mismatch control (MM), and 5'-TACCGCGTGCGACCCTCT-3' as a random control (RC). Phosphorothioate oligonucleotides corresponding to the initiation site of human
Bcl-2 described above were made with 5'-methylation of cytosine (m5C) residues in the two CpG motifs: 5'-TCTCCCAG
m5CGTG
m5CGCCAT-3'. The following
Bcl-xL target sequence was used: 5'-CCATCCCGGAAGAGTTCATT-3'. In addition, the following
Bcl-xL sequences were used as a sense control and a two-base MM: 5'-AATGAACTCTTCCGGGATGG-3' [
16] and 5'-CCATCCCAGAAGAGTTTATT-3', respectively. The
Bcl-2 and
Bcl-xL sequences were not homologous with
Bcl-xS or with any other known human gene sequences. All of the oligonucleotides were diluted to a concentration of 1 mM, filter-sterilized, and stored at -30°C in distilled water. Doxorubicin (DOX) and mitomycin C (MMC) were from Kyowa Hakko Co., Ltd (Tokyo, Japan), paclitaxel (TXL) was from Bristol-Myers K.K. (Tokyo, Japan), and docetaxel (TXT) was from Aventis Pharma (Tokyo, Japan). DOX, MMC, and TXT were prepared with saline solution, and TXL was dissolved in dimethyl sulfoxide (DMSO).
Cell extraction and Western blotting
Cells were washed twice with PBS, centrifuged at 2,700 g and 4°C, and lysed with lysis buffer containing 10 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM EDTA, 10 mM CHAPS, 10 μg/ml aprotinin, and 0.02 mM phenylmethylsulfonyl fluoride. Lysate was incubated for 15 min on ice and centrifuged for 15 min at 2,700 g. Supernatant was collected and the protein quantity was estimated with Bio-Rad protein assay dye (Bio-Rad, Hercules, CA, USA). Samples containing equal amounts of protein (15 μg) were subjected to electrophoresis on a 12.5% sodium dodecyl sulfate-polyacrylamide gel, and transferred to a poly(vinylidene difluoride) membrane. After being blocked overnight with PBS containing 5% nonfat milk powder, the membrane was incubated with primary antibody (1:200 dilution) for 1 hour at room temperature at 25°C. Antibodies used for specific immune blotting included anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-pAkt, anti-poly(ADP-ribose) polymerase (anti-PARP), and anti-β-actin. All antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membrane was washed three times with PBS and then incubated with anti-rabbit or anti-mouse IgG antibody (1:1,000 dilution; Sigma Chemical, St Louis, MO, USA) for 1 hour at room temperature. After three washes with PBS, specific protein bands were detected with an enhanced chemiluminescence western blot detection system (ECL; Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK), and detected after exposure to Hyper film ECL (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Each protein signal was quantified with Scion image software (Scion Corporation, Frederick, MA, USA).
MTT assays and cell viability
Cells were seeded into 96-well plates at 104 viable cells per well and left to attach to the plate for 24 hours. After 24 hours, cells were treated with anticancer drugs for 48 hours. The final volume was 200 μl per well. Subsequently, 200 μl of medium containing 0.25 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma Chemical, Tokyo, Japan) was added to each well for 3 hours. The medium was then removed and 150 μl of DMSO (Wako Pure Chemical Industries, Osaka, Japan) was added to each well for 30 min at room temperature. The absorbance of each well was measured with a microculture plate reader at 540 nm. Growth inhibition was expressed as a ratio of the mean absorbance of drug-treated cells to that of control cells. Experiments were performed in triplicate, and growth inhibition rates and IC50 values were calculated. Cell viability was also assessed with the trypan blue dye-exclusion test.
Transfection
pZip
neo plasmid expression vectors containing human
bcl-2 cDNA were used [
21]. MDA-MB-453 cells in the exponential phase of growth were transfected with the pZip
neo plasmid expression vector with the use of Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA), in accordance with the instructions provided by the manufacturer. After this, cells were selected with 400 μg/ml Geneticin Liquid (Gibco BRL, New York, USA), and the resulting clones were pooled. The expression of Bcl-2 by MDA-MB-453 cells was determined by Western blotting with a monoclonal antibody against Bcl-2.
Antitumor activity in vivo
Four-week-old female athymic mice (Balb/c, nu/nu) were obtained from Clea Japan (Tokyo, Japan). The research protocol was in accordance with the institutional guidelines of the Hiroshima University Animal Care and Use Committee. BT-474 (estrogen receptor (ER)-positive), ZR-75-1 (ER-positive), and MDA-MB-231 (ER-negative), cells were established as subcutaneous xenografts by the injection of 107 cells suspended in 150 μl of Matrigel (Becton Dickinson Labware, Bedford, MA, USA) in the lateral back region.
Mice were implanted subcutaneously with pellets engineered to give a controlled release of 0.72 mg of 17β-estradiol over 60 days (Innovative Research, Sarasota, FL, USA) 1 week before estrogen-dependent cell implantation. When palpable tumors arose, three or four pieces (1 to 2 mm3) of nonnecrotic tissue were subcutaneously transplanted into other mice with the use of a biomedical stainless steel needle (implant needle) under anesthesia. Once tumors had reached a volume of about 100 mm3, mice were randomized to receive one of the following treatments: no treatment (control group); treatment with AS Bcl-2 or AS Bcl-xL ODNs; treatment with various anticancer agents; or combined treatment with AS ODNs and various anticancer agents. AS Bcl-2 and AS Bcl-xL ODNs (5 mg/kg) were administered by intraperitoneal injection 6 days a week, every other week for 4 weeks. The anticancer agents were administered once a week for 4 weeks as 10 mg/kg TXL or TXT, or 2 mg/kg MMC, by intraperitoneal injection, or 6 mg/kg doxorubicin by bolus injection into the tail vein. Serial measurements of tumor diameter were made with calipers, and tumor volumes were calculated as volume = width2 × length/2.
Serum levels of IL-12
Changes in serum levels of IL-12 after exposure to AS Bcl-2 ODNs, as well as AS Bcl-2 ODNs with methylated CpG motifs (synthetic CpG AS Bcl-2 ODNs), were measured by enzyme-linked immunosorbent assay (SRL, Tokyo, Japan); the results were compared with control values. Changes in splenic weight and serum IL-12 levels were evaluated after treatment with AS Bcl-2 or synthetic CpG AS Bcl-2 ODNs.
Statistical analysis
All of the linear regression was performed with Microsoft Excel (Seattle, WA, USA). Student's t-test was used to measure statistical significance between two treatment groups. Multiple comparisons were performed with a one-way analysis of variance (ANOVA). Data were considered significant if P < 0.05.
Discussion
In the present study we showed that treatment with AS Bcl-2 and AS Bcl-xL ODNs produced sequence-specific decreases in protein levels, thereby enhancing the chemosensitivity of BT-474, ZR-75-1, and MDA-MB-231 breast cancer cells to various anticancer drugs both in vitro and in vivo. Treatment with AS Bcl-2 caused greater enhancement of chemosensitivity than treatment with AS Bcl-xL. A number of factors might explain the different effects of AS Bcl-2 and AS Bcl-xL on chemosensitivity, despite the fact that both Bcl-2 and Bcl-xL inhibit apoptotic cell death through the mitochondrial pathway. One determining factor might be the differential expression of Bcl-2 and Bcl-xL in breast cancer cells. Overexpression of Bcl-2 is observed more frequently than overexpression of Bcl-xL (70% versus 40%) in breast cancer tissue, which suggests a more important role for Bcl-2 in conferring drug resistance.
Another influencing factor might be a difference in the ability of sequence-specific AS ODNs to suppress Bcl-2 and Bcl-xL expression. Although our results indicate that greater suppression of Bcl-2 than Bcl-xL was achieved, the
in vitro and
in vivo data obtained in this study suggest that differences in Bcl-2 and Bcl-xL suppression do not fully explain their differing effects on chemosensitivity. The differential effects of Bcl-2 and Bcl-xL on drug sensitivity might be unique to breast cancer. In addition, despite similarities in function in the Bcl-2 family proteins, there is evidence to suggest that Bcl-2 and Bcl-xL are subject to different regulatory mechanisms. Bcl-2 inhibits Bid-induced apoptosis at the mitochondrial level by blocking cytochrome
c release, whereas Bcl-xL does not affect the insertion of tBid into mitochondrial membranes [
23,
24]. Some reports suggest that Bcl-xL, but not Bcl-2, is capable of modulating apoptosis induced by tumor necrosis factor-related apoptosis ligand (TRAIL) [
25].
With regard to the effects of AS
Bcl-2 on chemosensitivity, sensitivity to DOX and taxanes
in vitro was increased to a greater extent than sensitivity to MMC in BT-474, ZR-75-1, and MDA-MB-231 cells. Moreover, enhanced sensitivity to DOX and taxanes was more pronounced in BT-474 cells than in ZR-75-1 cells. These findings suggest that the downregulation of Bcl-2 expression by AS
Bcl-2 enhances drug sensitivity by modulating the apoptotic signal transduction pathway of Bcl-2. The apoptotic signal transduction pathway commonly induced by anticancer agents is associated with the induction of Bax and cleaved PARP, and the downregulation of Bcl-2 and pAkt. Bcl-2 expression is regulated by the ER-responsive element of the promoter region of the
bcl-2 gene [
26], such that overexpression of Bcl-2 might be expected to confer greater drug resistance on ER-positive breast cancer cells. Increases in chemosensitivity to DOX and taxanes
in vitro did not correlate well with antitumor activity
in vivo, suggesting that other factors might influence the response of athymic mice to chemotherapeutic agents. The converse was observed for combined treatment of MDA-MB-231 cells with AS
Bcl-xL and MMC, for which far greater efficacy was observed
in vivo than
in vitro.
The role of Bcl-2 in determining the chemosensitivity of breast cancer cells was tested in MDA-MB-453 cells expressing low levels of Bcl-2. Transfection of the
bcl-2 gene into MDA-MB-453 cells decreased their sensitivity to DOX and MMC but not to taxanes such as TXL and TXT. Several studies indicate that
in vitro treatment with taxanes induces phosphorylation and inactivation of the Bcl-2 protein as well as apoptosis [
27], which might explain why Bcl-2-transfected cells retained their sensitivity to taxanes in the present experiment. Because the enforced overexpression of Bcl-2 can act as an antioxidant in response to DNA damage, the decreased chemosensitivity of the Bcl-2-transfected breast cancer cells to DNA-damaging agents, including DOX and MMC, may be explained by this effect.
Akt is another antiapoptotic protein that belongs to the serine/threonine kinase family. Bcl-2 is activated by Akt through a cyclic-AMP-responsive element-binding protein (CREB) [
28]. An Akt- and Bcl-2-dependent pathway might mediate the prevention of anticancer drug-induced cell death through CREB and NF-κB. Because downregulation of Bcl-2 by AS
Bcl-2 might result in downregulation of pAkt, downregulation of pAkt after treatment with DOX might be augmented by concurrent treatment of ZR-75-1 cells with AS
Bcl-2.
Previous reports have suggested that AS ODNs have immunostimulatory effects due to their CpG motifs, in addition to their AS activity [
29]. In the present study we observed immune stimulation by CpG motifs, resulting in significant spleen enlargement and elevated serum IL-12 levels. However, the role of immune stimulation in mediating antitumor activity remains uncertain because AS
Bcl-2 ODNs with methylated CpG motifs, when transplanted into athymic mice, demonstrated antitumor activities that were similar to those of their nonmethylated counterparts with immunostimulatory activity. We therefore suggest that the therapeutic activity of AS
Bcl-2 is due to the AS–mRNA interaction and not to immunostimulatory effects in this model. Similarly, methylated AS
Bcl-2, with 5'-methylation of CpG motif cytosine residues, had the same antitumor effect as unmethylated AS
Bcl-2 ODNs in human melanoma xenografts transplanted into mice with severe combined immunodeficiency [
30]. However, given that AS
Bcl-2 stimulates IL-12 secretion and results in the development of splenomegaly, effects that are not observed with methylated AS
Bcl-2, Th1-mediated immunostimulation may have antitumor effects in solid tumors in humans. Further studies are required to determine whether the immunostimulatory effects of the CpG motifs in AS
Bcl-2 have antitumor effects in the clinical setting.
Phase III clinical trials using Genasense (known as G3139) in the treatment of patients with chronic lymphocyctic leukemia, malignant melanoma, and multiple myeloma are complete, and are nearly complete for non-small cell lung cancer [
31]. Although a phase III trial of G3139/dacarbazine versus dacarbazine alone in advanced malignant melanoma does not show a significant increase in overall survival by the addition of G3139, combination treatment with G3139 and dacarbazine shows a significant increase in progression-free survival and response rate, compared with dacarbazine alone [
32]. The results of other phase III trials are eagerly anticipated. In addition, phase I and II trials of G3139 in advanced esophageal, gastric, and colon cancer are ongoing, as are trials in hepatocellular carcinoma, metastatic breast cancer, and hormone refractory prostate cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ME carried out the study design, animal feeding, data collection (Western blotting, drug sensitivity assay in vitro and in vivo, transfection, ELISA), statistical analysis, data interpretation, manuscript preparation, and literature search. RK participated in the design and coordination of the study, in the data interpretation, and manuscript preparation. KT and YU participated in the data collection (Western blotting, transfection). TT organized the study as the director, manuscript preparation, and funding the collection. All authors read and approved the final manuscript.