The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH2-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.
We previously cloned the reduced expression in immortalized cells (REIC) gene and reported that the expression is downregulated in many human cell lines and tumors.1, 2, 3, 4, 5, 6, 7, 8 The REIC gene is identical to Dickkopf-3 (Dkk-3) gene and REIC/Dkk-3 has been considered as a tumor suppressor gene to provide a new means of gene therapy for some of the human malignant tumors. The forced expression of REIC/Dkk-3, using a plasmid vector, inhibited cell growth in HeLa and liver cancer cell lines.9 Adenovirus vector carrying REIC/Dkk-3 selectively induced apoptosis in prostate cancer and testicular cancer cells through the activation of c-Jun-NH2-kinase (JNK) and c-Jun.1, 5
Breast cancer is one of the most common female malignancies in the world. Although a definite therapeutic effect has been achieved by existing treatment modalities of surgery, hormonal therapy, chemotherapy and radiation, a considerable number of patients experience disease progression and recurrence. In particular, multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer with anticancer drugs such as adriamycin. One of the most important mechanisms by which tumor cells resist cytotoxic effects of anticancer agents is overexpression of the MDR1 gene and its product, P-glycoprotein.10 P-glycoprotein plays a drug efflux role in MDR cancer cells and augments cell survival against anticancer drugs.11 Elucidation of the molecular mechanisms underlying MDR results in the development of new therapeutic strategy. Recent studies have showed that activated JNK and c-Jun is accompanied with the downregulation of MDR1 gene and P-glycoprotein expression.12, 13
Gene therapy has been used in clinical trials for human diseases and it provides an innovative and attractive therapeutic potential. The feasibility of using adenoviral vectors to deliver therapeutic genes to animals and humans has been demonstrated.14, 15, 16, 17 This study utilized an adenoviral vector encoding human REIC gene and demonstrated that REIC/Dkk-3 overexpression sensitized multidrug-resistant breast cancer MCF7/ADR cells to the doxorubicin (adriamycin) with decreased level of P-glycoprotein. In addition, the adenovirus-mediated overexpression of REIC/Dkk-3 induces apoptosis in multiple breast cancer cell lines, thus indicating the therapeutic potential of REIC/Dkk-3 gene transfer in breast cancer.
Materials and methods
Primary human mammary epithelial cells (HMEC) were purchased from Cambrex (Baltimore, MD) and cultured according to the manufacturer's instructions. Human fibroblast, OUMS24, was established in this university.18 Human breast cancer cell lines MCF7/wild type (MCF7/Wt), MDA-MB-231, SK-BR-3, HCC1806 and the human cervical cancer cell line HeLa were obtained from the American Type Culture Collection (Rockville, MD). The multidrug-resistant breast cancer cell line MCF7/ADR was kindly provided by Professor KH Cowan (Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center).19 Human fibroblast, OUMS24 was grown in Dulbecco's Modified Eagle Medium, (Invitrogen, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IUml−1) and streptomycin (100 μgml−1) and incubated in 5% CO2. MCF7/ADR was grown in RPMI-1640 medium (Sigma, St. Louis, MO) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IUml−1), streptomycin (100 μgml−1) and 10 μM doxorubicin (ADRIACIN, Kyowa Hakkoh Co.). Other cell lines were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IUml−1), and streptomycin (100 μgml−1).
Western blotting analysis
The cells were washed twice with phosphate buffered saline (PBS) and then lysed with lysis buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM dithiothreitol, 1 mM PMSF, 5 μgml−1 leupeptin, 5 μgml−1 aprotinin, 2 mM Na3VO4, 1 mM NaF and 10 mM β-GP) and the proteins were extracted. After centrifugation, the supernatants were adjusted to equal protein concentration in each experiment and diluted with the same volume of 2 × SDS sample buffer, and heated for 5 min at 95 °C. Samples (10 μg of protein) were separated on a 7.5% SDS-polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane. The blots were blocked for 1 h with 10% non-fat milk powder, 6% glycine and 0.1% Tween-20 in Tris-buffered saline at room temperature. The proteins were then identified with the use of the following primary antibodies; rabbit antihuman REIC/Dkk-3 antibody raised in this laboratory (1:1000), sc-571 (1:500) for JNK, sc-1694 (1:500) for c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), no. 9255 (1:500) for phosphorylated JNK, no. 9261 (1:500) for phosphorylated c-Jun, no. 9661 (1:500) for cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA), T5168 (1:8000) for tubulin (Sigma), C219 (1:250) for P-glycoprotein (Calbiochem, San Diego, CA). After extensive washing with 0.1% Tween-20 in Tris-buffered saline, the blots were exposed to the appropriate horseradish peroxidase-conjugated secondary antibody. After extensive washing with Tween-20 in Tris-buffered saline, development was done using the enhanced chemiluminescence detection method (ECL kit, Amersham Pharmacia Biotech, Chandler, AZ). In some experiments, 1 μM JNK inhibitor (SP600125, AG Scientific, Inc.) was added to inhibit the kinase activity of JNK.
Adenovirus vector carrying REIC/Dkk-3 (Ad-REIC)
A full-length cDNA of REIC/Dkk-3 was integrated into the cosmid vector, pAxCAwt, and then it was transferred into an adenovirus vector by the COS-TPC method (Takara Bio, Shiga, Japan).1 An adenovirus vector carrying the LacZ gene (Ad-LacZ) was used as a control.
To examine in vitro apoptosis induction after treatment, cells were seeded in flat-bottom 6-well plates and incubated for 24 h. The cells were treated with the Ad-LacZ and Ad-REIC at the indicated multiplicity of infection (MOI) in serum-free medium for 2 h and the medium was exchanged with fresh complete medium. After 48 h incubation, Hoechst 33342 stock solution was added into the medium to the 2 μgml−1 concentration and the cells were incubated in the dark for 10 min. Hoechst 33342 is an intercalating dye that allows determination of total chromatin quantity variations and the degree of chromatin condensation.20, 21 Using fluorescence microscopy, apoptotic cells were identified by the presence of highly condensed or fragmented nuclei. Apoptotic cells were counted at three to five different fields in microscopic observations. One hundred cells were judged in one field.
To detect apoptotic cells in vivo, the in situ cell death detection kit, Fluorescein was used (Roche, Penzberg, Germany) for terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Briefly, the tumor tissue was cut, placed in the OCT compound and snap frozen in liquid nitrogen. The frozen section (10 μm) samples were fixed with methanol for 30 min at room temperature, washed and permeabilized with PBS containing 0.1% Triton X-100, and then they were stained with the transferase biotin-dUTP nick end labeling reaction mixture.
Cell viability assay
Cells were seeded at the concentration of 1000 cells per well in flat-bottom 96-well microplates. After 24 h incubation, the cells were treated with the Ad-LacZ and Ad-REIC at 100 MOI in serum-free medium for 2 h and the medium was exchanged to the fresh complete medium. After 48 h, the floating dead cells were removed by changing the medium and attached cells were cultivated with doxorubicin at the indicated concentration for 72 h. At the end of incubation, the viability of cells was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp, Madison, WI), according to the manufacturer's instructions.
Detection and quantification of intracellular doxorubicin accumulation
The cells were seeded in flat-bottom 6-well plates and incubated for 24 h. The cells were treated with the Ad-LacZ and Ad-REIC at 100 MOI in serum-free medium for 2 h and the medium was exchanged to the fresh complete medium. After 48 h, the floating dead cells were removed by changing the medium and attached cells were cultivated with 10 μM doxorubicin for 36 h. At the end of the incubation, red auto-fluorochrome of doxorubicin was observed and scanned. The scanned image was subjected to analysis for the quantification of doxorubicin's integrated density using Scion Image (Scion Corp, Frederick, MD).
Xenograft model and treatments
Female athymic (nu/nu) mice (5–6 weeks old) were purchased from Charles River Laboratories (Yokohama, Japan). 17β-Estradiol pellets (SE-121, Innovative Research of America, Sarasota, FL) were subcutaneously implanted into the right shoulder area of mice 3 days before tumor cell injection. MCF7/Wt cells (5 × 106 cells/0.1 ml PBS) were subcutaneously injected into the left thigh of mice. The tumors were allowed to reach 3–6 mm in size and the mice were randomized into groups of seven animals each. Next, 1.2 × 108 plaque-forming units of adenovirus vector (Ad-LacZ and Ad-REIC) adjusted to 0.1 ml volume with PBS buffer were injected intratumorally. As a negative control, the same volume of PBS was injected. The size of the tumors was measured twice a week. The tumor volume was calculated using an empirical formula: 1/2 (w1 × w2 × w2), where w1 represents the largest tumor diameter and w2 represents the smallest tumor diameter. Approval of all animal procedures was obtained according to the guidelines established at our university.
The data are shown as the mean±s.e. The unpaired Student's t-test was performed for statistical analysis between the two groups and the difference was considered significant if P<0.05. The analyses were performed using the Statview 4.5 software program (Abacus Concepts, Berkeley, CA).
Reduced expression of REIC/Dkk-3 in human breast cancer cell lines
The REIC/Dkk-3 protein expression was first examined in various cell lines. Human fibroblast, OUMS24, was used as a positive control for the expression.1 In primary HMEC, REIC/Dkk-3 protein was clearly recognized at the molecular size between 60 and 70 kDa, (Figure 1a) as previously reported that REIC/Dkk-3 protein made plural bands of different molecular size.1, 4, 9 The band of REIC/Dkk-3 expression could not be observed in all the cancer cell lines that were examined (Figure 1a).
Apoptosis induction in MCF7/Wt and MCF7/ADR cells by treatment with Ad-REIC
To examine the possible use of REIC/Dkk-3 as a mean for gene therapy in breast cancer, REIC/Dkk-3 was overexpressed in the cell lines using Ad-REIC.
To optimize the MOI for the apoptotic induction by Ad-REIC treatment, we examined the protein expression at 10 and 100 MOI. The expression level of REIC/Dkk-3 protein was weak at 10 MOI by western blotting analysis, but it was strong at 100 MOI in both MCF7/Wt and MCF7/ADR cells (Figure 1b).
In an apoptosis assay with Hoechst 33342, apoptotic cells were frequently observed in Ad-REIC-treated MCF7/Wt and MCF7/ADR cells (Figure 2a), but not in HMEC (data not shown). The incidence of apoptosis at 100 MOI was 5.5, 21.1, 25.0 and 15.2% in HMEC, MCF7/Wt, MCF7/ADR and SK-BR-3 cells, respectively (Figure 2b). In comparison with the control Ad-LacZ treatment, Ad-REIC at 100 MOI significantly induced apoptosis in MCF7/Wt and MCF7/ADR cells. In MDA-MB-231 cells, no significant change in apoptosis induction was observed between Ad-REIC and the control treatment (data not shown).
Ad-REIC treatment sensitizes multidrug-resistant MCF7/ADR cells to doxorubicin
To further investigate the utility of Ad-REIC in the cancer treatment, the MDR of MCF7/ADR cells was investigated. MCF7/Wt and MCF7/ADR cells were divided into three groups of no treatment, Ad-LacZ and Ad-REIC. The ability of the REIC/Dkk-3 overexpression to reverse drug resistance was then evaluated by exposing the cells to increasing concentrations of doxorubicin. In MCF7/Wt cells, no significant change was observed between the treatment groups. After the Ad-REIC treatment, the dose–response curve for doxorubicin toxicity was significantly shifted to lower concentrations in MCF7/ADR cells (Figure 3). In 10 μM doxorubicin, the cell survival rate was 88.1, 85.6 and 32.3% in the no treatment, Ad-LacZ and Ad-REIC groups, respectively (P<0.05; no treatment and Ad-LacZ vs Ad-REIC groups). In 25 μM doxorubicin, the cell survival rate was 45.9, 32.1 and 14.3% in the no treatment, Ad-LacZ and Ad-REIC groups (P<0.05; no treatment group vs Ad-REIC group).
Ad-REIC treatment downregulates P-glycoprotein expression in MCF7/ADR cells in activated JNK-c-Jun-dependent manner
Western blotting was used to examine the expression level of the indicated proteins under REIC/Dkk-3 overexpression (Figure 4). The cells were treated with the Ad-LacZ and Ad-REIC at 100 MOI, and after 48 h, the floating dead cells were removed and attached cells were lysed for the sample. With Ad-REIC treatment, both MCF7/Wt and MCF7/ADR cells strongly expressed REIC/Dkk-3 protein. No significant change in the levels of JNK was observed in both cell lines and the treatment groups. The c-Jun protein level in MCF7/ADR cells was higher than that in MCF7/Wt. The activation of JNK in MCF7/Wt and MCF7/ADR cells after Ad-REIC treatment was confirmed using a phosphorylated JNK-specific antibody and by detecting the phosphorylation of c-Jun. Notably, an overexpression of REIC/Dkk-3 significantly downregulated P-glycoprotein in MCF7/ADR cells. SP600125, a JNK inhibitor, is known to inhibit the kinase activity of JNK but not the phosphorylation of JNK itself.22 Combined treatments with Ad-REIC and SP600125 did not change the activation of JNK, but they did decrease the expressional level of phosphorylated c-Jun and reversed the level of P-glycoprotein in MCF7/ADR cells. In contrast, the expressional change of P-glycoprotein, the level of cleaved caspase-3 in MCF7/Wt and MCF7/ADR cells was upregulated by Ad-REIC treatment, whereas it was reversed by SP600125.
Increased accumulation of intracellular doxorubicin by Ad-REIC treatment
To examine the effects of Ad-REIC treatment on the doxorubicin dynamics in each cell type, the intracellular accumulation of doxorubicin was analyzed at a concentration of 10 μM. In MCF7/ADR cells, Ad-REIC treatment significantly increased the intracellular accumulation of doxorubicin, which was more than twice of that in the control group (Figure 5). In MCF7/Wt cells, no significant change was observed between the treatment groups.
Intratumoral Ad-REIC treatment inhibits tumor growth in a breast cancer xenotransplantation model
As in vitro apoptosis induction owing to the overexpression of REIC/Dkk-3 was observed in MCF7/Wt cells, the in vivo antitumor effect of Ad-REIC was next investigated using a subcutaneous tumor model. In PBS and Ad-LacZ-treated groups, the tumor size progressively increased during a 3-week observation period (Figure 6a). In contrast, the tumor volume in the Ad-REIC-treated group remained almost unchanged during 2 weeks after the treatment, and then the tumors began to grow slowly. A significant difference was observed from day 7 to the end of 3 weeks, between the no treatment, Ad-LacZ and the Ad-REIC groups. To confirm the REIC/Dkk-3 expression and the apoptotic effect of Ad-REIC treatment, tumor tissues were resected 3 days after the vector injection. In the Ad-REIC-treated tumor, an overexpression of REIC/Dkk-3 was confirmed by western blotting (data not shown). Thereafter, the tumor sections were examined by transferase biotin-dUTP nick end labeling staining. Very few apoptotic cells were observed in the tumor treated with Ad-LacZ, whereas many transferase biotin-dUTP nick end labeling positive cells were observed in the Ad-REIC-treated tumor (Figure 6b).
Cancer progression is often accompanied by an inhibition of apoptotic cell death23 and increased metastatic activity, such as invasion and motility.24 In cancer cells, the apoptotic and metastatic processes are modified by the production of both positive and negative effectors,25, 26 and a number of proteins exhibit potent anticancer effects involved in these processes.17, 27, 28, 29 REIC/Dkk-3, a member of the Dkk gene family known to interfere with Wnt signaling through Wnt receptors,30, 31 has been reported to play a distinct role in the induction of apoptosis1, 9 and in the inhibition of invasion/motility.32, 33 The expression of Dkk gene was initially found to be suppressed in various human cancer cells in comparison with the levels in normal cells.4, 6, 7, 8 Similar findings were documented when analyzing normal human tissue and the cancer specimens.1, 34 In addition, the REIC/Dkk-3 expression in prostate cancer consistently decreases at the critical transition from noninvasive disease to highly invasive disease.1, 34 These findings strongly indicate that REIC/Dkk-3 is a tumor suppressor gene and it may therefore be an attractive therapeutic protein with an ability to inhibit both oncogenesis and cancer progression.
This study has demonstrated that REIC/Dkk-3 has the potential utility as a gene therapeutic agent against breast cancer. The adenovirus-mediated overexpression of REIC/Dkk-3 induced apoptotic cell death in the breast cancer cells, and which occurred in JNK phosphorylation-dependent manner. The intratumoral apoptotic effect by in vivo Ad-REIC administration explains the MCF/Wt tumor growth inhibition in the mouse model. Moreover, the overexpression of REIC/Dkk-3 also downregulated the P-glycoprotein expression through the JNK activation in drug-resistant breast cancer MCF7/ADR cells, and sensitized its drug resistance against doxorubicin.
JNK activation by Ad-REIC treatment plays a major role in the apoptosis induction in several cancer cell lines.1, 2, 5 In prostate cancer PC-3 cells, the overexpression of REIC/Dkk-3 activates JNK, reduces the Bcl-2 protein level, induces the mitochondrial translocation of Bax protein, releases cytochrome c into the cytoplasm, thus finally leading to apoptotic cell death.1 This study first demonstrated that cleaved caspase-3 was involved in the Ad-REIC-induced apoptotic pathway. In the case of breast cancer cells, the activation of JNK may link to apoptosis induction in the reported mitochondrial cascade.
This study demonstrated that the Ad-REIC treatment induced apoptosis in multiple breast cancer cell lines, MCF7/Wt, MCF7/ADR and SK-BR-3, but not in primary HMEC. In addition, the REIC/Dkk-3 protein expression was barely detected in the breast cancer cell lines, but it was abundant in the primary epithelial cells. These results indicate that the overexpression of REIC/Dkk-3 selectively induces apoptotic cell death in breast cancer cells that lack endogenous REIC/Dkk-3 expression. This phenomenon was consistently observed in earlier studies,1, 2, 5 and the lack or absence of REIC protein expression seems to be important for the induction of apoptosis in the cancer cells. Although it is not elucidated as to how Ad-REIC treatment upregulates JNK activity, these findings might give some clues to resolve the question. Ongoing studies disclosed that Ad-REIC administration in the REIC absent cancer cells caused endoplasmic reticulum stress and induced apoptosis in the JNK-dependent manner (data not shown). Interestingly, the overexpression of some kinds of proteins induces endoplasmic reticulum stress-triggered JNK activation and apoptosis.35, 36 Thus, one possible mechanism is that the REIC protein overexpression itself might trigger significant endoplasmic reticulum stress and then activate JNK at the endoplasmic reticulum of cancer cells in which REIC protein expression has become almost absent and could not be done smoothly. Further studies will be required to clarify the molecular stream of Ad-REIC-induced endoplasmic reticulum stress–JNK activation axis.
P-glycoprotein plays a central role in the anticancer drug efflux to augment cell survival against the drug, and its upregulation is one of the most important mechanisms by which cancer cells resist the cytotoxic effects of anticancer agents.10, 11 Here we considered that one mechanism to explain the reversible effects of drug resistance by Ad-REIC treatment may be the JNK –activation-dependent downregulation of P-glycoprotein. This theory is well supported by the current findings, which show the intracellular accumulation of doxorubicin in the drug-resistant MCF7/ADR cells to be significantly higher after the Ad-REIC treatment. Regarding this point, we also confirmed the effect of Ad-REIC on the drug resistance of another cell line, MCF-7/MDR, which ectopically expresses P-glycoprotein as the sole mediator of resistance.37 As expected, the Ad-REIC-mediated REIC overexpression in the MCF-7/MDR cells reduced the P-glycoprotein level and significantly improved drug resistance against doxorubicin (data not shown). On the basis of these findings, we concluded that the role of Ad-REIC in the reversal of drug resistance is because of the suppression of P-glycoprotein, at least in part. In addition, the reversal of doxorubicin resistance by Ad-REIC treatment is not complete in MCF7/ADR cells. Previously published studies demonstrated MCF7/ADR cells to have multiple mechanisms of drug resistance other than P-glycoprotein overexpression.19, 38 As a result, it is quite conceivable that REIC/Dkk-3 might only affect one or a subset of these mechanisms and the reversal of drug resistance is therefore only partial.
Previously published studies demonstrated transcription factor c-Jun to be a principal determinant in the downregulation of P-glycoprotein.12, 13 In this study, the REIC/Dkk-3 overexpression in MCF7/ADR cells caused the c-Jun activation and P-glycoprotein downregulation in a JNK-dependent manner. Using the other adriamycin-resistant cancer cell line, bladder cancer KK47/ADM, we also confirmed the overexpression of REIC/Dkk-3 because of Ad-REIC treatment to downregulate the P-glycoprotein expression in a JHK and c-Jun-dependent manner while also improving drug resistance (data not shown). On the basis of these findings, the phosphorylation of c-Jun by JNK activation therefore seems to be a consistent molecular event to inhibit the P-glycoprotein expression after Ad-REIC treatment.
In conclusion, these experiments demonstrated the significant therapeutic effects of in vitro and in vivo Ad-REIC treatment in breast cancer cells. It is notable that not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using a single agent, Ad-REIC. These results indicate REIC/Dkk-3 to thus be a novel target for breast cancer treatment and Ad-REIC might therefore be an attractive agent against the drug-resistant cancer in combination with conventional antineoplastic agents.
This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology's FY2006 ‘Creation of Innovation Centers for Advanced Interdisciplinary Research Areas’ Scheme in JAPAN. We thank Prof. Kenneth H. Cowan (Derector, Eppley Institute for Research in Cancer and Allied Diseases, UNMC) for the MCF7/ADR cell donation and Hideo Ueki (Okayama University) for his technical assistance. We also thank Prof. Yoshikazu Sugimoto (Department of Chemotherapy, Kyoritsu University of Pharmacy, Tokyo, JAPAN) who kindly provided the MCF-7/MDR cell line.
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Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene
Molecular Biotechnology (2014)