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Cancer Research 67, 1203, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-2310
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Evidence that Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Induction by 5-Aza-2'-Deoxycytidine Sensitizes Human Breast Cancer Cells to Adriamycin

Jing Xu, Jun-Ying Zhou, Michael A. Tainsky and Gen Sheng Wu

Program in Molecular Biology and Genetics, Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan

Requests for reprints: Gen Sheng Wu, Program in Molecular Biology and Human Genetics, Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201. Phone: 313-833-0715, ext. 2328; Fax: 313-831-7518; E-mail: wug{at}karmanos.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) inhibits DNA methyltransferase activity and sensitizes cancer cells to chemotherapy, but the mechanisms of its sensitization are not fully understood. Here, we show that 5-aza-CdR induces tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in the human breast cancer MDA-231 cells. Induction of TRAIL by 5-aza-CdR correlated with inactivation of Akt. Furthermore, we show that overexpression of the active form of Akt by adenovirus infection or inhibition of the Akt downstream target glycogen synthase kinase 3 by its pharmacologic inhibitors abolishes TRAIL induction by 5-aza-CdR. Importantly, we show that the combined treatment of breast cancer cells with 5-aza-CdR and Adriamycin significantly increases apoptotic cell death compared with the treatment with either agent alone. Moreover, the combined treatment activated both death receptor and mitochondrial apoptotic pathways, whereas Adriamycin alone activated only the mitochondrial pathway while 5-aza-CdR failed to activate either. More importantly, down-regulation of TRAIL by small interference RNA silencing decreased 5-aza-CdR–mediated Adriamycin-induced caspase activation and apoptosis, thus conferring Adriamycin resistance. Taken together, our results suggest that induction of TRAIL by 5-aza-CdR is critical for enhancing chemosensitivity of breast cancer cells to Adriamycin. [Cancer Res 2007;67(3):1203–11]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), also known as Apo2 ligand, is a member of the TNF ligand family (1, 2). TRAIL induces apoptosis in a variety of transformed or tumor cells but not normal cells (3, 4). Importantly, TRAIL (under the name of Apo2L/TRAIL) is being tested in phase I clinical trials (2). There are four membrane-bound TRAIL receptors: DR4 (5), DR5 (6–9), TRAIL-R3 (10, 11), and TRAIL-R4 (12, 13). Because both DR4 and DR5 are proapoptotic receptors, activation of these two receptors leads to activation of caspases (14). On the other hand, because TRAIL-R3 lacks an intracellular domain, and because TRAIL-R4 has a truncated death domain, both TRAIL-R3 and TRAIL-R4 can antagonize TRAIL-induced apoptosis by competing for ligand binding (14). Upon activation of DR4 and DR5 by TRAIL binding, caspase-8 is recruited via Fas-associated death domain protein (FADD) to the death-inducing signaling complex (DISC) and is subsequently activated (14). Activated caspase-8 directly activates caspase-3, caspase-6, and caspase-7 or activates the intrinsic mitochondria-mediated pathway through caspase-8–mediated Bid cleavage, which indirectly activates caspase-3, caspase-6, and caspase-7 (15–17).

5-Aza-2'-deoxycytidine (5-aza-CdR) is an inhibitor of DNA methyltransferases, which can inhibit DNA methyltransferases and reverse DNA methylation (18). 5-Aza-CdR was originally synthesized as an antimetabolite agent that induces differentiation of acute myelogenous leukemia (19). Studies showed that 5-aza-CdR can inhibit cancer cell growth, particularly leukemia cells (19). The mechanisms underlying its anticancer activity are not fully understood, but it is believed to reactivate genes, including the tumor suppressor genes p16, E-cadherin, and hMLH1 in cancer cells (20). Reactivation of these genes is associated with cell cycle arrest and apoptosis, which leads to inhibition of tumor cell growth (20). By using microarray screen technology, it has been found that there are a limited number of genes that are reactivated by 5-aza-CdR in tumors (21). Furthermore, it has been shown that 5-aza-CdR can activate the p53 pathway, independent of demethylation of the p53 promoter (22).

Despite its promising anticancer activity, the early clinical trials showed that 5-aza-CdR has low anticancer activity and high toxicity. Recent studies showed that low doses of 5-aza-CdR exhibit promising clinical activity against hematologic malignancies (23). In addition, a study showed that 5-aza-CdR in combination with other anticancer agents has a synergistic effect on activation of silenced tumor suppressor gene (24). Extending this finding to the clinical setting has found that the combination of 5-aza-CdR with histone deacetylase (HDAC) inhibitors has better efficacy than use of either agent alone (25). Thus, combination regimens have the advantage over a single agent to treat cancer. In addition, it has been shown that 5-aza-CdR sensitizes lung cancer cells to cisplatin and paclitaxel, but the underlying mechanisms are not fully understood (26).

Here, we show that 5-aza-CdR induces TRAIL via the Akt pathway. Induction of TRAIL by 5-aza-CdR enhanced Adriamycin-induced apoptosis in the human breast cancer cells. Importantly, down-regulation of TRAIL by small interference RNA (siRNA) silencing decreased caspase activation and abrogated 5-aza-CdR–mediated sensitization of breast cancer cells to Adriamycin-induced cell death. Our data indicate that induction of TRAIL by 5-aza-CdR plays a critical role in sensitization of breast cancer cells to Adriamycin and suggest that TRAIL ligand is a potential target for cancer therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. The DNA methylation inhibitor 5-aza-CdR, cycloheximide, LiCl, and anti-actin antibody (AC-74) were purchased from Sigma (St. Louis, MO). The glycogen synthase kinase 3 (GSK3) inhibitor Ro-318220 was obtained from Calbiochem (San Diego, CA). MS275 was obtained from Alexis Biochemicals (San Diego, CA). Adriamycin was obtained from the Oncology Outpatient Pharmacy at the Karmanos Cancer Institute. Monoclonal anti-human TRAIL, DR4, and polyclonal DR5 antibodies were purchased from Imgenex (San Diego, CA). Rabbit anti-caspase-9, anti-caspase-8, anti-caspase-7, anti-caspase-3, and anti–poly (ADP-ribose) polymerase (PARP) polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). The adenovirus vectors encoding the myristoylated active form of Akt (AxCA-Myr-Akt) and control viruses encoding galactosidase (AdCA-LacZ) were kindly provided by Dr. Wataru Ogawa (Kobe University School of Medicine, Chuo-ku, Japan), as described previously (27).

Cell lines, culture conditions, and treatment. The human breast cancer MCF-7 cells were obtained from the Karmanos Cancer Institute. The human breast cancer MDA-231 and T47-D cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM. These cells were supplemented with either 10% fetal bovine serum (FBS) for MDA-231 and T47-D or 5% FBS for MCF-7 and antibiotics at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Cells were treated with various concentrations of 5-aza-CdR and/or Adriamycin for different intervals of time as indicated in each figure legend. Cells were infected with adenovirus encoding AxCA-Akt or control LacZ viruses, as described previously (28).

Isolation of RNA, reverse transcription-PCR, and Northern blot analysis. Total cellular RNA was purified from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After reverse transcription of 2 µg of total RNA by oligo(dT) priming, the resulting single-strand cDNA was amplified using Taq DNA polymerase (Promega, Madison, WI) and specific primers directed against human TRAIL (5-CAATGACGAAGAGAGTATGA-3' and 5-TGGGAATAGATGTAGTAAAA-3'). PCR conditions were 94°C/2 min; 34 cycles at 94°C/30 s, 50°C/30 s, and 72°C/30 s followed by 72°C/7 min for the final extension. Aliquots (10 µL) of the amplified cDNA were separated by 1.5% agarose gel electrophoresis, visualized by ethidium bromide staining, and compared with the expression level of glyceraldehyde-3-phosphate dehydrogenase (5-AGTCGGATACACACATATTCATCA and 5-ATGGTGGGGTAGATCTTCTTCT-3'). For Northern blot analysis, total RNA (10 µg) was separated in a 1.5% formaldehyde agarose gel and blotted to Hybond-N+ membrane (Amersham Pharmacia, Biotech, Piscataway, NJ). The blots were hybridized with radiolabeled human TRAIL cDNA, as described previously (29). Radioactive signals were analyzed by autoradiography.

siRNA transfection for knockdown of TRAIL. siRNA duplex oligonucleotides were purchased from Dharmacon Research (Lafayette, CO). The targeted sequences for TRAIL siRNA were 5-AACGAGCUGAAGCAGAUGCAGdTdT-3' (sense) and 5-CUGCAUCUGCUUCAGCUCGUUdTdT-3' (antisense), as described previously (30). The transfection was done as suggested by Dharmacon, with slight modifications. Briefly, MDA-231 cells were plated at 3 x 105 per well in six-well plates. The next day, cells were transfected with TRAIL siRNA oligonucleotides or scrambled oligonucleotides (20 nmol/L) using Oligofectamine (Invitrogen). After 3 days, transfected cells were harvested for examining the expression of TRAIL protein by Western blot analysis, or subjected to the treatments. To determine chemosensitivity, transfected cells were placed at 4,000 per well in 96-well plates and then treated with or without 5-aza-CdR (10 µmol/L) in the presence or absence of Adriamycin (0.1 µg/mL) for 72 h, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.

MTT assays. MTT assay was previously described (31). Briefly, cells were treated with 5-aza-CdR at different doses for various times. In the experiments involving Adriamycin, cells were treated with Adriamycin (0.1 µg/mL) in the presence or absence of 5-aza-CdR (10 µmol/L). After incubation with MTT solution, Isopropanol was added to dissolve the formazan crystals. Absorbance was measured using a Vmax Microplate Reader (Molecular Devices, Sunnyvale, CA) at 600 nm. The survival was calculated from the mean of pooled data from three separate experiments with four wells (31).

Western blot analysis. Whole-cell lysates were prepared as described previously (32), and protein concentration was determined using the Protein Assay kit (Bio-Rad, Hercules, CA). Cell lysates (100 µg) were electrophoresed through 15% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The blots were probed or reprobed with the antibodies, and bound antibody was detected using enhanced chemiluminescence (ECL) reagent or ECL Plus reagent (Amersham Pharmacia, Biotech) according to the manufacturer's protocol.

Determination of half-life of TRAIL protein. MDA-231 cells were left untreated or treated with 10 µmol/L 5-aza-CdR for 24 h, and cycloheximide (5 µg/mL) was added to the cells. Cells were harvested at the indicated time points. The levels of TRAIL protein were determined by Western blot analysis.

Assay of caspase-3 activity. The enzymatic activity of caspase-3 was assayed using the caspase-3 colorimetric assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. Briefly, cells were left untreated or treated with 5-aza-CdR, Adriamycin, or in combination for 72 h and then lysed in lysis buffer for 10 min on ice. The lysed cells were centrifuged at 14,000 rpm for 5 min, and 150 µg protein was incubated with 50 µL of reaction buffer and 5 µL of caspase-3 substrate at 37°C for 1 h, and the absorbance was measured at a wavelength of 405 nm on a plate reader.

Flow cytometry. Subconfluent cells were left untreated or treated with 5-aza-CdR (10 µmol/L) in the presence or absence of Adriamycin (0.1 µg/mL) for 72 h. Cells were then harvested and fixed with ice-cold 70% (v/v) ethanol for 24 h. After centrifugation at 200 x g for 5 min, the cell pellet was washed with PBS (pH 7.4) and resuspended in PBS containing propidium iodide (50 µg/mL), Triton X-100 (0.1%, v/v), and DNase-free RNase (1 µg/mL). Cells were then incubated at room temperature for 1 h, and DNA content was determined by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Construction of reporter vectors. TRAIL reporter vectors were constructed previously (33). Briefly, pGL3-TRAIL1 containing a 2.196-kb fragment of the human TRAIL promoter, including upstream of the translational start site, was amplified from human placental DNA using GC-Rich PCR system (Roche Molecular Biochemicals, Indianapolis, IN) and the following primers: 5 CCGCTCGAGCGATCTCTTGACCTCATGAT-3' and 5-CCCAAGCTTGATCCTGTCAGAGTCTGACTGCTG-3'. The PCR conditions for amplifying the fragment were as follows: 95°C/3 min; 30 cycles at 95°C/30 s, 65°C/30 s, and 68°C/2.25 min, followed by 68°C/7 min for the final extension. The amplified fragment was isolated from 1% agarose gel, digested with XhoI and HindIII, and subcloned into pGL3-Basic (Promega). The insert was verified by DNA sequencing. Using pGL3-TRAIL1 as a template, several deletion constructs were generated by PCR using the above reverse primer and the following forward primers: pGL3-TRAIL2 (5-CCGCTCGAGGAGGCAGGAGAATTGCTT-3'), pGL3-TRAIL3 (5-CCGCTCGAGGACATTCAAGATGGAATTATG-3'), and pGL3-TRAIL4 (5-CCGCTCGAGGAGAAATGGGCTTGAGGTGAG-3'). The PCR conditions were the same as used for amplifying pGL3-TRAIL1.

Luciferase reporter assays. Transfections for luciferase assays were carried out as described previously (28). Briefly, MDA-231 cells were plated at 6 x 105 per well in six-well plates. The next day, the cells were transfected with 5 µg of reporter constructs and 5 ng of pRLSV40 (Promega) using LipofectAMINE 2000 reagent (Invitrogen). After 24 h, transfected cells were treated with or without 5-aza-CdR (10 µmol/L) or 10 µmol/L MS275 for 24 h. Firefly luciferase activities were assayed using the dual-luciferase reporter assay system (Promega) in a Turner TD20/20 luminometer and normalized to Renilla luciferase activity.

Statistical analysis. Statistical analysis was done using Student's t test. The data were presented as the mean ± SD, and P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5-Aza-CdR sensitizes the human breast cancer cells to Adriamycin. 5-Aza-CdR has been shown to inhibit leukemia and breast cancer cell growth (23, 34). To determine whether 5-aza-CdR can effectively inhibit breast cancer cell growth, we treated three breast cancer cell lines (MDA-231, MCF-7, and T47-D) with various doses of 5-aza-CdR for 72 h, and the effects of 5-aza-CdR on growth inhibition were determined by MTT assays. As shown in Fig. 1A , 5-aza-CdR inhibited the growth of all three cell lines in a dose-dependent manner; MDA-231 was the most sensitive line, whereas both MCF-7 and T47-D cells were equally sensitive to 5-aza-CdR (Fig. 1A). We chose to focus on MDA-231 cells in this study because this line was more sensitive than the other lines to 5-aza-CdR. It has been shown that 5-aza-CdR can enhance conventional chemotherapeutic agent–induced anticancer activity (26). To determine whether 5-aza-CdR–mediated growth inhibition can be enhanced by chemotherapeutic agents, MDA-231 cells were treated with 5-aza-CdR in combination with Adriamycin. Figure 1B shows that the growth inhibition was about 76% in cells treated with 5-aza-CdR in combination with Adriamycin (0.1 µg/mL), compared with ~54% in cells treated with Adriamycin alone, whereas 5-aza-CdR alone caused ~48% of growth inhibition. These data suggest that 5-aza-CdR could enhance sensitivity of MDA-231 cells to Adriamycin.


Figure 1
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  		Figure 1. Role of 5-aza-CdR, Adriamycin, or in combination on cell death. A, effects of 5-aza-CdR on growth inhibition. The human breast cancer cell lines MDA-231, T47-D, and MCF-7 were left untreated or treated with various doses of 5-aza-CdR for 72 h. Cell viability was determined by MTT assays. Representative of three independent experiments. B, 5-aza-CdR sensitizes MDA-231 cells to Adriamycin. MDA-231 cells were treated with 10 µmol/L 5-aza-CdR, 0.1 µg/mL Adriamycin, or in combination for 72 h. Cell viability was determined by MTT assays. Representative of three independent experiments. **, P < 0.001, statistical significance. C, activation of the apoptotic pathways by 5-aza-CdR, Adriamycin, or in combination. MDA-231 cells were left untreated or treated with 10 µmol/L 5-aza-CdR, 0.1 µg/mL Adriamycin, or in combination, and total protein was extracted at 72 h. Cleavage of caspase-9, caspase-8, caspase-7, caspase-3, and PARP was determined by Western blot analysis. ß-Actin was used as a loading control. D, effects of the treatments with 5-aza-CdR, Adriamycin, or in combination on caspase-3 activity. MDA-231 cells were treated as in (C) and then lysed in lysis buffer. The lysed cells were centrifuged, and 150 µg protein was incubated with 50 µL of reaction buffer and 5 µL of caspase-3 substrate, and the absorbance was measured at a wavelength of 405 nm on a plate reader. Representative of three independent experiments. **, P < 0.001, statistical significance. E, effects of the treatments with 5-aza-CdR, Adriamycin, or in combination on apoptotic cell death. MDA-231 cells were treated as in (D) for 72 h, and cells were then harvested and fixed. After centrifugation, the cell pellet was washed with PBS and resuspended in PBS containing propidium iodide, Triton X-100, and DNase-free RNase. DNA content was determined by flow cytometry. 5-Aza, 5-aza-2'-deoxycytidine; Adr, Adriamycin.

 
It has been known that both 5-aza-CdR and Adriamycin can kill cells in part by apoptosis. To determine the mechanisms underlying 5-aza-CdR–mediated sensitization, we examined the activation of the apoptotic pathways. As shown in Fig. 1C, Adriamycin treatment resulted in modest cleavage of caspase-9, caspase-3, and PARP, which indicates that the mitochondrial apoptotic pathway is activated. Minimal cleavage of caspase-8 by Adriamycin treatment (Fig. 1C) is consistent with the fact that chemotherapeutic agents, including Adriamycin, mainly target the mitochondrial apoptotic pathway. In contrast, the combination of 10 µmol/L 5-aza-CdR with 0.1 µg/mL Adriamycin significantly enhanced cleavage of caspase-9, caspase-7, and PARP, compared with cells treated with Adriamycin alone, whereas such changes were not observed in cells treated with 5-aza-CdR (10 µmol/L) alone (Fig. 1C). Importantly, the combined treatment of 5-aza-CdR and Adriamycin resulted in a significant increase in cleavage of caspase-8, which was not significant in cells treated with Adriamycin alone (Fig. 1C). Cleavage of caspase-8 and caspase-9 in cells treated with both 5-aza-CdR and Adriamycin indicates that both the death receptor and mitochondrial apoptotic pathways are activated. In addition, combined treatment also enhanced cleavage of caspase-3 (Fig. 1C) and caspase-3 activity (Fig. 1D) relative to the treatments with single agent alone. Furthermore, we analyzed apoptotic cell death by fluorescence-activated cell sorting analysis. Figure 1E shows that 5-aza-CdR and Adriamycin treatments resulted in ~11% and ~23% of the cell population in sub-G1 (apoptotic cells), respectively. In contrast, the combined treatments resulted in ~39% of the cell population in sub-G1 (Fig. 1E). Taken together, these results suggest that the enhanced effects of the combined treatments on cell death are attributable to the augmented induction of apoptosis.

5-Aza-CdR induces TRAIL expression in MDA-231 cells. We previously showed that 5-aza-CdR induces TRAIL in human fibroblasts (35). Because TRAIL is a potent apoptotic inducer, we suspected that induction of TRAIL by 5-aza-CdR may be responsible for chemosensitization. To test this possibility, we treated the breast cancer cell MDA-231 cells with various doses of 5-aza-CdR for 24 h, and induction of TRAIL was determined by Western blot. As shown in Fig. 2A , TRAIL protein was induced by different doses of 5-aza-CdR. Consistent with induction of TRAIL protein, TRAIL mRNA was induced by 5-aza-CdR, determined by both Northern blot and reverse transcription-PCR, respectively (Fig. 2B). To determine whether 5-aza-CdR affects the expression of TRAIL receptors, we examined the levels of DR4 and DR5 proteins. Figure 2A shows that 5-aza-CdR treatment does not increase DR4 and DR5 protein, which suggests that such induction is TRAIL ligand specific.


Figure 2
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  		Figure 2. Induction of TRAIL protein by 5-aza-CdR in MDA-231 cells. A, effects of 5-aza-CdR on TRAIL, DR4, and DR5 proteins. MDA-231 cells were left untreated or treated with various doses of 5-aza-CdR for 24 h, and the total protein was then extracted for assaying TRAIL, DR4, and DR5 by Western blot analysis. ß-Actin was used as a loading control. B, induction of TRAIL mRNA by 5-aza-CdR in MDA-231 cells. Cells were treated as in (A), and total RNA was extracted to assay for TRAIL mRNA expression by Northern blot analysis (top) and reverse transcription-PCR (bottom), respectively. rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls for Northern blot and reverse transcription-PCR, respectively.

 
5-Aza-CdR and HDACI regulate TRAIL expression by different mechanisms. It has been shown that inhibitors of HDAC can induce TRAIL expression via activation of the TRAIL promoter in the human leukemia cells (36, 37). Because the DNA methyltransferase inhibitors, including 5-aza-CdR, couple with inhibitors of HDACs to activate gene expression (24), it was possible that induction of TRAIL by 5-aza-CdR may be through the same mechanism as mediated by HDACIs (36, 37). To test this possibility, we transfected MDA-231 with either the TRAIL luciferase reporter constructs pGL3-TRAIL1, pGL3-TRAIL2, pGL3-TRAIL3, and pGL3-TRAIL4 or the empty vector pGL3-basic and then treated cells with 5-aza-CdR (10 µmol/L) or MS275 (10 µmol/L). Cells were harvested after 24 h, and luciferase activity was assayed using the dual-luciferase reporter assay system. As shown in Fig. 3B , luciferase activity was not stimulated in cells transfected with pGL3-TRAIL1, pGL3-TRAIL2, pGL3-TRAIL3, and pGL3-TRAIL4 upon 5-aza-CdR treatment, compared with untreated cells. In contrast, the HDACI MS275 was able to stimulate the TRAIL promoter activity in the breast cancer cell line MDA-231 (Fig. 3B), which is consistent with two previous studies showing that HDACI induces TRAIL expression through the transcriptional activation of the TRAIL promoter (36, 37). Therefore, our results suggest that 5-aza-CdR–induced TRAIL is independent of the activation of TRAIL promoter, which is different from HDACI-mediated TRAIL expression.


Figure 3
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  		Figure 3. Effects of 5-aza-CdR on the TRAIL promoter and TRAIL protein stability. A, schematic depiction of luciferase reporter constructs as detailed in Materials and Methods. The translation start site (arrow) is designated at + 1. B, activation of the TRAIL promoter by HDACIs but not by 5-aza-CdR. MDA-231 cells were transfected with pGL3-TRAIL1, pGL3-TRAIL2, pGL3-TRAIL3, pGL3-TRAIL4, and pGL3-Basic. pRLSV40 was added in transfections for normalization. The next day, cells were left untreated or treated with 10 µmol/L 5-aza-CdR or 10 µmol/L MS275. Luciferase activity was determined 24 h later. Relative firefly luciferase activities normalized to Renilla luciferase activities. **, P < 0.01, statistical significance. C, 5-aza-CdR treatment increased TRAIL protein half-life. MDA-231 cells were left untreated or treated with 10 µmol/L 5-aza-CdR for 24 h and then treated with 5 µg/mL cycloheximide (CHX) for 0, 30, 60, and 120 min. Top, TRAIL and actin levels were examined by Western blotting. Bottom, quantification of remaining TRAIL.

 
Because 5-aza-CdR did not activate the TRAIL promoter (Fig. 3B), we suspected that 5-aza-CdR–induced TRAIL expression may be through stabilization of TRAIL protein. To test this possibility, MDA-231 cells were left untreated or treated with 5-aza-CdR (10 µmol/L) for 24 h and then treated with cycloheximide (5 µg/mL) for 0 to 120 min, and TRAIL level was examined by Western blotting. As shown in Fig. 3C, the basal half-life of TRAIL was ~30 min. In contrast, 5-aza-CdR treatment significantly increased the half-life of TRAIL; there was about 85% of TRAIL remaining at the end of the 120-min cycloheximide treatment (Fig. 3C). Thus, these results suggest that 5-aza-CdR–induced TRAIL ligand may be through stabilization of TRAIL protein.

5-Aza-CdR induces TRAIL expression via inhibition of Akt. A previous study suggested that the phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays an important role in regulation of TRAIL expression (38). To determine whether Akt is involved in 5-aza-CdR–mediated TRAIL induction, we treated MDA-231 cells with 5-aza-CdR for various times and then examined the level of phosphorylated Akt. As shown in Fig. 4A , 5-aza-CdR treatment resulted in a decrease in the level of phosphorylated Akt, which was accompanied with an increase in TRAIL protein. In contrast, total Akt remained unchanged. These data suggest that Akt may be involved in the negative regulation of TRAIL, which can be relieved by 5-aza-CdR treatment. Because Akt can be inhibited by the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we examined the effects of 5-aza-CdR treatment on the levels of PTEN. Figure 4A shows that 5-aza-CdR treatment has no effect on the levels of PTEN protein. In addition, because nuclear factor-{kappa}B (NF-{kappa}B) and Akt can functionally interact (39), we examined the levels of total and phosphorylated I{kappa}B-{alpha} because activation of NF-{kappa}B requires phosphorylation and ubiquitin-mediated degradation of I{kappa}B-{alpha} by the I{kappa}B kinase complex. As shown in Fig. 4A, 5-aza-CdR treatment had no effect on the level of I{kappa}B-{alpha}. Thus, these results establish a correlation between induction of TRAIL and inactivation of Akt.


Figure 4
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  		Figure 4. Role of the active form of Akt in 5-aza-CdR–induced TRAIL. A, effects of 5-aza-CdR treatment on the levels of phosphorylated Akt (phospho-Akt). MDA-231 cells were treated with 10 µmol/L 5-aza-CdR for 0, 1, 2, 4, 8, 16, and 24 h, and total protein was extracted. The levels of TRAIL, phosphorylated Akt, total Akt, PTEN, phosphorylated I{kappa}B-{alpha} (phospho-I{kappa}B-{alpha}), and total I{kappa}B-{alpha} were determined by Western blot analysis. ß-Actin was used as a loading control. B, overexpression of the active form of Akt abolishes TRAIL induction by 5-aza-CdR. MDA-231 cells were infected with the adenoviruses encoding the active form of Akt or the control viruses and then left untreated or treated with 10 or 20 µmol/L 5-aza-CdR for 24 h. Total protein was extracted for assaying for TRAIL and phosphorylated Akt proteins. ß-Actin was used as a loading control. C and D, inhibition of GSK3 by Ro-318220 and LiCl abolishes TRAIL induction by 5-aza-CdR. MDA-231 cells were left untreated or pretreated with 2 µmol/L Ro-318220 (C) or 20 mmol/L LiCl (D) for 30 min and then treated with 10 µmol/L 5-aza-CdR in the presence of either inhibitor for 24 h. Total protein was extracted for assaying the levels of TRAIL and total Akt by Western blot analysis. ß-Actin was used as a loading control.

 
To determine a direct role of Akt in negatively regulating TRAIL expression upon 5-aza-CdR treatment, we infected MDA-231 cells with either the control adenoviruses encoding LacZ, or the adenoviruses encoding the myristoylated active form of Akt (27). As shown in Fig. 4B, cells infected with adenoviruses encoding the active form of Akt overexpressed phosphorylated Akt, compared with cells infected with the control viruses. To determine the effects of overexpression of the active form of Akt on TRAIL expression in response to 5-aza-CdR treatment, cells infected with the LacZ viruses or viruses encoding the active form of Akt were treated with 5-aza-CdR for 24 h, and the levels of TRAIL were then determined. Figure 4B shows that 5-aza-CdR induces TRAIL protein in cells infected with the control LacZ viruses. In contrast, induction of TRAIL by 5-aza-CdR was abolished in cells infected with the viruses encoding the myristoylated active form of Akt (Fig. 4B). These results suggest that relief of Akt-mediated inhibition may be responsible for TRAIL induction by 5-aza-CdR.

We have shown that overexpression of the active form of Akt can inhibit TRAIL induction by 5-aza-CdR (Fig. 4B). Because Akt exerts its activity by phosphorylating its downstream substrates, including GSK3, one would expect that blockade of its substrate activity could affect Akt function. It has been shown that blockade of the Akt downstream substrate GSK3 can inhibit TRAIL induction by wortmannin (38). To test whether inhibition of GSK3 has the same effect on TRAIL induction by 5-aza-CdR as observed with wortmannin, we pretreated MDA-231 cells with the GSK3 inhibitor Ro-318220 for 30 min and then treated these cells with 5-aza-CdR in the presence of this inhibitor for 24 h, and induction of TRAIL was then determined. As shown in Fig. 4C, 5-aza-CdR induced TRAIL expression, compared with untreated cells. In contrast, Ro-318220 abolished 5-aza-CdR–induced TRAIL (Fig. 4C). Consistent with the results obtained with Ro-318220, LiCl, another GSK inhibitor (40), was able to abrogate TRAIL induction by 5-aza-CdR (Fig. 4D). Abrogation of TRAIL induction by the GSK3 inhibitors suggests that blockade of Akt-mediated signaling may be responsible for TRAIL induction by 5-aza-CdR. Thus, our data clearly indicate that Akt plays an important role in the negative regulation of TRAIL expression, which can be relieved by 5-aza-CdR.

Induction of TRAIL by 5-aza-CdR is required for sensitization of MDA-231 cells to Adriamycin-induced death. We have shown that 5-aza-CdR induces TRAIL, and the combination of 5-aza-CdR with Adriamycin enhances cell death relative to either agent alone. Because TRAIL is a potent apoptotic inducer, we sought to determine whether induction of TRAIL by 5-aza-CdR is required for this enhanced killing effect. To this end, MDA-231 cells were transfected with either control oligos or oligos against TRAIL, and the effects of siRNA-mediated TRAIL down-regulation on cell death were determined. As shown in Fig. 5A , the levels of TRAIL in cells transfected with TRAIL siRNA were decreased compared with cells transfected with control oligos. Importantly, induction of TRAIL by 5-aza-CdR was also decreased compared with cells transfected with control oligos (Fig. 5A), indicating that TRAIL siRNA is sufficient to down-regulate TRAIL expression. To determine the effects of down-regulation of TRAIL on cell survival, MDA-231 cells transfected with either TRAIL siRNA or control siRNA were treated with 5-aza-CdR, Adriamycin, or in combination for 72 h, and cell viability was then determined. As shown in Fig. 5B, cells transfected with TRAIL siRNA were resistant to 5-aza-CdR–mediated growth inhibition, compared with cells transfected with non-target oligos; growth inhibition was ~7% in cells transfected with TRAIL siRNA compared with ~36% in cells transfected with non-target oligos upon 5-aza-CdR treatment. Similarly, down-regulation of TRAIL increased cell resistance to Adriamycin compared with cells transfected with control oligo, although such resistance was not as significant as observed in cells treated with 5-aza-CdR (Fig. 5B). More importantly, enhanced growth inhibition by the combination was abrogated in cells transfected with TRAIL siRNA, compared with cells transfected with control oligos (Fig. 5B). Survival rates increased from ~20% in cells transfected with non-targeted siRNA to ~41% in cells transfected with TRAIL siRNA following the combined treatment of 5-aza-CdR with Adriamycin. Furthermore, in cells transfected with TRAIL siRNA, 5-aza-CdR no longer enhanced Adriamycin-induced cell death; cell viability was ~51% in cells treated with Adriamycin alone compared with ~41% in cells with combined treatment (Fig. 5B). Collectively, these results suggest that TRAIL plays a critical role in enhancing Adriamycin-induced cell death.


Figure 5
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  		Figure 5. Down-regulation of TRAIL abolishes 5-aza-CdR–mediated sensitization of MDA-231 cells to Adriamycin. A, down-regulation of TRAIL by siRNA silencing. MDA-231 cells were plated at 3 x 105 per well in six-well plates. The next day, cells were transfected with TRAIL siRNA or control oligonucleotides using Oligofectamine. After 3 d, cells were left untreated or treated with 5-aza-CdR (10 µmol/L) for 24 h, and induction of TRAIL was determined by Western blot. B, effects of silencing TRAIL expression on cell survival. MDA-231 cells were transfected with TRAIL siRNA or control oligonucleotides as described in (A). After 3 d, cells were left untreated or treated with 5-aza-CdR (10 µmol/L) in the absence or presence of Adriamycin (0.1 µg/mL) for 72 h. Cell viability was determined by MTT assays. Cell survival data are expressed as percentage of untreated cells. Representative of three independent experiments. **, P < 0.001; *, P < 0.05, statistical significance. C, effects of down-regulation of TRAIL by siRNA on the caspase cascade. MDA-231 cells were transfected with TRAIL siRNA or control oligonucleotides and treated with 5-aza-CdR (10 µmol/L) in the presence or absence of Adriamycin (0.1 µg/mL) for 72 h as described in (A). Total protein was extracted for assaying cleavage of caspase-9, caspase-8, caspase-7, caspase-3, and PARP by Western blot analysis. ß-Actin was included as a loading control.

 
To investigate the effects of down-regulation of TRAIL on 5-aza-CdR–mediated Adriamycin-induced apoptosis, cells transfected with TRAIL siRNA or control oligos were treated with either 5-aza-CdR (10 µmol/L), Adriamycin (0.1 µg/mL), or in combination for 72 h, and activation of caspases was then examined. As expected, in cells transfected with control oligos, activation of caspase-9, caspase-8, caspase-7, and caspase-3, as well as cleavage of PARP, was significant in cells treated with both 5-aza-CdR and Adriamycin, compared with untreated or cells treated with 5-aza-CdR alone, whereas such changes was minimal in Adriamycin-treated cells (Fig. 5C). In contrast, cleavage of caspase-9, caspase-8, caspase-3, and PARP was abrogated in cells transfected with TRAIL siRNA following the combined treatment (Fig. 5C), indicating that down-regulation of TRAIL abrogates activation of the caspase cascade induced by the combined treatment, leading to cell survival. Taken together, these results suggest that TRAIL plays a critical role in 5-aza-CdR–mediated Adriamycin-induced caspase activation and apoptosis.

Overexpression of the active form of Akt increases resistance of breast cancer cells to both 5-aza-CdR and Adriamycin. We have shown that 5-aza-CdR–mediated inhibition of Akt is responsible for TRAIL induction. To determine whether overexpression of the active form of Akt affects cell death induced by 5-aza-CdR, Adriamycin, or in combination, we infected MDA-231 cells with either the control adenoviruses or the adenoviruses encoding the myristoylated active form of Akt, and survival cells were then counted. In general, cells infected with Akt-adenoviruses proliferated more rapidly and were more resistant to either agent alone or in combination, compared with cells infected with the control viruses (Fig. 6A ). We consistently found that there was less caspase-3 activity in cells infected with the viruses encoding the active form of Akt over cells infected with control viruses (Fig. 6B), suggesting that constitutive expression of the active form of Akt renders MDA-231 cells resistant to anticancer drug-induced apoptosis. Thus, our data suggest that overexpression of the active form of Akt gives cells growth advantage and enhances resistance to anticancer agents in part through suppression of TRAIL-mediated apoptosis.


Figure 6
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  		Figure 6. Overexpression of phosphorylated Akt renders MDA-231 cells resistant to Adriamycin,5-aza-CdR,or in combination. A, effects of overexpression of phosphorylated Akt on cell survival. MDA-231 cells were infected with either the adenoviruses encoding the active form of Akt or the control viruses and then treated with 5-aza-CdR (10 µmol/L), Adriamycin (0.1 µg/mL), or in combination for 72 h. Viable cells were counted. B, effects of overexpression of the active form of Akt on caspase-3 activity. MDA-231 cells were treated as in (A), and caspase-3 activity was determined as described in Materials and Methods. **, P < 0.01 (A); **, P < 0.001 (B); *, P < 0.05 (A and B), statistical significance.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that 5-aza-CdR enhances Adriamycin-induced cell death. We also show that 5-aza-CdR induces TRAIL in the human breast cancer cell MDA-231. Furthermore, we find that induction of TRAIL by 5-aza-CdR is abrogated by overexpression of the active form of Akt. Importantly, down-regulation of TRAIL expression by siRNA silencing abrogated 5-aza-CdR–mediated sensitization of breast cancer cells to Adriamycin. Thus, our findings suggest for the first time that induction of TRAIL by 5-aza-CdR is critical for sensitizing human cancer cells to chemotherapeutic agents.

5-Aza-CdR is a DNA methyltransferase inhibitor that activates gene expression primarily through a demethylation-dependent mechanism (41). It has also been shown that 5-aza-CdR can induce gene expression through a demethylation-independent mechanism including activation of the p53/p21 pathway (22). We have shown that TRAIL is induced within 1 h upon 5-aza-CdR treatment, suggesting that induction of TRAIL by 5-aza-CdR occurs before DNA is demethylated. We believed that induction of TRAIL is an early event, and that cell death is a later event, which was abolished by transfection of TRAIL siRNA. Therefore, we conclude that TRAIL induction by 5-aza-CdR is through a DNA demethylation-independent mechanism.

TRAIL has been shown to selectively kill tumor or transformed cells while sparing normal cells, but its regulation is not fully understood. Previous studies suggested that TRAIL induction by both IFN and retinoid is through the transcription factor IFN-stimulated response elements (42–44). In addition, a recent study showed that inhibitors of HDACs induce TRAIL through activation of the TRAIL promoter mediated by the transcription factors Sp1 and Sp3 in leukemia cells (36). Because activation of gene expression could involve both DNA demethylation and histone acetylation, it is possible that induction of TRAIL by 5-aza-CdR is through the same mechanism as operated by HDACIs. We have found that 5-aza-CdR does not activate the TRAIL reporter constructs, whereas HDACIs were able to activate the TRAIL reporter construct (Fig. 3B), suggesting that 5-aza-CdR and HDACIs regulate TRAIL expression by different mechanisms. Importantly, we have shown that 5-aza-CdR treatment increased the half-life of TRAIL protein (Fig. 3C). This may explain why induction of TRAIL is not in a dose-dependent manner (Fig. 2A).

It has been shown that the PI3K inhibitor wortmannin can induce TRAIL in the human colon cancer cell lines (38), and that overexpression of the active form of Akt can abolish TRAIL induction by wortmannin, suggesting that Akt plays a negative role in TRAIL expression. Consistent with this, we have shown that 5-aza-CdR treatment results in a decrease in phosphorylated Akt, which is correlated with induction of TRAIL (Fig. 4A). More importantly, we have shown that overexpression of the active form of Akt abolishes TRAIL induction by 5-aza-CdR (Fig. 4B). Because active Akt phosphorylates its downstream targets, including GSK3, to initiate its function, it is expected that inhibition of GSK3 activity might affect TRAIL induction. Indeed, we have shown that the GSK inhibitors Ro-318220 and LiCl can inhibit TRAIL induction by 5-aza-CdR (Fig. 4C). Taken together, our results strongly suggest that 5-aza-CdR–induced TRAIL is through suppression of the Akt pathway.

Although 5-aza-CdR itself can inhibit cancer cell growth, recent studies suggested that 5-aza-CdR could serve as a sensitizer to enhance the anticancer activity of the conventional anticancer drugs. It has been shown that 5-aza-CdR induces caspase-8 in both neuroblastoma and Ewing tumor cells, and that induction of caspase-8 by 5-aza-CdR sensitizes tumor cells to anticancer agents including TRAIL, etoposide, and cisplatin (45, 46). Moreover, a study suggested that 5-aza-CdR induces caspase-9 and subsequently sensitizes lung cancer cells to p53-induced apoptosis (26). Induction of caspase-9 by 5-aza-CdR also sensitized lung cancer cells to cisplatin and paclitaxel (26). Thus, activation of either death receptor or mitochondrial pathway is likely the mechanism underlying its sensitization. In this study, we have shown that cotreatment of the human breast cancer cell line MDA-231 with 5-aza-CdR and Adriamycin results in a significant activation of caspase-8, which was not obvious in cells treated with either 5-aza-CdR or Adriamycin alone (Fig. 1C), indicating that 5-aza-CdR sensitizes MDA-231 cells to Adriamycin, and that activation of caspase-8 involves in this sensitization. Because activation of caspase-8 can result from activation of death receptors, it is likely that the death receptor pathway is activated in this combined treatment. Consistent with this, we showed that TRAIL is induced upon 5-aza-CdR treatment. Because activation of the TRAIL pathway can induce apoptosis, it is expected that activation of the TRAIL pathway by 5-aza-CdR can activate caspase-8 and caspase-3 and induce apoptosis. Although 5-aza-CdR can induce TRAIL, it is possible that such induction is not relevant to this sensitization. Through down-regulation of TRAIL expression by siRNA silencing, we found that 5-aza-CdR–mediated sensitization is abrogated (Fig. 5B), suggesting TRAIL is indeed involved in this sensitization. However, we could not exclude the possibility that other members of the apoptotic pathways, including caspase-8 or caspase-9, are involved in this sensitization as indicated in previous studies (26, 45). Thus, our data suggest that the enhanced effects of the combination on cell death are attributable to the activation of both the TRAIL death receptor apoptotic pathway by 5-aza-CdR and the mitochondrial apoptotic pathway by Adriamycin, leading to strong induction of apoptosis and cell death.

In summary, we show that 5-aza-CdR induces TRAIL via inhibition of the Akt pathway. Furthermore, we show that the treatments of breast cancer cells with 5-aza-CdR enhance Adriamycin-induced anticancer activity via activation of both the death receptor and mitochondrial apoptotic pathways. More importantly, we show that induction of TRAIL is critical for 5-aza-CdR–mediated sensitization of breast cancer cells to Adriamycin because knockdown of TRAIL by siRNA silencing abrogates such sensitization. Therefore, our results identify a novel mechanism by which the DNA demethylating agent 5-aza-CdR sensitizes the breast cancer cells to Adriamycin via inhibiting the Akt pathway. Because both TRAIL and 5-aza-CdR are being tested in clinical trials, their combination may provide therapeutic gain for treating solid tumors.


   Acknowledgments
 
Grant support: NIH grant R01 CA100073.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Wataru Ogawa for providing the adenovirus vectors encoding the myristoylated active form of Akt.

Received 6/23/06. Revised 10/20/06. Accepted 11/ 7/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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