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Cancer Research 66, 10092-10099, October 15, 2006. doi: 10.1158/0008-5472.CAN-06-1633
© 2006 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Is Required for Tumor Necrosis Factor {alpha}–Mediated Sensitization of Human Breast Cancer Cells to Chemotherapy

Jing Xu, Jun-Ying Zhou and Gen Sheng Wu

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

Requests for reprints: Gen Sheng Wu, Program in Molecular Biology and Human Genetics, Karmanos Cancer Institute, Department of Pathology, 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
 
Tumor necrosis factor {alpha} (TNF{alpha}) induces apoptosis and sensitizes cancer cells to chemotherapy, but the mechanism underlying its sensitization is not fully understood. Here, we report that TNF{alpha}-mediated sensitization of cancer cells to chemotherapy involves activation of the TRAIL pathway. We show that the combined treatment of breast cancer cells with TNF{alpha} and Adriamycin significantly increases cell death compared with the treatment with either agent alone. The combined treatment activated both death receptor and mitochondrial apoptotic pathways, whereas Adriamycin alone activated only the mitochondrial pathway, and TNF{alpha} failed to activate either. Furthermore, we show that TNF{alpha} induces TRAIL through a transcriptional mechanism. Using reporter gene assays in conjunction with chromatin immunoprecipitation assays, we show that TRAIL induction by TNF{alpha} is regulated via both nuclear factor-{kappa}B and Sp1 binding sites. Importantly, down-regulation of TRAIL by small interfering RNA silencing decreased TNF{alpha}-mediated Adriamycin-induced caspase activation and apoptosis, and thus enhanced breast cancer cell resistance to Adriamycin. Collectively, our results suggest that induction of TRAIL by TNF{alpha} is critical for sensitization of breast cancer cells to chemotherapy. (Cancer Res 2006; 66(20): 10092-9)


   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 of type II transmembrane proteins (1). TRAIL induces apoptosis in a variety of transformed or tumor cells but not normal cells (2, 3). To date, five human TRAIL receptors have been identified: DR4 (4), DR5 (KILLER, TRAIL-R1, TRICK2; refs. 5–9), TRID (TRAIL-R3, DcR1, or LIT; refs. 7, 10–12), TRUNDD (DcR2 or TRAIL-R4; refs. 13–15), and osteoprotegerin (16). In addition, a mouse TRAIL receptor has been identified (17). Both DR4 and DR5 are proapoptotic receptors, and activation of these two receptors leads to activation of caspases (18). Unlike DR4 and DR5, TRID lacks an intracellular domain, and TRUNDD has a truncated death domain. Thus, these two receptors act as decoy receptors to antagonize TRAIL-induced apoptosis by competing for ligand binding (18). TRAIL-mediated apoptosis is initiated by binding to DR4 and DR5, which then recruits caspase-8 via Fas-associated death domain protein (FADD). 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 (19–21). Although TRAIL seems to be a tumor-selective, apoptosis-inducing cytokine and is a promising new agent for the treatment of cancer (2, 3), the mechanisms by which TRAIL is regulated, including the transcriptional mechanism, are poorly understood. In fact, DR5 is the first gene in the TRAIL pathway that has been shown to be a DNA damage–inducible gene (5).

In addition to TRAIL, there are several other members of the TNF ligand family, including TNF{alpha}. TNF{alpha} is a pleiotropic proinflammatory cytokine that plays an important role in a variety of cellular responses, including inflammation and apoptosis (22). Like TRAIL, TNF{alpha} exerts its biological activity via interaction with its two cognate membrane receptors, TNF-R1 and TNF-R2 (22). TNF-R1 is the major mediator of TNF signaling, whereas TNF-R2 can only be activated by membrane bound TNF, but not by soluble TNF (1). Binding of TNF{alpha} to TNF-R1 recruits caspase-8 via both TRADD and FADD, and then activates caspase-8, leading to apoptosis (22). In addition, recruitment of TRAF2 and RIP to the receptors activates the c-Jun-NH2-kinase or nuclear factor-{kappa}B (NF-{kappa}B) pathways, which activates a diverse of cellular responses, including apoptosis and cell survival (22). Therefore, the net outcome of activation of TNF signaling is likely to be dependent on stimuli and cell types. In spite of being a potent apoptotic inducer, TNF{alpha} is not currently used as an anticancer agent for the treatment of human cancer because it causes serious toxicity to normal cells at pharmacologically achievable doses. It has been shown that low doses of TNF{alpha} that are unable to trigger apoptosis can serve as sensitizers to enhance conventional chemotherapeutic agent–induced anticancer activity in some cancer cells (23), but the mechanisms underlying its sensitization are poorly understood.

In this article, we showed that TNF{alpha} enhances Adriamycin-induced apoptosis in the human breast cancer cell lines via activation of both death receptor and mitochondrial apoptotic pathways. We also showed that TNF{alpha} induces TRAIL through a transcriptional mechanism via both NF-{kappa}B and Sp1 binding sites. More importantly, down-regulation of TRAIL by small interfering RNA (siRNA) silencing decreased caspase activation and abrogated TNF{alpha}-mediated sensitization of breast cancers to Adriamycin-induced cell death. Thus, our data indicate that induction of TRAIL by TNF{alpha} 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
 
Cell lines, culture conditions, and treatment. The human breast cancer MCF-7 cell was obtained from the Karmanos Cancer Institute. The human breast cancer MDA-231 and T47-D cells were obtained from American Type Culture Collection (Manassas, VA). These cells were maintained in DMEM and supplemented with either 10% fetal bovine serum (FBS; MDA-231 and T47-D) or 5% FBS (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 TNF{alpha} and/or Adriamycin for different intervals of time as indicated in each figure legend.

Reagents. TNF{alpha} was purchased from R&D Systems (Minneapolis, MN). Anti-actin antibody (AC-74) were purchased from Sigma (St. Louis, MO). Adriamycin was obtained from the Oncology Outpatient Pharmacy at the Karmanos Cancer Institute. Monoclonal anti-human TRAIL and DR4 antibodies and rabbit polyclonal anti-human DR5 antibody were purchased from Imgenex (San Diego, CA). Mouse anti-caspase-8, rabbit anti-caspase-9, anti-caspase-3, anti-poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Isolation of RNA and Northern blot analysis. Total cellular RNA was purified from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. 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 (24). 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 (25). The transfection was done as suggested by Dharmacon with slight modifications. Briefly, MDA-231 cells were plated at 2 x 105 per well in six-well plates. The next day, cells were transfected with TRAIL siRNA oligonucleotides or scrambled oligonucleotides 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 TNF{alpha} (10 ng/mL) in the presence or absence of Adriamycin (0.1 µg/mL) for 3 days, 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 (26). Briefly, MDA-231 cells were treated with TNF{alpha} (10 ng/mL) in the presence or absence of Adriamycin (0.1 µg/mL) for 72 hours and then incubated 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 six wells (26).

Western blot analysis. Whole cell lysates were prepared as described previously (27), 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 Reagent (Amersham Pharmacia Biotech) according to the protocol of the manufacturer.

Assay of caspase-3 activity. The enzymatic activity of caspase-3 was assayed using the caspase-3 colorimetric assay kit (R & D Systems) according to the protocol of the manufacturer. Briefly, cells left untreated or treated with TNF{alpha} (10 ng/mL), Adriamycin (0.1 µg/mL), or in combination for 72 hours, and then lysed in lysis buffer for 10 minutes on ice. The lysed cells were centrifuged at 14,000 rpm for 5 minutes, and 150 µg protein was incubated with 50 µL of reaction buffer and 5 µL of caspase-3 substrate at 37°C for 2 hours, and the absorbance was measured at a wavelength of 405 nm on a plate reader.

Construction of reporter vectors. 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) with the forward primer 5'-CCGCTCGAGCGATCTCTTGACCTCATGAT-3' and the reverse primer 5'-CCCAAGCTTGATCCTGTCAGAGTCTGACTGCTG-3'. The PCR product, TRAIL1, was cloned into TA vector (Invitrogen) and confirmed by DNA sequencing. After digesting with XhoI and HindIII, TRAIL1 was cloned into pGL3-Basic (Promega, Wisconsin, WI), which was designed as pGL3-TRAIL1. 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'-CCGCTCGAGGACATTCAAGATGGAATTATG-3'), pGL3-TRAIL3 (5'-CCGCTCGAGGAGAAATGGGCTTGAGGTGAG-3'), pGL3-TRAIL4 (5'-CCGCTCGAGTTCCCTCCTTTCCAACGACT-3'), pGL3-TRAIL5 (5'-CCGCTCGAGAGGAAAGGGGAGGGACAGTT-3'), and pGL3-TRAIL6 (5'-CCGCTCGAGAGTTGCAGGTTCAATAGATG-3'). The PCR conditions for amplifying all the fragments were the same as follows: 95°C/3 minutes; 30 cycles at 95°C/30 seconds, 65°C (62°C for pGL3-TRAIL6)/30 seconds, and 68°C/2 minutes, followed by 68°C/7 minutes for the final extension.

Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) was done using the ChIP Assay kit (Upstate Biotechnology, Lake Placid, NY) according to the protocol of the manufacturer. Briefly, 3.5 x 106 cells were used for each ChIP assay. Formaldehyde was added to a final concentration of 1% and incubated at 37°C for 10 minutes. Sonication was done on ice using a Sonics and Materials Vibra-Cell sonicator with a 3-mm tip set at 30% maximum power using 5 x 10-second pulses with a 10-second interval between sonications. Samples were diluted a 10-fold in ChIP dilution buffer, and a 10 µL aliquot (1% of total) was removed to serve as an input sample. Chromatin was precleared with 60 µL protein A/protein G at 4°C with rotation for 2 hours, followed by the addition of 2 µg Sp1 antibody or 2 µg unrelated Wee1 antibody. Antibodies used for ChIP analysis were as follows: Sp1 (Upstate Biotechnology) and Wee 1 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitations were done at 4°C overnight with rotation. To collect immune complexes, 60 µL protein A/protein G was added and incubated at 4°C with rotation for 2 hours. After washing, formaldehyde cross-link was reversed in immunoprecipitated samples and the input chromatin samples by the addition of 8 µL of 5 mol/L NaCl, and incubation at 65°C for 4 hours. Following proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation, DNA was resuspended in 50 µL water. The PCR primers used were as follows: 5'-AATGGGCTTGAGGTGAGTGCAGAT-3' and 5'-ATGAGTTGTTTTTCTGGGTTCTGT-3' for a region containing two Sp1 sites in the TRAIL promoter. The PCR conditions were as follows: 94°C for 2 minutes; 31 cycles at 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 s; followed by 72°C for 7 minutes. The PCR products were analyzed on agarose gels (2%) with ethidium bromide staining.

Luciferase reporter assays. Transfections for luciferase assays were carried out as previously described (28). Briefly, MDA-231 cells were plated at 2 x 105 per well in six-well plates. The next day, the cells were transfected with 5 µg reporter constructs and 5 ng pRLSV40 (Promega) using LipofectAMINE 2000 reagent (Invitrogen). After 24 hours, transfected cells were left untreated or treated with TNF{alpha} (10 ng/mL) for 24 hours. 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.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} enhances cell death induced by Adriamycin in the human breast cancer cells. To determine whether TNF{alpha} can sensitize cells to Adriamycin-induced death, we examined the effects of TNF{alpha} on MDA-231 cell growth. As shown in Fig. 1A , TNF{alpha} inhibited cell growth; ~62% of TNF{alpha}-treated cells survived compared with untreated cells. To determine whether TNF{alpha} plays a role in chemotherapeutic agent–mediated sensitivity, MDA-231 cells were treated with TNF{alpha} in combination with Adriamycin. Figure 1A shows that the survival rate was ~10% in cells treated with TNF{alpha} (10 ng/mL) in combination with Adriamycin (0.1 µg/mL), compared with that of 46% in cells treated with Adriamycin alone. These data suggest that TNF{alpha} could sensitize MDA-231 cells to Adriamycin.


Figure 1
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  		Figure 1. Induction of apoptosis by Adriamycin, TNF{alpha}, or in combination in the human breast cancer cell line MDA-231. A, TNF{alpha} sensitizes MDA-231 cells to Adriamycin (Adria)–induced growth inhibition. MDA-231 cells were left untreated or treated with TNF{alpha} (10 ng/mL), Adriamycin (0.1 µg/mL), or in combination for 72 hours. Cell viability was determined by MTT assays. Representative of three independent experiments. B, activation of the apoptotic pathways by TNF{alpha}, Adriamycin, or in combination. MDA-231 cells were treated as in (A). Total protein was extracted at 72 hours. Cleavage of caspase-9, caspase-8, caspase-3, and PARP was determined by Western blot analysis. Actin was used as a loading control. C, effects of treatments with TNF{alpha}, Adriamycin, or in combination on caspase-3 activity. MDA-231 cells were treated as in (A) for 72 hours and then lysed in lysis buffer for 10 minutes on ice. The lysed cells were centrifuged at 14,000 rpm for 5 minutes, and 150 µg protein was incubated with 50 µL of reaction buffer and 5 µL of caspase-3 substrate at 37°C for 2 hours, and the absorbance was measured at a wavelength of 405 nm on a plate reader. D, effects of the treatments with TNF{alpha}, Adriamycin, or in combination on apoptotic cell death. MDA-231 cells were treated as in (A) for 72 hours, and cells were then harvested and fixed with ice-cold 70% (v/v) ethanol for 24 hours. After centrifugation at 200 x g for 5 minutes, the cell pellet was washed with PBS (pH 7.4) and resuspended in PBS containing propidium iodide (50 µg/mL), Triton X (0.1%, v/v), and DNase-free RNase (1 µg/mL). Cells were then incubated at room temperature for 1 hour and DNA content was determined by flow cytometry.

 
It has been known that both TNF{alpha} and Adriamycin can kill cells by apoptosis. To determine the mechanisms underlying TNF{alpha}-mediated sensitization, we examined the activation of the apoptotic pathways. As shown in Fig. 1B, Adriamycin treatment resulted in modest cleavage of caspase-9, caspase-3, and PARP, but not caspase-8, which indicates that the mitochondrial apoptotic pathway is activated. Lacking of caspase-8 cleavage by Adriamycin treatment is consistent with the fact that chemotherapeutic agents, including Adriamycin, mainly target the mitochondrial apoptotic pathway (29). In contrast, the combination of 10 ng/mL TNF{alpha} with 0.1 µg/mL Adriamycin significantly enhanced cleavage of caspase-9, caspase-3, and PARP, compared with cells treated with Adriamycin alone, whereas such changes were not detected in cells treated with 10 ng/mL TNF{alpha} alone (Fig. 1B). Importantly, the combined treatment of TNF{alpha} and Adriamycin resulted in a significant increase in cleavage of caspase-8, which was not observed in cells treated with Adriamycin alone (Fig. 1B). Thus, cleavage of caspase-8 and caspase-9 in cells treated with both TNF{alpha} and Adriamycin indicates that both the death receptor and mitochondrial apoptotic pathways are activated. Consistent with cleavage of caspase-3, caspase-3 activity was also enhanced in cells treated with both agents relative to single agent alone (Fig. 1C).

Because activation of caspases can lead to apoptotic cell death, we analyzed cell death by fluorescence-activated cell sorting analysis. As shown in Fig. 1D, TNF{alpha} treatment at 10 ng/mL did not increase the sub-G1 population (apoptotic cells), whereas Adriamycin at 0.1 µg/mL resulted in 19% of the cell populations in sub-G1. In contrast, the combination of TNF{alpha} with Adriamycin resulted in a significant increase in the sub-G1 population; 41% of the cell populations were in sub-G1 (Fig. 1D). These data are in good agreement with enhanced caspase activation in cells cotreated with TNF{alpha} and Adriamycin. Taken together, these results suggest that the enhanced effect of the combined treatments on cell death is attributable to the augmented induction of apoptosis.

TNF{alpha} induces TRAIL expression in human breast cancer cells. Although TRAIL seems to be a tumor-selective, apoptosis-inducing cytokine (2, 3), its regulation is not fully understood. A previous study showed that TNF{alpha} induces TRAIL expression in Jurkat T cells (30). To determine whether TRAIL is involved in TNF{alpha}-mediated sensitization, we treated the breast cancer cell MCF-7 with various doses of TNF{alpha} for 24 hours, and induction of TRAIL was determined by Western blot. As shown in Fig. 2A , TRAIL was induced at as low as 1 ng/mL and then increased to the higher level in a dose-dependent manner. Induction of TRAIL by TNF{alpha} was also observed in MDA-231 cells (Fig. 2B) and T47-D (Fig. 2C). Consistent with induction of TRAIL protein, TRAIL mRNA was induced by TNF{alpha} in T47-D (Fig. 2D) and MCF-7 cells (Fig. 2E) in a dose-dependent manner, as well as in MDA-231 cells (data not shown), which indicates that induction of TRAIL by TNF{alpha} is through a transcriptional mechanism. To determine the effects of the treatments on TRAIL receptors, we analyzed the levels of DR4 and DR5 protein. Figure 2A and B shows that TNF{alpha} treatment does not increase DR4 and DR5 protein in both MDA-231 and MCF-7 cells, as well as in T47-D cells (data not shown), suggesting that such induction is TRAIL ligand specific.


Figure 2
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  		Figure 2. Induction of TRAIL protein by TNF{alpha} in MCF-7 (A), MDA-231 (B), and T47-D (C). Cells were left untreated or treated with various doses of TNF{alpha} for 24 hours, and the total protein was then extracted for assaying TRAIL by Western blot analysis. The levels of DR4 and DR5 were also determined in (A) and (B). Actin was used as a loading control. Induction of TRAIL mRNA by TNF{alpha} in T47-D cells (D) and in MCF-7 cells (E). Cells were left untreated or treated with various doses of TNF{alpha} for 24 hours, and total RNA was extracted to assay for TRAIL mRNA expression by Northern blot analysis. rRNA was visualized as a loading control.

 
To gain insight into induction of TRAIL by TNF{alpha}, we treated MDA-231, MCF-7, and T47-D cells with TNF{alpha} at 10 ng/mL for different time points, and induction of TRAIL was then analyzed. Figure 3A and B shows that in both MCF-7 and T47-D cells, induction of TRAIL was increased at 1 hour, significant at 4 hours, and then stayed at a higher level up to 24 hours upon TNF{alpha} treatment. Such induction was also detected in MDA-231 cells (Fig. 3C). Of note, we detected two bands, ~32 and 29 kDa (Fig. 2A-C), by Western blot analysis using anti-TRAIL antibody from Imgenex. We believed that these bands are two isoforms of TRAIL because they can be down-regulated by TRAIL siRNA (Fig. 5A). Interestingly, the 29 kDa band was induced significantly in MDA-231 cells (Fig. 2B and 3C), whereas both bands were equally induced in both MCF-7 and T47-D cells (Fig. 3A and B). It is not clear whether each isoform plays a different role in TRAIL-mediated apoptosis, which is under investigation. Nevertheless, our data clearly show that TNF{alpha} specifically induces TRAIL ligand through the transcriptional mechanism.


Figure 3
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  		Figure 3. Induction of TRAIL protein by TNF{alpha} is in a time-dependent manner. MCF-7 (A), T47-D (B), and MDA-231 (C) cells were treated with 10 ng/mL TNF{alpha} for 0, 1, 2, 4, 8, 16, and 24 hours, and total protein was extracted for assaying TRAIL by Western blot analysis. Actin was included as a loading control.

 

Figure 5
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  		Figure 5. Down-regulation of TRAIL abolishes TNF{alpha}-mediated sensitization of MDA-231 cells to Adriamycin. A, down-regulation of TRAIL by siRNA silencing. MDA-231 cells were plated at 2 x 105 per well in six-well plates. The next day, cells were transfected with TRAIL siRNA or control oligonucleotides using Oligofectamine. After 3 days, cells were left untreated or treated with TNF{alpha} (10 ng/mL) for 24 hours, and induction of TRAIL was determined by Western blot. B, effects of down-regulation of TRAIL expression by siRNA on cell survival. MDA-231 cells were transfected with TRAIL siRNA or control oligonucleotides as described in (A). After 3 days, cells were left untreated or treated with TNF{alpha} in the absence or presence of Adriamycin (0.1 µg/mL) for 72 hours. Cell viability was determined by MTT assays. Columns, percentage of untreated cells. Representative of three independent experiments. 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 TNF{alpha} (10 ng/mL) in the presence or absence of Adriamycin (0.1 µg/mL) for 72 hours as described in (A). Total protein was extracted for assaying cleavage of caspase-9, caspase-8, caspase-3, and PARP by Western blot analysis. Actin was included as a loading control.

 
Induction of TRAIL by TNF{alpha} is mediated by both NF-{kappa}B and Sp1. To determine the mechanisms underlying TNF{alpha}-induced TRAIL expression, we generated the TRAIL promoter luciferase reporter pGL3-TRAIL1 that contains the TRAIL promoter, including immediate upstream of the translational start site (Fig. 4A ). To determine the responsiveness of this construct to TNF{alpha}, we transfected MDA-231 cells with either pGL3-TRAIL1 or the empty vector pGL3 and then treated cells with TNF{alpha} (10 ng/mL). Cells were harvested after 24 hours, and luciferase activity was assayed using the dual-luciferase reporter assay system. As shown in Fig. 4B, luciferase activity was stimulated ~2.3-fold in cells transfected with pGL3-TRAIL1 following TNF{alpha} treatment, compared with untreated cells. Modest activation of the TRAIL promoter by TNF{alpha} in MDA-231 cells is consistent with a previous study showing that TNF{alpha} weakly activates the TRAIL promoter in the absence of additional stimulation of NF-{kappa}B activity (30).


Figure 4
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  		Figure 4. Transcriptional regulation of TRAIL induction by TNF{alpha} involves both NF-{kappa}B and Sp1 binding sites. A, schematic depiction of luciferase reporter constructs as detailed in Materials and Methods. The translation start site (arrow) is designated at +1. Two Sp1 binding sites and one NF-{kappa}B binding site were also indicated. B, involvement of the NF-{kappa}B binding site in TNF{alpha}-induced luciferase reporter activity. MDA-231 cells were transfected with pGL3-TRAIL1, pGL3-TRAIL2, pGL3-TRAIL3, and pGL3-Basic. pRLSV40 was added in transfections for normalization. The next day, cells were left untreated or treated with 10 ng/mL TNF{alpha}. Luciferase activity was determined 24 hours later. Luciferase activity in cells transfected with control vector was arbitrarily given as 1. C, an Sp1-binding site is required for transactivating the TRAIL promoter by TNF{alpha}. MDA-231 cells were transfected with several deletion constructs as described in (A), along with pRLSV40. The next day, cells were left untreated or treated with 10 ng/mL TNF{alpha}, and luciferase activity was determined 24 hours later. Columns, relative firefly luciferase activities, normalized to Renilla luciferase activities. D, in vivo binding of Sp1 to the TRAIL promoter by ChIP assays. Top, immunoprecipitations were done using rabbit polyclonal antibodies against Sp1. Immunoprecipitations with unrelated Wee1 and no antibody (NoAb) were used as negative controls. Primer pairs targeting two Sp1 sites of the TRAIL gene were used for PCR analysis of the immunoprecipitated DNA samples. Bottom, quantification of the experiments (top). Columns, percentages of DNA immunoprecipitated relative to the input.

 
To define regulation of TRAIL by TNF{alpha}, several deletion constructs were made and then tested for their ability to be transactivated by TNF{alpha}. As shown in Fig. 4B, transfections of pGL3-TRAIL2, which contains a 504 bp fragment upstream of translational start site, including a NF-{kappa}B–binding site, resulted in an ~2.9-fold increase in luciferase activity. It has been shown that activation of the TRAIL promoter by TNF{alpha} is mediated by NF-{kappa}B (30). To confirm the role of NF-{kappa}B in transactivating the TRAIL promoter in breast cancer cells, we transfected pGL3-TRAIL3 that lacks this NF-{kappa}B binding site (Fig. 4A) into MDA-231 cells and then assayed for luciferase activity upon TNF{alpha} treatment. As shown in Fig. 4B, there was an ~1.9-fold increase in luciferase activity in cells transfected with pGL3-TRAIL3, compared with untreated cells. A decrease in luciferase activity in cells transfected with pGL3-TRAIL3 without the NF-{kappa}B binding site suggests a role of NF-{kappa}B in regulating the TRAIL promoter activity, which is consistent with a previous study (30).

Because pGL3-TRAIL3 could be activated by TNF{alpha}, although the activation was not as strong as pGL3-TRAIL2 that contains a NF-{kappa}B binding site (Fig. 4B), we suspected that there might be an additional regulatory element responsible for TNF{alpha}-mediated transcription. To this end, we transfected three additional deletion constructs into MDA-231 cells and found that pGL3-TRAIL6 containing one Sp1 binding site is not longer to be activated by TNF{alpha}, whereas pGL3-TRAIL5 containing two Sp1 sites was activated by TNF{alpha} (Fig. 4C). These data suggest that the second Sp1 site is required for TNF{alpha}-mediated promoter activity. Of note, the deletion construct pGL3-TRAIL5 had high basal activity (Fig. 4C), which suggests that an Sp1 site may be important for maintaining the basal transcriptional activity of the TRAIL gene. To determine whether Sp1 in fact binds to the TRAIL promoter, we did ChIP assay of the TRAIL promoter with anti-Sp1 antibody. MDA-231 cells were left untreated or treated with TNF{alpha} (10 ng/mL) and then harvested 24 hours later for ChIP analysis after immunoprecipitation with an antibody against Sp1 or an unrelated antibody against Wee1. Figure 4D shows that PCR amplification of the immunoprecipitated DNA with Sp1 antibody resulted in single bands of a size consistent with the region of the TRAIL promoter. Importantly, there were higher levels of amplified DNA in TNF{alpha}-treated cells than untreated cells, whereas there was a little amplification using immunoprecipitated DNA with Wee1 antibody, which served as negative control (Fig. 4D, bottom). These results suggest that TNF{alpha} activates the TRAIL promoter via an Sp1 site.

Induction of TRAIL by TNF{alpha} is required for sensitization of MDA-231 cells to Adriamycin-induced death. We have shown that TNF{alpha} induces TRAIL, and the combination of TNF{alpha} with Adriamycin enhances cell death relative to either agent alone. Because TRAIL is a potent apoptotic inducer, we asked whether induction of TRAIL by TNF{alpha} is required for this enhanced killing effect. To this end, MDA-231 cells were transfected with either control oligonucleotides or oligonucleotides 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 tranfected with control oligonucleotides, and induction of TRAIL by TNF{alpha} was also decreased compared with cells transfected with control oligonucleotides, indicating that TRAIL siRNA is sufficient to down-regulate TRAIL expression. To determine the effects of down-regulation of TRAIL on cell viability, MDA-231 cells transfected with either TRAIL siRNA or control siRNA were treated with TNF{alpha}, Adriamycin, or in combination for 72 hours, and cell viability was then determined. As shown in Fig. 5B, cells transfected with TRAIL siRNA were completely resistant to TNF{alpha}-mediated growth inhibition, whereas TNF{alpha}-mediated growth inhibition was 60% in cells transfected with nontarget oligonucleotides. Similarly, down-regulation of TRAIL increased cell resistance to Adriamycin compared with cells transfected with control oligonucleotides, although such resistance was not as significant as observed in cells treated with TNF{alpha} (Fig. 5B). More importantly, enhanced killing effect by the combination was abrogated in cells transfected with TRAIL siRNA, compared with cells transfected control oligonucleotides (Fig. 5B). Survival rates increased from 9% in cells transfected with nontargeted siRNA to 49% in cells transfected with TRAIL siRNA following the combined treatment of TNF{alpha} with Adriamycin. Furthermore, in cells transfected with TRAIL siRNA, TNF{alpha} was no longer to enhance Adriamycin-induced cell death; cell viability was 56% in cells treated with Adriamycin alone compared with 49% in cells treated with both TNF{alpha} and Adriamycin (Fig. 5B). Collectively, these results suggest that TRAIL plays a critical role in enhancing Adriamycin-induced cell death.

Activation of TRAIL-mediated caspase cascade by TNF{alpha} is critical for enhancing cell death by Adriamycin. To investigate the effects of down-regulation of TRAIL on TNF{alpha}-mediated, Adriamycin-induced caspase activation, cells transfected with TRAIL siRNA or control oligonucleotides were treated with TNF{alpha} (10 ng/mL), Adriamycin (0.1 µg/mL), or in combination for 72 hours, and activation of caspases was then examined. As expected, in cells transfected with control oligonucleotides, activation of caspase-9, caspase-8, and caspase-3, as well as cleavage of PARP, was significant in cells treated with both TNF{alpha} and Adriamycin, compared with untreated or cells treated with TNF{alpha} alone, whereas such changes was minimal in Adriamycin-treated cells (Fig. 5C). In contrast, cleavage of caspase-9, caspase-8, and caspase-3, and PARP was abrogated in cells transfected with TRAIL siRNA following the combined treatment (Fig. 5C), indicating that through down-regulation of TRAIL, caspases are no longer to be activated by both TNF{alpha} and Adriamycin, thus abrogating apoptosis. Taken together, these results suggest that TRAIL plays a central role in TNF{alpha}-mediated Adriamycin-induced caspase activation and apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that TNF{alpha} enhances Adriamycin-induced cell death. We also showed that TNF{alpha} induces TRAIL via both NF-{kappa}B and Sp1. Importantly, we showed that induction of TRAIL by TNF{alpha} is required for enhancing Adriamycin-induced breast cancer cell death because knockdown of TRAIL by siRNA silencing abolished TNF{alpha}-mediated sensitization. Thus, our findings suggest that TRAIL plays a critical role in sensitizing human cancer cells to chemotherapeutic agents.

TRAIL has been shown to selectively kill tumor or transformed cells while sparing normal cells, but its regulation is poorly understood. By analyzing the genomic sequence of the TRAIL gene, it has been found that the TRAIL promoter contains a number of transcription regulatory elements, including ISRE, NF-{kappa}B, and Sp1 (31, 32). The best-characterized regulatory element in the TRAIL promoter is ISRE, an IFN-response element. It has been shown that IFN is able to directly induce TRAIL in both human leukemia Jurkat and colon cancer cell line HT29 (31, 32). Further studies showed that IFN acts as a mediator to induce TRAIL in response to retinoid treatment (33) and paramyxovirus infection (34). Induction of TRAIL is believed to be through binding of IFN to an IFN-stimulated response element, leading to induction of TRAIL and apoptosis (33, 34). In addition to its regulation by IFN, it has been shown that the TAX oncoprotein encoded by human T-cell leukemia virus induces TRAIL that is dependent on NF-{kappa}B signaling (35). Furthermore, a study indicated that TRAIL is induced by T-cell receptor mimetics in human T cells and that such induction is linked to a c-Rel binding site in the proximal TRAIL promoter (30). Consistent with this, we showed that deletion of the NF-{kappa}B binding site results in a partial decrease in luciferase activity induced by TNF{alpha} (Fig. 4B), suggesting that NF-{kappa}B is involved in activation of the TRAIL promoter. It has been shown that the transcription factor Sp1 plays an important role in the initiation gene transcription (36, 37). It has also been shown that Sp1 interacts with NF-{kappa}B to enhance the transcription reinitiation of TNF{alpha}-induced genes (37). Consistently, we have shown that deletion of one Sp1 binding site results in a complete loss of TRAIL promoter activity (Fig. 4C), indicating that this Sp1 site is required for transactivation of the TRAIL promoter by TNF{alpha}. In addition, we have shown that pGL3-TRAIL5 that contains 80 bp upstream of the translational start site has a higher level of luciferase activity in the presence or absence of TNF{alpha} treatment (Fig. 4C). Furthermore, we have found by ChIP assays that Sp1 can be bound to the TRAIL promoter even in the absence of TNF{alpha} treatment (Fig. 4D). Taken together, these observations suggest that one Sp1 site is important for maintaining a higher basal level of TRAIL promoter activity.

The ability of TNF{alpha} to induce apoptosis suggests that TNF{alpha} is a potential agent for the treatment of human cancers, but pharmacologic doses of TNF{alpha} that have anticancer activity cause serious toxicity to normal cells, thus limiting its application. However, it has been shown that low doses of TNF{alpha}, which are unable to induce tumor cell apoptosis, are potential sensitizers to enhance conventional chemotherapeutic agent–induced anticancer activity (23). Consistent with this, it has been shown that low dose of TNF{alpha} enhances Adriamycin-mediated anticancer activity in soft tissue sarcoma in rats (38). Moreover, in the human Hodgkin cell line HD-MyZ cells, TNF{alpha} pretreatment enhanced activation of caspase-3 and cell death induced by several anticancer drugs, including etoposide and epirubicin, and the underlying mechanism was believed to be caspase-9 dependent and NF-{kappa}B independent (23). In this study, we have found that either TNF{alpha} at 10 ng/mL or Adriamycin at 0.1 µg/mL has limited effect on MDA-231 cell death. In contrast, the combination of TNF{alpha} with Adriamycin significantly enhances cell death (Fig. 1A). We believe that the underlying mechanism involves the activation of both death receptor and mitochondrial pathways. This hypothesis was supported by the following observations. First, Adriamycin treatment (0.1 µg/mL) weakly activated the mitochondrial pathway, as evidenced by modest cleavage of caspase-9 (Fig. 1B), which is consistent with the notion that anticancer drugs, including Adriamycin, mainly target the mitochondrial apoptotic pathway. Second, TNF{alpha} had little effects on either activation of caspase-8 or caspase-9 (Fig. 1B), which is in agreement with the notion that low doses of TNF{alpha} are unable to induce apoptosis in the absence of RNA or protein synthesis inhibitors. Last, the combined treatment significantly enhanced activation of caspase-8 and caspase-9, leading to apoptotic cell death (Fig. 1B), suggesting that activation of both mitochondrial and death receptor pathways is critical for TNF{alpha}-mediated, Adriamycin-induced cell death. However, our results differ from a previous study showing that activation of caspase-8 is not important in TNF{alpha}-mediated Adriamycin-induced anticancer activity (23). The reason for this discrepancy is not clear, but we believe that this difference may be due to differences in the cell lines used. Nevertheless, our results clearly indicate that both death receptor and mitochondrial pathways are activated in combined treatment in the human breast cancer cell line MDA-231.

What is the mechanism underlying TNF{alpha}-mediated sensitization of breast cancer cells to Adriamycin? Although TNF{alpha} treatment commonly activates the NF-{kappa}B pathway, which leads to apoptosis or cell survival in part dependent on stimuli and cell types, a previous study indicated that NF-{kappa}B is not involved in TNF{alpha}-mediated sensitization of the human Hodgkin cell line HD-MyZ to etoposide and epirubicin (23). Further, it has been shown that TNF{alpha}-mediated sensitization is independent of caspase-8 (23). In this study, we have shown that the treatment of MDA-231 breast cancer cells with both TNF{alpha} and Adriamycin results in a significant activation of caspase-8, which was not obvious in cells treated with either TNF{alpha} or Adriamycin alone (Fig. 1B), indicating that activation of caspase-8 involves in this sensitization. Because activation of caspase-8 can result from activation of death receptors, it is very likely that the death receptor pathway is activated in this combined treatment. Consistent with this, we have shown that TRAIL ligand is induced upon TNF{alpha} treatment (Fig. 2A-C). However, we have also found that Adriamycin does not induce TRAIL receptors in MDA-231 cells (data not shown), which excludes the possibility that an increase in the TRAIL receptors by Adriamycin involves in enhancing cell death in the combined treatment. It is well documented that activation of the TRAIL pathway can induce apoptosis. Therefore, it is expected that activation of the TRAIL pathway by TNF{alpha} can activate caspase-8 and caspase-3 and induce apoptosis. Although TNF{alpha} can induce TRAIL, it is possible that such induction is not relevant to this sensitization. To exclude this possibility, we used siRNA silencing to down-regulate TRAIL expression and found that TNF{alpha} no longer sensitizes MDA-231 cells to Adriamycin (Fig. 5B), indicating TRAIL is indeed involved in this sensitization. In addition, down-regulation of TRAIL enhances cell survival in response to either TNF{alpha} or Adriamycin treatment, suggesting that TRAIL may play a role in cell death induced by either agent alone. Thus, these data strongly suggest that induction of TRAIL by TNF{alpha} is critical for enhancing Adriamycin-induced cell death in the breast cancer cells.

In summary, we show that TNF{alpha} induces TRAIL via both NF-{kappa}B and Sp1. We also show that the treatment of breast cancer cells with TNF{alpha} enhances Adriamycin-induced anticancer activity via activation of both the death receptor and mitochondrial apoptotic pathways. More importantly, we provide evidence that induction of TRAIL is critical for TNF{alpha}-mediated sensitization of breast cancer cells to Adriamycin because down-regulation of TRAIL by siRNA silencing abrogates such sensitization. Therefore, our findings have an important implication to target TRAIL in combination with conventional anticancer drugs as new regimens for the treatment of human cancer.


   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.

Received 5/ 3/06. Revised 7/26/06. Accepted 8/15/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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