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Clinical Cancer Research 14, 3617, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-2060
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

Matrix metalloproteinase-9 Inhibition Down-Regulates Radiation-Induced Nuclear Factor-{kappa}B Activity Leading to Apoptosis in Breast Tumors

Sateesh Kunigal1, Sajani S. Lakka1, Pushpa Joseph2, Norman Estes3 and Jasti S. Rao1,4

Authors' Affiliations: Departments of 1 Cancer Biology and Pharmacology, 2 Pathology, 3 Surgery, and 4 Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois

Requests for reprints: Jasti S. Rao, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605. Phone: 309-671-3445; Fax: 309-671-3442; E-mail: jsrao{at}uic.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases (MMP) and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (MMP-9 adenoviral construct, Ad-MMP-9), affects breast cancer sensitivity to radiation.

Experimental Design: In the present study, we used antisense Ad-MMP-9 to down-regulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro before irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were done with orthotopic breast tumors, and radiosensitivity was evaluated both in vitro and in vivo.

Results: Ad-MMP-9 infection resulted in down-regulation of radiation-induced levels of hypoxia-inducible factor 1{alpha} and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9, in combination with radiation, decreased levels of the transcription factors nuclear factor-{kappa}B and activator protein 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas–Fas ligand apoptotic cascade, which resulted in the cleavage of PARP-1 and caspase-10, caspase-3, and caspase-7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model.

Conclusions: In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.

Radiotherapy provides a key management strategy for many epithelial tumor types, forms part of the multidisciplinary approach for breast cancer treatment, and is now of routine value after conservative surgery to reduce locoregional tumor recurrence. However, the significant limitations of radiotherapy make it inefficient as the sole treatment for breast and many other cancers. Some cancer cells are intrinsically resistant to damage by ionizing radiation, and treatment can actually induce tumor cell proliferation and repopulation, resulting in a diminished response to radiation, resistant growth, and poor local control (1). Hypoxia-driven cellular modifications have been shown to contribute to this poor prognostic outlook, giving rise to more aggressive locoregional disease, invasive capacity, and angiogenesis (2).

Intratumoral hypoxia plays a pivotal role in activating the key transcription factor, hypoxia-inducible factor (HIF), which mediates activation of the "survival machinery" in cancer cells. Specifically, HIF1 regulates the expression of numerous genes and controls glycolysis, erythropoiesis, apoptosis, and angiogenesis (3). Studies have shown that HIF1{alpha} activity promotes tumor growth in vivo (4). Hypoxic tumor regions show increased gene expression caused by hypoxia-induced activation of transcription machinery (5, 6). Several of the gene products induced or up-regulated under hypoxic conditions play pivotal roles in the metastatic process, and studies have suggested that hypoxia could promote metastasis in human cancer (5). Hypoxia also activates nuclear factor-{kappa}B (NF-{kappa}B) (7), a transcription factor important in the promotion and progression of tumor development and survival (8). As such, inhibition of these pathways represents a potentially important cancer therapeutic.

Cancer cells often develop radioresistance mechanisms related to the DNA repair response. By combining chemotherapy and radiation therapy, radiation efficiency may be strengthened by inhibiting DNA repair and overcome resistance to apoptosis. NF-{kappa}B is activated by DNA-damaging agents and could be involved in cell cycle arrest and prevention of apoptosis, allowing DNA repair (9). However, sustained NF-{kappa}B activation could permit cells with accumulated radiation-induced DNA damage to escape elimination by apoptosis (10). Indeed, high constitutive NF-{kappa}B activity prevents cancerous cells from apoptosis (11), resulting in a more aggressive potential seen in prostate cancer (12), malignant melanomas, Hodgkin's disease, leukemia, breast cancer, and cutaneous T-cell lymphoma cancer cell lines (13).

The transcription factor activator protein 1 (AP-1) comprises members of the Jun and Fos families and has been implicated in the regulation of apoptosis and cell proliferation (14). Studies suggest that Jun B and c-Jun may trigger apoptosis and promote proliferation of erythroid cells, respectively (15). Activation of Sp1, another key transcription factor, occurs under hypoxic conditions in various cancer cells, including breast carcinoma (16, 17). Thus, agents that enhance apoptosis in irradiated tumor cells could have significant therapeutic benefits.

Degradation of the extracellular matrix plays an important role in tumor metastasis. Matrix metalloproteinases (MMP) have been primarily associated with matrix remodeling, a necessary component of invasion. It has been reported that preoperative, short-course radiotherapy decreases local recurrence rates (e.g., rectal cancer) and, combined with optimal surgery, improves patient survival. Although radiotherapy has proved benefits, several reports show an increase in expression and activation of gelatinase MMPs (18). Initially, MMPs were thought to simply breakdown components of the extracellular matrix, allowing for invasion and metastasis. However, recent studies confirmed that MMP activity involves precise MMP localization on a cell's invasive front, exposure of key components in the extracellular matrix transform it from a barrier into a scaffold for invasion, and cleavage of free insulin-like growth factors result in cell growth, division, and inhibition of apoptosis (19).

Hypoxia-targeted gene therapy presents a number of interesting developments and applications in modern oncology. Attempts are currently being made to overcome the adverse effects and limitations of radiation and exploit resistant hypoxic tumor cells using combination gene therapy and radiotherapy. In this study, we combined radiation with down-regulated MMP-9, which reduced hypoxia by using a replication-deficient recombinant adenovirus containing a 528-bp antisense expression segment for human MMP-9 (Ad-MMP-9AS or Ad-MMP-9; ref. 20) to treat breast cancer tumors. Our results indicated that, after the combined treatment, transcription factor activity controlling the expression of the oncoproteins was reduced and proapoptotic molecules were up-regulated, resulting in increased apoptosis and tumor suppression.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments. MDA MB 231 human breast cancer cells were purchased from the American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum in a humidified CO2 incubator at 37°C. MDA MB 231 cells were serum-starved for 12 to 18 h and treated with 100 multiplicity of infection of the empty vector (Ad-CMV) or antisense MMP-9 adenoviral construct (Ad-MMP-9). Either Ad-MMP-9 or Ad-CMV was added to the cell monolayer (1.0 mL/60-mm dish or 3 mL/100-mm dish), and cells were incubated at 37°C for 60 min with brief agitation every 5 min. The necessary amount of culture medium was then added, and cells were returned to the incubator. For the radiation treatment, the cells were starved of serum, exposed to 5 Gy, and returned to the incubator after the addition of the necessary amount of complete culture medium. For the treatment combination of virus and radiation, the cells were first infected with 100 multiplicity of infection of Ad-MMP-9 or Ad-CMV, and 24 h later, the cells were irradiated with 5 Gy. After the above-described treatments, the cells were incubated in hypoxic condition for a period of 12 to 16 h. The hypoxic condition was accomplished with the Anaerocult mini setup (EM Science).

Reverse transcription–PCR. Total RNA was isolated from cells in all treatment conditions using TRIzol per standard protocol. Total RNA was treated with DNase I (Invitrogen) to remove contaminating genomic DNA. PCR analysis was done using the one-step reverse transcription–PCR kit (Invitrogen). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The following primers were used:

HIF1{alpha} 5'-AGTCTGCAACATGGAAGG-3' sense,
5'-CACGACTTGATTTTCTCCC-3'antisense;
MMP-9 5'-TGGACGATGCCTGCAACGTG-3'sense,
5'-GTCGTGCGTGTCCAAAGGCA-3'antisense;
GAPDH 5'-CGGAGTCAACGGATTTGGTCGTAT-3'sense,
5'-AGCCTTCTCCATGGTGGTGAAGAC-3'antisense.

The PCR conditions were as follows: 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, annealing temperature set according to the AT and GC content of the primers, respectively, for 1 min and 72°C for 1 min. The final extension was at 72°C for 5 min.

Electromobility shift assay. Nuclear extracts were prepared from MDA MB 231 cells that were treated with Ad-MMP-9, radiation, or both. Cells were detached with EDTA, resuspended in buffer A [10 mmol/L HEPES (N-2-hydroxyethylenepiperazine-N'-2-ethanesulfonic acid; pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT] containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L iodoacetamide, 0.1 mmol/L quercetin, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 0.3 mmol/L sodium vanadate), and incubated on ice for 15 min. After homogenization in a Wheaton 0.1-mL homogenizer, the nuclei were collected by centrifugation. The pellet was resuspended in buffer B [20 mmol/L HEPES (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT] containing protease inhibitors and incubated on ice for 30 min, followed by centrifugation at 13,000 x g (5 min at 4°C). The supernatant was dialyzed against buffer C (20 mmol/L HEPES, 20% glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT) containing protease inhibitors for 2 h at 4°C, followed by centrifugation at 13,000 x g (5 min at 4°C). The supernatant proteins were used immediately or aliquoted and stored at –80°C.

Binding reaction was done for 30 min on ice in a volume of 20 µL, containing 4 µg nuclear protein extracts, 40 ng poly(dI-dC), 4 µL 5x binding buffer [1x binding buffer: 20 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L DTT, 10% glycerol] with or without 20-fold to 50-fold excess of cold competitor or unrelated competitor and a 32P-labeled probe (3x104 cpm). For supershift electrophoretic mobility shift assay, protein extracts were incubated with 6 µg SP1 monoclonal antibody or isotype control before the addition of the 32P-labeled probe. DNA-protein complexes were separated on 5% polyacrylamide gel in Tris/glycine buffer at 4°C. The following double-stranded oligonucleotides (Santa Cruz Biotechnology) were used in this study:

NF-{kappa}B 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5';
AP-1 5'-CGC TTG ATG ACT CAG CCG GAA-3' and 3' GCG AAC TAC TGA GTC GGC CTT-5'.

End-labeled probes were prepared with 40 µCi (1,480 MBq) [{gamma}-32P] ATP using T4 polynucleotide kinase and were gel-purified on NAP-5 Sephadex G-25 DNA-grade columns.

Western blot analysis. MDA MB 231 cells were treated with Ad-CMV, Ad-MMP-9, radiation of 5 Gy, or a combination (Ad-MMP-9 plus radiation) and incubated under hypoxic conditions as described earlier. After the incubation period, the cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer containing protease inhibitors. Whole-cell extracts were subjected to SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were blocked with 7% nonfat dry milk and probed with antibodies for the following molecules: MMP-9, HIF1{alpha}, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK, NF-{kappa}B, p50, p65, c-fos, jun D, AP-1, Fas, Fas ligand (Fas-L), caspase-10, caspase-3, caspase-7, and PARP-1. Appropriate antibody conjugate with horseradish peroxidase was used as the secondary antibody. Membranes were developed according to an enhanced chemiluminescence protocol as per the manufacturer's instructions (Amersham Biosciences). Nuclear and cytoplasmic fractions for Western blotting were prepared as described elsewhere (21).

Gelatin zymography. Gelatin-substrate gel electrophoresis was done as described previously (22). MDA MB 231 cells were transfected with Ad-CMV, Ad-MMP-9, radiation of 5 Gy, or a combination (Ad-MMP-9 plus radiation) and incubated under hypoxic conditions as described earlier. To collect conditioned media, cells were washed once with serum-free medium and incubated with fresh serum-free medium. After 12 to 14 h, conditioned medium was collected and centrifuged to remove cellular debris, and protein concentrations were determined. Equal amounts of protein were subjected to 0.1% gelatin SDS-PAGE under nonreducing conditions. Gels were washed in 2.5% Triton X-100 and incubated overnight in Tris-CaCl2 buffer. The gels were then stained with 0.2% Coomassie blue for 30 min and destained in 20% methanol and 10% acetic acid. The clear bands represent gelatinase activity.

Clonogenic survival assay. A clonogenic survival assay was used to investigate the sensitivity of MDA MB 231 cells infected with Ad-MMP-9 to radiation therapy as described previously (23). Briefly, the cells were treated as described earlier. The MDA MB 231 cells were trypsinized and plated in 100-mm dishes to assay for their colony-forming ability immediately after irradiation under hypoxic condition. Colonies were counted 10 to 15 d later. Survival curves were plotted using the GraphPad Prism 3.0 software program.

Animal experiments. MDA MB 231 cells were cultured in complete medium until a 70% to 80% density was obtained. At this point, cells were trypsinized, washed once with serum-free medium, and counted. Cells were injected bilaterally into the second mammary fat pads of athymic, female, 4-wk-old to 6-wk-old nu/nu mice (5-6 x 106/100 µL serum-free culture medium). Tumor growth was monitored daily. Once the tumor reached ~6 to 8 mm in size, the animals were divided into five groups of five animals each. Group 1 received PBS injections, group 2 received three doses of 5 x 108 plaque-forming units intratumorally on alternate days, group 3 received three doses of 5 x 108 plaque-forming units intratumorally on alternate days, group 4 was irradiated with two doses of 5 Gy on alternate days, and group 5 received Ad-MMP-9 and radiation treatments. In the case of the combined treatment, the tumors were first given intratumoral injections of Ad-MMP-9 on alternate days. Then, after the third dosage, the tumors were given two doses of 5 Gy on alternate days. The regression in the orthotopic tumor growth was followed for up to 8 wk. Mice were euthanized when the tumor diameter in control mice measured between 1.2 and 1.5 cm were removed and further processed. Additionally, as there was no tumor in animal treated with the Ad-MMP-9 infection in combination with irradiation, we included one more group of five animals that received combined treatment. The animals with tumor were euthanized after 15 d post combined treatment. Finally, the tumor volume was calculated using the formula V = {pi} / 6 (a x b x c).

Immunohistochemistry. Tumor samples fixed in 10% neutral buffered formalin were embedded in paraffin using automatic embedding equipment, after which 5-µm sections were prepared. Immunohistochemical analysis for MMP-9, HIF1{alpha}, and caspase-3 was done on paraffin-embedded breast tumor sections of mice treated with Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9 infection in combination with irradiation.

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was done with paraformaldehyde-fixed, paraffin-embedded breast tumor sections as per the manufacturer's protocol. Briefly, the TUNEL method identifies apoptotic cells in situ by using terminal deoxynucleotidyl transferase to transfer biotin-dUTP to the free 3'-OH of cleaved DNA. The biotin-labeled cleavage sites are then visualized by reaction with fluorescein-conjugated avidin (avidin-FITC). The cells were visualized using a fluorescent microscope with appropriate filter sets. DNA fragmentation in these treated tumors is indicative of apoptotic cell population.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Ad-MMP-9 infection inhibits radiation-induced MMP-9 and HIF1{alpha} expression at both the mRNA and protein levels in breast cancer cells. We analyzed the effect of Ad-MMP-9 (adenoviral construct of antisense to MMP-9 gene) on the MDA MB 231 cell line, which is the most aggressive breast cancer cell line for MMP-9 and HIF1{alpha} expression at the mRNA level. Reverse transcription–PCR analysis showed that Ad-MMP-9 infection inhibited MMP-9 and HIF1{alpha} at the mRNA level when compared with control or Ad-CMV (empty vector)–infected MDA MB 231 cells under hypoxic conditions (Fig. 1A ). In contrast, radiation alone augmented the expression level of these molecules. Treatment with Ad-MMP-9 plus radiation inhibited expression levels more than Ad-MMP-9 infection alone. The level of MMP-9 was reduced by nearly 40% in the Ad-MMP-9–treated cells when compared with mock and Ad-CMV treatments; MMP-9 level was reduced by >50% when cells were treated with Ad-MMP-9 plus radiation. Conversely, in the cells that were treated with radiation alone, the level of MMP-9 was 50% higher than in the control cells (Fig. 1A).


Figure 1
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  		Fig. 1. Ad-MMP-9 infection inhibits radiation-induced MMP-9 and HIF1{alpha} expression at both the mRNA and protein levels in breast cancer cells. A, briefly, MDA MB 231 cells were infected with Ad-CMV (100 multiplicity of infection), Ad-MMP-9 (100 multiplicity of infection), irradiation (IR; 5 Gy), or a combination of Ad-MMP-9 (100 multiplicity of infection) and irradiation (5 Gy), and incubated under hypoxic conditions as described in Materials and Methods. After 12 to 16 h of incubation, total RNA was extracted using TRIzol reagent and quantitated, and reverse transcription–PCR was done for assessment of MMP-9 and HIF1-{alpha} levels. Expression of GAPDH was verified for the equal loading of cDNA. B, densitometric analysis of MMP-9 and HIF1-{alpha} expression at the mRNA level (mean ± SE; n = 3). C, after incubation, cell lysates were prepared and used for Western blot analysis to determine the levels of MMP-9 and HIF1{alpha}. GAPDH was also used as a control to confirm equal loading of cell lysates. D, densitometric analysis of MMP-9 and HIF1-{alpha} expression at the protein level (mean ± SE; n = 3). E, MMP-9 activity was analyzed by gelatin zymography using equal amounts of protein from the conditioned medium as described in Materials and Methods. F, densitometric analysis of MMP-9 activity (mean ± SE; n = 4).

 
Similarly, the expression level of HIF1{alpha} at the mRNA level in the irradiated cells was increased by 30% compared with the control and Ad-CMV–infected cells. Ad-MMP-9 treatment alone inhibited the expression of HIF1{alpha} by 25% compared with the controls; combined treatment of Ad-MMP-9 and radiation resulted in a 60% reduction of HIF1{alpha} compared with the controls. These results support the earlier findings of Moeller et al. (24) that radiation increased the levels of active HIF1 and that the hypoxic condition is responsible for the poor response of the tumors to radiotherapy (Fig. 1A and B). Western blot analyses using respective antibodies against MMP-9 and HIF1{alpha} support the reverse transcription–PCR analysis results, where the level of MMP-9 was reduced nearly 40% to 45% in the Ad-MMP-9–treated cells compared with the control and Ad-CMV treatment and by more than 60% to 70% when cells were infected with Ad-MMP-9 in combination with radiation. Conversely, in the cells treated with radiation alone, the level of MMP-9 was 30% more than in the control cells (Fig. 1C and D).

In addition, analysis of MMP-9 activity by gelatin zymography using conditioned medium from the treated cells revealed decreased levels of MMP-9 activity in Ad-MMP-9–infected cells compared with the controls. MMP-9 activity was even further reduced with the combined treatment. In contrast, we observed an almost 2-fold increase in MMP-9 activity in the irradiated cells when compared with the control and Ad-CMV–treated cells (Fig. 1E and F).

Treatment with Ad-MMP-9 and radiation decreases the binding activity of NF-{kappa}B and AP-1. Ionizing radiation is reported to induce activation of NF-{kappa}B and AP-1 (25). Poynter et al. have reported on the phosphorylation and dephosphorylation of members of the ERK family of the mitogen-activated protein kinase cascade and the events leading to activation of the transcription factor NF-{kappa}B. These cascades are critical for the transcriptional up-regulation of genes important for cell survival, apoptosis, proliferation, transformation, and inflammation (26). Furthermore, it is known that ionizing radiation augments phosphorylation of ERK (27). Western blot analysis using whole-cell extracts of the cells treated with Ad-MMP-9 and Ad-MMP-9 in combination with radiation showed reduced phosphorylation of ERK, whereas irradiated cells showed increased phosphorylation when compared with the controls (Fig. 2A ). Activation of the binding activities of these transcription factors in tumor cells contributes to MMP-9 transcription and cell invasion (28). Western immunoblot analysis using the nuclear fractions from the treated cells showed reduced translocation of p50 and p65 (subunits of NF-{kappa}B) and c-fos and Jun D (subunits of AP-1) to the nucleus in the Ad-MMP-9–treated cells, and further reduction in the cells was treated with Ad-MMP-9 in combination with radiation compared with the controls. In contrast, analysis of the cytoplasmic fractions showed reduced signals for the above-said molecules in the Ad-MMP-9–treated cells, and further reduction in the cells treated with Ad-MMP-9 in combination with radiation compared with the controls (Fig. 2B). However, a slight increase of NF-{kappa}B and AP-1 translocation to the nucleus was seen in radiated cells alone when compared with the controls. Furthermore, we analyzed the DNA-binding activities of NF-{kappa}B and AP-1 in the nuclear extracts using NF-{kappa}B and AP-1–specific oligonucleotide probes (electrophoretic mobility shift assay). In the cells treated with Ad-MMP-9 or Ad-MMP-9 plus radiation, the DNA-protein complex was decreased when compared with the control and irradiated cells. The specificity of the NF-{kappa}B–DNA and AP-1–DNA complexes was confirmed by supershift assay using specific antibodies (Fig. 2C).


Figure 2
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  		Fig. 2. The combined treatment of Ad-MMP-9 and radiation decreases binding activity of NF-{kappa}B and AP-1. A, Western blot results for ERK1/2 and phosphorylated ERK. GAPDH was used as a control to confirm equal loading of cell lysates. B, Western blot analysis of subcellular fractions showing the translocation of the p50 and p65 subunits of NF-{kappa}B and c-fos and Jun D subunits of AP-1. C, the binding activities of NF-{kappa}B and AP-1 to DNA were determined using the electromobility shift assay. Oligonucleotide consensus probes for NF-{kappa}B and AP-1 were end labeled with 32P and used in the shift assay. SS, supershift.

 
Down-regulation of MMP-9 and radiation induces apoptosis in breast cancer cells. Because radiation-induced NF-{kappa}B activation leads to cell survival, we next examined whether Ad-MMP-9 treatment in combination with radiation could cause apoptosis. Western immunoblot analysis showed the up-regulation of Fas in the Ad-MMP-9, radiation, and combination treatments. However, Fas-L was up-regulated only in the Ad-MMP-9–treated and Ad-MMP-9 plus radiation-treated cells compared with the controls and radiation alone treatment (Fig. 3A ). The densitometric analysis also revealed an increase of Fas (~1.5-fold) and Fas-L (~1.8-fold) expression in the Ad-MMP-9–infected cells, in combination with irradiation compared with the irradiation alone. We also looked downstream of this cascade. Western blot analysis revealed the cleavage of PARP-1 and caspase-10, caspase-3, and caspase-7 in Ad-MMP-9, radiation, and combination treatments compared with the control and Ad-CMV–treated cells (Fig. 3B). To confirm these initial observations, we analyzed apoptosis by looking for DNA fragmentation (TUNEL) assay. TUNEL-positive, apoptotic MDA MB 231 cells were meagerly present in control or Ad-CMV–infected cells. Ad-MMP-9 infection, in combination with irradiation, resulted in a distinct increase of TUNEL-positive cells compared with the Ad-MMP-9 alone–treated and irradiation alone–treated cells (Fig. 3C). Quantitation of TUNEL-positive cells indicated that Ad-MMP-9 infection in combination with irradiation (~85%) had 2-fold more compared with irradiation alone (~40%; Fig. 3D). A clonogenic survival assay was done to determine the influence of the down-regulation of MMP-9 in breast tumor cell to ionizing radiation. Figure 3E indicates that down-regulation of MMP-9 in MDA-MB-231 cells increased the radiosensitivity than uninfected and controls in terms of clonogenic survival. Statistical analysis shows that clonogenic survival in irradiated MDA-MB-231 cells that has reduced MMP-9 activity is significantly reduced compared with irradiated control MDA-MB-231 cells. These data confirm that the down-regulation of active MMP-9 increase sensitivity to ionizing radiation.


Figure 3
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  		Fig. 3. Down-regulation of MMP-9 and radiation induces apoptosis in breast cancer cells. A, Western blot results for Fas and Fas-L. Representation of densitometric analysis of the expression of Fas and Fas-L (mean ± SE; n = 4). B, Western blot results for caspase-10, caspase-3, and caspase-7 show active caspase and PARP cleavage. C and D, TUNEL assay was done with MDA MB 231 cells with the earlier said treatments as per the manufacturer's protocol and the densitometric analysis of percentage of TUNEL-positive cells are represented (mean ± SE; n = 3). *, significant difference between irradiated alone cells and combination treatment of Ad-MMP-9 infection with irradiation (P ≤ 0.05). E, clonogenic survival was assessed between 15 d after radiation exposure. Each experiment was done at least four times, and triplicates were done for each experiment. Clonogenic survival was assessed between 15 d after radiation exposure. Each experiment was done at least four times, and triplicates were done for each experiment. Statistical analysis was done using PRISM and ANOVA. *, significant difference between irradiated alone cells and combination treatment of Ad-MMP-9 infection with irradiation (P ≤ 0.01).

 
MMP-9 and HIF1{alpha} mediate the inhibition of tumor growth after treatment with Ad-MMP-9 plus radiation. MDA MB 231 cells were chosen for animal studies because these cells form aggressive primary tumors. We compared the effect of the three treatments (Ad-MMP-9, radiation, and a combination) on tumor growth. Tumors that received radiation (two doses of 5 Gy) showed tumor regression of >50% when compared with the control and Ad-CMV–treated tumors. Analysis of tumor size revealed that tumors injected with 5 x 108 plaque-forming units of Ad-MMP-9 displayed a more delayed tumor growth than those of control mice. These tumors were suppressed by nearly 40% to 45% when compared with the controls. However, the combined treatment of Ad-MMP-9 and radiation completely regressed tumor growth in all of the mice by the end of the experiment (after 8 weeks; Fig. 4A ). Furthermore, to show the efficacy of the combined treatment, we also did another set of experiment where the animals were sacrificed after 15 days of the combined treatment.


Figure 4
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  		Fig. 4. MMP-9 and HIF1{alpha} mediate the inhibition of tumor growth after treatment with Ad-MMP-9 plus radiation. A, representative tumors from animals treated with Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9 in combination with irradiation. Growth delay curve of tumors from animals treated with Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9 in combination with irradiation. Tumor size was measured using calipers as described in Materials and Methods. Finally, the tumor volume was calculated using the formula V = {pi} /6 (a x b x c). The data represent the mean ± SD (n = 5) for each treatment at the said time point. B, MMP-9 activity was analyzed using gelatin zymography by loading equal amounts of protein from the tissue lysates from the tumors treated with Ad-MMP-9, irradiation, and controls. Additionally, the second panel in the zymogram shows MMP-9 activity in the tumor harvested after 15 d of the combination treatment compared with the control. MMP-9 activity is represented densitometrically. C, H&E staining and immunohistochemical analysis for MMP-9 and HIF1{alpha}was done on paraffin-embedded breast tumor sections of mice treated with Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9 plus radiation using specific antibodies for these molecules. Paraffin-embedded breast tumor sections of mice treated with Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9 plus radiation were immunostained for proteolytically cleaved caspase-3 active subunits, which exist only when cells undergo apoptosis. TUNEL assay was done with paraformaldehyde-fixed, paraffin-embedded breast tumor sections as per the manufacturer's protocol.

 
Additionally, to show the efficiency of the treatments, we analyzed the activity of MMP-9 in the tumor tissues using zymography. The densitometric analysis showed that the irradiated tumors had a 2-fold to 3-fold increase in MMP-9 activity compared with the controls. In contrast, Ad-MMP-9 treatment reduced activity by 2-fold to 2.5-fold than the control. MMP-9 activity was further reduced by 4-fold to 5-fold even after only 15 days of treatment with Ad-MMP-9 plus radiation (Fig. 4B). Furthermore, we analyzed the tumor sections from control, Ad-CMV, Ad-MMP-9, irradiation, and Ad-MMP-9, plus radiation treatment groups for MMP-9 and HIF1{alpha} using immunohistochemistry. We observed more apoptotic cells and necrotic areas in the sections from treated tumors compared with the control tumors. The sections from control and Ad-CMV–treated tumors revealed a highly aggressive tumor nature and many mitotic dividing cells (Fig. 4C). We observed significant expression of MMP-9 and HIF1{alpha} in control, Ad-CMV, and radiation-treated tumor sections. However, expression levels were drastically reduced in breast tumor sections of mice treated with Ad-MMP-9 and Ad-MMP-9 plus radiation. After the in vitro studies for apoptosis, we analyzed the effect of these treatments in vivo. Paraffin-embedded breast tumor sections of mice treated with Ad-CMV, Ad-MMP-9, radiation, and Ad-MMP-9 plus radiation were immunostained for proteolytically cleaved active subunits of caspase-3, which exist only when cells undergo apoptosis. The combination-treated tumors showed more active subunits of caspase-3 compared with the controls and irradiation alone treated tumors, thereby suggesting more apoptosis in the combined treated tumors even at 15 days after treatment (Fig. 4C). In addition, TUNEL assay indicated DNA fragmentation, which is indicative of apoptotic cell population, in Ad-MMP-9–treated and irradiated tumors (Fig. 4C). In tumors treated with Ad-MMP-9 and radiation, we observed a significantly higher presence of DNA fragmentation compared with the controls. This high level of apoptosis accounts for the 50% to 60% regression of tumors after treatment with Ad-MMP-9 plus radiation.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The goal of this study was to investigate the effect of combining MMP-9 inhibition along with radiation on orthotopic breast tumors. We found that Ad-MMP-9 treatment before radiation augmented the effects of radiation and successfully regressed tumors. In recent years, there has been a consensus that hypoxia can influence a broad spectrum of physiologic and pathologic cellular mechanisms (29). In particular, the combination of hypoxia gene therapy with ionizing radiation represents an exciting and promising approach to overcome and exploit resistant hypoxic tumor cells. Here, we also show that Ad-MMP-9 infection in irradiated cells decreased HIF 1, which is associated with reduction in sensitivity to radiation and causes disease failure after radiation therapy (30). We further show that Ad-MMP-9 infection augmented apoptosis in irradiated cells in vitro and in vivo. In our in vitro studies, we observed increases in HIF1{alpha}, both at the mRNA expression and protein levels, in control and irradiated cells. However, in cells treated with Ad-MMP-9 alone and Ad-MMP-9 plus radiation, levels of HIF1{alpha} were drastically reduced. Furthermore, the immunohistochemical analysis of tumor tissue sections supported the in vitro results. HIF1 is a key transcription factor that regulates the expression of a variety of genes, which control glycolysis, erythropoiesis, apoptosis, and angiogenesis (3). A direct correlation between tumor grade and HIF-1 expression in breast tumors has been shown (31). The role of HIF-1 in solid tumor growth is still not entirely clear, but previous work suggests that this transcription factor is necessary for the growth and angiogenesis of these tumors.

Radiation-induced MMP-9 leads to enhanced tumor growth and metastasis (32). Recent studies have implicated MMPs in multiple roles, including tumor growth (33), regulation of apoptosis (34), and angiogenesis (35). Thus, the observed radiation-induced augmentation in MMP-9 activity is not only integral to tumor invasion, but may also aid survival in a relatively hostile setting. Our results show down-regulation of MMP-9 in irradiated breast cancer cells decreased MMP activity at both the mRNA and protein levels.

Ionizing radiation acts through the induction of double-strand breaks to DNA to induce elimination of cancerous cells via apoptosis (36). The efficiency of radiotherapy for cancer treatment is limited by toxic side effects, which impede dose escalation. Moreover, cancer cells often develop radioresistance mechanisms that are related to the DNA repair response. The aim of combining gene therapy and radiation is to strengthen the efficiency of radiation by inhibition of DNA repair, overcoming the clonogenic survival in irradiated cells and in turn apoptotic resistance. Transcription factors like NF-{kappa}B and AP-1 are activated by DNA-damaging agents and could be involved in cell cycle arrest and prevention of apoptosis to allow DNA repair (9, 37). Not only does NF-{kappa}B promote survival of cancer cells, but it also contributes to abnormal proliferation and metastasis (25, 38–40). Our Western immunoblot analysis showed a decrease in the translocation of NF-{kappa}B subunits (p50 and p65) and AP-1 subunits (c-fos and Jun D) in the cells that received the combined treatment of Ad-MMP-9 and radiation. Furthermore, we did the electrophoretic mobility shift assay to analyze the protein-DNA interaction for NF-{kappa}B and AP-1. The results indicated that in the case of cells treated with Ad-MMP-9 alone and Ad-MMP-9 combined with radiation, the protein-DNA interaction was reduced compared with the control and Ad-CMV–treated cells. This observation was supported by the supershift assay using specific antibodies against NF-{kappa}B and AP-1, respectively (41).

Apoptosis is induced by different stimuli, such as death ligands and chemotherapeutic drugs that lead to the activation of caspases (42). Recently, researchers have shown that inhibition of NF-{kappa}B activity restores sensitivity to Fas-mediated apoptosis (43, 44). Treatment of breast cancer cells with Ad-MMP-9, radiation, or both induced caspase-dependent apoptosis, which is associated with the activation of several individual caspases. Our results show that the caspase-10 pathway may be responsible for induction of apoptosis where Fas and Fas-L are involved at the cell membrane. The Fas–Fas-L death pathway is an important mediator of apoptosis. Deregulation of the Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anticancer drugs (45). It has been reported that the resistance of leukemic eosinophils to Fas-mediated apoptosis is due to induced NF-{kappa}B activation (46). The results of the present study corroborate the findings of these studies; we observed that the reduction in NF-{kappa}B activation led to increased expression of Fas-L and directed apoptosis via the Fas–Fas-L mediated pathway. This led to the activation of caspase-10, a death effector domain–containing initiator caspase, which, in turn, cleaves or activates caspase-3 and caspase-7 (effector caspases capable of cleaving PARP-1). Caspase-3 immunofluorescent staining and TUNEL assay revealed the increased apoptosis in the tumors treated with Ad-MMP-9 and radiation compared with the control and irradiation alone–treated tumors. The TUNEL assay using the tissue sections shows synergistic effect when Ad-MMP-9 was given in combination with radiation. Furthermore, the clonogenic survival assay supported the results obtained involving activation of apoptotic cascade.

We suggest a new strategy for improving the radiosensitivity of breast tumors in treating breast cancer through down-regulation of MMP-9 using adenoviral constructs of antisense MMP-9 before radiation (Fig. 5 ). The decreased MMP-9 activity of the tumors inhibited phosphorylation of ERK, which reduced the transcriptional activity of NF-{kappa}B and AP-1. This in turn led to increased apoptosis, thereby regressing the tumor. The precise and rapid propagation of this signaling cascade demands strict and flexible regulatory processes that still remain unexplored. The nature of the regulators involved may have therapeutic implications. Our schematic is based on our in vitro and in vivo model data showing an increase in apoptosis and tumor reduction.


Figure 5
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  		Fig. 5. Schematic representations of the effect of irradiation (A) and Ad-MMP-9 in combination with irradiation (B) on breast tumor growth.

 
In summary, the present study indicates that MMP-9 might be a potential target candidate as an inducer of the HIF-regulated molecular cascade. Down-regulating MMP-9 activity augments the effect of radiotherapy by directly or indirectly reducing HIF transcription machinery. Because HIF1{alpha} is involved in the pathogenesis of several human diseases (e.g., myocardial and cerebral ischemia, pulmonary hypertension), Ad-MMP-9 may have a future therapeutic role as an HIF1{alpha} activity inhibitor.


   Acknowledgments
 
We thank Shellee Abraham for preparing the manuscript, Diana Meister and Sushma Jasti for reviewing the manuscript, and Noorjehan Ali for technical assistance.


   Footnotes
 
Grant support: National Cancer Institute grants CA 75557, CA 92393, CA 95058, CA 116708, National Institute of Neurological Disorders and Stroke grants NS47699 and NS57529, Caterpillar, Inc., and OSF Saint Francis, Inc. (J.S. Rao).

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 8/21/07; revised 12/14/07; accepted 1/22/08.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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