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 prior to 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 physiological and pathological 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 HIF1α, 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 HIF1α were drastically reduced. Further, 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 downregulation 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 in order 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-κ B and AP1 are activated by DNA damaging agents and could be involved in cell cycle arrest and prevention of apoptosis in order to allow DNA repair (9
). Not only does NF-κB promote survival of cancer cells, but it also contributes to abnormal proliferation and metastasis (25
). Our Western immunoblot analysis showed a decrease in the translocation of NF-κB subunits (p50 and p65) and AP1 subunits (c-fos and Jun D) in the cells that received the combined treatment of Ad-MMP-9 and radiation. Furthermore, we performed the electrophoretic mobility shift assay (EMSA) to analyze the protein-DNA interaction for NF-κB and AP1. 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 as compared to the control and Ad-CMV-treated cells. This observation was supported by the supershift assay using specific antibodies against NF-κB and AP1 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 of inhibition of NF-κB activity restores sensitivity to Fas-mediated apoptosis (43
). 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 ligand (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 anti-cancer drugs (45
). It has been reported that the resistance of leukemic eosinophils to Fas-mediated apoptosis is due to induced NF-κB activation (46
). The results of the present study corroborate the findings of these studies; we observed the reduction in NF-κB 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 (DED)-containing initiator caspase, which, in turn, cleaves or activates caspases 3 and 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 as compared to 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. Further, 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 downregulation of MMP-9 using adenoviral constructs of antisense MMP-9 before radiation (). The tumors’ decreased MMP-9 activity inhibited phosphorylation of ERK, which reduced the transcriptional activity of NF-κB and AP1. 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.
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. Downregulating MMP-9 activity augments the effect of radiotherapy by directly or indirectly reducing HIF transcription machinery. Since HIF1α 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 HIF1α activity inhibitor.