The cellular response to combined treatment with the microtubule stabilizing agent patupilone and ionizing radiation: regulation of matrix metalloproteinase activity

The combined application of the novel, clinically relevant microtubule stabilizing agent (MTSA) patupilone and ionizing radiation (IR) is a very promising treatment modality in cancer therapy as previously investigated at a preclinical level in our laboratory. Patupilone (epothilone B) is a compound of bacterial origin, which binds to the same site on beta-tubulin as taxanes, but has number of advantages over the later, e.g. potent cytotoxicity in multi-drug resistant tumors and – due to its relatively simple chemical structure – the availability of derivatives with improved pharmacological properties. Patupilone has a strong supra-additive inhibitory effect on the growth of tumor xenografts in mice when applied together with IR. Interestingly, the additive cytotoxic effect was much less pronounced in vitro indicating that an additional effect on the level of tumor microenvironment must exist. While the initial hypothesis was that patupilone targets neovascular endothelial cells directly, further investigations revealed that the additive effect in vivo could only be observed in tumors xenografts derived from patupilone sensitive cancer cells. Thus the effect on the tumor microenvironment might be induced in an indirect way, mediated by tumor cells, for example via the secretion of biologically active molecules. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with a broad variety of substrates including components of the extracellular matrix and growth factors (e.g. the transforming growth factor β (TGF- β). MMPs play an important role in the regulation of cell survival, apoptosis, angiogenesis, matrix remodelling and metastasis. The function of MMPs is strictly regulated by a complex activation processes (MMPs are expressed as zymogens) and natural inhibitors of their enzymatic activity. The tissue inhibitors of MMPs (TIMPs) represent a group of the most important players in the MMP regulation cascade currently known. TIMPs are a family of small proteins (size of 20-30 kDa), that contains 4 members: TIMP-1-4. All 4 TIMPs are potent inhibitors of the MMP enzymatic activity; TIMP-2 was also shown to be a co-activator of MMP-2 zymogen. Additionally, TIMPs have MMP independent biological functions, for example mitogenic, pro- and anti-apoptotic. MMPs and TIMPs are secreted both by tumor-associated stromal cells and by cancer cells and are important components of the tumor microenvironment. Overexpression of MMPs and TIMPs was reported for almost all tumor types and associated with a bad survival prognosis. In the present study we focused our investigations on the effect of the microtubule stabilizing agent patupilone on the MMP function in tumor cells after treatment with ionizing radiation. The results presented herein further confirmed the radiosensitizing capacity of patupilone to enhance IR-induced cytotoxicity in a wide range of human cancer cells (HCT116 colon carcinoma cells, HT1080 fibrosarcoma cells, U251 glioma cell and medulloblastoma ii cell lines DAOY, D425, D341). It is also interesting to note that patupilone sensitized to ionizing radiation already in a very low dose range. At these doses patupilone was not cytotoxic itself and did not alter microtubule cytoskeletal structures (0.2 nM patupilone in HT1080 cells). The combined treatment modality with patupilone and IR is more potent in vivo than in cellular model systems. Most probably, this is due to the additional treatment effects on the level of the tumor microenvironment, which may be mediated by tumor cells. Herein, we investigated an effect of the indicated treatment regimens on one of the important components of the tumor microenvironment, namely MMPs. We identified that the activity of secreted MMP was upregulated after treatment with IR and that pre-treatment with patupilone could diminish this increase. MMP activity is required for tumor cell invasion as well as for other cellular processes. In this study tumor cell invasiveness was indeed increased after IR and, in line with the events on the level of MMP activity, patupilone could counteract this effect of IR. In an attempt to uncover mechanisms that contribute to the counteracting effect of patupilone on IR-induced MMP activity, we dissected the MMP regulation cascade on vaious levels. Patupilone did not influence IR-induced events on the level of cell cycle distribution and gene transcription. Namely, patupilone did not interfere with the accumulation of cells in G2/M phase after IR and did not alter IR-induced transcription of MMPs and TIMPs. Both MMP and TIMP protein levels were elevated in the cell lysate and conditioned cell culture media (CM) after treatment with 10 Gy of IR. Patupilone did not influence intracellular and extracellular protein levels of MMPs. However, the amount of extracellular TIMP-1 and TIMP-2 proteins after combined treatment with patupilone and IR was significantly reduced when compared to treatment with IR alone. Furthermore, the counteracting effect of patupilone on IR-induced MMP activity was not observed in TIMP-1 and TIMP-2 depleted cells. After TIMP depletion the IR-increased MMP activity did not exceed the MMP activity level after combined treatment in non-depleted cells, indicating that the proMMP activation processes after IR requires TIMPs. These findings strongly suggest that patupilone counteracted IR-induced MMP activity due to the interference with the MMP activation processes by the depletion the secreted TIMP-1 and TIMP-2 proteins. To our knowledge, we demonstrate for the first time that patupilone can counteract IRinduced MMP-related processes. It is particularly interesting is that the counteracting effect of patupilone was observed in human glioma cells. Glioblastoma (or grade IV glioma) is one of the most treatment resistant tumors in adults. The primary reasons associated with the poor prognosis are: extensive infiltration of surrounding brain tissue by tumor cells and the blood-brain barrier as an obstacle for the adequate delivery of chemotherapeutics. The ability of patupilone to cross the blood-brain barrier makes patupilone a very potent and promising chemotherapeutical agent for the treatment of brain malignancies. The capacity of patupilone to inhibit IR-induced glioma cell invasion, as shown in the present study, increases the value iii of the compound as a candidate for a combination with radiation therapy for glioblastoma treatment. MMPs play an important role in cancer progression by remodelling the extracellular matrix and regulating multiple biologically active molecules. Overexpression of MMPs and TIMPs in multiple tumor types positively correlates with the aggressiveness of the disease. There is a strong indication that radiation therapy can promote MMP expression and tumor invasiveness. Thus, therapeutic agents that cleverly target members of MMP and TIMP families in selective and disease/function-specific way can represent an interesting class of agents for a combination with ionizing radiation. This study demonstrates that the MTSA patupilone is a compound of great interest. It is a potent inhibitor of tumor cell proliferation under hypoxic and normoxic conditions, a very promising anti-metastatic and anti-angiogenic agent, a potent radiosensitizer in vivo and in vitro (as described elsewhere). In addition, the present study demonstrated that patupilone inhibits IR-induced activity of secreted MMPs and IR-induced cell invasion. Since IR-induced MMP activity may be relevant to cancer progression, the combined treatment modality of IR and patupilone might, indeed, be of great clinical benefit for cancer therapy.