Vasilios Panagopoulos

Hypoxia-activated pro-drug TH-302 exhibits potent tumor suppressive activity and cooperates with chemotherapy against osteosarcoma

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS.

Pharmacologic inhibition of bone resorption prevents cancer-induced osteolysis but enhances soft tissue metastasis in a mouse model of osteolytic breast cancer.

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

Uncovering a new role for peroxidase enzymes as drivers of angiogenesis.

Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzymes catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.

Anticancer efficacy of the hypoxia‐activated prodrug evofosfamide (TH‐302) in osteolytic breast cancer murine models

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH‐302) is a prodrug created by the conjugation of 2‐nitroimidazole to bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross‐linking effector, Br‐IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA‐MB‐231‐TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer‐induced bone destruction was measured using micro‐CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Peroxidase Enzymes Regulate Collagen Extracellular Matrix Biosynthesis

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

Anticancer efficacy of the hypoxia‐activated prodrug evofosfamide is enhanced in combination with proapoptotic receptor agonists against osteosarcoma

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia also leads to treatment opportunities as demonstrated by the development of compounds that target regions of hypoxia within tumors. Evofosfamide is a hypoxia‐activated prodrug that is created by linking the hypoxia‐seeking 2‐nitroimidazole moiety to the cytotoxic bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions of tumors, the DNA cross‐linking toxin, Br‐IPM, is released leading to cell death. This study assessed the anticancer efficacy of evofosfamide in combination with the Proapoptotic Receptor Agonists (PARAs) dulanermin and drozitumab against human osteosarcoma in vitro and in an intratibial murine model of osteosarcoma. Under hypoxic conditions in vitro, evofosfamide cooperated with dulanermin and drozitumab, resulting in the potentiation of cytotoxicity to osteosarcoma cells. In contrast, under the same conditions, primary human osteoblasts were resistant to treatment. Animals transplanted with osteosarcoma cells directly into their tibiae developed mixed osteosclerotic/osteolytic bone lesions and consequently developed lung metastases 3 weeks post cancer cell transplantation. Tumor burden in the bone was reduced by evofosfamide treatment alone and in combination with drozitumab and prevented osteosarcoma‐induced bone destruction while also reducing the growth of pulmonary metastases. These results suggest that evofosfamide may be an attractive therapeutic agent, with strong anticancer activity alone or in combination with either drozitumab or dulanermin against osteosarcoma.

Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

In vivo toxicological assessment of electrochemically engineered anodic alumina nanotubes: a study of biodistribution, subcutaneous implantation and intravenous injection

Electrochemically engineered anodic alumina nanotubes (AANTs) have recently shown good in vitro biocompatibility. However, in vivo toxicological and pathological studies are required to clarify the bio-safety of this novel nanomaterial. Herein, we present a pioneering pilot toxicity study on AANTs in immune-competent murine models (Balb/c mice, 8 weeks). AANTs were administered by intravenous (IV) injection and subcutaneous (SC) implantation routes considering the future toxicological implications associated with this nanomaterial for potential biomedical applications. AANTs, 736 nm long and 90 nm in outer diameter, were chosen as a nanomaterial model. We demonstrate that IV injected AANTs do not have any effect on the mortality or body weight of these animal models within 28 days at three different doses (20, 50, 100 mg kg-1). The biodistribution of AANTs characterized by fluorescence imaging and inductively coupled plasma revealed the accumulation of AANTs in the liver and spleen after IV injection. When AANTs were injected intravenously, the highest dose of 100 mg kg-1 caused moderate hepatotoxicity, identified by histopathological analysis. The implantation of AANTs subcutaneously and directly under the skin leads to an inflammatory response, which is a typical foreign body reaction. Taken together, this work provides new insights into the toxicity patterns of new nanomaterials such as AANTs and establishes a rationale for the design of functional AANTs for future biomedical applications.

Zoledronate Enhances the Cytotoxicity of Gamma Delta T Cell Immunotherapy in an Orthotopic Mouse Model of Osteolytic Osteosarcoma

Objective: Osteosarcoma is the most common primary tumor of the bone, predominantly affecting children and adolescents. While localized osteosarcoma can be readily treated with the use of pre-operative chemotherapy in combination with surgery, patients who develop metastatic disease and tumor-induced osteolysis continue to have a poor prognosis. Many cancer cells express tumor-specific antigens, rendering them vulnerable to immune effector T cell killing. There is increasing evidence that highly cytotoxic gamma delta (Vγ9Vδ2) T cells together with the bone anti-resorptive drug zoledronate may hold significant clinical benefit in the treatment of a variety of tumor types. Methods: Ex vivo expanded Vγ9Vδ2 T cells were used to assess effector-mediated killing of osteosarcoma cells (BTK-143 and K-HOS) in response to zoledronate pre-treatment. An orthotopic mouse model of osteolytic osteosarcoma was used to verify Vγ9Vδ2 T cell cytotoxicity in combination with zoledronate on tumor growth, osteolysis and metastasis. Results: Pre-treatment of osteosarcoma cells with zoledronate enhanced Vγ9Vδ2 T cell rapid killing compared to untreated cells in vitro via blockade of the mevalonate pathway. When adoptively transferred into osteosarcoma bearing NOD/SCID mice in vivo, Vγ9Vδ2 T cells in combination with zoledronate potentiated the anti-cancer efficacy of Vγ9Vδ2T cells and inhibited tumor induced osteolysis. Importantly, Vγ9Vδ2 T cells alone reduced both the incidence and burden of lung metastases. Conclusion: This study demonstrated the dual-action of zoledronate to enhance the immunogenicity of osteosarcoma cells to Vγ9Vδ2 T cell cytotoxicity and provide protection against tumor-induced osteolysis.