Lirija Alili

Combined cytotoxic and anti-invasive properties of redox-active nanoparticles in tumor–stroma interactions

Tumor-stroma interaction plays an important role in tumor progression. Myofibroblasts, pivotal for tumor progression, populate the microecosystem of reactive stroma. The formation of myofibroblasts is mediated by tumor derived transforming growth factor β1 (TGFβ1) which initiates a reactive oxygen species cell type dependent expression of alpha-smooth muscle actin, a biomarker for myofibroblastic cells. Myofibroblasts express and secrete proinvasive factors significantly increasing the invasive capacity of tumor cells via paracrine mechanisms. Although antioxidants prevent myofibroblast formation, the same antioxidants increase the aggressive behavior of the tumor cells. In this study, the question was addressed of whether redox-active polymer-coated cerium oxide nanoparticles (CNP, nanoceria) affect myofibroblast formation, cell toxicity, and tumor invasion. Herein, nanoceria downregulate both the expression of alpha-smooth muscle actin positive myofibroblastic cells and the invasion of tumor cells. Furthermore, concentrations of nanoceria being non-toxic for normal (stromal) cells show a cytotoxic effect on squamous tumor cells. The treatment with redox-active CNP may form the basis for protection of stromal cells from the dominating influence of tumor cells in tumor-stroma interaction, thus being a promising strategy for chemoprevention of tumor invasion.

Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species

Myofibroblasts, pivotal for tumor progression, populate the microecosystem of reactive stroma. Using an in vitro tumor-stroma model of skin carcinogenesis, we report here that tumor-cell-derived transforming growth factor β1 (TGFβ1) initiates reactive oxygen species-dependent expression of α-smooth muscle actin, a biomarker for myofibroblastic cells belonging to a group of late-responsive genes. Moreover, protein kinase C (PKC) is involved in lipid hydroperoxide-triggered molecular events underlying transdifferentiation of fibroblasts to myofibroblasts (mesenchymal-mesenchymal transition, MMT). In contrast to fibroblasts, myofibroblasts secrete large amounts of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), resulting in a significant increase in the invasive capacity of tumor cells. The thiol N-acetyl-L-cysteine, the micronutrient selenite as well as selenoprotein P and the lipid peroxidation inhibitors α-tocopherol and butylated hydroxytoluene significantly lower both the number of TGFβ1-initiated myofibroblasts and the secretion of HGF, VEGF and IL-6, correlating with a diminished invasive capacity of tumor cells. This novel concept of stromal therapy, namely the protection of stromal cells against the dominating influence of tumor cells in tumor-stroma interaction by antioxidants and micronutrients, may form the basis for prevention of MMT in strategies for chemoprevention of tumor invasion.

Selenoprotein P expression is controlled through interaction of the coactivator PGC‐1α with FoxO1a and hepatocyte nuclear factor 4α transcription factors

Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine‐tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor‐γ coactivator 1α (PGC‐1α) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)‐4α, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO‐responsive element daf16‐binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC‐1α. Moreover, the PGC‐1α‐inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC‐1α and SeP caused by dexamethasone treatment. In a brain‐derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC‐1α together with FoxO1a, and overexpression of HNF‐4α potentiated this effect. Conclusion: High‐level expression of SeP in liver is ensured by concerted action of the coactivator PGC‐1α and the transcription factors FoxO1a and HNF‐4α. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose‐6‐phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC‐1α as a key regulator of selenium homeostasis. (HEPATOLOGY 2008;48:1998‐2006.)

Selenoprotein P protects endothelial cells from oxidative damage by stimulation of glutathione peroxidase expression and activity

A major fraction of the essential trace element selenium circulating in human blood plasma is present as selenoprotein P (SeP). As SeP associates with endothelial membranes, the participation of SeP in selenium-mediated protection against oxidative damage was investigated, using the human endothelial cell line Ea.hy926 as a model system. Hepatocyte-derived SeP prevented tert-butylhydroperoxide (t-BHP)-induced oxidative cell death of Ea.hy926 cells in a similar manner as did sodium selenite, counteracting a t-BHP-induced loss of cellular membrane integrity. Protection was detected after at least 10 h of SeP supplementation and it peaked at 24 h. SeP time-dependently stimulated the expression of cytosolic glutathione peroxidase (cGPx) and increased the enzymatic activities of glutathione peroxidase (GPx) and thioredoxin reductase (TR). The cGPx inhibitor mercaptosuccinate as well as the γ-glutamylcysteine synthetase inhibitor buthionine sulfoximine counteracted the SeP-mediated protection, while the TR inhibitors cisplatin and auranofin had no effect. The presented data suggest that selenium supplementation by SeP prevents oxidative damage of human endothelial cells by restoring expression and enzymatic activity of GPx.

Downregulation of tumor growth and invasion by redox-active nanoparticles.

AIMS Melanoma is the most aggressive type of malignant skin cancer derived from uncontrolled proliferation of melanocytes. Melanoma cells possess a high potential to metastasize, and the prognosis for advanced melanoma is rather poor due to its strong resistance to conventional chemotherapeutics. Nanomaterials are at the cutting edge of the rapidly developing area of nanomedicine. The potential of nanoparticles for use as carrier in cancer drug delivery is infinite with novel applications constantly being tested. The noncarrier use of cerium oxide nanoparticles (CNPs) is a novel and promising approach, as those particles per se show an anticancer activity via their oxygen vacancy-mediated chemical reactivity. RESULTS In this study, the question was addressed of whether the use of CNPs might be a valuable tool to counteract the invasive capacity and metastasis of melanoma cells in the future. Therefore, the effect of those nanoparticles on human melanoma cells was investigated in vitro and in vivo. Concentrations of polymer-coated CNPs being nontoxic for stromal cells showed a cytotoxic, proapoptotic, and anti-invasive capacity on melanoma cells. In vivo xenograft studies with immunodeficient nude mice showed a decrease of tumor weight and volume after treatment with CNPs. INNOVATION In summary, the redox-active CNPs have selective pro-oxidative and antioxidative properties, and this study is the first to show that CNPs prevent tumor growth in vivo. CONCLUSION The application of redox-active CNPs may form the basis of new paradigms in the treatment and prevention of cancers.

Combination of Conventional Chemotherapeutics with Redox-Active Cerium Oxide Nanoparticles—A Novel Aspect in Cancer Therapy

Nanotechnology is becoming an important field of biomedical and clinical research and the application of nanoparticles in disease may offer promising advances in treatment of many diseases, especially cancer. Malignant melanoma is one of the most aggressive forms of cancer and its incidence is rapidly increasing. Redox-active cerium oxide nanoparticles (CNP) are known to exhibit significant antitumor activity in cells derived from human skin tumors in vitro and in vivo, whereas CNP is nontoxic and beyond that even protective (antioxidative) in normal, healthy cells of the skin. As the application of conventional chemotherapeutics is associated with harmful side effects on healthy cells and tissues, the clinical use is restricted. In this study, we addressed the question of whether CNP supplement a classical chemotherapy, thereby enhancing its efficiency without additional damage to normal cells. The anthracycline doxorubicin, one of the most effective cancer drugs, was chosen as reference for a classical chemotherapeutic agent in this study. Herein, we show that CNP enhance the antitumor activity of doxorubicin in human melanoma cells. Synergistic effects on cytotoxicity, reactive oxygen species generation, and oxidative damage in tumor cells were observed after co-incubation. In contrast to doxorubicin, CNP do not cause DNA damage and even protect human dermal fibroblasts from doxorubicin-induced cytotoxicity. A combination of classical chemotherapeutics with nongenotoxic but antitumor active CNP may provide a new strategy against cancer by improving therapeutic outcome and benefit for patients. Mol Cancer Ther; 13(7); 1740–9. ©2014 AACR.

Post-translational processing of selenoprotein P: implications of glycosylation for its utilisation by target cells

Abstract Selenoprotein P (SeP) is a highly glycosylated plasma protein containing up to 10 selenocysteine residues. It is secreted by hepatocytes and also by the human hepatoma cell line HepG2. Pharmacological inhibitors interfering with N-glycosylation, intracellular trafficking and calcium homeostasis were applied to examine post-translational processing and secretion of SeP by HepG2 cells. In parallel, the prototypic secretory glycoprotein α1-antitrypsin was used as technical control. Secretion of SeP was stimulated by increasing the extracellular calcium concentration and by inhibiting the release of sequestered calcium through dantrolene or U-73122. In contrast, brefeldin A and thapsigargin suppressed SeP secretion. Tunicamycin and monensin induced the synthesis of truncated non-glycosylated and partially glycosylated forms of SeP, which were secreted in spite of their impaired glycosylation. Both non-glycosylated and partially glycosylated SeP is utilised as selenium donor by target cells: impaired glycosylation affected neither the ability of SeP to induce the synthesis of the selenoenzyme cytosolic glutathione peroxidase nor its capacity to protect endothelial cells from oxidative stress.

Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species.

Tumor-stroma interaction is a prerequisite for tumor progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumor-associated fibroblasts to myofibroblasts by growth factors, for example transforming growth factor beta (TGFβ). In this study, the question was addressed of whether fibroblast associated NAD(P)H oxidase, known to be activated by TGFβ1, is involved in the fibroblast-to-myofibroblast switch. The up regulation of alpha smooth muscle actin (αSMA), a biomarker for myofibroblasts, is mediated by a TGFβ1-dependent increase in the intracellular level of reactive oxygen species (ROS). The source of these ROS was identified to be NAD(P)H oxidase, showing two activity peaks over time with an early and late activity peak after treatment with the growth factor. The late (second) activity peak of NAD(P)H oxidase was identified to be responsible for the downstream signaling resulting in reactive oxygen species triggered activation of the stress kinase p38 and expression of αSMA. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-myofibroblast switch and may be important for chemoprevention.

A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3‐dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co‐culturing skin fibroblasts and keratinocytes on a collagen–glycosaminoglycan–chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin‐C (MMC). Recently published data showed that MMC treatment resulted in a drug‐induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.

Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage

Recently, it has been published that cerium (Ce) oxide nanoparticles (CNP; nanoceria) are able to downregulate tumor invasion in cancer cell lines. Redox-active CNP exhibit both selective pro-oxidative and antioxidative properties, the first being responsible for impairment of tumor growth and invasion. A non-toxic and even protective effect of CNP in human dermal fibroblasts (HDF) has already been observed. However, the effect on important parameters such as cell death, proliferation and redox state of the cells needs further clarification. Here, we present that nanoceria prevent HDF from reactive oxygen species (ROS)-induced cell death and stimulate proliferation due to the antioxidative property of these particles.