Peter Brenneisen

Ultraviolet-B Irradiation and Matrix Metalloproteinases

Abstract: Effects of sunlight have fascinated researchers for decades because nearly every living thing on earth is likely to be exposed to sunlight and the ultraviolet (UV) fraction of it. In addition to detrimental long‐term effects such as immunosuppression and skin cancer, premature aging of the skin (photoaging) is a well‐documented consequence of exposure to UVA and UVB. Photoaged skin is biochemically characterized by an overgrowth of abnormal elastic fibers in the dermis and by a dramatic decrease of distinct collagen types. Ultraviolet irradiation induces delayed UV‐responsive genes, among them matrix metalloproteinases, which degrade macromolecules of the extracellular matrix, a hallmark in carcinogenesis and aging. We are interested in UVB‐triggered initial events and in subsequent signaling resulting in enhanced expression of two major members of the matrix metalloproteinase family, the interstitial collagenase (MMP‐1) and stromelysin‐1 (MMP‐3), in human dermal fibroblasts. Especially, these skin cells play a central role in connective tissue breakdown in photoaging and as stromal cells in tumor invasion and metastasis by means of their capability to produce matrix metalloproteinases. In this review, we will focus on UVB‐triggered induction of matrix metalloproteinases, the so far identified components of the UVB‐modulated signal transduction pathway(s), and the UVB irradiation‐associated generation of reactive oxygen species (ROS). Finally, a potentially novel aspect in UVB irradiation‐mediated expression of interstitial collagenase and stromelysin‐1—namely, the involvement of reactive nitrogen species (RNS)—is discussed.

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.

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.

AIMSnMelanoma 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.nnnRESULTSnIn 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.nnnINNOVATIONnIn 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.nnnCONCLUSIONnThe 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.

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes.

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.

Vascular endothelial growth factor causally contributes to the angiogenic response upon ultraviolet B irradiation in vivo

Summary Backgroundu2003Ultraviolet (UV)‐B irradiation has been shown to be an inducer of vascular endothelial growth factor (VEGF) in primary keratinocytes and epidermal cell lines in vitro.

Activation of protein kinase CK2 is an early step in the ultraviolet B-mediated increase in interstitial collagenase (matrix metalloproteinase-1; MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts.

Enhanced expression of matrix metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in skin fibroblasts and subsequent damage of dermal connective tissue in the context of sun-induced premature aging and skin tumour progression is causally linked to UVB irradiation. Here, we were interested in identifying components of the complex signal-transduction pathway underlying UVB-mediated up-regulation of these delayed UV-responsive genes and focused on components maximally activated early after irradiation. A 2.3-fold increase in protein kinase CK2 activity was measured at 20-40 min after low-dose UVB irradiation (at 10 mJ/cm2) of dermal fibroblasts. This UVB-mediated increase in CK2 activity was abrogated by pharmacological approaches using non-toxic concentrations of the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB). Preincubation of fibroblasts with DRB prior to UVB irradiation lowered MMP-1 by 49-69% and MMP-3 protein levels by 55-63% compared with UVB-irradiated controls. By contrast, the CK2 inhibitor did not affect the UVB-triggered transcription of MMPs. Furthermore, UVB irradiation of fibroblasts overexpressing a kinase-inactive mutant of CK2 (CK2alpha-K68A-HA) resulted in lowering of the protein levels of MMP-1 by 25% and MMP-3 by 22% compared with irradiated fibroblasts transfected with the vector control. This reduction in MMP protein levels correlated with the transfection efficiency. Taken together, we describe a novel aspect of protein kinase CK2, namely its inducible activity by UVB irradiation, and provide evidence that CK2 is an early mediator of the UVB-dependent up-regulation of MMP-1 and MMP-3 translation, whereas their major tissue inhibitor of matrix metalloproteinase-1 is not affected by CK2.

Protective and determining factors for the overall lipid peroxidation in ultraviolet A1-irradiated fibroblasts: in vitro and in vivo investigations.

Summaryu2003 Backgroundu2003Lipid peroxidation (LPO) is one major effector mechanism by which ultraviolet (UV) A contributes to photoageing and the promotion of skin cancer. It is a fingerprint of photo‐oxidative stress within the skin, and is initiated by several pathways, with different reactive oxygen species (ROS) and iron ions being involved.

Long‐term growth arrest of PUVA‐treated fibroblasts in G2/M in the absence of p16INK4a, p21CIP1 or p53

Abstract:u2002 Premature aging of the skin is a prominent side‐effect of psoralen photoactivation, a therapy used for different skin disorders. Recently, we demonstrated that treatment of fibroblasts with 8‐methoxypsoralen and ultraviolet A irradiation resulted in growth arrest with morphological and functional changes reminiscent of replicative senescence. To further elucidate the underlying molecular mechanisms, we analysed the cell‐cycle phases of the growth‐arrested fibroblasts. After PUVA treatment, fibroblasts arrested in G2/M, in contrast to spontaneously senesced fibroblasts arresting in a cell‐cycle phase with many features similar to G1. To address the role of the cell‐cycle controlling genes p16INK4a, p21CIP1 and p53, we analysed the expression of these genes. p16INK4a, p21CIP1 and p53 protein levels increased substantially with different time kinetics in growth‐arrested fibroblasts. Because p16INK4a, p21CIP1 and p53 are involved in replicative senescence, we applied the PUVA regimen to fibroblasts deficient in either of these genes. p16INK4a, p21CIP1 and p53 null mutant fibroblast strains underwent growth arrest with a senescent phenotype similar to wild‐type human fibroblasts. Based on these results, we propose that redundant or alternate pathways are involved in the response of dermal fibroblasts to PUVA treatment resulting in a phenocopy of replicative senescence in vitro.