Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes.

The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100 mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade III and IV malignant gliomas.

PURPOSE: Glioblastoma multiforme (GBM) remains one of the most devastating diseases known to man and affects more than 17,000 patients in the United States alone every year. This malignancy infiltrates the brain early in its course and makes complete neurosurgical resection almost impossible. Recent years have brought significant advances in tumor biology, including the discovery that many cancers, including gliomas, appear to be supported by cells with stem-like properties. In the current study we have investigated the effects of combining metformin with the standard treatment-of-care, as this drug, already used in the treatment of diabetes mellitus, has shown surprising results in the treatment of breast cancer, being also associated with lower mortality in several other malignancies. METHODS: The subjects of the current study were 8 patients with newly diagnosed high-grade gliomas, operated at the Department of Neurosurgery - Clinical University Emergency Hospital, Cluj Napoca. Tumor tissue cultures were established and characterized using immunofluorescence microscopy and PCR analysis and the sensitivity to metformin, epidermal growth factor (EGF) and temozolomide (TMZ) was tested. Microvascular density (MVD) assay was performed on the tumor samples. RESULTS: Seven of the 8 cases had a positive correlation between the number of endothelial cells, the phenotype of isolated tumor cells and the response to adjuvant chemoradiotherapy. The isolated tumor cells had a stem-like behavior, being resistant to conventional drugs. In most cases there was no statistical significant difference between TMZ alone and TMZ plus EGF arms, but there was a important difference between TMZ alone and TMZ plus metformin arms in 6 of the cases. CONCLUSION: New drugs and targeted molecular therapies are important for future therapeutics, but sometimes we must not exclude drugs already used in the clinic that might have remarkable results. Such is the case of metformin, a drug used for decades in the treatment of type 2 diabetes mellitus that has proven to enhance the effect of TMZ in the treatment of breast cancer and, starting with this paper, of brain cancer.

Mesenchymal stem cell irradiation in culture engages differential effect of hyper-fractionated radiotherapy for head and neck cancers.

PURPOSE: The purpose of this study was to challenge current knowledge on the potential therapeutic advantages of stem cells in radiotherapy by developing an in vitro model of the healthy tissue surrounding or replacing the widely resected tumor. After radical surgery, the start of radiotherapy is often delayed due to wound healing process, with potential loss of the opportunity for treating microscopic disease instead of macroscopic early recurrence. Hyperfractionated radiotherapy, contrary to the standard one, can extend the limits of radical surgery and shorten the gap before the onset of postoperative radiotherapy, with potential improvement in local control. METHODS: By using both mesenchymal stem cells and pre-differentiated osteoblasts, cultured in proper pro-osteogenic media after cell irradiation, we investigated both the differences in the response to DNA damage between lineages undergoing differentiation in culture and the intensity of the mineralization process. RESULTS: Ionizing radiation stimulated stem cell proliferation and differentiation at 0.5 Gy and 1 Gy, thus confirming in vitro the clinical results of hyperfractionated irradiation randomized trials in head and neck cancers -HNCs-. CONCLUSION: To our knowledge, this study is the first to investigate the biophysics of low dose gamma irradiation on stem cell culture, focusing on the potential applications in radiation oncology. For advanced oral cavity and oropharyngeal cancers, as radical surgery often implies major bone resection, the use of mesenchymal stem cells as bone reconstruction vectors might shorten the onset of adjuvant hyperfractionated radiotherapy which enhances the mineralization process. As postoperative radiotherapy has recently being revisited for osteosarcoma, this scenario could impact also on bone reconstruction process in this pathology.

Comparative study of two evaluation methods for the genotoxic effects of environmental heavy metals on normal cells.

Genetic modifications caused by chronic exposure to low levels of toxic metals may activate stress-signaling pathways, thus increasing cancer incidence among affected individuals. The aim of this study was to evaluate the relationship between exposure to heavy metals and the incidence of chromosomal aberrations and DNA lesions in a chronically exposed population by using specific biomarkers. The study included 156 subjects divided into two major groups: exposed individuals (in a heavy metal contaminated region, Maramures, Romania) and non-exposed population, as control group (Cluj, Romania). We compared the results of two cytogenetic methods for the detection and quantification of DNA lesions and chromosomal aberrations in normal human cells: Single Cell Gel Electrophoresis or Comet assay and Cytokinesis Block Micronucleus assay. The methods were performed on lymphocytes isolated from whole blood in density gradient. The basal DNA lesions and chromosomal aberrations were evaluated, as well as the repair capacity of the supplementary lesions induced by genotoxic agents such as ionizing radiations. Our results showed a great interindividual variability in the basal level of the DNA lesions and chromosomal aberrations, between and within the groups, the most affected being the heavy metals-exposed groups. Non-exposed subjects from rural area Cluj appeared to be more susceptible to the induction of supplementary DNA lesions and chromosomal aberrations by irradiation. The most efficient repair capacity of the radio-induced DNA lesions was observed in the non-exposed Cluj urban group. Both cytogenetic assays (as tools for detection of DNA lesions and chromosomal aberrations) may be used in human biomonitoring studies as indicators of early biological effects induced by exposure to heavy metals.