| Přispěvatelé: |
Ferrante, MARIA CARMELA, Tafuri, S., Scibelli, A., Naso, B., Cardone, M., Staiano, N., Lucisano, A., Ferrante, M. C., Lucisano, Antonia, Ferrante, Mc, Tafuri, Simona, Scibelli, A, Naso, B, Cardone, M, Staiano, Norma, DELLA MORTE, Rossella, Belisario, Ma |
| Popis: |
Ochratoxin A (OTA), produced by Aspergillus and Penicillium strains, is a secondary fungal metabolite widespread in a variety of animal feed and human food, such as cereals, pork and poultry meat, coffee, wine, grape juice,and milk. OTA is a derivative of the isocumarin linked to L--phenylalanine and it is classified as a pentaketide. Epidemiological studies correlate OTA with Balkan endemic nephropathy (BEN), immunosuppression, hepatotoxicity, carcinogenicity, teratogenicity and mutagenicity. The molecular mechanisms involved in OTA-induced toxicity are not clearly defined. The aim of the present study was to assess the ability of OTA to affect cell-matrix adhesion and signaling in monkey kidney COS cell line. COS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented by 10% foetal bovine serum (FBS) and 1% glutamine. Cells were harvested for propagation or cell attachment experiments by treatment with 0.25 % trypsin / 0.02 % ethylenediamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS) and with 5 mM EDTA in PBS, respectively. The viability of cells exposed to increasing concentrations of OTA for different time intervals was measured by the trypan blue exclusion method. Cell adhesion assays both in the presence and in the absence of increasing concentrations of OTA were performed as previously described. Apoptosis induced by OTA was evaluated on cell lysates incubated with N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) by measuring the increase in fluorescence due to the cellular caspase-3 activity. In order to evaluate whether OTA exerted a toxic effect, the cells were exposed to increasing concentrations of OTA (0-100 M) for different time intervals (1-72 h). The mycotoxin was not cytotoxic at concentrations lower than 100. A significant cytotoxic effect with 100 OTA was detected only after prolonged cell exposure. Then, we evaluated the percentage of cell detachment from immobilized collagen or fibronectin induced by non-cytotoxic concentrations of OTA. After 24 h of cell exposure to OTA we observed an 80 % and 55 % of cell detachment from immobilized collagen and fibronectin, respectively. Previous studies have demonstrated that OTA induces apoptosis in different cell systems. We investigated whether OTA induces apoptosis in COS cells and how the kinetics of OTA-induced apoptotic response correlates with the kinetics of cell detachment. After 9h of cell exposure to OTA (10 M), the mycotoxin induced apoptosis in COS cells adherent to collagen or fibronectin. This result indicates that the kinetics of cell detachment and apoptosis induced by OTA were not related. Since these events are associated with cell signaling, we have evaluated the effect of cell exposure to OTA on tyrosine phosphorylation of the focal adhesion associated proteins FAK (focal adhesion kinase) and paxillin. Our results show that OTA causes a reduction of tyrosine phosphorylation of pp125FAK and paxillin in adherent COS cells. Since either FAK or Shc pathways are implicated in the activation of the MAPK (mitogen-activated protein kinase) cascade Further investigations are in progress to evaluate the effect of OTA on Shc pathway. |
