Ochratoxin A Sequentially Activates Autophagy and the Ubiquitin-Proteasome System.

Ochratoxin A (OTA) is a carcinogenic mycotoxin, which is produced by Aspergillus and Penicillium genera of fungi and commonly contaminates food and feed. We and others have previously shown that OTA causes sustained activation of PI3K/AKT and MAPK/ERK1-2 signaling pathways in different cell types and animal models. Given the close relationship between cellular signaling activity and protein stability, we were curious whether increased PI3K/AKT and MAPK/ERK1-2 signaling may be the result of OTA-stimulated alterations in proteolytic activity. We show that both of the major proteolytic systems, autophagy, and the ubiquitin-proteasome system (UPS), are activated upon OTA exposure in human kidney proximal tubule HK-2 and mouse embryonic fibroblast (MEF) cells. OTA stimulates transient autophagic activity at early time points of treatment but autophagic activity subsides after 6 h even in the sustained presence of OTA. Interestingly, OTA exposure also results in increased cell death in wild-type MEF cells but not in autophagy-halted Atg5-deficient cells, suggesting that autophagy exerts a pro-death effect on OTA-induced cytotoxicity. In addition, prolonged OTA exposure decreased ubiquitinated protein levels by increasing proteasomal activity. Using purified and cellular proteasomes, we observed enhanced chymotrypsin-, caspase-, and trypsin-like activities of the 26S but not the 20S proteasome in the presence of OTA. However, in the cellular context, increased proteasomal activity depended on prior induction of autophagy. Our results suggest that autophagy and subsequent UPS activation are responsible for sustained activation of PI3K/AKT and MAPK/ERK1-2 pathways through regulating the levels of critical phosphatases VHR/DUSP3, DUSP4, and PHLPP, which are known to be involved in OTA toxicity and carcinogenicity.

Water soluble molybdenocene complexes: synthesis, cytotoxic activity and binding studies to ubiquitin by fluorescence spectroscopy, circular dichroism and molecular modeling.

Four new molybdenocene complexes, Cp2Mo(l-ascorbato), Cp2Mo(6-O-palmitoyl-l-ascorbato), [Cp2Mo(ethyl maltolato)]Cl and Cp2Mo((2S)-2-amino-3-methyl-3-thiolato-butanoato), were synthesized and structurally characterized by standard analytical methods. The cytotoxicity of these complexes was assessed on colon HT-29 and breast MCF-7 cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A higher cytotoxic activity was shown by all the new complexes on the MCF-7 cells over the Cp2MoCl2 complex. The complexes Cp2Mo(l-ascorbato), Cp2Mo(6-O-palmitoyl-l-ascorbato) and [Cp2Mo(ethyl maltolato)]Cl displayed a stronger cytotoxic activity on colon cancer HT-29 cell line, over the molybdenocene dichloride (Cp2MoCl2). In contrast, Cp2Mo((2S)-2-amino-3-methyl-3-thiolato-butanoato) exhibited proliferative properties on this cell line. Ubiquitin (Ub)-molybdenocene interactions were investigated using cyclic voltammetry, fluorescence quenching spectroscopy, circular dichroism (CD) and molecular modeling. The thermodynamic parameters (ΔH and ΔS) obtained using fluorescence quenching spectra and van't Hoff plot indicate the Ub-molybdenocene interactions are mainly hydrophobic. The CD data also support hydrophobic interactions with conformational changes in the Ub protein. Docking studies using molecular modeling revealed the amino acids involved in the Ub-molybdenocene interactions and corroborated the hydrophobic nature of the binding combined with hydrogen bonding.