Loredana Urso

[Pt(O,O′‐acac)(γ‐acac)(DMS)], a new Pt compound exerting fast cytotoxicity in MCF‐7 breast cancer cells via the mitochondrial apoptotic pathway

We showed previously that a new Pt complex containing an O,O′‐chelated acetylacetonate ligand (acac) and a dimethylsulphide in the Pt coordination sphere, [Pt(O,O′‐acac)(γ‐acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′‐acac)(γ‐acac)(DMS)] is also cytotoxic in a MCF‐7 breast cancer cell line relatively insensitive to cisplatin, and to gain a more detailed analysis of the cell death pathways.

Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.

Sublethal concentrations of the platinum(II) complex [Pt(O,O′‐acac)(γ‐acac)(DMS)] alter the motility and induce anoikis in MCF‐7 cells

Background and purpose:  We showed previously that a new Pt(II) complex ([Pt(O,O′‐acac)(γ‐acac)(DMS)]) exerted high and fast apoptotic processes in MCF‐7 cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′‐acac)(γ‐acac)(DMS)] is also able to exert anoikis and alter the migration ability of MCF‐7 cells, and to show some of the signalling events leading to these alterations.

Cisplatin reduces endothelial cell migration via regulation of type 2-matrix metalloproteinase activity.

Aims: In this study we investigated the effect of cisplatin on endothelial cell migration, an essential process for vascular remodeling and regeneration in several physiological and pathological situations. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were treated with cisplatin and endothelial cell migration analyzed by fluorescence and scratch-wound migration assay. MMP2 and MMP9 activity were determined by zymographic assay, and MAPK activation by Western blotting analysis. Results: We demonstrated that cisplatin provoked a time- and dose-dependent decrease of HUVEC migration; this effect was clearly independent from its well known cytotoxic activity. In addition, cisplatin markedly reduced MMP2 activity in both conditioned media and cell lysates, increased p38 MAPK and JNK phosphorylation, but did not affect ERK phosphorylation. Endothelial cell migration was attenuated by treatment of cells with GM6001, a non-specific inhibitor of MMPs, or by a selective anti-MMP2 antibody. However, treatment of cells with SB202190 or SP600125, inhibitors of p38 MAPK and JNK respectively, did not affect HUVEC migration. Conclusion: These results suggested that cisplatin induced a reduction of endothelial cell migration through an inhibition of MMP2 activity by downstream signal transduction pathways independent of JNK and p38 MAPK activation.

Anti‐apoptotic effects of protein kinase C‐δ and c‐fos in cisplatin‐treated thyroid cells

Background and purpose:  We showed previously that cisplatin inititates a signalling pathway mediated by PKC‐δ/extracellular signal‐regulated kinase (ERK), important for maintaining viability in PC Cl3 thyroid cells. The studies described herein examined whether c‐fos was associated with cisplatin resistance and the signalling link between c‐fos and PKC‐δ/ERK.

Functions of epidermal growth factor receptor in cisplatin response of thyroid cells

Epidermal growth factor receptor (EGFR) signal transduction pathway has been reported to play a vital role in the biologic progression of several tumours and as targets for therapeutic intervention. We have investigated the role of EGFR in the thyroid PC Cl3 cells response to the chemo-therapeutic agent cisplatin. It was found that cisplatin provoked (1) the activation (phosphorylation) and internalization of EGFR, (2) the phosphorylation of mitogen-activated protein kinase (MAPK)/p38, (3) the activation of PKC-epsilon, (4) the enhancement of matrix metalloproteinase-2 (MMP-2) expression and activity, (5) the generation of reactive oxygen species (ROS) and (6) the activation of the apoptotic intrinsic pathway. Inhibition or down regulation of EGFR reduced (1) the phosphorylation of MAPK/p38, (2) the cisplatin-provoked activation of PKC-epsilon, and (3) the activation of caspase-7 and PARP cleavage and the overall cells sensitivity to cisplatin. PKC-epsilon inhibition achieved by siRNA blocked MAPK/p38 activation and significantly increased the cell resistance to cisplatin. Finally, when the cisplatin-induced ROS generation was blocked by using NAD(P)H oxidase inhibitors, a decrease in cisplatin-induced MMP-2 enhancement, MAPK/p38 and EGFR activation, and caspase-7 proteolysis occurred. In conclusion, these findings supported a model in which cisplatin provokes an oxidant-induced MMP-2-dependent EGFR transactivation responsible for the induction of cell apoptosis, a process ascribable to the intracellular signalling of PKC-epsilon and MAPK/p38.

Effects of cisplatin on matrix metalloproteinase-2 in transformed thyroid cells

We investigated the effects of cisplatin (cisPt) on matrix metalloproteinase-2 (MMP-2) gelatinolitic activity in transformed PC E1Araf rat thyroid cells. Cells incubated with increasing cisPt concentrations showed dose- and time-dependent decrease of the MMP-2 protein and activity. CisPt provoked the translocation from the cytosol to the plasma membrane of atypical protein kinase C-zeta (PKC-zeta) and the activation of PKB/AKT. The effect of cisPt on MMP-2 was dependent on PKC-zeta activation since it was potentiated by a myristoylated PKC-zeta pseudo substrate peptide or by PKC-zeta down-regulation by siRNA. Moreover, MMP-2 activity modulation by cisPt was also dependent on PKB/AKT activation since it was decreased by PKB/AKT down-regulation by siRNA or by pharmacological inhibition of PI3K, thus indicating the importance of the balance of PKB/AKT and PKC-zeta in regulating the cisPt effect on MMP-2 activity. The PC E1Araf cells displayed a migratory capacity that was blocked by MMP-2 down-regulation using siRNA or pharmacological inhibition. The inhibition of cell migration was also obtained with cisPt; in cisPt-treated cells the administration of MMP-2 active protein was able to restore cell migration capacity. In conclusion, the decrease of MMP-2 secretion after cisPt was allowed by PKB/AKT and counteracted by PKC-zeta; the cisPt-provoked inhibition of MMP-2 secretion ended in reduction of cell migration.