Peptide Antagonists of the PED-hPLD1 Binding Viparelli, F.; Doti, N.; Monti, S.M. ...
Advances in experimental medicine and biology,
2009, Letnik:
611
Book Chapter, Journal Article
Along with insulin resistance and increased risk of type 2 diabetes (T2D), lean first-degree relatives of T2D subjects (FDR) feature impaired adipogenesis in subcutaneous adipose tissue (SAT) and ...subcutaneous adipocyte hypertrophy well before diabetes onset. The molecular mechanisms linking these events have only partially been clarified. In the present report, we show that silencing of the transcription factor
(
) in human preadipocytes impaired differentiation in mature adipose cells in vitro. The reduced adipogenesis was accompanied by inappropriate
-signaling activation. Importantly, in preadipocytes from FDR individuals,
expression was attenuated, with hypermethylation of the
promoter region found responsible for its downregulation, as revealed by luciferase assay. Both
gene expression and DNA methylation were significantly correlated with SAT adipose cell hypertrophy in FDR, whose increased adipocyte size marks impaired adipogenesis. In preadipocytes from FDR, the low
expression negatively correlated with enhanced transcription of the
signaling downstream genes
and
. In silico evidence indicated that
and
were directly controlled by
. The
promoter region also was hypermethylated in peripheral blood leukocytes from these same FDR individuals, which was further revealed in peripheral blood leukocytes from an independent group of obese subjects. Thus,
controlled adipogenesis in humans by suppressing
signaling. Altered DNA methylation of the
promoter contributed to restricted adipogenesis in the SAT of lean subjects who were FDR of type 2 diabetics and in obese individuals.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can trigger apoptosis in some tumor cells but not other tumor cells. To explore the signal transduction events in TRAIL-triggered ...apoptosis and its modulation in nontransfected tumor cells, we analyzed TRAIL-induced death-inducing signaling complex (DISC) in TRAIL-sensitive and -resistant glioma cells. Caspase-8 and caspase-10 were recruited to the DISC, where they were proteolytically activated to initiate apoptosis in TRAIL-sensitive glioma cells. Caspase-8 and caspase-10 were also recruited to the DISC in TRAIL-resistant cells, but their further activation was inhibited by two antiapoptotic proteins termed cellular Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15kDa (PED/PEA-15). Both long and short forms of c-FLIP were recruited to the DISC, where the long form c-FLIP was cleaved to produce intermediate fragments. Of the three isoforms of PED/PEA-15 proteins, only the doubly phosphorylated form was expressed and recruited to the DISC in TRAIL-resistant cells, indicating that the phosphorylation status of PED/PEA-15 determines its recruitment in the cells. Treatment with calcium/calmodulin-dependent protein kinase inhibitor rescued TRAIL sensitivity in TRAIL-resistant cells, providing a potential new approach to sensitize the cells to TRAIL-induced apoptosis.
Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells ...infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.
GnRH agonist therapy is known to reduce uterine leiomyoma volume, although the molecular mechanisms responsible for this effect remain poorly understood. In this study, we have investigated the ...molecular mechanisms involved in the anti‐proliferative effect of a GnRH agonist, leuprolide acetate (LA), in uterine leiomyomas obtained from six patients treated with LA for 3 months before surgery (group B), compared with tumours from six untreated patients (group A). To this end, we have evaluated the expression and the activity of molecules involved in the regulation of cell survival and proliferation. In group B, the total activity of PI3K was reduced by 60% compared with control samples. Furthermore, LA caused a reduction of PKB activation of ∼50%, measured as serine 473 phosphorylation. In parallel with PKB reduction in LA samples, we observed a 60% reduction in the phosphorylation of its substrate BAD. While Bcl‐xL/BAD association was not significantly modified in LA‐treated leiomyomas, BAD/14.3.3 interaction was reduced, due to a 50% decreased 14.3.3 expression. In addition, LA was able to reduce the expression of the antiapoptotic proteins FLIP and PED/PEA15 by 70 and 50% respectively, compared with control samples. We next evaluated the activation of MAP kinases in leiomyomas. Activation of p42 and p44 MAP kinase isoforms was increased by 30% in group B. However, the phosphorylation of the transcription factor Elk1 was not increased in a similar fashion in LA‐treated leiomyomas compared with group A. Thus, these data suggest that LA reduction of leiomyoma volume is mediated at least in part by a decreased activation of the PI3K/PKB survival pathway and by the suppression of antiapoptotic factors.
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