Release of Gelatinase A (Matrix Metalloproteinase 2) Induced by Photolysis of Caged Phosphatidic Acid in HT 1080 Metastatic Fibrosarcoma Cells

Abstract

Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic. We have synthesized a photolyzable derivative of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events. 1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of PA. Photolysis of the compound by ultraviolet light resulted in the formation of phosphatidic acid. The structure of the compound and of its photolytic products was verified by NMR spectroscopy. The utility of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which the formation of PA by phospholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after 120 min of incubation. Ultraviolet illumination of cPA-loaded cells for 5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to the medium, whereas nonphotolyzed cPA caused no change in MMP-2 release. Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly introduced PA. Thus, the effect of laminin on MMP-2 secretion can be mimicked by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis. These results indicate that cPA could serve as a unique tool for studying the cellular roles of PA.

Photoactivated Spatiotemporally-Responsive Nanosensors of in Vivo Protease Activity

“…Thus, we designed a veiled, MMP-sensitive nanosensor by conjugating the photolabile small molecule 1-(4,5-dimethoxy-2-nitrophenyl) diazoethane (DMNPE) to protease cleavable substrates (Figure ). DMNPE reacts with acidic groups , and, by coupling it to an MMP substrate sequence containing free carboxylic acid side chains, serves as a removable barrier to block enzymatic cleavage. Furthermore, we hypothesized, based on previous studies, that DMNPE should be located within a few amino acids from the putative cleavage site in order to effectively block protease activity by steric hindrance.…”

Expression and Clinical Role of Protein of Regenerating Liver (PRL) Phosphatases in Ovarian Carcinoma

“…Our results show a significant dose-dependent expression of PRL-3 in response to exogenous laminin (Figure 3A). In previous studies, we have shown that laminin signaling involves activation of phospholipase D (PLD) enzymes [25,26]. We repeated the experiments in the presence of primary and secondary alcohol, known modulators of PLD signal transduction.…”

Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness

“…In addition to participating in cell survival pathways, PLD is thought to contribute to cancer metastasis via multiple mechanisms, including cytoskeletal rearrangement and cell migration 31 . Recent reports suggest PLD may also contribute to metastasis through the induction of matrix metalloproteinase (MMP) secretion and extracellular matrix degradation 41–45 . PLD has also been suggested to have pro-angiogenic properties via interactions with sphingosine kinase-1 and sphingosine-1-phosphate (S1P) 46 .…”