The human asparaginase enzyme (ASPG) inhibits growth in leukemic cells

<div><p>The human protein ASPG is an enzyme with a putative antitumor activity. We generated in bacteria and then purified a recombinant GST-ASPG protein that we used to characterize the biochemical and cytotoxic properties of the human ASPG. We demonstrated that ASPG possesses asparaginase and PAF acetylhydrolase activities that depend on a critical threonine residue at position 19. Consistently, ASPG but not its T19A mutant showed cytotoxic activity in K562, NALM-6 and MOLT-4 leukemic cell lines but not in normal cells. Regarding the mechanism of action of ASPG, it was able to induce a significant apoptotic death in K562 cells. Taken together our data suggest that ASPG, combining different enzymatic activities, should be considered a promising anti-cancer agent for inhibiting the growth of leukemia cells.</p></div

SDS Page of purified GST-fusion proteins.

<p>Line 1: marker, line 2: GST, line 3: GST-ASPG, line 4: GST-ASPG (T19A), line 5–7: 50, 100 and 250 ng of bovine serum albumin respectively.</p

PAF-AH activity of GST-ASPG.

<p>(A) The PAF-AH activity of recombinant GST-ASPG (●) and its point mutant GST-ASPG (T19A) (▲) was measured as function of enzyme concentration using Abcam's PAF acetylhydrolase Assay Kit as reported in Materials and Methods. Data points are represented as means ± SD of triplicate sample measurements and were fitted with a second order polynomial equation in GraphPad Prism software. (B) The residual PAF-acetylhydrolase activity of GST-ASPG was measured after pre-incubation for 10 min of 1.5 μg of GST-ASPG with 40 mM and 200 mM of D-asparagine using Abcam's PAF-AH assay. Data are shown as means ± SD of triplicate measurements. ** (<i>p</i> < 0.01); *** (<i>p</i> < 0.001).</p

Schematic representation of ASPG action on the growth and survival of leukemia cells.

<p>ASPG with its L-asparaginase activity could deprive leukemia cells of L-asparagine, which is an essential metabolite for its malignant growth, inducing apoptosis and blocking cell proliferation. ASPG could also inducing the arrest of cell proliferation converting the active PAF in the inactive form Lyso-PAF and down-regulating the expression of the Epithelial Sodium Channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref026" target="_blank">26</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref027" target="_blank">27</a>].</p

Cytotoxicity of GST-ASPG on human leukemia cell lines and normal cells.

<p>The percentage of cell survival was evaluated by CCK8 assay in K562 (A), NALM-6 (B) and MOLT-4 (C) cell lines after 24h of treatment with GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). HDFA (□) and PBMCs (◊) were used to analyze the cytotoxicity of GST-ASPG in normal cells (D). The results were fitted using GraphPad Prism software and represent the average and the standard deviation of three independent experiments. * (<i>p</i> < 0.05); *** (<i>p</i> < 0.001).</p

Caspase assay on K562.

<p>(A) Representative Guava caspase assay graphs of K562 cells lines treated with 0.7 μg of GST, GST-ASPG or GST-ASPG (T19A) for 6, 12 and 24h. (B). Bar graphs representing the percentage of caspase and/or 7AAD positive cells after treatment with 0.7 μg of GST, GST-ASPG or GST-ASPG (T19A) for 6, 12 and 24h. (C) Table representing the statistical significances between groups. *(p < 0.05); **(<i>p</i>< 0.01); ***(<i>p</i>< 0.001).</p

Cell growth inhibition by GST-ASPG.

<p>(A) K562 cells after 24h of incubation with 1000 ng (115 nM) of GST-ASPG and its inactive mutant GST-ASPG (T19A). (B) Time course of K562 cells treatment with 115 nM of GST-ASPG (T19A) (▲), 115 nM of GST ASPG (●) and after further addition of 115 nM of GST-ASPG at 12h (ο).Viability of K562 (C) and NALM-6 (D) cells after 24h of treatment with increasing concentrations of GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). The results (the average and the standard deviation of three independent experiments) were evaluated by Trypan Blue exclusion assay and fitted using GraphPad Prism software. * (<i>p</i> < 0.05); ** (<i>p</i> < 0.01); *** (<i>p</i> < 0.001).</p

Characterization of L-asparaginase activity of GST-ASPG.

<p>L-Asparaginase activity of GST-ASPG (●) and its catalytically inactive mutant GST-ASPG (T19A) (▲) was evaluated by Nessler’s method in a time dependent manner (A), as a function of enzyme concentration (B) and substrate concentration (C). A hill slope of 6.9 and an S_0.5 value of ≈13 mM were estimated. Ammonia release of GST-ASPG was measured using 15 mM of L-asparagine alone or in presence of 15 mM of D-asparagine; No detectable enzymatic activity was found using 15 mM of D-asparagine or 15 mM of L-glutamine as substrates (D). Steady state kinetic of recombinant GST-ASPG was calculated in presence of 0 mM (●), 10 mM (■) and 30 mM (▲) of D-asparagine and a Ki value of 71 mM was estimated (E). All the experiments were performed at 37°C as reported in Materials and Methods using 1.5 μg of each protein; data points are represented as means ± SD of triplicate sample measurements. *** (<i>p</i> < 0.001).</p

Discovery of PTPRJ Agonist Peptides That Effectively Inhibit <i>in Vitro</i> Cancer Cell Proliferation and Tube Formation

PTPRJ is a receptor protein tyrosine phosphatase involved in both physiological and oncogenic pathways. We previously reported that its expression is strongly reduced in the majority of explored cancer cell lines and tumor samples; moreover, its restoration blocks <i>in vitro</i> cancer cell proliferation and <i>in vivo</i> tumor formation. By means of a phage display library screening, we recently identified two peptides able to bind and activate PTPRJ, resulting in cell growth inhibition and apoptosis of both cancer and endothelial cells. Here, on a previously discovered PTPRJ agonist peptide, PTPRJ-pep19, we synthesized and assayed a panel of nonapeptide analogues with the aim to identify specific amino acid residues responsible for peptide activity. These second-generation nonapeptides were tested on both cancer and primary endothelial cells (HeLa and HUVEC, respectively); interestingly, one of them (PTPRJ-19.4) was able to both dramatically reduce cell proliferation and effectively trigger apoptosis of both HeLa and HUVECs compared to its first-generation counterpart. Moreover, PTPRJ-pep19.4 significantly inhibited <i>in vitro</i> tube formation on Matrigel. Intriguingly, while ERK1/2 phosphorylation and cell proliferation were both inhibited by PTPRJ-pep19.4 in breast cancer cells (MCF-7 and SKBr3), no effects were observed on primary normal human mammary endothelial cells (HMEC). We further characterized these peptides by molecular modeling and NMR experiments reporting, for the most active peptide, the possibility of self-aggregation states and highlighting new hints of structure–activity relationship. Thus, our results indicate that this nonapeptide might represent a great potential lead for the development of novel targeted anticancer drugs

Isolation and Functional Characterization of Peptide Agonists of PTPRJ, a Tyrosine Phosphatase Receptor Endowed with Tumor Suppressor Activity

PTPRJ is a receptor-type protein tyrosine phosphatase whose expression is strongly reduced in the majority of investigated cancer cell lines and tumor specimens. PTPRJ negatively interferes with mitogenic signals originating from several oncogenic receptor tyrosine kinases, including HGFR, PDGFR, RET, and VEGFR-2. Here we report the isolation and characterization of peptides from a random peptide phage display library that bind and activate PTPRJ. These agonist peptides, which are able to both circularize and form dimers in acqueous solution, were assayed for their biochemical and biological activity on both human cancer cells and primary endothelial cells (HeLa and HUVEC, respectively). Our results demonstrate that binding of PTPRJ-interacting peptides to cell cultures dramatically reduces the extent of both MAPK phosphorylation and total phosphotyrosine levels; conversely, they induce a significant increase of the cell cycle inhibitor p27^Kip1. Moreover, PTPRJ agonist peptides both reduce proliferation and trigger apoptosis of treated cells. Our data indicate that peptide agonists of PTPRJ positively modulate the PTPRJ activity and may lead to novel targeted anticancer therapies