The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

<p>Abstract</p> <p>Background</p> <p>Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit <it>in vitro </it>and <it>in vivo </it>the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.</p> <p>Methods</p> <p>A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. <it>In vitro</it>, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. <it>In vivo</it>, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. <it>In vivo </it>anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.</p> <p>Results</p> <p>Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. <it>In vitro</it>, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an <it>in vivo </it>Matrigel™ plug assay in mice</p> <p>Conclusions</p> <p>Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on <it>in vitro </it>and <it>in vivo </it>growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). <it>In vivo</it>, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.</p

Proteomic Analysis of Fusarium solani Isolated from the Asian Longhorned Beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process

Effect of oxidative stress on in vivo ADP-ribosylation of eukaryotic elongation factor 2

Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon. The investigation revealed that the endogenous ADP-ribosylation of eEF2 is complex and can take place in K562 cell lysates either under the action of endogenous transferase from [adenosine-C-14]NAD or by direct binding of free [C-14]ADP-ribose. These two types of ADP-ribosylation were distinguished by use of different treatments based on the chemical stability of the respective bonds formed. Under standard culture conditions, in vivo labeling of eEF2 in the presence of [C-14]adenosine was reversed to about 65% in the presence of diphtheria toxin and nicotinamide. This finding implied that the modification that took place under physiological circumstances was, mainly, of an enzymic nature. On the other hand, H2O2-promoted oxidative stress gave rise to a nearly two-fold increase in the extent of in vivo labeling of eEF2. This was accompanied by a loss of eEF2 activity in polypeptide chain elongation. Oxidative stress specifically inhibited the subsequent binding of free ADP-ribose to eEF2. The results thus provide evidence that endogenous ADP-ribosylation of eEF2 can also take place by the binding of free ADP-ribose. This nonenzymic reaction appears to account primarily for in vivo ADP-ribosylation of eEF2 under oxidative stress. (C) 2004 Elsevier Ltd. All rights reserved