Anita Ferraretto

Casein-derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells

Casein phosphopeptides β‐CN(1–25)4P and αs1‐CN(59–79)5P, from β‐ and αs1‐casein, respectively, both carrying the characteristic ‘acidic motif’ Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu, were chemically synthesized and administered to HT‐29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca2+]i due to influx of extracellular Ca2+. The response was quantitatively higher with β‐CN(1–25)4P than αs1‐CN(59–79)5P. The synthetic peptide corresponding to the ‘acidic motif’ was ineffective and the dephosphorylated form of β‐CN(1–25)4P almost inactive. The lack of the N‐terminally located five amino acids, or sequence modifications within the N‐terminal segment of β‐CN(1–25)4P, caused a total loss of activity, whereas the lack of the C‐terminal segment preserved activity. In conclusion, the influx of calcium into HT‐29 cells caused by β‐CN(1–25)4P appears to depend on the phosphorylated ‘acidic motif’ and the preceding N‐terminal region.

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons.

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain‐derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol‐12‐myristate‐13‐acetate (1 μM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain‐derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.

Caseinphosphopeptide-induced calcium uptake in human intestinal cell lines HT-29 and Caco2 is correlated to cellular differentiation.

Caseinphosphopeptides (CPPs) are considered as mineral carriers because of their ability to bind and solubilize calcium ions, with the possible role, yet to be definitely assessed, of improving calcium absorption at the intestinal level. Previous works demonstrated that CPPs improve calcium uptake, with increasing intracellular calcium concentration, by human differentiated tumor HT-29 cells, and that this effect correlates with the supramolecular structure of CPPs in the presence of calcium ions. The aim of the present study was to establish whether the CPP effect on calcium uptake is specific for HT-29 cells and depends on the differentiated state of the cells. To this purpose, HT-29 and Caco2 cells, two models of intestinal cells, were differentiated following appropriate protocols, including treatment with 1,25-(OH)2 vitamin D3. The CPP-dependent intracellular calcium rises were monitored at the single-cell level through fura2-fluorescence assays, and cell differentiation was assessed by biochemical and morphological methods. Results clearly showed that the ability to take up extracellular calcium ions under CPP stimulation is exhibited by both HT-29 and Caco2 cells, but only upon cell differentiation. This evidence adds novel support to the notion that CPPs favour calcium absorption, thus possibly acting as cellular bio-modulators and carrying a nutraceutical potential.

Casein phosphopeptide promotion of calcium uptake in HT‐29 cells − relationship between biological activity and supramolecular structure

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT‐29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs β‐CN(1–25)4P and αs1‐CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated β‐CN(1–25)4P, [β‐CN(1–25)0P]. The biological effect was found to be: (a) maximal with β‐CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol·L−1 Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by β‐CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+‐free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with β‐CN(1–25)0P. With both β‐CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol·L−1 Ca2+.

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

Characterisation of biotinylated liposomes for in vivo targeting applications

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550‐DPPE or PEG2000‐DPFE in liposomes containing biotin‐DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin‐DPPE liposomes containing GM1. Biodistribution studies in C26 tumour‐bearing mice showed that GM1‐liposomes containing small amounts of biotin‐DPPE have long circulation life‐times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin‐DPPE were detected within extra‐vascular spaces in tumours. In addition it was shown that biotin‐DPPE in GM1‐liposomes bound streptavidin in situ. These results suggest that GM1‐liposomes containing biotin‐DPPE have potential use as diagnostic or therapeutic reagents in pre‐targeting applications dependent on the high‐affinity interaction of biotin with streptavidin.

Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor

Previous studies have demonstrated a gain-of-function of the calcium-sensing receptor (CASR) gene R990G polymorphism. In this study, activation of the R990G CASR stably transfected in HEK-293 (HEK-990G) cells compared with that of the common variant (HEK-wild-type (WT)) by increasing concentrations of CaCl(2) or calcimimetic R-568 caused significantly higher intracellular free calcium concentration ([Ca(2+)](i)) and lower Ca-EC(50). Moreover, the [Ca(2+)](i) oscillation percentage was higher with a larger sinusoidal pattern in HEK-990G. R-568 induced a shift of the oscillatory events from 4 to 2  mmol/l extracellular calcium concentration in HEK-990G cells and increased the sinusoidal oscillation percentage in comparison with HEK-WT. Preincubation with thapsigargin or phospholipase C inhibitors completely prevented oscillations in both cell lines, consistent with the involvement of the inositol trisphosphate pathway, while protein kinase C inhibitor prevented oscillations in HEK-WT cells only. Finally, CaCl(2) and R-568 caused a significant increase in p44/42 extracellular signaling-regulated kinase phosphorylation, with the mean Ca-EC(50) values being significantly lower in HEK-990G. Our findings demonstrated that the 990G allele is associated with high sensitivity to R-568, which provided new evidence for differences in CASR signaling.

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin—along with ganglioside—within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.

Sphingosine Kinase 2 and Ceramide Transport as Key Targets of the Natural Flavonoid Luteolin to Induce Apoptosis in Colon Cancer Cells.

The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC). Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P), two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER)-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK) 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

Sphingosine-1-Phosphate and Calcium Signaling in Cerebellar Astrocytes and Differentiated Granule Cells

S1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum.