New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development

The influence of caseinphosphopeptides on intracellular calcium changes in primary human osteoblasts : a nutrient dependent modulation of bone cell metabolism

Caseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. They possess a sequence of three phosphorylated serines followed by two glutamic acids, the acidic motif, able to bind minerals such as calcium. These nutritional compounds display the ability to increase calcium solubility in the digestive tract. Thus, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization. Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect, analogous to that reported for human intestinal cells, is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. The 24 hours cell incubation with CPPs induced the increase of the activity of alkaline phosphatase, a marker of osteoblast differentiation, reaching a level similar to that produced by vitamin D. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. Taken together these results indicate that CPPs are endowed of a bone specific effect which underlying mechanism requires further evaluation. CPPs may act not only as a mere carrier for improving calcium absorption and utilization, but also as a trophyc compound for bone health by enhancing osteoblast differentiation and activity

Prognostic factors associated with survival and recurrence in resectable gastroesophageal cancer: retrospective analysis of 338 patients operated at the Hospital of Cremona in ten years' time

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L'importanza del calcio e i caseinofosfopeptidi: dalla nutrizione all'ingegneria tissutale

Bone is characterized by a physiologic turnover, in adult too: 0.05% of calcium, about 500 mg/die, is daily removed (osteoclastic activity) and hidden (osteoblastic activity). This mean that, in the adult, in about six years all bone minerals is changed. Bone mass level remain the same when deposition rate is equal to reabsorption rate, that is when osteoclastic and osteoblastic activity are in equilibrium. This turnover is most fast in the infant, in this population all bone minerals is changed in about one year. About the total bone mass (Peak Bone Mass, PBM) is stored up within 18-20 years old. An inadequate intake of calcium in this period may lead to a low bone mineral density, which may have implications for bone health, notably risk of osteoporosis, in later life. The PBM attainment constitutes a very important goal: grater bone mass in adult life means lower fractures in old age. Calcium is an essential aliment: all calcium in the body comes from diet. Many foods contain calcium, but the amount of calcium, provided per 100 g or per serving, and is bioavailability vary considerably. The major source of calcium is milk and milk products. The high bioavailability of calcium in milk is essential due to the presence of caseinphosphopeptides, CPPs, bioactive peptides which are formed by proteolytic digestium of casein in the ileum. Thanks to the presence, in their aminoacidic sequence, of an “acidic motive”, constitutes by three phosphorilated serine and two glutammate, Ser(P)-Ser(P)-Ser(P)-Glu-Glu, these peptides are able to prevent the formation of insoluble Ca-phosphate precipitates in the intestinal by complexing ionized calcium in soluble chelates which enhance the amount of intraluminal calcium available for transport across the mucosa by the non-saturable pathway. Many searchers have proved their ability to increase the amount of calcium absorption and its utilization in bone tissue. Nevertheless, specially in humans searchers have not only obtained positive but also non-significant results. Long term clinical studies are necessary to better investigate CPPs role in humans. More positive results are obtained in oral health: pre-formed CPPs and amorphous calcium phosphate nanocomplex (CPP-APC) have the potential to provide superior clinical efficacy in preventing dental caries, treating and reparing early stage of disease. In present study we are demostrated that CPPs are able to iduce calcium uptake in osteoblasts, likewise they do in intestinal cells, both cell types involved in calcium metabolism in vivo. CPPs have instead no action in other cell types, like fibroblasts or neuronal cells. In particular, in osteoblast, kinetics and morphology CPPs cells response are different versus intestinal cells. This difference can be explained by different functions that they are assigned in vivo: intestinal cells are delegated to the calcium absorption instead osteoblast cells are delegated to the right calcemia maintenance. Moreover, in osteoblasts, CPPs action is mediated by L-type calcium channel activation and this calcium influx in response to CPPs somministration activates cellular function, such as differenziating activity to mature osteoblasts stadium, likewise in the presence of vitamin D3, osteoblasts lineage physiological growth factor. This results allow to suppose a role for CPPs not only as a functional food, as integration in prevention strategy of these pathologys and life styles characterized by an inadeguate calcium absorption and intake; but also as possible candidates in bone tissue engineering clinical strategies, for example by their integration in scaffolds. Nevertheless this is only the first step and more studies are necessary to better understand CPPs biological role and effect in vivo. In last years many scientific reports have open the doors to a new research area, in which bioactive components from foods are the protagonists. They not only contribute to as adequate nutritional value, but generate advantages for health, reducing the risk to untle several pathologys, without the contribution of body foreign chemical substances

Casein phosphopeptides induces intracellular calcium changes and cell function modulation in primary human osteoblasts

Caseinphosphopeptides (CPPs) are a family of peptides originating from in vivo and in vitro hydrolysis of casein. These nutritional compounds display the ability to bind and solubilize minerals such as calcium. The importance to enhance the mineral uptake by the intestine relies on two findings: i) the inverse correlation found between the colon tumor incidence and the absorption of calcium from diet as put in evidence by recent epidemiologic studies; ii) minerals, especially calcium and zinc, are fundamental for improving and maintaining a correct bone mass. On this basis, CPPs were hypothesized to increase the calcium absorption and retention in vivo, with potential effects on bone mineralization (1). Notwithstanding, there are controversial reports on CPP action. The methodological approach used by different laboratories to study calcium absorption and bone mineralization resulted unable to out light whether the peptides have a specific effect on bone metabolism besides the enhancement of calcium availability. We have therefore designed the following study to evaluate a possible direct role of CPPs in bone cell metabolism. Primary human osteoblasts were established in culture using trabecular bone samples obtained from waste materials during orthopedic surgery of patients without metabolic or malignant bone disease. Cytosolic calcium changes were measured by video-microscopy using the fura-2 method on single cells. A mixture of CPPs of commercial origin as well as pure synthetic CPPs were used. The administration of CPPs to human osteoblasts caused an immediate but transient intracellular calcium change in a dose dependent manner. This CPP-induced effect is not cytotoxic and is triggered by an influx of the extracellular ions through the cell plasma membrane. The osteoblast pre-treatment with the active form of vitamin D, known to differentiate human osteoblast, does not affect the cell responsiveness to CPP administration. 4 hours of cell incubation with CPPs induced an increase of the expression of alkaline phosphatase, while 24 hours of cell incubation induced the increase of activity of alkaline phosphatase and of Runx2 expression, both markers of osteoblast differentiation. The same CPP treatment caused a small but significative reduction in cell rate proliferation and a slight increase in apoptosis activity. These results open the possibility to use CPPs, a nutrient component of the western diet, as tools for improving osteoblast mineralization and differentiation. Further studies are in progress to develop the way to use CPPs in bone tissue repairing and associated pathology