Research articleCasein phosphopeptides modulate proliferation and apoptosis in HT-29 cell line through their interaction with voltage-operated L-type calcium channels
Introduction
A new promising field in nutrition is the identification of bioactive compounds, that is to say molecules naturally occurring in food, which can exert physiologically relevant actions either directly, or upon enzymatic hydrolysis, when inactive within the parent molecules, carried on by digestive enzymes or by proteolytic microorganisms. All these proteolytic processes can be achieved in vitro, for instance, using proteolytic enzymes extracted by plants or microorganisms, but also in vivo, during the gastrointestinal digestion and the fermentation of food by the gut bacteria (for a review, see Korhonen and Pihlanto [1]). A lot of effects exerted by these bioactive compounds are now recognized at multiple levels in the body, such as at cardiovascular, nervous, gastrointestinal and immune level. The goal of all the studies devoted to discover new bioactive molecules in food is to improve human health simply by consuming a “normal diet” that naturally contains a consistent amount of these bioactive molecules or by using them as additives in food.
Milk and dairy products are well-known sources of bioactive peptides originating during milk fermentation with the starter cultures by the dairy industry or by cheese ripening [1]. A family of bioactive peptides derived from the digestion of casein, the milk main protein fraction, is named casein phosphopeptides (CPPs), due to their high content of phosphorylated regions [2]. In contrast to casein, which does not possess any bioactivity and serves as a source of amino acids for human nutrition, CPPs are characterized by the ability to bind and solubilize cations, especially calcium [3]. Numerous studies arose in the last decades trying to address to CPPs also the aptitude to enhance calcium absorption in the intestine, but the difficulties of the both human and animal studies have provided conflicting results, as reviewed in Ferraretto and Fiorilli [4].
Instead, studies using in vitro cell cultures have revealed new and interesting potentialities for CPPs, such as the modulation of the intestinal immune system by triggering cytokine secretion and stimulating immunoglobulin A production [5], [6], the release of interleukin-6 cytokine in human epithelial intestinal cell lines [7], the protective antiproliferative effects on cultured mammalian intestinal cells [8] and the modulation of cell viability, that is, proliferation and apoptosis, in different human cell cultures [9], [10]. In this context, the recent findings that CPPs can induce intracellular calcium increases in intestinal human tumor cell lines HT-29 and Caco2 but only upon cell differentiation [11] can be considered not only from the calcium absorption process point of view, since the calcium ion influx induced by some mitogenic factors can play a role as messengers of cellular events linked to control of the proliferation, thus cell viability [12]. In fact, abnormal cell proliferation is often associated to pathological conditions such as inflammation and tumor [13], [14]. The calcium ion entry mechanisms in cells can be more than one — voltage-operated calcium channels, receptor-operated channels and store-operated calcium entry — and all of them have been associated with cell growth in tumor and normal tissues [12]. More recently, it was demonstrated that in HT-29 cells, as well as in a primary human colon cancer cell line (AZ-97), the activation of voltage-activated L-type calcium channels, which mediate the calcium influx according to the depolarization state of the cell, is correlated to apoptosis, and their blockade may promote the growth of colon cancer cells [15].
The human HT-29 cell line, derived from an intestinal adenocarcinoma, is frequently used as an intestinal in vitro cell model due to its ability to undergo different patterns of cell differentiation, in dependent of the modifications in their growth medium [16]. When cultivated in Dulbecco's modified Eagle's medium (DMEM), HT-29 cells do not display any signs of differentiation, apart from rare apical microvilli and desmosomes; on the contrary, when cultivated in Roswell Park Memorial Institute 1640 (RPMI) medium, HT-29 cells display a differentiated morphological phenotype characterized by a complete junctional apparatus (tight junctions, adherens junctions, desmosomes) and very abundant microvilli [11], [17]. Despite these morphological differences, no significative variation in the activities of alkaline phosphatase and sucrase–isomaltase, known enzymatic markers of intestinal cell differentiation, between HT-29 DMEM and RPMI cells was observed, as already reported [11], [17], and HT-29 RPMI cells are considered a heterogeneous cell population.
The aims of the present work are therefore to explore the possibility that the CPP-mediated calcium influx in differentiated HT-29 cells could involve the L-type calcium channels known to be expressed in this cell line [15], [18] and to study the possible correlated modulation of cell proliferation and apoptosis. Moreover, the same studies were carried on in undifferentiated tumor HT-29 cells, in order to verify the possibility that CPPs, due to their property to complex calcium ions, may behave as ethyleneglycol-O, O′-bis(2-aminoethyl)-N, N, N′, N′-tetraacetic acid (EGTA), the known calcium-chelating agents, affecting the extracellular medium and influencing cell functions [19], [20].
All the experiments were done at physiological condition, the calcium concentration present in the growing medium, and at calcium overload. The supraphysiological concentrations of calcium used in this work — 6 mM for undifferentiated DMEM cells, 2 and 6 mM for differentiated RPMI cells — were chosen in order to mimic the situation at intestinal lumen after a meal, since it is reported in literature that in this particular condition, the extracellular calcium concentration can reach values as high as 10 mM [21], [22], [23]. Since tumor DMEM cells grow in 2 mM calcium concentration, while differentiated RPMI cells require small amounts of calcium ions to survive [24], [25] (0.4 mM calcium in RPMI medium), the experiments with RPMI cells were carried on both at 2 and 6 mM calcium, in order to compare results with DMEM cells and to reproduce more than one step in the increasing calcium concentration after a meal.
Section snippets
Materials and reagents
Cell culture media, Bay-K8644, Nifedipine, Nimodipine and all other reagents, unless otherwise specified, were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum was from EuroClone Ltd. (West Yorkshire, UK). Fura-2 acetoxymethyl ester was from Calbiochem (La Jolla, CA, USA). Bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), was obtained from Molecular Probes (Eugene, OR, USA).
Cell culture
The human colon carcinoma cell line HT-29 (BS TCL 132) was obtained from Istituto
CPP effects on proliferation rate and apoptosis in undifferentiated/differentiated HT-29 cells
In HT-29 DMEM cells, the proliferation rate both at physiological [Ca2+]o (2 mM) in the growth medium and in calcium overload (6 mM) was not affected by the cell incubation with 1280 μM CPPs, a dose at which they are demonstrated to exert the maximal bioactivity [11], [17], or 500 μM EGTA (Fig. 1A). On the contrary, the apoptotic activity was increased by CPP administration both at 2 mM [Ca2+]o (caspase 3/7 activity and apoptotic nuclei staining; Fig. 1B, C) and at 6 mM [Ca2+]o (apoptotic
Discussion
At the intestinal level, the extracellular calcium concentration can modulate processes such as proliferation and apoptosis, cellular events that are deeply and directly involved both in the maintaining of a cell-differentiated phenotype and in the development of a cancer phenotype [24]. Intestinal cells require small amounts of calcium ions to survive, about 0.05–0.1 mM [24], [25], and when an overload of calcium takes place in the intestinal lumen, for instance, after a meal, they stop
Acknowledgments
This work was supported in part by the EU FAIR Programme Project CT98-3077 [Casein phosphopeptide (CPP): Nutraceutical/functional food ingredients for food and pharmaceutical applications] and by Fondazione Romeo and Enrica Invernizzi (CPP: role in the calcium intestinal absorption and its utilization. A perspective study on their possible usage as nutraceuticals or functional food to favour calcium bioavailability).
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