Epidemiological evidence suggests that people who regularly consume fermented dairy foods have a reduced risk of developing certain cancers (van't Veer et al. Reference van't Veer, Dekker, Lamers, Kok, Schouten, Brants, Sturmans and Hermus1989). Milk proteins, which have long been known for their nutritional and technological benefits, have also been shown to possess bioactive properties in addition to their nutritive value (Hartmann et al. Reference Hartmann, Wal, Bernard and Pentzien2007; Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009). For instance, cytochemical studies have provided evidence that milk protein-derived peptides can affect the viability and growth of cancer cells (Mac Donald et al. Reference MacDonald, Thornton and Marshall1994; Hartmann & Meisel, Reference Hartmann and Meisel2004).
Casein and other milk proteins provide a rich source of bioactive peptides that have physiological roles in the human body (Korhonen & Pihlanto, Reference Korhonen and Pihlanto2006). These peptides affect the major systems including the cardiovascular, immune, nervous and digestive systems (for review see Korhonen & Pihlanto, Reference Korhonen and Pihlanto2006). Furthermore, there is a considerable amount of scientific evidence from both in vitro and in vivo studies to suggest that bioactive peptides regulate specific physiological functions. Milk-derived peptides are inactive within the sequences of the parent protein, such as casein, but can be released during gastrointestinal digestion, fermentation and food processing (Korhonen & Pihlanto-Leppälä, Reference Korhonen and Pihlanto-Leppälä2001; FitzGerald & Meisel, Reference FitzGerald, Meisel, Fox and McSweeney2003). Unravelling the mechanisms through which dietary factors alter the gastrointestinal environment to prevent or promote tumour formation is a major challenge and may never be completely performed using in vivo experimentation. However, in vitro models may provide clues to help us understand these mechanisms (Cameron, Reference Cameron, Moyer and Poste1990).
Although Jing and Kitts (Reference Jing and Kitts2004) showed that casein does not affect cellular enzymes, Laparra et al. (Reference Laparra, Alegría, Barberá and Farré2008) reported that caseinophosphopeptides reduced glutathione (GSH) concentration and increased GSH-reductase activity in Caco-2 cells. More recently, we demonstrated that eight distinct casein hydrolysates may exert specific antioxidant and immunomodulatory effects on human Jurkat T cells (Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009). Furthermore, studies have provided increasing evidence that food-derived bioactive peptides modulate the viability (e.g. proliferation, differentiation, and apoptosis) of different cell types such as Int-407 cells (Jing & Kitts, Reference Jing and Kitts2004), HL-60 cells (Hartmann & Meisel, Reference Hartmann and Meisel2004) and Jurkat T cells (Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009). Therefore, the aim of the present study was to further investigate the effects of sodium caseinate and eight distinct casein hydrolysates on the viability and growth of both human Jurkat T cells and intestinal Caco-2 cells. Cell viability was assessed using the MTT (mitochondrial activity), lactate dehydrogenase (LDH) release (membrane integrity) and Trypan Blue (membrane integrity) assays. Cell growth was monitored using the MTT, Trypan Blue and Bromodeoxyuridine (BrdU) proliferation (DNA synthesis) assays. Hence, these assays give a broad insight into the effects of the test samples on cell membrane integrity, mitochondrial activity, and DNA synthesis of different cell lines.
Materials and Methods
Materials
Human Jurkat T cells and human adenocarcinoma Caco-2 cells were purchased from the European Collection of Animal Cell Cultures (Salisbury,Wiltshire, SP4 0JG, UK). Foetal bovine serum (FBS) was purchased from Invitrogen (Paisley, PA4 9RF, Scotland). Cell culture plastics were supplied by Cruinn Diagnostics (Greiner Bio-One, Frickenhausen, Germany). All other cell culture reagents and chemicals were purchased from Sigma-Aldrich Chemical Co. (Airton Road, Tallaght, Dublin, Ireland) unless otherwise stated.
Generation of casein hydrolysates
Sodium caseinate (NaCN, 85·92% w/w protein) was provided by Arrabawn Co-op Society Ltd., Tipperary, Ireland. The protein content of the NaCN substrate was determined using the macro-Kjeldahl method (IDF, 2001), a Kjeldahl conversion factor of 6·38 was used. NaCN hydrolysates were generated using different commercially available food-grade enzyme preparations from mammalian, bacterial and plant sources. For hydrolysis with neutral proteinase preparations, aqueous solutions of NaCN ranging in concentration from 9·3 to 10·6% (w/v) protein were incubated at 50°C at pH 7. The pH was maintained constant using a pH stat (Titrino 718, Metrohm, Herisau, Switzerland) charged with 2 m-NaOH as previously described (Flanagan & FitzGerald, Reference Flanagan and FitzGerald2002). In the case of hydrolysis with acid proteinase, the NaCN concentration was 5% (w/v) while the pH was maintained constant at pH 2·4 using 1 m-HCl. Enzyme inactivation was by heat treatment at 80°C for 20 min. Hydrolysate samples were stored at −18°C prior to subsequent analysis. The reversed-phase HPLC profile of each hydrolysate has been outlined elsewhere (Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009).
Cell culture
Jurkat cells were grown in RPMI medium supplemented with 5% (v/v) FBS. Caco-2 cells were grown in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) non-essential amino acids. Both cell lines were cultured in an atmosphere of CO2-air (5:95, v/v) at 37°C and were maintained in the absence of antibiotics. The casein hydrolysates were filter sterilised (0·2 μm diameter) and diluted directly with sterile RPMI medium. Samples a, c, d, h and NaCN were diluted 1/10 prior to filtration as the concentrated samples could not pass through the filter membranes (0·2 and 0·45 μm diameter) undiluted. Control cultures comprised of cells grown in media only.
Incubation of cells with test compounds
For all experiments, Jurkat cells were seeded at a density of 2×105 cells/ml and Caco-2 cells were seeded at 3×104 cells/cm2. Jurkat cells were supplemented with increasing concentrations (0·5–10% v/v) of NaCN and its hydrolysates (a–h) for 24 h. Caco-2 cells were allowed adhere for 24 h in complete media. After 24 h, the culture media was removed and replaced with growth medium containing 2·5% (v/v) FBS, 1% (v/v) NEAA and the test samples (0·5–10% v/v) for 24 h.
MTT assay
Measurement of mitochondrial activity can be carried out by assessing the activity of the enzyme mitochondrial reductase (Mosmann, Reference Mosmann1983; Ciapetti et al. Reference Ciapetti, Cenni, Pratelli and Pizzoferrato1993). Mitochondrial activity was determined using the MTT assay (MTT I proliferation kit, Roche Diagnostics; West Sussex, RH15 9RY, UK). The assay is based on the conversion of the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its purple formazan product (Mosmann, Reference Mosmann1983). At the time of assay, 10 μl pre-warmed MTT labelling reagent was added to each well of a 96-well plate and then incubated for a further 4 h. Solubilising solution (100 μl) was then added to all wells and incubated overnight at 37°C. Absorbance was measured at 570 nm, with a reference wavelength of 690 nm, using a microplate reader.
LDH Release assay
Release of LDH into the medium is an indication of compromised membrane integrity (Koh & Choi, Reference Koh and Choi1987), which was measured using an in vitro LDH release assay kit (Biogenesis, Dorset, BH17 7DA, UK). At the time of assay, 100 μl media was collected from all test wells of a 96-well plate. Substrate mix (100 μl) was added to each sample and incubated at room temperature for 1 h. HCl (1 m, 50 μl) was then added to each well and the plate was centrifuged at 716×g for 5 min. Absorbance was measured at 492 nm using a microplate reader. LDH release was expressed as fold difference relative to the control.
Trypan Blue Exclusion assay
This assay is based on the principle that viable cells will exclude trypan blue dye, whereas dead or dying cells absorb the dye and, thus, appear blue (Aras et al. Reference Aras, Hartnett and Aizenman2008). At the time of assay, 1·5 ml Jurkat cells in media was centrifuged at 100×g for 5 min. The supernatant was removed and the cell pellet was re-suspended in PBS. Trypan blue (50 μl) was mixed with 50 μl cell suspension (1:1) and incubated at room temperature for 3 min. The mixture was loaded onto a haemocytometer and cells were analysed using a microscope.
BrdU Cell proliferation assay
DNA synthesis was assessed using the BrdU incorporation assay (ELISA kit; Roche, DE-68305, Mannheim, Germany), which is based on the incorporation of BrdU into the newly synthesised DNA, substituting for thymidine during DNA replication (Motobu et al. Reference Motobu, El-Abasy, Na and Hirota2002). Briefly, at the time of assay, 10 μl BrdU labelling solution was added to each well of a 96-well plate and the plates were incubated for a further 24 h. The cells were denatured and fixed using Fix/Denat solution for 30 min at 37°C. Anti-BrdU-POD was then added to the wells and incubated for a further 120 min at 20°C. Lastly, 100 μl substrate solution was added to each well and the plates were incubated at 20°C until colour development was sufficient. Twenty-five microliters of 1 m H2SO4 was added as a stopping solution to each well and absorbance was measured at 450 nm, using a microplate reader, with a reference wavelength of 690 nm.
Statistical analysis
Results are presented as mean values±se. Statistical analysis was evaluated by one-way ANOVA followed by Dunnett's test (GraphPad Prism 4.0, GraphPad Inc, San Diego, CA, USA). The level of statistical significance was taken at P<0·05 or P<0·01.
Results
Cell Viability
MTT assay:
In our previous study (Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009) we mentioned that the viability of Jurkat cells decreased significantly following incubation with increasing concentrations of the casein hydrolysates (a–h), with the exception of hydrolysate e (data not shown). Caco-2 cell viability significantly (P<0·05) decreased in cells that were supplemented with increasing concentrations of samples b, c, and f for 24 h (Table 1), although viability did not decrease below 65%.
Viability of Caco-2 cells incubated with casein hydrolysates (a–h) and sodium caseinate for 24 hFootnote †
Values are the mean±se for n=4 independent experiments
NaCN, sodium caseinate (which was only available at the time when using Caco-2 cells)
† Caco-2 cells (3×104 cells/cm2) were supplemented with increasing concentrations of casein hydrolysates (a–h) and sodium caseinate for 24 h
‡ Cell viability was determined by the MTT assay and expressed as a percentage of the control
* P<0·05 compared with control (0% v/v): one-way ANOVA, Dunnett's test
LDH Release assay:
The presence of the casein hydrolysates did not induce LDH release from Jurkat cells (data not shown). LDH release significantly (P<0·01) decreased in Caco-2 cells supplemented with casein hydrolysates a–h for 24 h (Table 2).
Lactate dehydrogenase (LDH) release from Caco-2 cells incubated with casein hydrolysates (a–h) and sodium caseinate for 24 hFootnote †
Values are the mean±se for n=3 independent experiments
NaCN, sodium caseinate
† Human Caco-2 cells (3×104 cells/cm2) were supplemented with increasing concentrations of casein hydrolysates (a–h) and sodium caseinate for 24 h
‡ Cytotoxicity was determined by the LDH release assay. LDH release is expressed as fold difference relative to control
* P<0·05 compared with control (0% v/v): one-way ANOVA, Dunnett's test
Trypan Blue Exclusion assay:
After 24 h incubation, membrane integrity, as measured by this assay, was not affected by the presence of NaCN and its hydrolysates in Jurkat cells (data not shown). There was good correlation between the Trypan Blue assay and the LDH release assay in Jurkat cells, hence membrane integrity was not assessed in Caco-2 cells using the Trypan Blue assay.
Cell growth
MTT assay:
There was a significant decrease in the growth of Jurkat cells supplemented with casein hydrolysates a–d, and f for 24 h (Table 3; Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009). Growth in Caco-2 cells supplemented with increasing concentrations of samples a, b, c, and f significantly decreased (P<0·05, P<0·01) after 24 h incubation (Table 3).
Growth of Caco-2 and Jurkat T cells supplemented with casein hydrolysates (a–h) and sodium caseinate for 24 hFootnote †
Values are the mean±se for n=4 independent experiments
NaCN, sodium caseinate (which was only available at the time when using Caco-2 cells)
† Cells were supplemented with increasing concentrations of samples for 24 h
§ Data that was not shown from a previous study (Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009)
‡ Cell growth was determined by the MTT assay, and expressed as MTT reduction index
* P<0·05, **P<0·01 compared with control: one-way ANOVA, Dunnett's test
Trypan Blue Exclusion assay:
After 24 h, cell density was not affected by the presence of casein hydrolysates in Jurkat cells (data not shown). As mentioned earlier, there was good correlation between the Trypan Blue assay and the LDH release assay in Jurkat cells; hence, cell density was not assessed in Caco-2 cells using the Trypan Blue assay.
BrdU Incorporation assay (DNA synthesis):
Casein and its hydrolysates exerted different effects on DNA synthesis in Jurkat cells. After 24 h supplementation, casein hydrolysates a and h, at a level of 0·5% (v/v) only, significantly (P<0·05) reduced DNA synthesis to 45·6% and 55·6% (Table 4). Following 24 h treatment of Caco-2 cells with casein hydrolysates, BrdU incorporation was not affected compared with control (Table 4).
Effects of casein hydrolysates (a–h) and sodium caseinate on DNA synthesis in Jurkat cells incubated for 24 hFootnote †
Values are the mean±se for n=4 independent experiments
NaCN, sodium caseinate
† Cells were supplemented with increasing concentrations of casein hydrolysates (a–h) and sodium caseinate for 24 h
‡ Cell growth was determined by the BrdU incorporation assay and expressed as a percentage of control (cells)
* P<0·05 compared with control: one-way ANOVA, Dunnett's test
Discussion
There is evidence that milk protein-derived peptides may have a direct effect on the viability of cancer cells (Mac Donald et al. Reference MacDonald, Thornton and Marshall1994; Hartmann & Meisel, Reference Hartmann and Meisel2004; Phelan et al. Reference Phelan, Aherne-Bruce, O'Sullivan, FitzGerald and O'Brien2009), which offers potential for their inclusion into functional foods as these peptides are also of nutritional importance (Meisel & Schlimme, Reference Meisel and Schlimme1996).
When assessing the therapeutic potential of any novel sample in vitro, it is important to consider the cell as a whole and to study the molecular mechanisms underlying different modulating activities caused by the sample on individual components within the cell (Gülden & Seibert, Reference Gülden and Seibert2003). To determine if casein hydrolysates influence the growth and viability of Jurkat cells and Caco-2 cells, four different assays were selected, namely the MTT, LDH release, Trypan Blue, and BrdU incorporation assays.
Both LDH release and Trypan Blue assays are based on the principle that cell death cascades alter membrane permeability (Aras et al. Reference Aras, Hartnett and Aizenman2008). LDH is a soluble cytosolic enzyme that is released into the culture medium following loss of membrane integrity, whereas the Trypan Blue assay measures the ability of cells to exclude dye from being taken up into their cytoplasm (Aras et al. Reference Aras, Hartnett and Aizenman2008). The Trypan Blue assay is very accurate; however it is very time-consuming when used as a screening assay (Durrieu et al. Reference Durrieu, Degraeve, Carnet-Pantiez and Martial2005). In the Jurkat T cell model there was good correlation between the results of the LDH release and the Trypan Blue assays, hence the Trypan Blue assay was not carried out on the Caco-2 cell model. LDH release was not negatively affected by the presence of casein hydrolysates in both the Jurkat cells and Caco-2 cells. Hence, the casein hydrolysates did not have any major effect on membrane integrity in these selected cell models.
In the MTT assay, the reduction of MTT by a mitochondrial reductase takes place in viable cells when mitochondrial reductase enzymes are active, which is used as a measure of mitochondrial activity as well as viability and growth of cells (Mosmann, Reference Mosmann1983). The BrdU incorporation assay was selected to analyse DNA synthesis since this assay is based on the incorporation of BrdU instead of thymidine into DNA during DNA synthesis. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that are actively replicating their DNA (Motobu et al. Reference Motobu, El-Abasy, Na and Hirota2002).
In Jurkat cells, the MTT and BrdU assays correlated well as the same trend was seen from both assays. Samples a and h tended to reduce rates of cell division in Jurkat cells. This shows that some casein samples have specific effects on BrdU incorporation during DNA synthesis. Meisel & Günther (Reference Meisel and Günther1998) showed that bioactive sequences of casein and a lyophilized extract of Gouda cheese had both apoptotic and anti-proliferative activity in HL-60 cells at concentrations as low as 1 pmol/l. In addition, Roy et al. (Reference Roy, Wantanabe and Tamai1999) reported that bovine skimmed milk digested with the yeast Saccharomyces cerevisiae had an anti-proliferative activity toward human leukaemia HL-60 cells. On the other hand, Azuma et al. (Reference Azuma, Nagaune, Ishino, Mori, Kaminogawa and Yamauchi1989) found that some human casein tryptic fragments could stimulate DNA synthesis in BALB/c3T3 cells. MacDonald et al. (Reference MacDonald, Thornton and Marshall1994) showed that bacterial hydrolysis of casein using commercial yogurt starter cultures yielded bioactive peptides that reduced [3H]thymidine incorporation in IEC-6 cells and increased DNA synthesis in Caco-2 cells, thus suggesting cell-specific effects. Furthermore, Ringseis et al. (Reference Ringseis, Matthes, Lehmann, Becker, Schöps, Ulbrich-Hofmann and Eder2005) showed that certain hydrolysates from casein inhibited endothelial cell (HaoEC) proliferation whereas other hydrolysates had no effect.
Recently, Ramos-Mandujano et al. (Reference Ramos-Mandujano, Weiss-Steidera, Meloa, Córdovaa, Ledesma-Martíneza, Bustos, Silvestre, Aguiňiga, Sosa, Martínez, Sánchez, García and Santiago-Osorio2008) showed that NaCN and its individual casein components displayed different inhibitory characteristics on the proliferation of 32Dcl3 and WEHI-3 myeloid cells. At 0·1 mg/ml, NaCN exerted the strongest growth inhibition on 32Dcl3 cells but the inhibition was not as potent on WEHI-3 cell growth. This variation in findings may be explained by the different protein concentrations and the different cell lines used in these studies (Hagiwara et al. Reference Hagiwara, Shinoda, Fukuwatari and Shimamura1995; Ramos-Mandujano et al. Reference Ramos-Mandujano, Weiss-Steidera, Meloa, Córdovaa, Ledesma-Martíneza, Bustos, Silvestre, Aguiňiga, Sosa, Martínez, Sánchez, García and Santiago-Osorio2008). In addition, the types and sources of peptide can have individual effects on cells (Hagiwara et al. Reference Hagiwara, Shinoda, Fukuwatari and Shimamura1995; Meisel & Günther, Reference Meisel and Günther1998). In the present study, an explanation for the activity profiles of the hydrolysates may be that they relate to the different proteolytic enzyme preparations used in their manufacture (manuscript in preparation).
In conclusion, NaCN had no significant effect on the viability and growth of Caco-2 whereas its associated casein hydrolysates (a–h) had differing effects on cell viability and proliferation. Hydrolysate a had an inhibitory effect on proliferation while e had a promotive effect, which was cell-specific. The casein hydrolysates seemed to be more toxic to Jurkat cells compared with Caco-2 cells. A vast majority of tumour promoters are potent inhibitors of apoptosis (Wright et al. Reference Wright, Zhong and Larrick1994) and therefore apoptosis-inducing peptides can be classified as probable anti-carcinogens. According to Meisel & FitzGerald (Reference FitzGerald, Meisel, Fox and McSweeney2003), effects on both cell viability and immune function may be a mechanism by which bioactive peptides exert protective effects in cancer development. Our data add to our previous findings and suggest that the eight distinct casein hydrolysates have varying effects on two different cell culture models. Further research is warranted to assess the effects of the casein hydrolysates on the cell cycle of these two cell lines (Elion, Reference Elion2001).
Funding for this research was provided under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries & Food, Ireland. The authors have no conflicts of interest, financial or otherwise.
