Article

Anticancer Activity of Buttermilk Against SW480 Colon Cancer Cells is Associated with Caspase-Independent Cell Death and Attenuation of Wnt, Akt, and ERK Signaling:

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Abstract

Buttermilk is a rich source of milk fat globule membrane (MFGM) fragments assembled from bioactive polar lipids and proteins that originate from bovine mammary epithelial cells. The objective of this study was to examine growth-modulatory effects of experimental buttermilks varying in sphingolipid and phospholipid composition on a colon cancer cell line of human origin. Buttermilks were prepared from washed and unwashed cream using gravity or centrifugation. Compositional analysis showed that sphingomyelin (SM) (10.4-29.5%) and lactosylceramide (LacCer) (1.2-44.3%) were the predominant sphingolipids detected. Experimental samples inhibited in vitro growth of SW480 colon cancer cells in a dose-dependent manner. Antiproliferative activity was selective toward cancer cells. A fraction enriched in LacCer (44.3%), obtained by microfiltration induced caspase-independent cell death as evident by phosphatidylserine externalization, increased percentage of degraded DNA, and loss of mitochondrial membrane potential in SW480 cells. This fraction downregulated growth-signaling pathways mediated by β-catenin, phosphorylated Akt (serine/threonine-specific protein kinase), ERK1/2 (extracellular signal-regulated kinase), and c-myc. This study is to our knowledge the first to screen buttermilk samples that vary in polar lipid composition for antiproliferative activity in vitro.

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... Buttermilk is a traditional dairy beverage which is a byproduct of butter manufacture. Buttermilk is a rich source of milk fat globule membrane fragments (155). ...
... A fraction enriched in lactosylceramide (44.3%), induced caspase-independent apoptosis as evident by phosphatidylserine exposure, increased levels of degraded DNA, and loss of mitochondrial membrane potential in SW480 cells. This fraction also downregulated b-catenin-mediated growth-signaling pathways, phosphorylated Akt (serine/threonine-specific protein kinase), ERK1/2 (extracellular signal-regulated kinase), and c-myc (155). ...
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Cancer is the second leading cause of death with profound socio-economic consequences worldwide. Growing evidence suggests the crucial role of diet on cancer prevention and treatment. In Traditional Persian Medicine (TPM) there is a major focus on contribution of special diet and foods to cancer management. In the present article, the cytotoxic and antitumor activities of several food items including plants and animal products recommended by TPM as anticancer agents are discussed. Strong evidence supports the anticancer effects of beetroot (Beta vulgris) and its major compound betanin, cinnamon and cinnamaldehyde, barley (H. vulgare) and its products, extra-virgin olive oil, black pepper (P. nigrum) and its piperine, grapes (V. vinifera) and its compound resveratrol, ginger and its compound 6-gingerol, whey protein, fish, and honey. However, additional pharmacological studies and clinical trials are needed to elucidate their molecular and cellular mechanisms of actions, frequency, and amount of consumption, possible adverse effects, and optimum preparation methods. Moreover, studying mechanisms of actions of the bioactive compounds present in the discussed food items can be helpful in identifying and development of new anticancer agents.
... TGF-β is also regarded as a T cell regulator in colon cancer and may suppress tumor progression in colon cancer (33,34). In addition, previous studies have demonstrated that the anticancer activity of buttermilk against sw480 colon cancer cells is associated with caspase-independent cell death via the attenuation of the Wnt, protein kinase B (AKT) and ERK signaling pathways, and the reactivation of ERK and AKT confers the apoptotic resistance of colon cancer cells (35,36). Furthermore, a previous study also revealed that growth suppression in colon cancer cells is associated with RAS/ERK by bone morphogenetic protein-mothers against decapentaplegic homolog signaling (37). ...
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Non‑small‑cell lung cancer (NSCLC) is one of the most common malignancies that is responsible for a high level of cancer‑associated mortalities worldwide. Previous evidence has shown that Calotropin is an upstream activator of protein kinase B, which can further inhibit the growth and promote the apoptosis of NSCLC cells. In the present study, the efficacy of Calotropin on growth, aggressiveness and apoptosis of NSCLC cells was investigated, as well as the potential underlying mechanism. The results demonstrated that Calotropin inhibited H358 cell growth, migration and invasion. Flow cytometry assay showed that Calotropin promoted the apoptosis of H358 cells in vitro. Western blot analysis demonstrated that Calotropin inhibited fibronectin (FN), Vimentin (VIM) and E‑cadherin (Eca) protein expression levels in H358 cells in vitro. In addition, Calotropin treatment upregulated pro‑apoptosis gene expression, including caspase‑3, caspase‑8 and apoptotic protease activating factor‑1, and downregulated anti‑apoptosis gene expression, including P53, B‑cell lymphoma (Bcl) 2 and Bcl‑2‑like protein 2 in H358 cells. The results also revealed that the expression levels of cytotoxic T‑lymphocyte associated antigen 4 (CTLA‑4) were decreased by Calotropin treatment in H358 cells. Analyses of the underlying mechanism indicated that Calotropin inhibited transforming growth factor‑β (TGF‑β) and extracellular signal‑regulated kinase (ERK) expression. Overexpression of CTLA‑4 inhibited Calotropin‑mediated downregulation of TGF‑β and ERK expression in H358 cells. In vivo assay revealed that Calotropin administration significantly inhibited tumor growth and prolonged animal survival over the 120‑day observation period. Immunohistochemistry demonstrated that the number of apoptotic cells increased and the expression levels of CTLA‑4 were decreased in the Calotropin‑treated tumor group when compared with control. In addition, the expression levels of TGF‑β and ERK were downregulated in the Calotropin‑treated tumor group compared with control. In conclusion, the results of the present study indicated that Calotropin administration regulated NSCLC apoptosis by downregulating the CTLA‑4‑mediated TGF‑β/ERK signaling pathway, suggesting that Calotropin may be a potential anti‑cancer agent for the treatment of NSCLC.
... The fraction containing higher amounts of lactosylceramide (44.3%) attained by microfiltration, encouraged caspase-independent cell death as evidenced by phosphatidylserine externalisation, increased the proportion of degraded DNA and loss of mitochondrial membrane potential in SW480 cells. This fraction down controlled growth-signalling pathways mediated by bcatenin, phosphorylated ERK1/2 (extracellular signal-regulated kinase), Akt (serine/threonine-specific protein kinase), and c-Myc regulator gene (Kuchta-Noctor et al. 2016). ...
Article
Buttermilk is one of the most important by‐products of the dairy industry. Approximately 2 million tonnes were produced within the European Union in 2015. Recently, buttermilk has gained increasing attention due to its unique structure, characteristics, and promising applications. It is classified as a functional food because it contains water‐soluble components, polar lipids and milk fat globule membranes. This review is focused on buttermilk composition, global production, and applications in several food industries. Furthermore, the beneficial characteristics of buttermilk for human health are highlighted.
... Similarly, buttermilk polar lipids inhibited the growth of SW480 colon cancer cells in a dose-dependent manner. This study determined that a sphingolipid fraction mainly composed of lactosylceramide downregulated growth-signaling pathways mediated by βcatenin, extracellular signal-regulated kinase 1/2, phosphorylated serine/threonine-specific protein kinase, and cmyc (153). Extensive study of sphingomyelin and related compounds confirms that they are central to control of cell growth, differentiation, migration, and apoptosis, and may have therapeutic value (154,155). ...
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Cancer is the second leading cause of mortality worldwide. The role of unresolved inflammation in cancer progression and metastasis is well established. Platelet-activating factor (PAF) is a key proinflammatory mediator in the initiation and progression of cancer. Evidence suggests that PAF is integral to suppression of the immune system and promotion of metastasis and tumor growth by altering local angiogenic and cytokine networks. Interactions between PAF and its receptor may have a role in various digestive, skin, and hormone-dependent cancers. Diet plays a critical role in the prevention of cancer and its treatment. Research indicates that the Mediterranean diet may reduce the incidence of several cancers in which dietary PAF inhibitors have a role. Dietary PAF inhibitors such as polar lipids have demonstrated inhibitory effects against the physiological actions of PAF in cancer and other chronic inflammatory conditions in vitro and in vivo. In addition, experimental models of radiotherapy and chemotherapy demonstrate that inhibition of PAF as adjuvant therapy may lead to more favorable outcomes. Although promising, there is limited evidence on the potential benefits of dietary PAF inhibitors on cancer prevention or treatment. Therefore, further extensive research is required to assess the effects of various dietary factors and PAF inhibitors and to elucidate the mechanisms in prevention of cancer progression and metastasis at a molecular level.
... Furthermore, the numerous health and nutritional benefits associ- ated with PLs have been extensively reviewed (Contarini & Povolo, 2013;Dewettinck et al., 2008;Spitsberg, 2005) along with both in vivo and in vitro investigations of associated effects of PLs on health. PLs have been demonstrated to exhibit anticancer effects, particularly suppression of colon tumours (Berra, Colombo, Scottocornola, & Giacosa, 2002;Hertervig, Nilsson, Cheng, & Duan, 2003;Kuchta-Noctor, Murray, Stanton, Devery, & Kelly, 2016;Parodi, 2003). Other health benefits associated with PLs include myelination of the central nervous system (Oshida et al., 2003), development, activation and regulation of the immune sys- tem ( Cinque et al., 2003;Mills, Ross, Hill, Fitzgerald, & Stanton, 2011), and reduced absorption of cholesterol (Eckhart, Wang, & Donovan, 2002). ...
Article
A novel purified dairy phospholipid (PL) extract was generated from a buttermilk powder (BMP) substrate combining techniques of enzymatic hydrolysis, ultrafiltration (UF) and supercritical fluid extraction (SFE). This combined process yielded a purified lipid extract with 56.34 ± 0.15% total PL in dry matter, depleted of protein and lactose. Application of this PL enriched extract in vitro demonstrated a neurotrophic effect of dairy PLs on cortical neurite outgrowth. At a concentration of 150 µg mL⁻¹, a 43% stimulated increase in neurite outgrowth was observed compared with the control (0% stimulation). % stimulation decreased to 12% upon increasing the PL dose to 300 µg mL⁻¹. The results of this study demonstrate that a dairy PL extract from buttermilk promotes the development and outgrowth of cortical neurons and complements the outcomes of new research (Matsuo, 2016) demonstrating that PLs are further stimulated in this role by omega PUFAs present.
... In the butter-making process, the butter is churned from the cultured cream, and the remnant liquid is known as buttermilk [10]. Buttermilk contains more phospholipids than skim milk because it contains a milk fat globule membrane; phospholipids in buttermilk have potential cholesterol-and blood-pressure-lowering as well as anti-cancer effects [11,12]. Milk phospholipids have been shown to downregulate the expression of nuclear factor kappa-B (NF-κB) in UVB-irradiated hairless mice, demonstrating their skin-protective effect [13]. ...
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This study investigated the protective effects of glucocerebroside-containing buttermilk concentrated powder (GCBM) on oxidative stress and photoaging in ultraviolet B (UVB)-irradiated hairless mice. We measured antioxidant enzyme activities, collagen synthesis-related pathways, and moisturizing-related factors in the dorsal skin of mice. We observed that dietary supplementation with GCBM increased antioxidant enzyme activity and decreased pro-inflammatory cytokine expression in the UVB-irradiated dorsal skin. Furthermore, dietary supplementation with GCBM inhibited wrinkle formation by suppressing the JNK/c-FOS/c-Jun/MMP pathway and stimulating the TGF-βRI/Smad3/procollagen type I pathway. Dietary supplementation with GCBM also increased skin moisturization by stimulating hyaluronic acid and ceramide synthesis in the dorsal skin. Therefore, buttermilk powder supplementation helps prevent photoaging and can be used as an effective component in developing anti-photoaging products.
... PS is associated with cognitive function and releasing stress, and is replaced by inactive cholesterol as the brain ages [11,12]. SM has been found to be effective in inhibiting colon tumors [13]. Also, MPLs have been implicated in mitigating the risks of Alzheimer's disease and repairing cognitive ability [14], restoring immunological defenses, reducing the incidence of cardiovascular diseases [15,16], and reducing cholesterol absorption and total liver lipids [17]. ...
Article
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Milk phospholipids (MPLs) have been used as ingredients for food fortification, such as bakery products, yogurt, and infant formula, because of their technical and nutritional functionalities. Starting from either buttermilk or beta serum as the original source, this review assessed four typical extraction processes and estimated that the life-cycle carbon footprints (CFs) of MPLs were 87.40, 170.59, 159.07, and 101.05 kg CO2/kg MPLs for membrane separation process, supercritical fluid extraction (SFE) by CO2 and dimethyl ether (DME), SFE by DME, and organic solvent extraction, respectively. Regardless of the MPL content of the final products, membrane separation remains the most efficient way to concentrate MPLs, yielding an 11.1–20.0% dry matter purity. Both SFE and solvent extraction processes are effective at purifying MPLs to relatively higher purity (76.8–88.0% w/w).
... Several studies have been performed in vitro to evaluate the effects of milk phospholipids on cancer cell lines. Kuchta-Noctor et al. [92] examined the growth-modulatory effects of buttermilk phospholipids and sphingolipid fractions obtained by membrane filtration on SW480 colon cancer cells. Their results confirmed that all of the derived buttermilk fractions proved to be antiproliferative toward SW480 cells. ...
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p>Milk is one of the most important foods for mammals, because it is the first form of feed providing energy, nutrients and immunological factors. In the last few years, milk lipids have attracted the attention of researchers due to the presence of several bioactive components in the lipid fraction. The lipid fraction of milk and dairy products contains several components of nutritional significance, such as ω-3 and ω-6 polyunsaturated fatty acids, CLA, short chain fatty acids, gangliosides and phospholipids. Prospective cohort evidence has shown that phospholipids play an important role in the human diet and reinforce the possible relationship between their consumption and prevention of several chronic diseases. Because of these potential benefits of phospholipids in the human diet, this review is focused on the recent advances in phospholipids from colostrum, milk and dairy by-products. Phospholipid composition, its main determination methods and the health activities of these compounds will be addressed.</p
... Castro-Gómez et al. (2016) reviewed the biological roles of phospholipids and concluded that their biological actions can be exploited in the adjunct therapy of widely diffused pathologies, such as neurodegeneration or the metabolic syndrome. The protein constituents of MFGM are also reported to be antiproliferative (Bourlieua et al., 2018;Castro-Gómez et al., 2016;Kuchta-Noctor, Murray, Stanton, Deverya, & Kelly, 2016;Tomé-Carneiro et al., 2018). ...
Article
Enhancing the nutritional/economical value of small/medium size dairy enterprises product’s is important for their survival in a competitive market. Probiotic fermented dairy products is a trend driven by consumer´s acceptance of such products. This study intends the production of probiotic butter with the side possibility of buttermilk valorisation by confirming the minimum microbial counts required to claim the probiotic properties of the novel products. Pasteurized milk inoculated with commercial probiotic culture was added to cream, alone or combined with aromatic starters and fermented during 34 h. Probiotic microorganisms counts evaluated throughout cream fermentation present a final value of around 8Log CFUmL‐1. Butter presented values higher than 6Log CFUg‐1, within the reference values required to be considered as a probiotic, being softer and with higher humidity than conventional butter. Fermented buttermilk, having the potential commercialization as a probiotic dairy beverage, was also obtained representing a significant contribution to the circular economy.
... MPLs are relevant to human nutrition since their intake has shown beneficial effects on cognitive performance (Hellhammer, Waladkhani, Hero, & Buss, 2010). Moreover, the therapeutic ability of MPLs have been documented in experimental arthritis in rats (Hartmann et al., 2009), anticancer activity of colon cancer cells (Kuchta-Noctor, Murray, Stanton, Devery, & Kelly, 2016), antioxidant activity in vitro assays , and protection against gastrointestinal infections (Küllenberg, Taylor, Schneider, & Massing, 2012). Bioactivity of MPLs has been recently reviewed elsewhere (Küllenberg et al., 2012;Ortega-Anaya & Jiménez-Flores, 2019). ...
Article
We report improvements in the extraction of milk phospholipids (MPLs) from beta-serum, a dairy byproduct, by applying ultrasound prior to a tertiary amine extraction. Three acoustic intensities (15.53 ± 1.20, 31.76 ± 2.46, or 44.56 ± 3.47 W cm⁻²) were applied for 4 min before the amine extraction (N,N-dimethylcyclohexylamine, CyNMe2). The extracted lipids were fractionated by solid phase-microextraction, and the recovered MPLs were quantified by HPLC-CAD. An acoustic intensity of 44.56±3.47 W cm⁻² followed by CyNMe2 extraction (12/1, solvent to ratio ratio) yielded 69.67 ± 3.45% of MPLs, while only 7.57 ± 0.59% were recovered without ultrasound. The fraction of MPLs was made of phosphatidylinositol (32%), phosphatidylethanolamine (30%), and sphingomyelin (37%). Scanning electron images and particle size revealed significant disruption of the complex arrangement between membrane proteins and MPLs, which may help to release the MPLs into the aqueous medium.
... The ability of milk PLs (MPLs) to scavenge free radicals has been demonstrated in vitro by Huang et al. (2020a). Several other health claims have been investigated over the past years, including anti-inflammatory effect (Hartmann et al., 2009), protecting effect against gastrointestinal infections (Hellhammer et al., 2010), and improved immunological functions (Kuchta-Noctor et al., 2016). Most of the health claims of PLs have been related to PS and SM, whose concentration is relatively high in milk fat (12 and 24% of the total PLs, respectively) (Huang et al., 2020b). ...
Article
Dairy byproducts represent a rich source of phospholipids with the potential for isolation and further commercialization. In this study, we extracted phospholipids from two dairy byproducts (buttermilk and beta-serum) using a tertiary amine (N,N-dimethylcyclohexylamine, CyNMe2) as a switchable hydrophilicity solvent. For comparison, the phospholipids were extracted via Folch (chloroform: methanol). CyNMe2 extraction resulted in recovery values of 98.66 ± 0.89 and 7.67 ± 0.51% for buttermilk and beta-serum, respectively. Insights into the extraction mechanism were obtained by the analysis of protein profile, particle size, zeta potential, and microstructure. CyNMe2 generated microfractures and hollow openings in the solid matrix through ion pair formation that releases the phospholipids from the solid matrix. In comparison to Folch, CyNMe2 resulted in higher concentration of phospholipids (up to 9-fold increment), and it provided a different relative distribution, where phosphatidylcholine and phosphatidylinositol were the predominant phospholipids. The outcomes of this study help to gain insights into the extraction mechanism by which CyNMe2 acts, and develop extraction strategies for dairy byproducts.
... More specifically, cheese wheys have proteins of high biological value regulating various physiological functions in human body such as blood pressure, glycemic response, inflammatory processes, and improvement of immune system (Athira et al., 2015). In addition, ricotta wheys have high lactose content (Sansonetti et al., 2009), and butter wheys have less lactose and the of phospholipids, which play an important role in many metabolic processes in human organisms, including in vitro anticancer properties according to Kuchta-Noctor et al. (2016). ...
... (Table 3). It has been reported that a dairy-based preparation enriched in lactosylceramide was found to induce the demise of colon cancer cells [42]. ...
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For non-bovine milks, information regarding bioactive lipids is fragmented, unreliable or unavailable. The purpose of the current study was to analyse bioactive lipids in the milk of dairy animals using modern analytical methods to achieve the most reliable results. Bioactive lipids in human milk were also analysed and used as a reference. A suite of modern analytical methods was employed, namely High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS), Gas Chromatography (GC) and Nuclear Magnetic Resonance (NMR). The total lipid content was determined, and phospholipid, fatty acid, neutral glycosphingolipids and ganglioside (GM3 and GD3) levels were measured. Lipid classes in selected milks were reliably characterised for the first time, including gangliosides in deer, camel and sheep; cerebrosides in deer, camel and buffalo; plasmalogens in deer, buffalo and goat and phospholipids in deer. Our study demonstrated the advantage of utilising a range of analytical techniques in order to characterise a diverse set of bioactive lipids.
... (Zanabria et al. 2013). Mechanistic insights were given by Kuchta-Noctor et al. who demonstrate that a buttermilk-derived polar lipid fraction rich in SM and lactosylceramide induced caspase-independent cell death by a mechanism including downregulation of growth-signaling pathways mediated by β-catenin, phosphorylated Akt ERK1/2 and c-myc (Kuchta-Noctor et al. 2016). Antiproliferative activity of freshly isolated MFGM was sensitive to heat treatment, hydrolysis and/or phospholipase A2 treatment (Zanabria, Griffiths, and Corredig 2020), illustrating that processing may affect MFGM functionality and that overall antiproliferative activity cannot be explained by a single bioactive component. ...
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Polar lipids including glycerophospholipids and sphingophospholipids are important nutrients and milk is a major source, particularly for infants. This systematic review describes the human and bovine milk polar lipid composition, structural organization, sources for formulation, and physiological functionality. A total of 2840 records were retrieved through Scopus, 378 were included. Bovine milk is a good source of polar lipids, where yield and composition are highly dependent on the choice of dairy streams and processing. In milk, polar lipids are organized in the milk fat globule membrane as a tri-layer encapsulating triglyceride. The overall polar lipid concentration in human milk is dependent on many factors including lactational stage and maternal diet. Here, reasonable ranges were determined where possible. Similar for bovine milk, where differences in milk lipid concentration proved the largest factor determining variation. The role of milk polar lipids in human health has been demonstrated in several areas and critical review indicated that brain, immune and effects on lipid metabolism are best substantiated areas. Moreover, insights related to the milk fat globule membrane structure-function relation as well as superior activity of milk derived polar lipid compared to plant-derived sources are emerging areas of interest regarding future research and food innovations.
... These two diseases of the gut are linked, as patients with IBD have a greater risk of developing colorectal cancer [171]. The health effects of milk PLs on colorectal cancer and colitis are summarized in Table 7. Kutchta-Noctor et al. [173] observed the effects of buttermilk, containing SM, lactosylceramide (LacCer), and ceramide, on growth inhibition of SW480 human colon cancer cells and noncancerous fetal human colon (FHC) cells. They reported that buttermilk containing SM and LacCer led to growth inhibition of SW480 cells and was selective towards cancer cells, with no effect on FHC cell growth. ...
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Dairy phospho- and sphingolipids are gaining interest due to their nutritional and technological properties. A new HPLC method, using an evaporative laser light-scattering detector, was developed, which enabled excellent separation of glucosylceramide, lactosylceramide, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine in less than 21 min, including the regeneration of the column. No loss of column performance was observed after 1500 runs because an acid buffer was used. The output signal of the evaporative laser light scattering detector was highly dependent of the flow of the carrier gas and the temperature of the nebulizer, and was maximized by means of a response surface experimental design. Finally, raw milk, cream, butter, buttermilk, Cheddar whey, quarg, and Cheddar cheese were analyzed for their polar lipid content. The absolute values varied substantially (0.018 to 0.181 g/100 g of product). Significant differences were found in the relative content of each polar lipid class among the analyzed products.
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Sphingolipids are in all eukaryotic cells and modulate cell growth, differentiation, and transformation; however, little is known about the physiological effects of their consumption. Mice were fed diets supplemented with milk sphingomyelin to determine effects on colon carcinogenesis. Cancer was initiated in CF1 mice by 1,2-dimethylhydrazine. Mice were then fed AIN76A diets supplemented with 0.025 to 0.1 g sphingomyelin/100 g for 28 wk until the supply of sphingomyelin was depleted and then fed unsupplemented diet for 24 wk. Sphingomyelin did not affect weight gain. Mice fed sphingomyelin had a 20% incidence of colon tumors compared with 47% in controls (P = 0.08 for all sphingomyelin-fed mice vs. controls). Tumors were adenomas or adenocarcinomas and located in the distal third of the colon. In shorter-term studies, colonic epithelial cell proliferation was significantly greater than controls in mice fed 0.025 g sphingomyelin/100 g diet, but not in those fed higher amounts of sphingomyelin. The number of aberrant crypts was significantly lower in 1,2-dimethylhydrazine-treated mice fed 0.05 g sphingomyelin/100 g diet than in controls. These results demonstrate that consumption of sphingomyelin affects the behavior of colonic cells. Because sphingolipids are present in food, the reduction in 1,2-dimethylhydrazine-induced premalignant lesions and the incidence of colon tumors in CF1 mice implies that these compounds may be another important class of nutritional modulators of carcinogenesis.
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In vitro miniaturized colorimeteric assays are extensively used in the determination of a substance’s ability to enhance cell growth or promote cell death. The widespread use of colorimeteric endpoint assays in the determination of cell number, the most common measure of cell growth, is due to their simplicity and sensitivity and also to their ability to be scaled up using a semi-automated high-throughput system.
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The milk fat globule membrane (MFGM) surrounds fat globules, protects them against lipolysis and disperses the milk fat in the milk plasma. Besides their structural and emulsifying roles, in vivo and in vitro studies have demonstrated that phospholipids and sphingolipids of MFGM possess cancer risk‐reducing properties. Several reports attribute its chemopreventive activity to products of sphingomyelin hydrolysis, which affect multiple cellular targets that control cell growth, differentiation and apoptosis. With knowledge on the potential health benefits of MFGM lipids and proteins, dairy industries could in the future address their research in developing new functional dairy products enriched in beneficial MFGM components.
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The milk fat globule membrane (MFGM) has gained a lot of attention recently, due to the growing interest in its nutritional and technological properties. The whole membrane as well as the separate lipid and protein components have great potential for new product applications with unique nutritional and technological properties. This review focuses on the nutritional and technological aspects of the MFGM material, but also gives an overview of the gathered information about the composition, structure and isolation methods of the MFGM from different dairy sources.
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The effect of cream pasteurization on the composition and microstructure of buttermilk after pasteurization, evaporation and spray-drying was studied. The composition of milk fat globule membrane (MFGM) isolated from buttermilk samples was also characterized. Pasteurization of cream resulted in higher lipid recovery in the buttermilk. Spray-drying of buttermilk had a significant effect on phospholipid content and composition. After spray-drying, the phospholipid content decreased by 38.2% and 40.6%, respectively in buttermilk from raw or pasteurized cream when compared with initial buttermilks. Pasteurization of cream resulted in the highest increase in whey protein recovery in MFGM isolates compared with all other processing steps applied on buttermilk. A reduction in phospholipid content was also observed in MFGM isolates following spray-drying. Transmission electron microscopy of the microstructure of buttermilks revealed extremely heterogeneous microstructures but failed to reveal any effect of the treatments.
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The bovine milk fat globule membrane (MFGM) contains several antimicrobial components with proven efficacy in vitro, but in vivo evidence is scarce. The present study was performed to determine the efficacy of the bovine MFGM in vivo. Rats were fed diets based on bovine skimmed milk powder (low in MFGM) or bovine sweet buttermilk powder (high in MFGM). After dietary adaptation, rats were orally infected with Salmonella enteritidis or Listeria monocytogenes. Whereas sweet buttermilk powder did not protect rats against infection with S. enteritidis, it protected against L. monocytogenes, as shown by a lower colonisation and translocation of this pathogen. Protection coincided with higher listericidal capacity of gastric and caecal contents. The digestion products of phosphoglycerides and sphingomyelin are bactericidal in vitro. To study their role, rats were fed diets containing either 0·1 % phosphatidylcholine or sphingomyelin, or a control diet. After dietary adaptation, rats were infected with L. monocytogenes. Since Listeria colonisation was not affected by these diets, phosphoglycerides and sphingomyelin are not involved in the protective effect of sweet buttermilk. Additional in vitro experiments were performed to further explore the mechanism of the beneficial effects of sweet buttermilk. Inhibition of the adherence of L. monocytogenes to the intestinal mucosa is the most likely explanation, since sweet buttermilk powder inhibited the binding of L. monocytogenes in both a haemagglutination assay and a Caco-2 cell adherence assay. In conclusion, sweet buttermilk powder, which is rich in MFGM, protects against L. monocytogenes infection in rats, probably by preventing adherence of this pathogen to the intestinal mucosa.
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Milk fat globule membrane (MFGM) is a biopolymer composed primarily of membrane proteins and lipids that surround the fat globules in milk. Although it is considered to have potential as a bioactive ingredient, few feeding studies have been conducted to measure its potential benefits. The aim of this investigation was to determine if dietary MFGM confers protection against colon carcinogenesis compared to diets containing corn oil (CO) or anhydrous milk fat (AMF). Male, weanling Fischer-344 rats were randomly assigned to one of three dietary treatments that differed only in the fat source: (1) AIN-76A diet, corn oil; (2) AIN-76A diet, AMF; and (3) AIN-76A diet, 50% MFGM, 50% AMF. Each diet contained 50 g/kg diet of fat. With the exception of the fat source, diets were formulated to be identical in macro and micro nutrient content. Animals were injected with 1,2-dimethylhydrazine once per week at weeks 3 and 4, and fed experimental diets for a total of 13 weeks. Over the course of the study dietary treatment did not affect food consumption, weight gain or body composition. After 13 weeks animals were sacrificed, colons were removed and aberrant crypt foci (ACF) were counted by microscopy. Rats fed the MFGM diet (n = 16) had significantly fewer ACF (20.9 +/- 5.7) compared to rats fed corn oil (n = 17) or AMF (n = 16) diets (31.3 +/- 9.5 and 29.8 +/- 11.4 respectively; P < 0.05). Gene expression analysis of colonic mucosa did not reveal differential expression of candidate colon cancer genes, and the sphingolipid profile of the colonic mucosa was not affected by diet. While there were notable and significant differences in plasma and red blood cell lipids, there was no relationship to the cancer protection. These results support previous findings that dietary sphingolipids are protective against colon carcinogenesis yet extend this finding to MFGM, a milk fat fraction available as a food ingredient.
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Sphingolipid metabolites are generated throughout the intestinal tract after hydrolysis of orally administered complex sphingolipids and significantly suppress colon cancer in carcinogen-treated CF1 mice. In the present study, the mechanisms of tumor suppression by dietary sphingolipids were investigated. Changes in select genes that are critical in early stages of colon cancer were analyzed in the colonic mucosa of dimethylhydrazine-treated CF1 mice fed AIN76A diet with or without 0.05% sphingomyelin (SM). Supplementation with SM did not significantly alter mRNA levels of most of the selected genes. However, a downregulation of beta-catenin (p = 0.007) and increased protein levels of connexin-43 (p = 0.017) and Bcl-2 (p = 0.033) were observed in SM-fed animals. This suggests that sphingolipids may be regulating specific post-transcriptional events to reverse aberrant expression of individual proteins. Since the dysregulation of beta-catenin metabolism and its transcriptional activity in addition to a decreased intercellular communication has been causally linked to the development of colon cancer while a low Bcl-2 expression is associated with a worse prognosis in colon cancer, the reversal of these early changes may be important events in the prevention of colon cancer by orally administered sphingolipids, and may provide specific molecular biomarkers for sphingolipid efficacy in vivo.
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Sphingolipids are in all eukaryotic cells and modulate cell growth, differentiation, and transformation; however, little is known about the physiological effects of their consumption. Mice were fed diets supplemented with milk sphingomyelin to determine effects on colon carcinogenesis. Cancer was initiated in CF1 mice by 1,2-dimethylhydrazine. Mice were then fed AIN76A diets supplemented with 0.025 to 0.1 g sphingomyelin/100 g for 28 wk until the supply of sphingomyelin was depleted and then fed unsupplemented diet for 24 wk. Sphingomyelin did not affect weight gain. Mice fed sphingomyelin had a 20% incidence of colon tumors compared with 47% in controls (P = 0.08 for all sphingomyelin-fed mice vs. controls). Tumors were adenomas or adenocarcinomas and located in the distal third of the colon. In shorter-term studies, colonic epithelial cell proliferation was significantly greater than controls in mice fed 0.025 g sphingomyelin/100 g diet, but not in those fed higher amounts of sphingomyelin. The number of aberrant crypts was significantly lower in 1,2-dimethylhydrazine-treated mice fed 0.05 g sphingomyelin/100 g diet than in controls. These results demonstrate that consumption of sphingomyelin affects the behavior of colonic cells. Because sphingolipids are present in food, the reduction in 1,2-dimethylhydrazine-induced premalignant lesions and the incidence of colon tumors in CF1 mice implies that these compounds may be another important class of nutritional modulators of carcinogenesis.
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n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.
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The activation of beta-catenin to an oncogenic state can result from the inactivation of the tumor suppressor adenomatous polyposis coli (APC), by direct mutation in the beta-catenin gene, or by the activation of wnt receptors. Once activated, beta-catenin most likely promotes tumor progression through its persistent interaction with one or more of its numerous downstream targets.
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Eukaryotic organisms as well as some prokaryotes and viruses contain sphingolipids, which are defined by a common structural feature, i.e. , a "sphingoid base" backbone such as D-erythro-1,3-dihydroxy, 2-aminooctadec-4-ene (sphingosine). The sphingolipids of mammalian tissues, lipoproteins, and milk include ceramides, sphingomyelins, cerebrosides, gangliosides and sulfatides; plants, fungi and yeast have mainly cerebrosides and phosphoinositides. The total amounts of sphingolipids in food vary considerably, from a few micromoles per kilogram (fruits) to several millimoles per kilogram in rich sources such as dairy products, eggs and soybeans. With the use of the limited data available, per capita sphingolipid consumption in the United States can be estimated to be on the order of 150-180 mmol (approximately 115-140 g) per year, or 0.3-0.4 g/d. There is no known nutritional requirement for sphingolipids; nonetheless, they are hydrolyzed throughout the gastrointestinal tract to the same categories of metabolites (ceramides and sphingoid bases) that are used by cells to regulate growth, differentiation, apoptosis and other cellular functions. Studies with experimental animals have shown that feeding sphingolipids inhibits colon carcinogenesis, reduces serum LDL cholesterol and elevates HDL, suggesting that sphingolipids represent a "functional" constituent of food. Sphingolipid metabolism can also be modified by constituents of the diet, such as cholesterol, fatty acids and mycotoxins (fumonisins), with consequences for cell regulation and disease. Additional associations among diet, sphingolipids and health are certain to emerge as more is learned about these compounds.
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Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside G(D3) were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by > 40% (P < 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P < 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 microgram of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or G(D3)) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis.
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Colorectal cancer is the second leading cause of cancer death in the United States. Nonsteroidal anti-inflammatory drugs including sulindac are promising chemopreventive agents for colorectal cancer. Sulindac and selective cyclooxygenase (COX)-2 inhibitors cause regression of colonic polyps in familial polyposis patients. Sulindac induces apoptotic cell death in cancer cells in vitro and in vivo. In tumor cells, activation of extracellular-regulated kinase (ERK) 1/2 results in phosphorylation of several ERK1/2 effectors, including the proapoptotic protein Bad. Phosphorylation of Ser112 by ERK1/2 inactivates Bad and protects the tumor cell from apoptosis. Sulindac metabolites and other nonsteroidal anti-inflammatory drugs selectively inhibit ERK1/2 phosphorylation in human colon cancer cells. In this study we show that epidermal growth factor (EGF) strongly induces phosphorylation of ERK1/2 and Bad in HT29 colon cancer cells. EGF-stimulated phosphorylation of ERK and Bad is blocked by pretreatment with U0126, a selective MAP kinase kinase (MKK)1/2 inhibitor. Similarly, pretreatment with sulindac sulfide blocks the ability of EGF to induce ERK1/2 and Bad phosphorylation, but also down-regulates total Bad but not ERK1/2 protein levels. The ability of sulindac to block ERK1/2 signaling by the EGF receptor may account for at least part of its potent growth-inhibitory effects against cancer cells.
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Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases.
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For the last 15 yr, a great deal of knowledge has been accumulated on health beneficial factors, protein and nonprotein, of bovine milk fat globule membrane (MFGM). Among the health-beneficial components of the MFGM are cholesterolemia-lowering factor, inhibitors of cancer cell growth, vitamin binders, inhibitor of Helicobacter pylori, inhibitor of beta-glucuronidase of the intestinal Escherichia coli, xanthine oxidase as a bactericidal agent, butyrophilin as a possible suppressor of multiple sclerosis, and phospholipids as agents against colon cancer, gastrointestinal pathogens, Alzheimer's disease, depression, and stress. All of the above compel us to consider bovine MFGM as a potential nutraceutical.
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In the carcinogenesis of colorectal cancer (CRC) genetic instability and dysfunction of the Wnt-signalling pathway play important roles. Most Wnt-signalling dysfunctions lead to the nuclear accumulation of beta-catenin. The aim of the present study was to investigate whether nuclear accumulation of beta-catenin is associated with prognosis and genetic instability. We used immunohistochemistry to study nuclear beta-catenin expression in 67 CRCs. The expression was evaluated in the entire tumour section as mean values and in tumour budding at the invasive margin. We compared the results with chromosomal and microsatellite instability (CIN vs. MSI), p53 accumulation, and clinicopathological variables including survival. The nuclear accumulation of beta-catenin was significantly associated with abnormal p53 expression and aneuploidy, typically for CIN, whereas no tumour with nuclear beta-catenin expression at the invasive margin displayed MSI. The beta-catenin expression pattern did not correlate significantly with CRC patient prognosis when including all stages. However, in the clinically most interesting prognostic group, Dukes' stage B patients, high nuclear accumulation of beta-catenin was associated with a poor prognosis (p=0.01). Our results suggest that nuclear accumulation of beta-catenin in CRC is related to CIN and may be of prognostic importance. However, larger studies are needed to verify these findings.
Article
Intestinal cells are regularly exposed to sphingolipid metabolites, i.e., ceramide and sphingoid bases, after hydrolysis of complex sphingolipids from the diet. These metabolites are known regulators of cell growth, differentiation, and death. Non-pharmacological amounts in the diet have been shown to inhibit early stages of chemically induced colon cancer in mice. To distinguish between chemopreventive and chemotherapeutic effects of sphingomyelin supplements, mice were fed sphingomyelin before and after tumor initiation. Both applications drastically reduced tumor formation, without a significant difference among the groups, indicating that sphingolipids are as effective in the chemoprevention of tumors as in early intervention. The normalization of cell proliferation and rate of apoptosis, but not the induction of differentiation, seem to be key players in the suppression of tumor formation by dietary sphingomyelin. This may have implications for the development of a cancer prevention or treatment strategy with sphingolipids as an alternative to conventional drugs.
Milk fat globule membrane-a source of polar lipids for colon health?
  • Am Kuchta
  • Pm Kelly
  • C Stanton
  • Ra Devery
Yogurt and buttermilk in cancer management
  • R N Grant
Miniaturised in vitro assays in toxicity testing
  • O' Connor
  • R Heenan