The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5

Butyrate induces differentiation and alters cell proliferation in intestinal-epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium-dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose-free inosine-containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase-IV (DPP-IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS-TC2, BCS-TC2.2, HT-29, and Caco-2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco-2 cells. Inosine-differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco-2 cells. AnxA2 down-regulation is not due to proteasome activation and seems to be related to the butyrate-induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth-state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell-to-cell contacts. Butyrate induces changes in subcellular localization towards a vesicle-associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up-regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up-regulation of AnxA1 could contribute to the reported anti-inflammatory effects of butyrate in colon inflammatory diseases.     

Oxygen concentration modulates the differentiation of muscle stem cells toward myogenic and adipogenic fates

The physiological oxygen concentration of many tissues is far lower than that in which cells are typically cultured in vitro and this may inadvertently influence the proliferation and differentiation potential of many cell types. Muscle derived stem cells, known as satellite cells are responsible for the maintenance and repair of muscle tissue post-natally and in vivo would be exposed to oxygen concentrations of ～2-5%. Relatively few studies describe the function of these cells in large animal models and here we investigate the influence oxygen concentration has on modulating porcine muscle derived stem cell fate. We compared cells derived from two metabolically distinct muscles, the diaphragm and the hind limb semi-membranosus (SM) muscle. The two sub-populations responded differently to culture at atmospheric (～20%) and physiological (～5%) oxygen concentration. While myogenesis was enhanced in both populations at low oxygen, noticeably diaphragm derived cells exhibited greater myotube formation, than those from SM. The trans-differentiation of cells derived from these two sources was similarly affected, with considerable differences seen in adipogenic and neuronal tendencies. In addition to the effect of oxygen on cell phenotype, the expression of key signalling proteins varied between the two sub-populations during early time-points of induced differentiation, suggesting altered regulation of muscle specific stem cells under these conditions. While differences in muscle stem cell potential requires further investigation, the culture of cells in physiological oxygen concentration appears as fundamental to recreating the micro-environmental niche as routinely used factors such as cytokines, substrata and matrices.     

Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2

The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of intestinal cells.     

Protein Kinase-zeta inhibits collagen I-dependent and anchorage-independent growth and enhances apoptosis of human Caco-2 cells

Colonic carcinogenesis is accompanied by abnormalities in multiple signal transduction components, including alterations in protein kinase C (PKC). The expression level of PKC-zeta, an atypical PKC isoform, increases from the crypt base to the luminal surface and parallels crypt cell differentiation in normal colon. In prior studies in the azoxymethane model of colon cancer, we showed that PKC-zeta was down-regulated in rat colonic tumors. In this study, we showed that PKC-zeta is expressed predominantly in colonic epithelial and not stromal cells, and loss of PKC-zeta occurs as early as the adenoma stage in human colonic carcinogenesis. To assess the regulation of growth and differentiation by PKC-zeta, we altered this isoform in human Caco-2 colon cancer cells using stable constitutive or inducible expression vectors, specific peptide inhibitors or small interfering RNA. In ecdysone-regulated transfectants grown on collagen I, ponasterone A significantly induced PKC-zeta expression to 135% of empty vector cells, but did not alter nontargeted PKC isoforms. This up-regulation was accompanied by a 2-fold increase in basal and 4-fold increase in insulin-stimulated PKC-zeta biochemical activity. Furthermore, PKC-zeta up-regulation caused >50% inhibition of cell proliferation on collagen I (P < 0.05). Increased PKC-zeta also significantly enhanced Caco-2 cell differentiation, nearly doubling alkaline phosphatase activity, while inducing a 3-fold increase in the rate of apoptosis (P < 0.05). In contrast, knockdown of this isoform by small interfering RNA or kinase inhibition by myristoylated pseudosubstrate significantly and dose-dependently increased Caco-2 cell growth on collagen I. In transformation assays, constitutively up-regulated wild-type PKC-zeta significantly inhibited Caco-2 cell growth in soft agar, whereas a kinase-dead mutant caused a 3-fold increase in soft agar growth (P < 0.05). Taken together, these studies indicate that PKC-zeta inhibits colon cancer cell growth and enhances differentiation and apoptosis, while inhibiting the transformed phenotype of these cells. The observed down-regulation of this growth-suppressing PKC isoform in colonic carcinogenesis would be predicted to contribute to tumorigenesis.     

Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation

Objectives: Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. Materials and methods: After 3 days culture or after long‐term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT‐mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. Results: After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long‐term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Conclusions: Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long‐term culture of mES cells in vitro.     

Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer

The human intestinal Caco-2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new cell-maintenance protocol in which Caco-2 cells were subcultured at 50% of confluence instead of 80% of confluence, as usually suggested. Using this new protocol, Caco-2 cells retained a higher proliferation potential resulting in a cell population, which, on reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21-days differentiated monolayers by microarrays hybridization and by confocal microscopy. We then investigated if these differences could also modify the effects of toxicants on 21-days-differentiated cells. We analyzed the 2 h-acute toxicity of CuCl(2) in terms of actin depolymerization and metallothionein 2A (MT2A) and heat shock protein 70 (HSPA1A) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with culture protocol, the low-density growing cells showing a more homogeneous and stronger response. Our results suggest that cell growing density could influence a number of morphological and physiological properties of differentiated Caco-2 cells and these effects must be taken in account when these cells are used as intestinal model.     

The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.     

Effect of oxygen levels on the physiology of dendritic cells: implications for adoptive cell therapy

Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO(2) incubators under atmospheric oxygen conditions (21% O(2)), which differ from physiological oxygen levels of only 3-5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However, DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.     

Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion

Summary Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6). However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1β or tumor necrosis factor-α (TNF-α), and stimulation of these cells with IL-1β plus TNF-α induced a synergistic enhancement of IL-6 secretion. The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO₂ atmosphere as compared to cells grown in 5% CO₂, suggesting that environmental CO₂ levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC.     

All-trans retinoic acid enhances differentiation and influences permeability of intestinal Caco-2 cells under serum-free conditions

Vitamin A and retinoids are essential nutrients for the differentiation of epithelia. Vitamin A deficiency is accompanied by an impairment in intestinal integrity. We investigated whether retinoids influence the differentiation and permeability of Caco-2 cells under serum-free culture conditions as a model for the intestinal epithelium. Treatment of the Caco-2 cells with retinoic acids (RA) resulted in an increased specific activity, enhanced mRNA expression, and induction of the 5'-flanking promoter activity of the marker enzyme for the differentiation intestinal alkaline phosphatase. Surprisingly, permeability of the Caco-2 monolayer, as measured by transepithelial electric resistance and [3H]-mannitol flux, was found to be enhanced by RA. Treatment with RA had only a slight effect on the mRNA expression of the tight junction-associated proteins occludin, ZO-1, claudin-1, -3, and -4, but enhanced the expression of claudin-2, which was recently suggested to form a paracellular ion channel. The role of retinoids as potent inducers of epithelial differentiation was confirmed for the Caco-2 cells under serum-free culture conditions and it was concluded that IAP is a target gene of RA. The inverse regulation of the permeability by RA under these serum-free conditions showed that other mechanisms, which are essential to regulate intestinal epithelial integrity with respect to decreased permeability, have to be identified.     

Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE₂

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E? are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE? generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE? generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE? production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE? on VEGF production, Caco-2 cells were treated with cPLA? (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE? generation. The Caco-2 cells were also treated with a non selective PGE? receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE? or selective EP? receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE? appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl? was decreased by inhibition of concomitant PGE? generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE? plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP? activation.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

Teriflunomide – The common drug with underestimated oxygen - Dependent anticancer potential

Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect.The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21%oxygen level occurred in routine cell culture environment.The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels.We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced.Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.     

Effect of polyunsaturated fatty acid-enriched phosphatidylcholine and phosphatidylserine on butyrate-induced growth inhibition, differentiation and apoptosis in Caco-2 cells

Phospholipids are fascinating in terms of important bio-functional compounds. The present work investigated the effect of polyunsaturated phosphatidylcholine (PC) and phosphatidylserine (PS) on butyrate-induced growth inhibition, differentiation and apoptosis using Caco-2 cells. Growth inhibition of Caco-2 cells became apparent 24 h after addition of PC while it took 48 h with PS. Alkaline phosphatase activity of Caco-2 cells increased with combined PC or PS and sodium butyrate (NaBT) at 72 h, indicating that PC and PS enhanced cell differentiation in the presence of NaBT. An increased enrichment factor was also found when cells were treated with combinations of PC or PS and NaBT. These results suggest that marine PC and PS can be considered to be potentially useful colon cancer chemotherapy agents with high bio-availability.