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.     

MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.     

Inhibition of cdk2 activating phosphorylation by mevastatin

Phosphorylation of cdk2 on threonine 160 is essential for kinase activity. Mevastatin, an inhibitor of cholesterol synthesis, inhibits cell growth through inhibition of cdk2 and this has been suggested to be due to enhancement of p21 levels. In a prostate cancer cell line, PC3, mevastatin treatment led to elevated levels of p21 and caused a small increase in the p21 associated with cdk2. However, this increase in the associated p21 appeared out of proportion with the resulting dramatic inhibition of kinase activity. Using RNA interference we show that mevastatin inhibits cdk2 activity despite lack of induction of p21, p27, and p57. Instead the kinase was inhibited due to a decrease in activating phosphorylation. Phosphorylation of cdk2 from mevastatin-treated cells with exogenous cyclin-dependent kinase (cdk)-activating enzymes restored its functional activity. The only known mammalian cyclin H.cdk7.mat1 complex (cdk2-activating kinase, Cak), was not inhibited by mevastatin, suggesting either that a different CAK is responsible for cdk2 phosphorylation in vivo or that the regulation is at the level of substrate accessibility or of cdk2 dephosphorylation. These results suggest that mevastatin inhibits cdk2 activity in PC3 cells through the inhibition of Thr-160 phosphorylation of cdk2, providing a novel example of regulation of cdk2 at this level.     

PGE2 inhibits apoptosis in human adenocarcinoma Caco-2 cell line through Ras-PI3K association and cAMP-dependent kinase A activation

PGE2 plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2 synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2 has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2 on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2 is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2 on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.     

p38 MAPK signaling pathway is involved in butyrate-induced vitamin D receptor expression

Previously, we have demonstrated that the butyrate-induced differentiation in the human colon cancer cell line Caco-2 occurs via upregulation of the vitamin D receptor (VDR). However, the downstream pathways involved are unknown. The mitogen-activated protein kinases (MAPKs) have been shown to play an important role in regulation of cell differentiation, and may therefore be a potential target of butyrate action. To assess their role in butyrate-mediated cell differentiation and VDR expression, we used the specific p38-MAPK inhibitor SB203580 and the ERK1/2 MAPK-inhibitor PD98059. The p38-MAPK inhibitor abolished the butyrate effect on VDR expression and cell differentiation, while the ERK1/2 inhibitor did not influence the butyrate-mediated induction of cell differentiation and VDR expression. The essential role of the p38 pathway in up-regulation of VDR expression was further confirmed by using the p38 stimulator arsenite. These results imply an important role of the p38-MAPK in regulation of cellular differentiation through upregulation of VDR expression by butyrate.     

Butyrate-induced G2/M block in Caco-2 colon cancer cells is associated with decreased p34cdc2 activity

Butyrate, a short-chain fatty acid, has been reported to inhibit proliferation and stimulate differentiation in multiple cancer cell lines. Whereas the effects of butyrate on cellular differentiation are well documented, the relationship between butyrate-induced differentiation and its effect on cell cycle traverse is less well understood. The purpose of this study was to investigate the effects of butyrate on the regulatory proteins of the G2/M traverse in the Caco-2 colon cancer cell model. We demonstrated that the inhibition of proliferation and increased cellular differentiation after treatment of Caco-2 cells with butyrate were associated with a significant G2/M cell cycle block. Although protein levels of the major G2/M regulatory protein, p34cdc2, were unchanged, a decrease in p34cdc2 activity was noted. Despite this decrease in activity, the inhibitory tyrosine phosphorylation of p34cdc2 was decreased, suggesting that other factors are responsible for the decreased kinase activity. The reduced activity of p34cdc2 provides a possible mechanism for the accumulation of Caco-2 cells in the G2/M cell cycle compartment following exposure to butyrate. This cell system provides a new model for studies of G2/M cell cycle perturbations.     

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation anddifferentiation remain largely undefined. Previously, we showed anearly induction of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1 in Caco-2 cells, ahuman colon cancer line that spontaneously differentiates into a smallbowel phenotype. The purpose of our present study was to assess thetiming of cell cycle arrest in relation to differentiation in Caco-2cells and to examine the mechanisms responsible for CDK inactivation.Caco-2 cells undergo a relativeG₁/S block and cease toproliferate at day3 postconfluency; an increase in theactivity of terminally differentiated brush-border enzymes (sucrase andalkaline phosphatase) was noted at day6 postconfluency. Cell cycle block wasassociated with suppression of both CDK2 and CDK4 activities, which areimportant for G₁/S progression.Treatment of the CDK immune complexes with the detergent deoxycholate(DOC) resulted in restoration of CDK2, but not CDK4, activity atday 3 postconfluency, suggesting the presence of inhibitory protein(s)binding to the cyclin/CDK2 complex at this time point. An increasedbinding of p21^Waf1/Cip1 to CDK2complexes at day3 postconfluency was noted, suggesting a potential role for p21^Waf1/Cip1in CDK2 inactivation; however, immunodepletion ofp21^Waf1/Cip1 from Caco-2 proteinextracts demonstrated thatp21^Waf1/Cip1 is only partiallyresponsible for CDK2 suppression atday 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears tocontribute to the CDK2 inactivation noted atdays6 and12 postconfluency. Taken together, ourresults suggest that multiple mechanisms contribute to CDK suppressionduring Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leadsto G₁ arrest and inhibition ofproliferation that precede Caco-2 cell differentiation.

     

Does heme oxygenase-1 have a role in Caco-2 cell cycle progression?

Intestinal epithelium undergoes a rapid self-renewal process characterized by the proliferation of the crypt cells, their differentiation into mature enterocytes as they migrate up to the villi, followed by their shedding as they become senescent villus enterocytes. The exact mechanism that regulates the intestinal epithelium renewal process is not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role. Heme oxygenase-1 (HO-1) is involved in endothelial cell cycle progression, but its role in the intestinal epithelial cell turnover has not been explored. With its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process. In this study, we examined the role of HO-1 in the proliferation and differentiation of Caco-2 cells, a well-established in vitro model for human enterocytes. After confluence, Caco-2 cells undergo spontaneous differentiation and mimic the crypt to villus maturation observed in vivo. In preconfluent and confluent Caco-2 cells, HO-1 protein expression was determined with the immunoblot. HO-1 activity was determined by the ability of the enzyme to generate bilirubin from hemin. The effect of a HO-1 enzyme activity inhibitor, tin protoporphyrin (SnPP), on Caco-2 cell proliferation and differentiation was examined. In preconfluent cells, cell number was determined periodically as a marker of proliferation. Cell viability was measured with MTT assay. Cell differentiation was assessed by the expression of a brush border enzyme, alkaline phophatase (ALP). HO-1 was expressed in subconfluent Caco-2 cells and remained detectable until 2 days postconfluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the ALP enzyme activity of confluent cells. HO-1 may be involved in intestinal cell cycle progression.     

Physiological intestinal oxygen modulates the Caco-2 cell model and increases sensitivity to the phytocannabinoid cannabidiol

The Caco-2 cell model is widely used as a model of colon cancer and small intestinal epithelium but, like most cell models, is cultured in atmospheric oxygen conditions (～21%). This does not reflect the physiological oxygen range found in the colon. In this study, we investigated the effect of adapting the Caco-2 cell line to routine culturing in a physiological oxygen (5%) environment. Under these conditions, cells maintain a number of key characteristics of the Caco-2 model, such as increased formation of tight junctions and alkaline phosphatase expression over the differentiation period and maintenance of barrier function. However, these cells exhibit differential oxidative metabolism, proliferate less and become larger during differentiation. In addition, these cells were more sensitive to cannabidiol-induced antiproliferative actions through changes in cellular energetics: from a drop of oxygen consumption rate and loss of mitochondrial membrane integrity in cells treated under atmospheric conditions to an increase in reactive oxygen species in intact mitochondria in cells treated under low-oxygen conditions. Inclusion of an additional physiological parameter, sodium butyrate, into the medium revealed a cannabidiol-induced proliferative response at low doses. These effects could impact on its development as an anticancer therapeutic, but overall, the data supports the principle that culturing cells in microenvironments that more closely mimic the in vivo conditions is important for drug screening and mechanism of action studies.     

Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK

Mevastatin which is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, suppress cell proliferation and induce apoptosis. However, the molecular mechanism of apoptosis induction is not well understood. So, in the present study, we attempted to clarify the mechanism by which mevastatin induces apoptosis in HL60 cells. It was found that mevastatin induced apoptosis. At that time, we observed an increase in caspase-3 activity and morphological fragmentation of the nuclei. The apoptosis induced by mevastatin was not inhibited by the addition of farnesyl pyrophosphate (FPP), squalene, ubiquinone, and isopentenyladenine, but was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP). When we examined the survival signals at the time of apoptotic induction, we also observed that the administration of mevastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). However, other survival signals, such as nuclear factor kappa B (NF-kappaB), protein kinase B (Akt), and p38 mitogen-activated protein kinase (p38), exhibited no change. In addition, no quantitative change was observed in Bcl-2, which was an anti-apoptosis protein. It was also observed that apoptosis was induced when U0126, an MEK inhibitor, was added to the cells to inhibit ERK. These results suggested that mevastatin induced apoptosis when it inhibited GGPP biosynthesis and consequently decreased the level of phosphorylated ERK, which was a survival signal; moreover, at that time, there was no influence on NF-kappaB, Akt, p38, and Bcl-2. The results of this study also suggested that mevastatin could be used as an anticancer agent.     

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.     

Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation.

In many tissues, progenitor cells permanently withdraw from the cell cycle prior to commitment towards a differentiated phenotype. In the oligodendrocyte lineage a counting mechanism has been proposed, linking the number of cell divisions to growth arrest and differentiation. A direct prediction of this model is that an increase in the number of cell divisions would result in a delayed onset of differentiation. Since the cell cycle inhibitor p27Kip1 is an essential component of the machinery leading to oligodendrocyte progenitor growth arrest, we examined the temporal relationship between cell cycle withdrawal and expression of late differentiation markers in vivo, in mice carrying a targeted deletion in the p27Kip1 gene. Using bromodeoxyuridine to label proliferating cells, quaking (QKI) to identify embryonic glial progenitors, NG2 to identify neonatal oligodendrocyte progenitors, and myelin basic protein to label differentiated oligodendrocytes, we found an increased number of proliferating QKI- and NG2-positive cells in germinal zones of p27Kip1(-/-) mice at the peak of gliogenesis. However, no delay was observed in these mice in the appearance of the late differentiation marker myelin basic protein in the developing corpus callosum and cerebellum. Significantly, a decrease in cyclin E levels was observed in the brain of p27Kip1 null mice coincident with oligodendrocyte growth arrest. We conclude that two distinct modalities of growth arrest occur in the oligodendrocyte lineage: a p27Kip1-dependent mechanism of growth arrest affecting proliferation in early phases of gliogenesis, and a p27Kip1-independent event leading to withdrawal from the cell cycle and differentiation.     

Hepatocyte growth factor (HGF) promotes cardiac stem cell differentiation after myocardial infarction by increasing mTOR activation in p27<Superscript>kip</Superscript> haploinsufficient mice

Myocardial infarction (MI) is a major cause of death worldwide. The treatment of MI has improved during the last decade; however, the loss of myocytes remains a problem because the cells cannot be renewed. Cardiac stem cells were recently discovered and were thought to represent a promising treatment for MI. However, the efficiency of cardiac stem cell differentiation into myocyte is not sufficient. Hepatocyte growth factor (HGF) is related to the differentiation and proliferation of cardiac stem cells. p27^kip1 is a potent cell cycle inhibitor in most organs, especially the heart. In this study, we investigated the relationship among p27, HGF and cardiac stem cells in post-MI cardiac repair. We found that p27 haploinsufficient mice exhibited preserved cardiac function and improved cardiomyocyte renewal compared with wild-type mice. In addition, p27 haploinsufficiency may not increase cardiac stem cell proliferation but could improve their differentiation by increasing mammalian target of rapamycin expression.     

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.