Staurosporine synergistically potentiates the deoxycholate-mediated induction of COX-2 expression.

Colorectal cancer is a major cause of cancer-related death in western countries, and thus there is an urgent need to elucidate the mechanism of colorectal tumorigenesis. A diet that is rich in fat increases the risk of colorectal tumorigenesis. Bile acids, which are secreted in response to the ingestion of fat, have been shown to increase the risk of colorectal tumors. The expression of cyclooxygenase (COX)-2, an inducible isozyme of cyclooxygenase, is induced by bile acids and correlates with the incidence and progression of cancers. In this study, we investigated the signal transduction pathways involved in the bile-acid-mediated induction of COX-2 expression. We found that staurosporine (sts), a potent protein kinase C (PKC) inhibitor, synergistically potentiated the deoxycholate-mediated induction of COX-2 expression. Sts did not increase the stabilization of COX-2 mRNA. The sts- and deoxycholate-mediated synergistic induction of COX-2 expression was suppressed by a membrane-permeable Ca(2+) chelator, a phosphoinositide 3-kinase inhibitor, a nuclear factor-κB pathway inhibitor, and inhibitors of canonical and stress-inducible mitogen-activated protein kinase pathways. Inhibition was also observed using PKC inhibitors, suggesting the involvement of certain PKC isozymes (η, θ, ι, ζ, or µ). Our results indicate that sts exerts its potentiating effects via the phosphorylation of p38. However, the effects of anisomycin did not mimic those of sts, indicating that although p38 activation is required, it does not enhance deoxycholate-induced COX-2 expression. We conclude that staurosporine synergistically enhances deoxycholate-induced COX-2 expression in RCM-1 colon cancer cells.

Importance of uncharged polar residues and proline in the proximal two-thirds (Pro107-Ser128) of the highly conserved region of mouse ileal Na+-dependent bile acid transporter, Slc10a2, in transport activity and cellular expression.

BACKGROUND: SLC10A2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. Because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, SLC10A2 is important as the key transporter for understanding the in vivo kinetics of bile acids. SLC10A family members and the homologous genes of various species share a highly conserved region corresponding to Gly104-Pro142 of SLC10A2. The functional importance of this region has not been fully elucidated. RESULTS: To elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third (Thr130-Pro142) of the highly conserved region in mouse Slc10a2. In this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse Slc10a2 were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. Cell surface localization of Slc10a2 is necessary for bile acid absorption. Mutants in which Asp or Leu were substituted for Pro107 (P107N or P107L) were abundantly expressed, but their cell surface localization was impaired. The S126A mutant was completely impaired in cellular expression. The T110A and S128A mutants exhibited remarkably enhanced membrane expression. The S112A mutant was properly expressed at the cell surface but transport activity was completely lost. Replacement of Tyr117 with various amino acids resulted in reduced transport activity. The degree of reduction roughly depended on the van der Waals volume of the side chains. CONCLUSIONS: The functional importance of proline and uncharged polar residues in the highly conserved region of mouse Slc10a2 was determined. This information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or SLC10A2 inhibitors for hypercholesterolemia treatment.

Ursodeoxycholic acid protects colon cancer HCT116 cells from deoxycholic acid-induced apoptosis by inhibiting apoptosome formation.

We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (≥5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 µM DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways.

Mutational analysis of uncharged polar residues and proline in the distal one-third (Thr130-Pro142) of the highly conserved region of mouse Slc10a2.

Solute carrier family member 2 (SLC10A2) reabsorbs bile acids at the distal terminus of the ileum in an Na(+)-dependent manner. Alignment of deduced amino acid sequences of SLC10 family members and homologous genes in various species revealed a highly conserved region that corresponds to Gly(104)-Pro(142) of SLC10A2. To elucidate the functional importance of this region, uncharged polar residues and Pro in the distal one-third of this region in mouse Slc10a2 (mSlc10a2) were submitted to mutational analysis, and taurocholic acid uptake and cell surface localization were evaluated. In addition to mutations that abolished almost all of the transport activity with and without cellular localization failure (P142V and T130A respectively), a mutation that perhaps affected affinity for taurocholic acid was identified (T134A). These results suggest that the highly conserved region contains residues involved in the substrate interaction, function, and cellular localization of mSlc10a2.

Biphasic regulation of cell death and survival by hydrophobic bile acids in HCT116 cells.

A secondary bile acid, namely, deoxycholic acid (DCA), has been known to promote colon tumors; on the other hand, it also induces apoptosis in several human colon cancer cell lines. A hydrophobic primary bile acid, namely, chenodeoxycholic acid (CDCA), exhibits a similar property of apoptosis induction; DCA and CDCA also trigger some specific intracellular signal pathways in the human colon cancer cell line HCT116. In this article, we report that hydrophobic bile acids induce different cellular responses depending on their concentration, that is, a sublethal concentration of hydrophobic bile acids can suppress the apoptosis induced by a higher concentration of DCA. Pretreatment with DCA or CDCA at a concentration of < or = 200 microM for 8 h suppressed the apoptosis induced by 500 microM DCA in HCT116 cells. Under this condition, the association of caspase-9 and Apaf-1 and subsequent activation of caspase-9 were inhibited, but the release of cytochrome c from the mitochondria was not. At 200 microM, DCA and CDCA induced the phosphorylation of Akt and ERK1/2, although these phosphorylations do not appear to be indispensable for the cytoprotection. It is interpreted that prolonged exposure to sublethal concentrations of hydrophobic bile acids induces resistance to apoptosis, leading to promotion of colorectal tumorigenesis.

Soybean resistant protein elevates fecal excretion of cholesterol and bile acids and decreases hepatic cholesterol content in comparison with soybean protein isolate.

The effect of soybean resistant protein (RP) on serum and hepatic cholesterol levels and fecal excretion of steroids was examined. RP decreased cholesterol in the liver, probably due to the stimulated excretion of cholesterol and its metabolites, bile acids. The serum cholesterol level was not different as between RP and other soy-derived proteins.

Deoxycholic acid can induce apoptosis in the human colon cancer cell line HCT116 in the absence of Bax.

In the human colon cancer cells HCT116, deoxycholic acid (DCA) induces apoptosis via the mitochondrial pathway by triggering the release of mitochondrial factors such as cytochrome c. To elucidate if Bax, a proapoptotic member of the Bcl-2 family known to trigger cytochrome c release in response to various types of apoptotic stimuli, is involved in DCA-induced apoptosis in HCT116 cells, we analyzed DCA-induced apoptosis in Bax-knockout (Bax(-/-)) HCT116 cells. Cytochrome c release and caspase-9 activation were detectable after 5 min in both Bax(-/-) and Bax(+/-) HCT116 cells. Caspase-3 and caspase-8 activation was observed after 15 and 30 min, respectively. Bax(-/-) cells were protected from apoptosis by treating them with ursodeoxycholic acid for 12 h prior to DCA treatment. These results are consistent with our previous observations that were obtained by using wild-type HCT116 cells and suggest that Bax is not indispensable for DCA-induced apoptosis in HCT116 cells.

Excess leucine intake induces serine dehydratase in rat liver.

We have hypothesized that rat liver serine dehydratase (SDH) is induced in response to the amount of surplus amino acids from dietary protein. In the present study, we found that excess leucine intake strongly induced SDH activity in the liver but not in the kidney of rats. The increase in activity was accompanied by increases in the levels of SDH mRNA. On the other hand, isoleucine and valine had little effect on SDH induction. These results support our hypothesis and suggest that leucine is a signal for SDH induction.

Effects of Ala substitution for conserved Cys residues in mouse ileal and hepatic Na+-dependent bile acid transporters.

Although ileal and hepatic Na(+)-dependent bile acid transporters (SLC10A2 and SLC10A1 respectively) share structural similarities, the mutation of conserved amino acids often has distinct effects on them. We have identified two Cys residues in mouse Slc10a2 (Cys(51) and Cys(106)) the replacement of which by Ala remarkably reduces taurocholic acid (TCA) transport. Although Cys(51) is conserved in Slc10a1 as Cys(44), Ala substitution gave no apparent difference in TCA uptake. Here, we further analyzed the kinetics of TCA uptake and cell surface localization of these mutants. The C51A and C106A mutants of Slc10a2 showed significantly reduced TCA uptake, while no apparent difference in TCA uptake was observed for the Slc10a1-C44A mutant. The K(m) values for TCA uptake by these mutants were comparable, suggesting that these residues are not involved in the interaction with TCA.

Taurocholic acid does not induce apoptosis in HCT116 cells regardless of its intracellular concentration.

Hydrophobic bile acids but not hydrophilic bile acids induce apoptosis in HCT116 cells. We expressed sodium-dependent bile acid transporters in HCT116 cells, and the intracellular concentration of hydrophilic bile acids increased to that of the hydrophobic bile acids. But no sign of apoptosis was observed, which suggests a hydrophobic-bile acid-specific mechanism for the induction of apoptosis in HCT116 cells.

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion.

Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.

Characteristics of apoptosis in HCT116 colon cancer cells induced by deoxycholic acid.

Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.

Inverse correlation between the nitrogen balance and induction of rat liver serine dehydratase (SDH) by dietary protein.

Rats of different ages (3 to 15-wk-old) were fed on a 25% casein diet for one week, and the nitrogen balance and liver serine dehydratase (SDH, EC 4.2.1.13) activity were then determined. The value for nitrogen balance decreased with the age of the rats, while the liver SDH activity increased. A statistical analysis showed clear inverse correlation between the two factors (R(2) = 0.7372, p < 0.01). This result suggests that SDH was induced by response to the amount of surplus amino acids from dietary protein taken beyond the body's requirement. The increase in SDH activity was accompanied by an increase in the level of SDH mRNA. Since the half-life of this mRNA did not change significantly, the induction was mainly controlled at the level of transcription. In addition, the induction seems not to be related to gluconeogenesis, since the mRNA levels of tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), other gluconeogenic enzymes, were not changed under these experimental conditions.

ATP hydrolysis-dependent multidrug efflux transporter: MDR1/P-glycoprotein.

P-glycoprotein/MDR1 was the first member of the ATP-binding cassette (ABC) transporter superfamily to be identified in a eukaryote. In eukaryotes, ABC proteins can be classified into three major groups based on function: transporters, regulators, and channels. MDR1/P-glycoprotein is a prominent member of eukaryotic export-type ABC proteins. MDR1/P-glycoprotein extrudes a very wide array of structurally dissimilar compounds, all lipophilic and ranging in mass from approximately 300 to 2000 Da, including cytotoxic drugs that act on different intracellular targets, steroid hormones, peptide antibiotics, immunosuppressive agents, calcium channel blockers, and others. Nucleotide binding and hydrolysis by MDR1/P-glycoprotein is tightly coupled with its function, substrate transport. ATP binding and hydrolysis were extensively analyzed with the purified MDR1/P-glycoprotein. The vanadate-induced nucleotide trapping method was also applied to study the hydrolysis of ATP by MDR1/P-glycoprotein. When MDR1 hydrolyzes ATP in the presence of excess orthovanadate, an analog of inorganic phosphate, it forms a metastable complex after hydrolysis. Using this method, MDR1/P-glycoprotein can be specifically photoaffinity-labeled in the membrane, if 8-azido-[alpha(32)P]ATP is used as ATP. Visualization of the structure, as well as the biochemical data, is needed to fully understand how MDR1/P-glycoprotein recognizes such a variety of compounds and how it carries its substrates across the membrane using the energy from ATP hydrolysis. To do so, large amounts of pure and stable proteins are required. Heterologous expression systems, which have been used to express P-glycoprotein, are also described.

Postprandial changes in portal venous free amino acids and insulin/glucagon ratios as the result of protein over-intake are not directly linked to serine dehydratase induction in rat liver irrespective of age.

The activity of hepatic serine dehydratase (SDH) increases in tandem with its gene expression when the intake of protein greatly exceeds protein requirements. The actual conditions of plasma free amino acids and pancreatic hormones in weanling and mature rats when fed SDH-inducible and non-inducible diets were examined in relation to incentive factors to secure high SDH activity from a physiological standpoint. Both weanling and mature groups differing in protein requirements were allowed free access to respective diets diverse in protein content (i.e. 25% or 50% casein diet for the former and 6% or 25% casein diet for the latter) during the dark cycle (lights-out) over a period of 1 wk. Despite the difference in protein intake among these groups, there were no conspicuous changes in the plasma concentration of the urea or total or essential amino acids. Therefore, it appears that the individual amino acids did not up regulate the gene and function expressions of SDH merely by their superabundance and subsequent disposal. Portal venous insulin concentration was far higher in mature groups than in weanling groups, although there was little difference between the two groups of the same age in terms of insulin or glucagon concentration and their ratio in abdominal vena cava blood. Accordingly, it follows that the SDH gene undergoes transcriptional regulation through a combined signaling pathway triggered by perceiving surplus protein nutrition as a whole rather than directly through already-known plasma constituents such as free amino acids or pancreatic hormones in the circulatory system.

Quantity as well as quality of dietary protein affects serine dehydratase gene expression in rat liver.

Weanling rats were fed respective diets diverse in protein source and content for a full week, and hepatic serine dehydratase (SDH) was examined for its gene expression and activity induction attendant on high protein intake. The protein sources used were three kinds of milk casein, codfish meat, and wheat gluten. The body weight gain (% augmentation/wk) increased with increasing protein intake and reached a plateau in both milk casein- and codfish meat-fed rats by protein intake above 2.5 g/100 g BW/d; however, the body weight gain continued to increase albeit at a slower rate in wheat gluten-fed rats. Quite similar tendencies were also seen in nitrogen balance. The ascent of SDH activity induction and its causal gene expression were characterized as codfish meat>milk casein>>wheat gluten in order of response to protein intake near or more than 4 g/100 g BW/d. The difference in SDH gene expression among these dietary proteins was substantiated by a confirmation experiment in which six rats of each group were fed 25% or 50% protein diets under the same conditions as above. Hence, the quantity as well as quality of dietary protein turned out to have an influence on SDH gene expression.

Response of the induction of rat liver serine dehydratase to changes in the dietary protein requirement.

Growing and mature rats were examined for the effect of a change in dietary protein requirements on the induction of liver serine dehydratase (SDH). The rats were fed on diets varying in casein content, and the weight change and nitrogen balance was determined. SDH activity and its gene expression were induced in both growing and mature rats when their protein intake exceeded their nutritional requirements.

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.

Inhibition of the multidrug resistance protein 1 (MRP1) by peptidomimetic glutathione-conjugate analogs.

Inhibition of multidrug resistance protein 1 (MRP1) mediated cytostatic drug efflux might be useful in the treatment of drug resistant tumors. Because the glutathione (GSH) conjugate of ethacrynic acid (EA), GS-EA, is a good substrate of MRP1, GS-EA derivatives are expected to be good inhibitors of MRP1. To study structure-activity relationships of MRP1 inhibition, a series of novel GS-EA analogs was synthesized in which peptide bonds of the GSH backbone were replaced by isosteric groups [Bioorg Med Chem 10:195-205, 2002]. Several of these compounds were effective inhibitors of MRP1-mediated [(3)H]GS-EA and [(3)H]E(2)17betaG transport, as studied in membrane vesicles prepared from MRP1-overproducing Sf9 cells. The modifications of the peptide backbone have distinct implications for recognition by MRP1: the gamma-glutamyl-cysteine peptide bond is important for binding, whereas the cysteinyl-glycine amide does not seem essential. When the gamma-glutamyl-cysteine peptide bond (C-CO-N) is replaced by a urethane isostere (O-CO-N), an effective competitive MRP1-inhibitor (K(i) = 11 microM) is obtained. After esterification of this compound to improve its cellular uptake, it inhibited MRP1-mediated efflux of calcein from 2008 ovarian carcinoma cells overexpressing MRP1. This compound also partially reversed the resistance of these cells to methotrexate. Because the urethane isostere is stable toward gamma-glutamyl transpeptidase-mediated breakdown, it is an interesting lead-compound for the development of in vivo active MRP1 inhibitors.

Characterization of cloned mouse Na+/taurocholate cotransporting polypeptide by transient expression in COS-7 cells.

The mouse Na+/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 microM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.