Utility of human induced pluripotent stem cell-derived small intestinal epithelial cells for pharmacokinetic, toxicological, and immunological studies.

The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.

Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells.

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.

FF-10502, an Antimetabolite with Novel Activity on Dormant Cells, Is Superior to Gemcitabine for Targeting Pancreatic Cancer Cells.

In this paper, we report that 1-(2-deoxy-2-fluoro-4-thio-ß-d-arabinofuranosyl) cytosine (FF-10502), a pyrimidine nucleoside antimetabolite with a chemical structure similar to gemcitabine, shows beneficial anticancer activity via a novel mechanism of action on dormant cells. The growth inhibition of pancreatic cancer cell lines by FF-10502 (IC50, 60-330 nM) was moderately weaker than that by gemcitabine in vitro. In contrast, an in vivo orthotopic implantation model in mice with established human pancreatic cancer cell line, SUIT-2, revealed no mortality with FF-10502 intravenous treatment, which was related to regression of implanted tumor and little metastasis, whereas 75% of the mice treated with gemcitabine died by day 128. Two in vivo patient-derived xenograft models with gemcitabine-resistant pancreatic cancer cells also demonstrated complete tumor growth suppression with FF-10502, but only partial inhibition with gemcitabine. We also investigated the mechanism of action of FF-10502 by using dormant cancer cells, which are reportedly involved in the development of resistance to chemotherapy. In vitro serum starvation-induced dormant SUIT-2 cells developed resistance to gemcitabine even in combination with DNA damage inducers (DDIs; H2O2, cisplatin, and temozolomide). Interestingly, FF-10502 in combination with DDIs significantly induced concentration-dependent cell death in accordance with enhanced DNA damage. FF-10502 was far more potent than gemcitabine in inhibiting DNA polymerase ß, which may explain the difference in dormant cell injury, although further investigations for direct evidences are necessary. In conclusion, our study demonstrated the beneficial antitumor effects of FF-10502 in clinically relevant in vivo models, and suggests the importance of preventing DNA repair unlike gemcitabine.

Nuclear factor of activated T-cell activity is associated with metastatic capacity in colon cancer.

Metastatic recurrence is the leading cause of cancer-related deaths in patients with colorectal carcinoma. To capture the molecular underpinnings for metastasis and tumor progression, we performed integrative network analysis on 11 independent human colorectal cancer gene expression datasets and applied expression data from an immunocompetent mouse model of metastasis as an additional filter for this biologic process. In silico analysis of one metastasis-related coexpression module predicted nuclear factor of activated T-cell (NFAT) transcription factors as potential regulators for the module. Cells selected for invasiveness and metastatic capability expressed higher levels of NFATc1 as compared with poorly metastatic and less invasive parental cells. We found that inhibition of NFATc1 in human and mouse colon cancer cells resulted in decreased invasiveness in culture and downregulation of metastasis-related network genes. Overexpression of NFATc1 significantly increased the metastatic potential of colon cancer cells, whereas inhibition of NFATc1 reduced metastasis growth in an immunocompetent mouse model. Finally, we found that an 8-gene signature comprising genes upregulated by NFATc1 significantly correlated with worse clinical outcomes in stage II and III colorectal cancer patients. Thus, NFATc1 regulates colon cancer cell behavior and its transcriptional targets constitute a novel, biologically anchored gene expression signature for the identification of colon cancers with high risk of metastatic recurrence.

Effect of claudin expression on paracellular permeability, migration and invasion of colonic cancer cells.

Alteration in the expression of claudins, consisting of tight junctions (TJs), has been reported in various clinically isolated tumors. Claudins play an important role not only in the intercellular barrier function of TJs but also in migration and invasiveness of cancer cells. However, the use of different types of cells and different claudins in these studies has complicated the picture. In this study, we systematically examined the effect of claudin (claudin-1, -2, -3, -4 and -15) overexpression on the paracellular permeability, migration and invasiveness of Caco-2 colonic cancer cells. Overexpression of claudin-4 or claudin-2 increased or decreased, respectively, paracellular permeability. Overexpression of claudin-4 specifically stimulated the invasive activity of the Caco-2 cells. Furthermore, activation of matrix metalloproteinase (MMP)-2 and MMP-9 were observed in the claudin-4-overexpressing cells, suggesting that the invasive activity was stimulated through an increase in MMP activity. Overexpression of claudin-2 or claudin-3 and -4 stimulated or inhibited, respectively, the migration activity of the Caco-2 cells. Immunostaining analysis revealed that each of the overexpressed claudins localized at TJs under the conditions used to evaluate paracellular permeability. In contrast, they localized mainly in intracellular compartments under experimental conditions designed to assess cell invasion and migration. Overall, the results of this study show that the effect exerted by the claudins on the intercellular barrier function of TJs, as well as on cell migration and invasive activity, differs depending on the particular claudin species. Furthermore, the subcellular localization of the claudins varies according to the culture conditions.

Up-regulation of S100P expression by non-steroidal anti-inflammatory drugs and its role in anti-tumorigenic effects.

Epidemiological studies have revealed that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer. Various mechanisms, including induction of apoptosis and inhibition of the growth and invasion of cancer cells, have been implicated in this anti-tumorigenic activity. In this study we focused on S100P, which is known to be overexpressed in clinically isolated tumors and which functions through both intracellular and extracellular mechanisms. We showed the up-regulation of S100P expression in human gastric carcinoma cells treated with various NSAIDs, including celecoxib. The celecoxib-mediated up-regulation of S100P was suppressed by the transfection of cells with small interfering RNA for activating transcription factor 4 (ATF4), a transcription factor involved in the endoplasmic reticulum stress response. Furthermore, deletion of ATF4 binding consensus sequence located in the promoter of the S100P gene resulted in inhibition of celecoxibmediated transcriptional activation of the gene. These results suggest that celecoxib up-regulates the expression of S100P through an ATF4-mediated endoplasmic reticulum stress response. Celecoxib inhibited the growth and induced apoptosis, and these actions could be either suppressed or stimulated by transfection of cells with S100P overexpression plasmid or small interfering RNA, respectively. Celecoxib also inhibited the invasive activity of the cells. Cromolyn, which inhibits the binding of S100P to its receptor, enhanced the celecoxib-mediated inhibition of cell invasion and growth but did not affect apoptosis. These results suggest that S100P affects apoptosis, cell growth, and invasion through either an intracellular or an extracellular mechanism and that the up-regulation of S100P expression by NSAIDs reduces their anti-tumorigenic activity.

NSAIDs suppress the expression of claudin-2 to promote invasion activity of cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) show chemopreventive effects; however, the precise molecular mechanism of these effects is still unclear. On the other hand, the expression of proteins that form tight junctions (TJs) (such as claudins) in clinically isolated tumors is frequently altered relative to normal tissue. We previously reported that NSAIDs upregulate the expression of claudin-4 and that this upregulation contributes to NSAID-dependent inhibition of both migration activity and anchorage-independent growth of cancer cells. In the current study, we have systematically examined the effects of various NSAIDs on the expression of various TJ proteins and have found that NSAIDs specifically and drastically inhibit the expression of claudin-2. Overexpression or suppression of claudin-2 expression caused stimulation or inhibition, respectively, of the invasion and migration activity of cancer cells. Furthermore, NSAIDs inhibited the invasion and migration activity of cancer cells and this inhibition was suppressed by overexpression of claudin-2. In contrast, neither cell growth nor apoptosis induced by lack of anchorage of cancer cells was affected by overexpression or suppression of expression of claudin-2. These results suggest that inhibition of claudin-2 expression by NSAIDs contributes to NSAID-dependent inhibition of invasion of cancer cells in vitro and that it may be involved in the chemopreventive effects of NSAIDs by inhibiting metastasis in vivo.

Up-regulation of 150-kDa oxygen-regulated protein by celecoxib in human gastric carcinoma cells.

Induction of apoptosis by nonsteroidal anti-inflammatory drugs, such as celecoxib, is involved in their antitumor activity. An endoplasmic reticulum chaperone, 150-kDa oxygen-regulated protein (ORP150) is essential for the maintenance of cellular viability under hypoxia and is reported to be overexpressed in clinically isolated tumors. We here found that ORP150 was up-regulated by celecoxib in human gastric carcinoma cells. In conjunction with the suppression of tumor growth, orally administered celecoxib up-regulated ORP150 in xenograft tumors. Both the ATF4 and ATF6 pathways were activated by celecoxib, and suppression of ATF4 and ATF6 mRNA expression by small interfering RNA (siRNA) inhibited the celecoxib-dependent up-regulation of ORP150. Celecoxib administration led to an increase in the intracellular concentration of Ca2+, whereas 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca2+ chelator, inhibited the up-regulation of ORP150 and the activation of the ATF4 and ATF6 pathways. These results suggest that these Ca2+-activated pathways are involved in the celecoxib-mediated up-regulation of ORP150. Clones overexpressing ORP150 were less susceptible to celecoxib-induced, but not staurosporine-induced, apoptosis and displayed less up-regulation of C/EBP homologous transcription factor (CHOP), a transcription factor with apoptosis-inducing activity. In contrast, siRNA for ORP150 stimulated apoptosis and expression of CHOP in the presence of celecoxib but not staurosporine. These results suggest that up-regulation of ORP150 in cancer cells inhibits celecoxib-induced apoptosis, thereby decreasing the potential antitumor activity of celecoxib.

Geranylgeranylacetone protects membranes against nonsteroidal anti-inflammatory drugs.

Direct gastric mucosal cell damage mediated by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. We recently suggested that this direct cytotoxicity of NSAIDs is caused by their membrane-permeabilization activity. Geranylgeranylacetone (GGA), a clinically used antiulcer drug, can protect gastric mucosa against lesion formation mediated by NSAIDs. However, the mechanism by which this occurs is not fully understood. In this study, we show that GGA acts to stabilize membranes against NSAIDs. GGA suppressed NSAID-induced permeabilization of calcein-loaded liposomes and NSAID-induced stimulation of K(+)-efflux across the cytoplasmic membrane in cells. GGA was effective even when coadministered with NSAIDs and was also able to restore membrane fluidity that had been compromised by NSAIDs. This mechanism seems to play an important role in the antiulcer activity of GGA.

Induction of claudin-4 by nonsteroidal anti-inflammatory drugs and its contribution to their chemopreventive effect.

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca2+ concentration. Other drugs that increased the intracellular Ca2+ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca2+ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction-related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Molecular mechanism of adaptive cytoprotection induced by ethanol in human gastric cells.

Adaptive cytoprotection is the process by which the pretreatment of cells with low concentrations of a noxious agent prevents the damage caused by a subsequent exposure of those cells to higher concentrations of that same agent. In this study, a human gastric carcinoma cell line was used to examine the molecular mechanism of adaptive cytoprotection induced by ethanol. Pretreatment of cells with 1%-4% ethanol made cells resistant to a subsequent exposure to 8% ethanol. This adaptive cytoprotection was accompanied by an increase in prostaglandin E2 synthesis and was partially inhibited by inhibitors of cyclooxygenase-2, but not by an inhibitor of cyclooxygenase-1. Furthermore, the adaptive cytoprotection was not dependent on newly synthesized proteins and was inhibited by a protein tyrosine kinase inhibitor. Based on these results, it is proposed that the stimulation of cyclooxygenase-2-dependent prostaglandin E2 synthesis, which is regulated post-translationally by protein tyrosine phosphorylation, plays an important role in adaptive cytoprotection induced by ethanol in gastric cells.

Conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein are involved in DnaA-DnaA interaction.

We previously reported that a leucine-zipper-like structure (I26, L33 and L40) located in the N-terminal region of DnaA is essential for the duplex opening at oriC by DnaA. In this study, we focused on three other conserved hydrophobic amino acid residues, L3, L10 and L17, and examined the function of DnaA proteins mutated in these amino acid residues. DnaA427 (L17S) and DnaA413 (L3S, L10S and L17S) were inactive for oriC DNA replication both in vitro and in vivo. Although these mutant DnaA proteins maintained their binding activities for both ATP and oriC, they were unable to induce the opening of duplex DNA at oriC. Glutathione-S-transferase (GST)-fused wild-type DnaA interacted with wild-type DnaA but not with DnaA427 and DnaA413. Based on these results, we propose that conserved hydrophobic amino acid residues in the N-terminal region of DnaA are involved in DnaA oligomerization, in which DnaA-DnaA interaction is required.

Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372.

The DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. Acidic phospholipids decrease its affinity for adenine nucleotides, and re-activate the ADP-bound form to the ATP-bound form. We have previously reported that two mutant forms, DnaAR328E and DnaAK372E, have decreased affinity for cardiolipin (CL). In the present study, we constructed a mutant DnaA protein, DnaA435, with both R328E and K372E, and compared its biochemical characteristics with those of DnaAR328E and DnaAK372E. DnaA435 could bind to oriC DNA, but did not bind ATP or ADP. In DnaA435, compared with DnaAR328E and DnaAK372E, CL caused less inhibition of oriC DNA binding, suggesting that amino acids R328 and K372 are involved in the interaction of DnaA with acidic phospholipids. DnaA435 could initiate DNA synthesis on oriC both in vivo and in vitro. Based on these results, we propose that ATP activates DnaA protein by changing its higher order structure around R328 and K372.