Human Colon Adenocarcinoma-derived Cells Research Articles

Abstract 3791: Milk thistle as an inhibitor of PXR-mediated CYP3A4 induction

Abstract Introduction Many anticancer drugs, such as paclitaxel and erlotinib, are able to induce the expression of cytochrome P450 (CYP450) 3A4.1 Induction of intestinal and hepatic CYP3A4 can result in a decreased uptake and an increased metabolism of anticancer drugs. Consequently, systemic levels decrease, possibly resulting in a lower efficacy. The major mechanism behind this CYP3A4 induction is the activation of the pregnane X receptor (PXR)1 and therefore inhibition of PXR might prevent CYP3A4 induction. As 54-77% of the cancer patients use complementary and alternative medicine (CAM) including herbs and dietary supplements, these CAM are interesting to screen for their ability to inhibit PXR-mediated CYP3A4. For the current study, the following CAM were selected: α-carotene, Echinacea, garlic, Ginkgo biloba, P. ginseng, grape seed, green tea, milk thistle, saw palmetto, St. John's Wort, valerian, vitamin B6+12 and vitamin C.2,3 Aim The aim was to screen CAM for their ability to inhibit PXR-mediated CYP3A4 induction in LS180 cells, a human colon adenocarcinoma-derived cell line. Method The selected CAM were screened for possible inhibition of PXR-mediated CYP3A4 induction, using a CYP3A4 reporter gene assay and a real-time polymerase chain reaction (RT-PCR) assay. In both assays, LS180 cells were exposed to fresh medium containing 0.1% DMSO or model inducers of CYP3A4 (10 μM rifampicin, 20 μM erlotinib or 20 μM paclitaxel) with or without addition of known PXR inhibitors (10 μM ketoconazole and 10 μM A792611) or 50-100 μg/ml standardized CAM (table 1) for 24 hours. Data were analyzed by using the Student's unpaired t-test and were considered statistically significant when p< 0.05. Results Milk thistle was the most potent inhibitor of PXR-mediated CYP3A4 induction compared to the other CAM. The reporter gene assay demonstrated that 100 and 75 μg/ml milk thistle significantly inhibited PXR-mediated CYP3A4 induction by rifampicin by respectively 88% and 75%; by paclitaxel by respectively 90% and 81%; and by erlotinib by 82% and 49%. According to the RT-PCR assay 100 and 75 μg/ml milk thistle significantly inhibited PXR-mediated CYP3A4 induction by rifampicin by respectively 91% and 90%. Conclusion Milk thistle significantly inhibited PXR-mediated CYP3A4 induction at the transcriptional level, demonstrated by a reporter gene and RT-PCR assay. Thus, milk thistle is a suitable candidate for the inhibition of PXR-mediated CYP3A4 induction. Consequently, the concomitant use of milk thistle with anticancer drugs can possibly prevent the decrease in systemic levels of anticancer drugs resulting in a higher efficacy, but also a higher risk for toxicity.

PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line

PurposeThe development of multidrug resistance (MDR) is one of the major limitations in the treatment of cancer. Induction of P-glycoprotein (Pgp) has been regarded as one of the main mechanisms underlying anticancer drug-induced MDR. Since the induction of Pgp is (in part) regulated by the pregnane X receptor (PXR), the ability of several widely used anticancer drugs to activate PXR-mediated Pgp induction was investigated.MethodsA Pgp-reporter gene assay was employed to determine the ability of a panel of widely used anticancer drugs to induce Pgp. To further assess whether PXR could be involved in the induction of Pgp by anticancer drugs, Pgp protein expression after treatment with the anticancer drugs was determined in both wild-type and PXR-knocked down LS180 cells. Furthermore, the effect of the anticancer drugs on the intracellular accumulation of the Pgp-probes rhodamine 123 and doxorubicin was determined.ResultsOur study showed that vincristine, tamoxifen, vinblastine, docetaxel, cyclophosphamide, flutamide, ifosfamide and paclitaxel activate PXR-mediated Pgp induction, and were additionally shown to affect the intracellular accumulation of the Pgp probe rhodamine 123. Moreover, PXR activation was also shown to reduce the cytotoxic activity of the Pgp substrate doxorubicin in colon cancer cells.ConclusionOur results indicate that several anticancer drugs can activate PXR-mediated induction of Pgp and affect the accumulation of Pgp substrates.

The endocrine disrupting chemical, diethylhexyl phthalate, activates MDR1 gene expression in human colon cancer LS174T cells

Resistance to anticancer drugs is often mediated by the overexpression of P-glycoprotein encoded by the multi-drug resistance (MDR1) gene. The nuclear receptor, steroid and xenobiotic receptor (SXR), is one of the key transcriptional regulators of MDR1 gene expression. A variety of xenobiotics bind to SXR, and stimulate transcription on xenobiotic-response elements (XREs), located in the MDR1 gene promoter. Diethylhexyl phthalate (DEHP) is widely used as a plasticizer for polyvinyl chloride (PVC) medical devices. Previous studies have shown that a significant amount of DEHP leaches from PVC infusion bags and lines during interventions, such as total parenteral nutrition, blood transfusion, and cancer chemotherapy. Thus, the leaching of DEHP during parenteral chemotherapy for cancer patients may facilitate MDR1 expression in various tissues, including cancer cells, which may promote drug resistance. To examine such a hypothesis, the effect of DEHP on SXR-mediated transcription of the MDR1 gene was studied in the human colon adenocarcinoma-derived cell line, LS174T cells, which endogenously express SXR. DEHP increased the SXR-mediated transcription of the MDR1 gene in luciferase-reporter assays. The induction by DEHP was abrogated when a reporter plasmid containing mutated DR+4 motif in the XRE was used. In a mammalian two-hybrid assay, DEHP recruited steroid receptor co-activator-1 to the ligand-binding domain of SXR. Finally, using real-time reverse transcriptase-PCR, we showed that DEHP increased MDR1 gene expression in a dose-dependent manner. We conclude that DEHP is an inducer of the MDR1 gene in this cell line. As such, the leaching of DEHP from the PVC medical devices may influence the MDR1 expression, which may induce resistance to drugs in certain populations of cancer cells.

1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)(2)D(3) specifically activated protein kinase C-alpha (PKC-alpha) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)(2)D(3) activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)(2)D(3) caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1, 25(OH)(2)D(3) also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)(2)D(3) also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)(2)D(3) was mediated by ERK and JNK. Using a specific inhibitor of the Ca(2+)-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1, 25(OH)(2)D(3)-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)(2)D(3) stimulated AP-1 activation in CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to increases in cellular differentiation.

Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D 3 is mediated by mutual modulation of receptor expression

The human colon adenocarcinoma-derived cell line Caco-2 was used as a model system to study the interaction of epidermal growth factor (EGF) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) in control of colorectal cancer cell growth. The mitogenic stimulus of EGF was rapidly transduced via apical and basal membrane receptors alike into elevation of c- myc expression, causing a shift of Caco-2 cells from the G 0/G 1 into the S phase of the cell cycle. The stimulatory effect of EGF on cell division was effectively counteracted by 1,25(OH) 2D 3: the presence of the steroid hormone prevents the negative effect of EGF on vitamin D receptor abundance and concurrently minimises ligand-occupied EGF receptor numbers on both sides of Caco-2 cell monolayers. Our data suggest that EGF and 1,25-(OH) 2D 3 actions on mutual receptor levels represent a specific feature of the potent antimitogenic effect of the steroid hormone on colon cancer cells.

Differentiation-Related Pathways of 1α,25-Dihydroxycholecalciferol Metabolism in Human Colon Adenocarcinoma-Derived Caco-2 Cells: Production of 1α,25-Dihydroxy-3epi-cholecalciferol

We used the human colon adenocarcinoma-derived cell line Caco-2, which spontaneously differentiatesin vitro,as a model system to investigate the metabolism of 1α,25-dihydroxycholecalciferol in colon cancer cells. Subconfluent proliferating and confluent differentiating cells were incubated with 1 μM 1α,25-dihydroxycholecalciferol for a period of 24 to 48 h. HPLC analysis of the lipid extract of both cells and media was performed to isolate and identify the various metabolites of 1α,25-dihydroxycholecalciferol. Undifferentiated, highly proliferating Caco-2 cells metabolized 1α,25-dihydroxycholecalciferol into several side chain modified metabolites formed through the C-24 oxidation pathway. In contrast, no metabolites of the C-24 oxidation pathway were identified in differentiated Caco-2 cells. However, differentiated cells produced significant amounts of a metabolite which was less polar than 1α,25-dihydroxycholecalciferol on a straight phase HPLC system. This metabolite was identified as 1α,25-dihydroxy-3α-cholecalciferol by comigration with a synthetic standard on two different HPLC systems and gas chromatography–mass spectrometry. Thus, we were able to demonstrate that the state of differentiation has a profound influence on 1α,25-dihydroxycholecalciferol metabolism in colon cancer cells.

Growth Regulation of Human Colon Adenocarcinoma-Derived Cells by Calcium, Vitamin D and Epidermal Growth Factor

The Caco-2 cell line was utilized to analyze the role of nutrient factors such as calcium, vitamin D and epidermal growth factor (EGF) in epigenetic control of human colon carcinoma cell growth. Proliferative signals from either low extracellular calcium or EGF, respectively, are transduced in Caco-2 cells via an increase in c-myc proto-oncogene mRNA and nuclear protein expression levels. Activation of the EGF receptor is associated also with down-regulation of the cytoplasmic high-affinity vitamin D receptor (VDR). This would allow colon carcinoma cells to escape from the VDR-mediated anti-mitogenic action of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). However, Caco-2 cells have the unique property to synthesize the vitamin D hormone from 25-hydroxyvitamin D3. 1α,25(OH)2D3, in turn, counteracts the negative effect of EGF on VDR abundancy and slow down tumor cell proliferation through a c-myc-independent pathway.

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.