P-Glycoprotein (P-gp) Mediated Efflux in Caco-2 Cell Monolayers: The Influence of Culturing Conditions and Drug Exposure on P-gp Expression Levels

Anderle, Pascale; Niederer, Eva; Rubas, Werner; Hilgendorf, Constanze; Spahn‐Langguth, Hildegard; Wunderli‐Allenspach, Heidi; Merkle, Hans P.; Langguth, Peter

doi:10.1021/js970372e

Cited by 198 publications

(126 citation statements)

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“…1A). Next, we asked whether shear stress increased the expression of an endogenous PXR target gene MDR1, which is a known PXR target encoding p-glycoprotein or ATP-binding cassette B1 (ABCB1), a transporter responsible for drug efflux (12,13). Quantitative reverse-transcriptase PCR (qRT-PCR) showed that MDR1 expression was induced by LSS, but not OSS, in human umbilical vein ECs (HUVECs) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Shear stress activation of nuclear receptor PXR in endothelial detoxification

Wang

Fang

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Endothelial cells (ECs) are constantly exposed to xenobiotics and endobiotics or their metabolites, which perturb EC function, as well as to shear stress, which plays a crucial role in vascular homeostasis. Pregnane X receptor (PXR) is a nuclear receptor and a key regulator of the detoxification of xeno-and endobiotics. Here we show that laminar shear stress (LSS), the atheroprotective flow, activates PXR in ECs, whereas oscillatory shear stress, the atheroprone flow, suppresses PXR. LSS activation of PXR in cultured ECs led to the increased expression of a PXR target gene, multidrug resistance 1 (MDR1). An in vivo study using rats showed that the expression of MDR1 was significantly higher in the endothelium from the descending thoracic aorta, where flow is mostly laminar, than from the inner curvature of aortic arch, where flow is disturbed. Functionally, LSSactivated PXR protects ECs from apoptosis triggered by doxorubicin via the induction of MDR1 and other detoxification genes. PXR also suppressed the expression of proinflammatory adhesion molecules and monocyte adhesion in response to TNF-α and lipopolysaccharide. Overexpression of a constitutively active PXR in rat carotid arteries potently attenuated proinflammatory responses. In addition, cDNA microarray revealed a large number of the PXR-activated endothelial genes whose products are responsible for major steps of detoxification, including phase I and II metabolizing enzymes and transporters. These detoxification genes in ECs are induced by LSS in ECs in a PXR-dependent manner. In conclusion, our results indicate that PXR represents a flow-activated detoxification system to protect ECs against damage by xeno-and endobiotics.hemodynamics | endothelial homeostasis | nuclear hormone receptor | gene regulation

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Section: Resultsmentioning

confidence: 99%

Shear stress activation of nuclear receptor PXR in endothelial detoxification

Wang

Fang

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Similarly to the small intestinal epithelium, fully differentiated Caco-2 cells form a monolayer of polarized cells, develop tight junctions, and are surmounted by an apical brush border (Hidalgo et al, 1989;Vachon and Beaulieu, 1992). Another inherent property of the Caco-2 cells is the expression of many enterocytic transporter systems, the expression level of which often increases during cell differentiation (Anderle et al, 1998;Siissalo et al, 2007). The passage number (the number of times the cells in the culture have been detached, diluted, and grown again to near confluence) of Caco-2 cells also affects many of their properties, such as cell viability and growth in culture (Briske-Anderson et al, 1997;Anderle et al, 1998).…”

mentioning

confidence: 99%

“…Another inherent property of the Caco-2 cells is the expression of many enterocytic transporter systems, the expression level of which often increases during cell differentiation (Anderle et al, 1998;Siissalo et al, 2007). The passage number (the number of times the cells in the culture have been detached, diluted, and grown again to near confluence) of Caco-2 cells also affects many of their properties, such as cell viability and growth in culture (Briske-Anderson et al, 1997;Anderle et al, 1998). Thus, it is important to know and define the passage number of the cells under investigation, as well as interesting to examine its effect on UGT expression.…”

mentioning

confidence: 99%

The Expression of Most UDP-Glucuronosyltransferases (UGTs) Is Increased Significantly during Caco-2 Cell Differentiation, whereas UGT1A6 Is Highly Expressed Also in Undifferentiated Cells

Siissalo¹,

Zhang²,

Stilgenbauer³

et al. 2008

Drug Metab Dispos

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ABSTRACT:The human colon carcinoma cell line Caco-2 is often used as a model for intestinal drug absorption. To better understand xenobiotic glucuronidation in Caco-2 cells, we have examined the expression levels of different UDP-glucuronosyltransferases (UGTs) in them. The effects of two main factors were investigated, namely, passage number and cell differentiation. Hence, the mRNA levels of 15 human UGTs of subfamilies 1A and 2B were assessed in both undifferentiated and fully differentiated cells at four passage levels: P31, P37, P43, and P49. Quantitative reverse transcriptasepolymerase chain reaction was used to determine the mRNA levels of individual UGTs, and the values were normalized using ␤-actin as a reference gene. The results indicate that although passage number in the tested range exerts a mild effect on the expression level of several UGTs, the contribution of cell differentiation is much larger. The expression of nearly all the UGTs that were examined in this study was significantly, sometimes greatly, increased during cell differentiation. UGT1A6 was a distinct exception to this rule, however, because it was already highly expressed in the undifferentiated cells. The mRNA findings were confirmed at the enzyme activity level by measuring the glucuronidation of 1-naphthol, a very good substrate for UGT1A6, as well as estradiol that is not glucuronidated by this enzyme. The results revealed that 1-naphthol glucuronidation activity was high in both the differentiated and undifferentiated cells, whereas estradiol glucuronidation was only detected in the differentiated cells. Thus, Caco-2 cell differentiation plays a major role in UGT expression and ensuing metabolic reactions.

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“…A pure Caco-2 cell model also has a overexpression of P-glycoprotein which may lead to higher secretion rates and consequently lower permeability in the absorptive direction (Anderle et al 1998 ). The HT-29MTX cell line has less expression of tight junctions.…”

Section: Relevance To the Human In Vivo Situationmentioning

confidence: 99%

Co-cultivation of Caco-2 and HT-29MTX

Kleiveland

2015

The Impact of Food Bioactives on Health

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The intestinal epithelium is one of the body's largest mucosal surfaces and it generates a physical barrier against the external environment. The majority of cells lining the epithelium are absorptive enterocytes with mainly metabolic and digestive functions. Hence, the diversity of functions the intestinal epithelium carries out depends on the presence of additional specialized intestinal epithelial cells (IEC). Secretory IEC, goblet cells, enteroendocrine cells and Paneth cells are specialized cells that participate in maintaining the digestive and barrier functions of the epithelium. Goblet cells release mucins, which give rise to a mucus layer on the epithelial surface that functions as physical and biochemical barrier for luminal content. The presence of the different epithelial cell types in an in vitro model will affect how well the model refl ects the properties of the intestinal epithelium. We here describe a co-cultivation system of enterocytes and goblet cells, which are the two major epithelial cell types.

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P-Glycoprotein (P-gp) Mediated Efflux in Caco-2 Cell Monolayers: The Influence of Culturing Conditions and Drug Exposure on P-gp Expression Levels

Cited by 198 publications

References 19 publications

Shear stress activation of nuclear receptor PXR in endothelial detoxification

Shear stress activation of nuclear receptor PXR in endothelial detoxification

The Expression of Most UDP-Glucuronosyltransferases (UGTs) Is Increased Significantly during Caco-2 Cell Differentiation, whereas UGT1A6 Is Highly Expressed Also in Undifferentiated Cells

Co-cultivation of Caco-2 and HT-29MTX

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