Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines

Siissalo, Sanna; La, Laitinen; Koljonen, Maija; Vellonen, Kati‐Sisko; Kortejärvi, Hanna; Urtti, Arto; Hirvonen, Jouni; Kaukonen, Ann Marie

doi:10.1016/j.ejpb.2007.03.017

Cited by 44 publications

(27 citation statements)

References 29 publications

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“…A similar effect was recently reported on the expression of many sulfotransferases (Meinl et al, 2008) and earlier on glutathione-S-transferases (Peters and Roelofs, 1989). Cell differentiation has also been observed to affect the expression profile of efflux proteins in Caco-2 (Siissalo et al, 2007). Although the detailed knowledge on the individual factors that lead to such broad increase in phase II drug-metabolizing enzymes remains unknown, our findings have clear implications on drug uptake studies that use Caco-2 cells.…”

Section: Discussionsupporting

confidence: 51%

“…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. It is worth noting in this respect that Caco-2 cell passage number was previously reported to affect glucuronidation activity (Mizuma et al, 2004), but the range of passage numbers that was examined by these authors was considerably higher than in our drug transport studies (Siissalo et al, 2007).…”

mentioning

confidence: 38%

“…Improving the knowledge and understanding on mechanisms affecting drug permeability, such as biotransformation, will contribute significantly to the interpretation of the results obtained from Caco-2 studies. Several UGTs, as well as glucuronidation activity, were detected in Caco-2 cells before (Münzel et al, 1999;Mizuma et al, 2004;Ishida et al, 2007), and we have now examined the expression of nearly all the human UGTs in these cells, concentrating on passage numbers that have been validated in our laboratory for drug permeability and uptake studies (Koljonen et al, 2006;Siissalo et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionsupporting

confidence: 51%

mentioning

confidence: 38%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Confluent and differentiated Caco-2 cells have enterocyte-like properties (Hidalgo et al, 1989) and contain many enterocyte transporters (Siissalo et al, 2007), supporting their use for assessing both passive diffusion and active transport of drugs across the intestinal epithelium.…”

Section: Introductionmentioning

confidence: 99%

Effects of Cell Differentiation and Assay Conditions on the UDP-Glucuronosyltransferase Activity in Caco-2 Cells

Zhang¹,

Tolonen²,

Rousu³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Cell differentiation increases UDP-glucuronosyltransferase (UGT) gene expression in Caco-2 cells. Glucuronidation of 13 UGT substrates, 1-naphthol, diclofenac, epitestosterone, estradiol, ethinylestradiol, indomethacin, oxazepam, R-and S-propranolol, propofol, testosterone, trifluoperazine, and zidovudine, were studied to derive a broad view on the effect of cell differentiation on the glucuronidation activities of different human UGTs. In parallel, the glucuronidation of these compounds in human liver microsomes (HLM) and human intestinal microsomes (HIM) was analyzed. Because many of the substrates are highly lipophilic, the effects of dimethyl sulfoxide (DMSO) concentrations in the reaction mixture on glucuronidation rates were tested, as well as the effect of alamethicin, a pore-forming peptide. Large differences were observed in the effects of DMSO and alamethicin between recombinant UGTs and Caco-2 cells and HLM and HIM, and, therefore, the activity assays were performed under multiple conditions. Regardless of the assay conditions, however, the results clearly indicated that although differentiation increases glucuronidation activity, the rates in Caco-2 cells are mostly very low, much lower than those in either HLM or HIM. One clear exception was observed: substrates of UGT1A6, such as 1-naphthol, were glucuronidated at very high rates in both undifferentiated and differentiated Caco-2 cells. It may thus be concluded that Caco-2 cells, even differentiated ones, do not provide a good model system to assess first-pass drug glucuronidation in the intestine.

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In Vitro Assays to Predict In Vivo Interactions of P ‐Glycoprotein with Drugs

Volpe

Balimane

2017

Encyclopedia of Drug Metabolism and Interactions

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P‐glycoprotein (P‐gp) is an efflux transporter that pumps endogenous and exogenous compounds out of cells. P‐gp limits drug absorption from the intestines, aids in drug excretion from the liver and kidneys, and prevents entry of drugs into the brain. Given that interactions of a drug with P‐gp can play an important part in its pharmacokinetic properties, there are efforts in early discovery and development to identify compounds as P‐gp substrates or inhibitors. In vitro assays are based on cell membranes (ATPase, vesicles) or intact cells (accumulation, bidirectional) expressing the P‐gp transporter. This chapter reviews these in vitro assays along with their advantages and limitations. In determining whether a new compound is a substrate or inhibitor of P‐gp during drug discovery and development, a tier approach provides a scheme where higher throughput assays lead to those with greater complexity. An important aspect of using in vitro P‐gp assays during drug discovery and development is that they need to be optimized and validated in order to have confidence in their outcomes in determining whether a new compound is a substrate or inhibitor of the efflux transporter. The use of acceptance criteria and negative and positive control substrates and inhibitors is discussed along with how different assay parameters can effect outcomes. In conclusion, well‐established in vitro P‐gp interaction assays are reasonably predictive of the in vivo P‐gp effects in the clinic.

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Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines

Cited by 44 publications

References 29 publications

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

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

Effects of Cell Differentiation and Assay Conditions on the UDP-Glucuronosyltransferase Activity in Caco-2 Cells

In Vitro Assays to Predict In Vivo Interactions of P ‐Glycoprotein with Drugs

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