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

doi:10.1016/j.ejpb.2007.03.017

European Journal of Pharmaceutics and Biopharmaceutics

Volume 67, Issue 2, September 2007, Pages 548-554

https://doi.org/10.1016/j.ejpb.2007.03.017 Get rights and content

Abstract

The mRNA level expression of MDR1, MRP1-6, BCRP and CYP3A4 was determined by quantitative PCR in wild type (Caco-2WT) and vinblastine-treated (Caco-2VBL) Caco-2 cells at different passage levels (32–53). Differentiation increased the mRNA levels of MDR1, BCRP and all the MRPs except MRP4. Corresponding mRNA levels were observed in Caco-2WT and Caco-2VBL, except that the expression of MRD1 was higher in Caco-2VBL than in Caco-2WT cells. CYP3A4 was barely detected in either cell line.

MDR1 functionality was studied using rhodamine123 and verapamil as a substrate–inhibitor pair. Corresponding to the observed differences in mRNA levels, MDR1 activity was higher in the Caco-2VBL cells. In Caco-2WT, MDR1 functionality was elevated at low passage numbers (32–35) compared to higher ones (49–53). Verapamil inhibited MDR1 efflux except at higher passage Caco-2WT cells, where no MDR1 activity could be observed.

The results support the use of Caco-2VBL cells in MDR1 screening. The functional expression is higher than in Caco-2WT and remains consistent across the studied passages without major differences in mRNA levels of other efflux proteins. As both the passage number and the level of cell differentiation affect the expression profile of efflux proteins, short-term cell growth protocols should be evaluated accordingly.

Introduction

The epithelium of the intestinal mucosa is the first significant physical and biochemical barrier to limit drug absorption from the gastrointestinal tract. This barrier has an important role as a defense mechanism against potentially harmful substances, but it also limits the bioavailability of perorally administered drugs. In addition to passive permeation properties, active uptake and efflux systems as well as intestinal metabolic enzymes have been recognized to have a significant effect on the extent of absorption [1].

The multidrug resistance protein MDR1 (P-glycoprotein) is probably the most studied efflux transporter and, although initially discovered in tumor tissues, is also present in normal human intestinal epithelium, blood–brain barrier and hepatic canalicular membranes [2]. Its physiological function in the small intestine is to protect tissues from toxins or xenobiotics by limiting their absorption. MDR1 has also been observed to limit the oral bioavailability of several drugs by the same mechanism [3]. Inhibition and induction of MDR1 are potential mechanisms for drug interactions. The presence of MDR1 at important barrier epithelia that control drug absorption, distribution and excretion makes its substrates likely candidates for drug–drug interactions. There is also considerable interindividual variation in the expression of MDR1 as well as the other efflux proteins and metabolic enzymes and, thus, in their role in limiting oral absorption. This is due to polymorphism and/or variable exposure to inductive or inhibitory food components and medicines.

The clinical importance of the above-described defense mechanisms has led to the increased interest to develop in vitro methods that can be used during drug discovery and development in order to recognize e.g. MDR1 substrates. The Caco-2 is a widely used cell line derived from human colorectal carcinoma [4]. Permeability across fully differentiated Caco-2 monolayers is considered to model intestinal absorption, since the cells represent many of the characteristics and functions (i.e. transporters and metabolic enzymes) similar to the epithelium of the small intestine [5]. Caco-2 monolayers are used to identify drugs with potential absorption problems and to select clinical drug candidates based on absorption characteristics [6].

Expression of MDR1 has been demonstrated in Caco-2 cells, but several factors have been shown to influence its expression levels and/or functionality [7]. These factors include the age in culture and level of differentiation, passage number, and exposure to modulators, such as some MDR1 substrates [8], [9]. Typically, the expression may be quite high in the lower passages and then decline at higher passage numbers [8]. Induction of functional MDR1 expression in Caco-2 cells has been successful by adding e.g. vinblastine or vincristine to the culturing medium [10], [11]. Vinblastine-induced Caco-2 cell line has even been proposed as a platform for screening MDR1 substrates [12]. However, the expression of several other efflux proteins (MRP1-6, BCRP) has been observed in Caco-2 cell lines, but the results tend to vary between different laboratories and cell batches and even cultivation lots originating from the same batch [13], [14], [15]. This creates a need for careful characterization of the Caco-2 cells in use in terms of efflux protein expression and function in order to develop a reliable in vitro model to study efflux substrates.

In this study, we examined the effects of cell age (passage number) and level of differentiation on the mRNA expression levels of efflux proteins MDR1, MRP1-6 and BCRP in Caco-2 wild type (WT) and vinblastine-treated (VBL) cell models. As considerable overlap has been observed for substrates and inhibitors of efflux proteins, variability in the expression levels of efflux proteins would affect the suitability of the cell culture for efflux activity studies of any individual efflux protein. Of similar interest was the selectivity of the induction of MDR1 with vinblastine as the co-regulation of efflux proteins and metabolizing enzymes has been suggested [16], [17], [18]. Therefore, the mRNA levels of CYP3A4 were also studied. In addition, transport experiments with rhodamine123 across differentiated Caco-2 monolayers were performed to relate mRNA results with information on the functionality of the MDR1 in Caco-2WT and Caco-2VBL cells within the studied passage range.

Section snippets

Reagents and materials

Cell culturing reagents were purchased from Euroclone (Pero, Italy) except for fetal bovine serum and HBSS/PBS 10× concentrates from Gibco Invitrogen Corporation (Carlsbad, CA, USA) and Hepes from Sigma (St. Louis, MO, USA). All the plasticware were obtained from Corning B.V. Life Sciences (Schiphol-Rijk, Netherlands). Rhodamine123 and vinblastine were from Fluka (Buchs, Switzerland) and verapamil from MP Biomedicals (Aurora, OH, USA). Materials and reagents for qRT-PCR were purchased from

Expression of the efflux proteins in Caco-2WT and Caco-2VBL cells

The expression of MRP1 was at about similar level in all the samples (Table 1). The expression levels of MDR1, MRP2, MRP3 and MRP6 were higher in differentiated than undifferentiated cells (Fig. 1). Interestingly, the mRNA level expression of MRP4 was lower in differentiated than undifferentiated cells. Differentiation did not affect the expression of MRP5, but it was somewhat higher in older passages. The expression of BCRP was somewhat lower in the undifferentiated cells. In contrast to

Discussion

During the selection of clinical drug candidates, Caco-2 cell lines are often used as platforms for screening for potential interactions related to efflux mechanisms such as MDR1 (P-glycoprotein). However, considerable overlap has been reported in the specificity of substrates and inhibitors of different efflux proteins and metabolizing enzymes. Therefore, an essential part of designing study protocols is to elucidate the effect of the growth protocol on the transporters involved in the system.

Acknowledgements

TEKES (Finnish Funding Agency for Technology) and Orion Pharma are acknowledged for funding the work of Sanna Siissalo. Special thanks to Timo Korjamo and Mika Reinisalo at the University of Kuopio for help and qRT-PCR primers.

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

Abstract

Introduction

Section snippets

Reagents and materials

Expression of the efflux proteins in Caco-2WT and Caco-2VBL cells

Discussion

Acknowledgements

Pharmacol. Ther.

Adv. Drug Deliv. Rev.

J. Biol. Chem.

J. Pharm. Sci.

Eur. J. Pharm. Sci.

Adv. Drug Del. Rev.

Biochem. Pharmacol.

Eur. J. Pharm. Sci.

Eur. J. Pharm. Biopharm.

Eur. J. Cancer

J. Pharm. Sci.

J. Biomol. Screen.

Drug Metab. Pharmacokinet.

Role of transport proteins in drug absorption, distribution and excretion

Xenobiotica

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

J. Histochem. Cytochem.

Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture

Biol. Cell

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