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In vitro study of P-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in Caco-2 cells

  • Molecular Toxicology
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Abstract

The Caco-2 cell line is a reliable in vitro model for predicting drug intestinal absorption and P-glycoprotein (P-gp)-mediated excretion in humans. Recent in vivo studies suggested the induction of P-gp as a cellular protection tool against paraquat poisoning, through the increase in its pulmonary and intestinal excretion. Thus, the aim of the present work was to evaluate P-gp expression and activity in Caco-2 cells exposed to doxorubicin (a known P-gp inducer) and to correlate these changes with paraquat toxic effects. Cytotoxicity of doxorubicin (0–100 μM) and paraquat (0–1,000 μM) was evaluated for a maximum period of 96 h. In doxorubicin-exposed cells, P-gp expression and transport activity were evaluated by flow cytometry, using a fluorescein isothiocyanate–conjugated antibody and the P-gp fluorescent subtract rhodamine 123, respectively. A significant increase in P-gp expression was observed as soon as 6 h after exposure to 5 μM doxorubicin. P-gp activity also increased after 6 h, but only at higher doxorubicin concentrations (over 50 μM). Paraquat (0–5,000 μM) cytotoxicity was then evaluated with or without previous exposure of the cells to doxorubicin (5–100 μM, a concentration range causing both an increase in P-gp expression and activity). Under P-gp induction, a significant reduction in paraquat cytotoxicity was observed. Furthermore, when these cells were incubated with a specific P-gp inhibitor (UIC2 antibody) the doxorubicin protective effects were blocked, confirming the involvement of P-gp in the reduction in paraquat cytotoxicity. In conclusion, the human Caco-2 cell line model can be used for the study of P-gp induction as an antidotal pathway against substrates of this transporter system.

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Abbreviations

BCRP:

Breast cancer–resistant protein

DMEM:

Dulbecco’s modified Eagle’s medium

DOX:

Doxorubicin

EDTA:

Ethylenediamine tetraacetic acid

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

MTT:

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NEAA:

Nonessential aminoacids

NSAIDs:

Nonsteroidal anti-inflammatory drugs

PBS:

Phosphate-buffered saline solution

P-gp:

P-glycoprotein

PQ:

Paraquat

RHO 123:

Rhodamine 123

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Acknowledgments

This work was supported by the Fundação para a Ciência e Tecnologia (FCT) - project [PTDC/SAU-OSM/101437/2008] - QREN initiative with EU/FEDER financing through COMPETE - Operational Programme for Competitiveness Factors. Renata Silva acknowledges FCT for her PhD grant [SFRH/BD/29,559/2006]. Ricardo Dinis-Oliveira acknowledges FCT for his pos-Doc grant [SFRH/BPD/36,865/2007].

Caco-2 cells were kindly provided by Rosário Monteiro from the Biochemistry Department, Faculty of Medicine, University of Porto, Portugal.

Conflict of interest

The authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal

    Renata Silva, Helena Carmo, Ricardo Dinis-Oliveira, Félix Carvalho, Maria de Lourdes Bastos & Fernando Remião

  2. Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319, Porto, Portugal

    Ricardo Dinis-Oliveira

  3. Department of Clinical Analysis and Public Health, Center of Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicão, Rua José António Vidal, 81, 4760-409, Vila Nova de Famalicão, Portugal

    Ricardo Dinis-Oliveira

  4. Laboratory of Biochemistry, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal

    Anabela Cordeiro-da-Silva

  5. IBMC—Institute of Molecular and Cellular Biology, University of Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal

    Anabela Cordeiro-da-Silva & Sofia Costa Lima

Authors
  1. Renata Silva
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  2. Helena Carmo
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  3. Ricardo Dinis-Oliveira
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  4. Anabela Cordeiro-da-Silva
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  6. Félix Carvalho
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  7. Maria de Lourdes Bastos
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  8. Fernando Remião
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Correspondence to Renata Silva or Fernando Remião.

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Silva, R., Carmo, H., Dinis-Oliveira, R. et al. In vitro study of P-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in Caco-2 cells. Arch Toxicol 85, 315–326 (2011). https://doi.org/10.1007/s00204-010-0587-8

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  • DOI: https://doi.org/10.1007/s00204-010-0587-8

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