in vitro study of p-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in caco-2...

MOLECULAR TOXICOLOGY

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

Renata Silva • Helena Carmo • Ricardo Dinis-Oliveira •

Anabela Cordeiro-da-Silva • Sofia Costa Lima • Felix Carvalho •

Maria de Lourdes Bastos • Fernando Remiao

Received: 21 May 2010 / Accepted: 1 September 2010 / Published online: 21 September 2010

� Springer-Verlag 2010

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 cel-

lular 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 doxo-

rubicin (a known P-gp inducer) and to correlate these

changes with paraquat toxic effects. Cytotoxicity of

doxorubicin (0–100 lM) and paraquat (0–1,000 lM) 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 isothio-

cyanate–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 lM doxorubicin. P-gp activity also

increased after 6 h, but only at higher doxorubicin con-

centrations (over 50 lM). Paraquat (0–5,000 lM) cyto-

toxicity was then evaluated with or without previous

exposure of the cells to doxorubicin (5–100 lM, a con-

centration range causing both an increase in P-gp

expression and activity). Under P-gp induction, a signifi-

cant 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 involve-

ment of P-gp in the reduction in paraquat cytotoxicity. In

conclusion, the human Caco-2 cell line model can be used

R. Silva (&) � H. Carmo � R. Dinis-Oliveira � F. Carvalho �M. de Lourdes Bastos � F. Remiao (&)

REQUIMTE, Toxicology Department, Faculty of Pharmacy,

University of Porto, Rua Anıbal Cunha,

164, 4099-030 Porto, Portugal

e-mail: [email protected]

F. Remiao


H. Carmo


F. Carvalho


M. de Lourdes Bastos


R. Dinis-Oliveira

Faculty of Medicine, University of Porto,

Al. Prof. Hernani Monteiro,

4200-319 Porto, Portugal


R. Dinis-Oliveira

Department of Clinical Analysis and Public Health,

Center of Research in Health Technologies

(CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicao,

Rua Jose Antonio Vidal, 81,

4760-409 Vila Nova de Famalicao, Portugal

A. Cordeiro-da-Silva

Laboratory of Biochemistry, Faculty of Pharmacy,

University of Porto, Rua Anıbal Cunha, 164,



A. Cordeiro-da-Silva � S. C. Lima

IBMC—Institute of Molecular and Cellular Biology,

University of Porto, Rua do Campo Alegre, 823,


S. C. Lima


123

Arch Toxicol (2011) 85:315–326

DOI 10.1007/s00204-010-0587-8

for the study of P-gp induction as an antidotal pathway

against substrates of this transporter system.

Keywords P-glycoprotein induction � P-glycoprotein

transport activity � Paraquat toxicity � Caco-2 cells

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

Introduction

P-glycoprotein (P-gp; ABCB1) is an ATP-dependent efflux

pump encoded by the MDR1 gene in humans (Ambudkar

et al. 1999; Chang 2003; Gottesman et al. 2002; Shirasaka

et al. 2008; Silverman 1999). It belongs to the ATP-binding

cassette (ABC) superfamily of transporters (Ambudkar et al.

1999; Chang 2003; Gottesman et al. 2002; Shirasaka et al.

2008; Silverman 1999), which also includes the multidrug

resistance–associated proteins (MRP1, MRP2, MRP3,

MRP4 and MRP5), and the breast cancer–resistant protein

(BCRP/ABCG2), an ABC half-transporter (Chang 2003).

This efflux protein is highly expressed in neoplastic cells

from several cancer types, conferring a multidrug resis-

tance phenotype to those cells, which become resistant to

chemotherapy with anticancer drugs such as vinblastine,

actinomycin D and daunorubicin (Chang 2003; Gottesman

et al. 2002; Shirasaka et al. 2008; Silverman 1999).

In addition to the expression in tumor cells, P-gp is widely

distributed in the apical surfaces of normal human epithelial

tissues including the gastrointestinal tract, kidney, placenta,

testes and the blood–brain barrier (Thiebaut et al. 1987). In

these tissues, this 170-kDa protein plays an important role in

the excretion of a variety of structurally and pharmacolog-

ically unrelated hydrophobic compounds including vinca

alkaloids, colchicine, antibiotics, anthracyclines, cardiac

glycosides, organic cations and pesticides (Cordon-Cardo

et al. 1990; Gottesman et al. 2002). Given its cellular

polarized expression, broad substrate specificity and efflux

capacity, this protein may be suggested as an intracellular

protection mechanism against xenobiotics (Hunter et al.

1993b; Huynh-Delerme et al. 2005; Watanabe et al. 2005).

P-gp is inducible by many drugs including dexametha-

sone, rifampicin, the herbal antidepressant St John’s wort

(hyperforin and hypericin) and chemotherapeutic agents

namely, doxorubicin, daunorubicin and vinblastine (Zhou

2008). P-gp is induced not only by a number of chemical

compounds but also by physical stress, such as X-irradiation,

UV-irradiation and heat shock (Zhou 2008).

Caco-2 cells closely mimic the enterocytes of the small

intestine (Barta et al. 2008). This well-established human

carcinoma cell line, derived from human colorectal adeno-

carcinoma, exhibits spontaneous morphological and bio-

chemical enterocytic differentiation after confluence in

culture (Huynh-Delerme et al. 2005). These cells have been

widely accepted as a reliable in vitro model for predicting

drug intestinal absorption and excretion in humans (Barta

et al. 2008; Huynh-Delerme et al. 2005; Watanabe et al.

2005; Yamashita et al. 2000; Yamashita et al. 2002a;

Yamashita et al. 2002b). Caco-2 cells express P-gp (Hidalgo

and Jibin 1996; Hunter et al. 1993b; Shen et al. 2007;

Watanabe et al. 2005) as well as other transporters (Hirohashi

et al. 2000; Taipalensuu et al. 2001) and, except for BCRP, the

expression levels in these cells are in good agreement with

those of the normal human jejunum (Taipalensuu et al. 2001).

Moreover, the apical membrane localization of P-gp in Caco-

2 cells was confirmed, demonstrating its polarized expression

in this intestinal cell line (Hunter et al. 1993b).

Paraquat dichloride (methyl viologen; PQ), a known P-gp

substrate, is a widely used herbicide that is highly toxic upon

ingestion as a result of its pneumocyte accumulation through

active transport. Previous studies developed by our group

confirmed that P-gp induction was an effective antidotal

pathway against paraquat-induced lung toxicity in rats

exposed to high doses of the herbicide (Dinis-Oliveira et al.

2006a; Dinis-Oliveira et al. 2006b). The dexamethasone-

induced de novo synthesis of P-gp in intestine and lungs

resulted in a remarkable decrease in paraquat accumulation

in the lung, with an increase in its fecal excretion (Dinis-

Oliveira et al. 2006b). Additionally, there was an evident

decrease in lung damage, with lower lipid peroxidation and

carbonyl groups content, and a normalization of myeloper-

oxidase activity, as well as a significant enhancement in

survival time (Dinis-Oliveira et al. 2006a).

These results prompted the development of an in vitro

model for the screening and selection of potent and safe P-gp

inducers using the Caco-2 cell line. Although several

chemicals and clinically used drugs induce P-gp, no specific

P-gp inducer has yet been described. For this purpose, we

evaluated P-gp expression and transport activity in the

presence of doxorubicin, a known P-gp inducer.

316 Arch Toxicol (2011) 85:315–326

123

Additionally, we tested this model by effectively reducing

the toxic effect of a xenobiotic (paraquat) through P-gp

induction and correlated the changes in P-gp expression and

activity with the observed paraquat cytotoxic effects.

Materials and methods

Materials

Caco-2 cells were kindly provided by Rosario Monteiro from

the Biochemistry Department, Faculty of Medicine, University

of Porto, Portugal. Dulbecco’s modified Eagle’s medium

(DMEM) with 4.5 g/L glucose and GlutMAXTM, nonessential

amino acids (NEAA), fetal bovine serum (FBS), 0.25% tryp-

sin/1 mM EDTA, antibiotic (10,000 U/mL penicillin,

10,000 lg/mL streptomycin), fungizone (250 lg/mL ampho-

tericin B) and human transferrin (4 mg/mL) were purchased

from Gibco Laboratories (Lenexa, KS). AccuGENE�(1 9 PBS buffer) was purchased from Lonza Laboratories

(Verviers, Belgium). Doxorubicin (DOX), rhodamine 123

(RHO 123), paraquat (PQ), cyclosporine, verapamil and (4,5-

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

(MTT) were obtained from Sigma (St. Louis, MO, USA).

P-glycoprotein monoclonal antibody (clone UIC2) conjugated

with fluorescein isothiocyanate (FITC) was purchased from

Abcam (Cambridge, United Kingdom). Monoclonal anti-

human P-glycoprotein antibody (IOTest� CD243) used in the

P-gp inhibition studies was purchased from Beckman Coulter,

Inc. (Fullerton, USA). Flow cytometry reagents (BD Facs-

FlowTM and Facs CleanTM) were purchased from Becton,

Dickinson and Company (San Jose, CA). Mouse IgG2a-FITC

(negative moAb control to UIC2) was purchased from

ImmunoTools GmbH, (Friesoythe, Germany).

All the reagents used were of analytical grade or of the

highest grade available.

Caco-2 cell culture

Caco-2 cells were routinely cultured in 75-cm2 flasks using

DMEM medium supplemented with 10% FBS, 1% NEAA,

1% antibiotic, 1% fungizone and 6 lg/mL transferrin.

Cells were maintained in a 5% CO2–95% air atmosphere at

37�C, and the medium was changed every 2 days. Cultures

were passaged weekly by trypsinization (0.25% trypsin/

1 mM EDTA). The cells used for all the experiments were

taken between the 58 and 63th passages.

Doxorubicin cytotoxicity assays

For the in vitro evaluation of DOX cytotoxicity, the MTT

assay that measures mitochondrial activity was performed.

The cells were seeded onto 48-well plates at a density of

60,000 cells/cm2 to obtain confluent monolayers at the day

of the experiment. On the day of the experiment, the cells

were washed twice with PBS buffer (pH 7.4) and exposed

to DOX (0–100 lM) in fresh cell culture medium for 6, 12,

24, 48, 72 and 96 h.

For the MTT assay, at each selected time point, the cell

culture medium was removed, and the cells were washed

twice with PBS (pH 7.4), followed by the addition of fresh

cell culture medium containing 0.5 mg/L MTT and incu-

bation at 37�C in a humidified, 5% CO2–95% air atmo-

sphere for 1 h. After this incubation period, the cell culture

medium was removed, and the formed formazan crystals

dissolved in 100% DMSO. The absorbance was measured at

550 nm in a multi-well plate reader (BioTek Instruments,

Vermont, US). The percent cell viability relative to that of

the control cells was used as the cytotoxicity measure.

P-glycoprotein expression and activity

For the in vitro evaluation of P-gp expression and activity,

the cells were seeded onto 24-well plates at a density of

60,000 cells/cm2 to obtain confluent monolayers at the day

of the experiment. On the day of the experiment, the cells

were washed twice with PBS buffer (pH 7.4) and exposed

to DOX (0–100 lM) in fresh cell culture medium for 6, 12,

24, 48, 72 and 96 h. After the incubation period, the cells

were washed twice with PBS and trypsinized with 0.25%

trypsin /1 mM EDTA to obtain a cell suspension. The cells

were then divided into two aliquots of approximately

250,000 cells, for the evaluation of P-gp activity and

expression, respectively. Given the cytotoxicity data

obtained, for the 50 and 100 lM DOX concentrations, P-gp

expression and transport activity were only evaluated up to

24 h after exposure.

For the evaluation of P-gp expression, the cells were

centrifuged (300g /10 min) and suspended in PBS buffer

(pH 7.4) containing 10% FBS and P-gp antibody [UIC2]

conjugated with FITC. The antibody dilution in this

experiment was applied according to the manufacturer’s

instructions for flow cytometry. Mouse IgG2a_FITC was

used as an isotype-matched negative control to estimate

nonspecific binding of the FITC-labeled anti-P-glycoprotein

antibody [UIC2]. The cells were then incubated for 30 min

at 37�C in the dark. After this incubation period, the cells

were washed twice with PBS buffer (pH 7.4) containing

10% FBS, suspended in ice-cold PBS and kept on ice until

analysis. Fluorescence measurements of isolated cells were

taken with a flow cytometer (FACSCalibur, Becton-

Dickinson Biosciences). The green fluorescence of FITC-

UIC2 antibody was measured by a 530 ± 15 nm band-pass

filter (FL1). Acquisition of data for 10,000 cells was gated

to include viable cells based on their forward and side light

scatters and the propidium iodide (4 lg/mL) incorporation

Arch Toxicol (2011) 85:315–326 317

123

(propidium iodide interlaces with a nucleic acid helix with

a resultant increase in fluorescence intensity emission at

615 nm). Logarithmic fluorescence was recorded and dis-

played as a single parameter histogram. The geometric

mean of fluorescence intensity for 10,000 cells was the

parameter used for comparison (calculated as percentage of

control). Nonlabeled cells (with or without doxorubicin)

were analyzed in each experiment by a 530 ± 15 nm band-

pass filter (FL1) in order to detect a possible contribution

from cells autofluorescence to the analyzed fluorescence

signals.

The P-gp transport activity was also evaluated by flow

cytometry using 1 lM RHO 123 as a P-gp fluorescent sub-

strate and 10 lM cyclosporine as a P-gp inhibitor. Rhoda-

mine 123 and cyclosporine cytotoxicity (evaluated after a

30-min incubation period, the time necessary for RHO 123

accumulation) was previously determined by the propidium

iodide incorporation assay. The P-gp transport activity assay

consisted of two phases: (i) an accumulation phase, in which

P-gp activity was blocked with 10 mM NaN3 (to inhibit

energy production) and 10 lM cyclosporine (a known P-gp

inhibitor) in order to accumulate the substrate inside the cells

and (ii) an efflux phase where the energy-dependent P-gp

function was re-established due to removal of the P-gp

inhibitor (cyclosporine) and the addition of an energy source

(DMEM with 4.5 g/mol glucose). When P-gp activity

increases, the amount of RHO 123 effluxed from the cells is

higher and accompanied by a decrease in the fluorescence

intensity due to the corresponding decrease in intracellular

RHO 123. For the accumulation phase, the cells were cen-

trifuged (300g/ 10 min), suspended in PBS buffer (pH 7.4)

containing 10 mM NaN3, 10 lM cyclosporine and 1 lM

RHO123 and incubated at 37�C for 30 min. After the

accumulation of the fluorescent substrate, the cells were

washed twice with ice-cold PBS with 10% FBS and centri-

fuged (300g/10 min) at 4�C. For the efflux phase, the

obtained cell pellet was then suspended in DMEM medium

containing 4.5 g/L glucose and incubated for 30 min at

37�C. After this efflux period, the cells were washed twice

with ice-cold PBS with 10% FBS, suspended in ice-cold

PBS and immediately analyzed as described above for the

P-gp expression assay using the FACSCalibur. The green

intracellular fluorescence of Rho123 was measured by a

530 ± 15 nm band-pass filter (FL1).

Paraquat cytotoxicity assays

Paraquat (PQ) cytotoxicity was evaluated in Caco-2 cells by

the MTT assay. Briefly, the cells were seeded onto 48-well

plates, at a density of 60,000 cells/cm2 and incubated, after

confluence, with PQ (0–1,000 lM) for 6, 12, 24, 48, 72 and

96 h. At each time point, cytotoxicity was evaluated as

described above for the DOX cytotoxicity assay.

Based on the obtained results, PQ cytotoxicity was then

evaluated with or without previous exposure of the cells to

doxorubicin after a 24-h incubation period and at a larger

concentration range (0–5,000 lM). The cells were seeded

onto 48-well plates to obtain confluent monolayers at the

day of the experiment. On the day of the experiment, the

cells were washed twice with PBS buffer (pH 7.4) and

exposed to doxorubicin (0, 5, 10, 50 and 100 lM) in fresh

cell culture medium for 24 h. After this incubation period,

the cells were washed twice with PBS buffer (pH 7.4) and

exposed to PQ (0–5,000 lM) in fresh cell culture medium

for another 24 h. Cytotoxicity was evaluated by the MTT

assay, as described before. To confirm the involvement of

P-gp in the DOX protective effects, these incubations were

repeated in the presence of a P-gp specific inhibitor (20 lL

of the UIC2 antibody stock solution for 500,000 cells,

according to the manufacturer instructions). In this assay,

the protocol was similar to that described above but with

previous P-gp inhibitor incubation for 30 min before PQ

exposure. A schematic representation of this protocol is

illustrated in Fig. 1.

The nonspecific inhibitors verapamil (50 and 100 lM)

and cyclosporine (5 and 10 lM) were also tested for P-gp

inhibition. With these inhibitors, and in the absence of

doxorubicin exposure, the cytotoxicity of PQ decreased.

This occurs probably due to an inhibiting effect on PQ

transporter, blocking the PQ entrance into the cells. The

inhibiting effect of both cyclosporine and verapamil on the

carrier-mediated transport system for choline was already

reported (Crowe et al. 2002) and therefore, these inhibitors

were not further tested.

Statistical analysis

For each assay, all experiments were performed in tripli-

cate and SD values were always \ 10%.

Data obtained in the DOX cytotoxicity assays and P-gp

expression and transport activity assay are expressed as

mean ± SEM (standard error of the mean) from 4 inde-

pendent experiments. All statistical calculations were per-

formed with the GraphPad Prism version 5.00 for Windows

(GraphPad Software, San Diego California, USA). Nor-

mality of the data distribution was assessed by three tests

(KS normality test, D’Agostino and Pearson omnibus

Fig. 1 Schematic representation of the experimental protocol for the

evaluation of doxorubicin (DOX) protective effects against paraquat

(PQ) cytotoxicity

318 Arch Toxicol (2011) 85:315–326

123

normality test and Shapiro–Wilk normality test). Statistical

comparison between groups was estimated using the non-

parametric method of Kruskal–Wallis [one-way analysis of

variance (ANOVA) on ranks] followed by Dunn’s post hoc

test. In all cases, P values lower than 0.05 were considered

statistically significant.

Data obtained with the PQ cytotoxicity assays are

expressed as mean ± SEM from 3 independent experiments.

Statistical comparison between groups was estimated using

the parametric method of one-way ANOVA on ranks fol-

lowed by the Bonferroni’s post hoc test. In all cases, P values

lower than 0.05 were considered statistically significant.

For the PQ cytotoxicity assay performed with or without

previous exposure to DOX and in the presence or absence of

the P-gp inhibitor (UIC2 antibody), 3 independent experiments

were performed. Concentration–response curves were fitted

using least squares as the fitting method, and the comparisons

between curves (bottom, top and LOG EC50) were made using

the extra sum-of-squares F test. In all cases, P values lower

than 0.05 were considered statistically significant.

Results

Doxorubicin cytotoxicity assays

Previous to the evaluation of the effect of doxorubicin on

P-gp expression and activity, the cytotoxicity of this

inducer was determined at different concentrations and

time points. The conversion of MTT to formazan crystals

by mitochondrial dehydrogenases was used as an index of

mitochondrial viability and was evaluated up to a maxi-

mum period of 96 h. Figure 2 presents the cytotoxic effects

of 0–100 lM DOX in Caco-2 cells. Doxorubicin exposure

resulted in a concentration- and time-dependent cytotoxic

effect that was more pronounced after 48 h of incubation.

The obtained results show that up to a period of 6 h

incubation, no significant effect on mitochondrial viability

occurred at any of the tested DOX concentrations. For

DOX concentrations up to 1 lM, no significant cytotoxic

effect was observed over 72 h of incubation. For the

5–100 lM concentration range, after 12 and 24 h, a small

but significant decrease in mitochondrial viability was

observed (values between 85 and 91% compared to con-

trol), although at the highest 100 lM DOX concentration

and after 24 h of incubation, a higher cytotoxicity occurred

(80% cell viability compared to control). For exposure

times higher than 48 h, there was a significant and more

pronounced reduction in mitochondrial activity, mainly at

the 5–100 lM DOX concentration range. At the end of the

experiment (96 h), mitochondrial viability decreased to

values of 56–49% when compared to the control cells

within this concentration range.

P-glycoprotein expression and activity

The effect of DOX on P-gp expression in Caco-2 cells was

evaluated by flow cytometry, using a P-gp monoclonal

antibody [UIC2] conjugated with FITC. Nonspecific

binding of the FITC-labeled–anti-P-glycoprotein antibody

[UIC2] was not observed as estimated by the fluorescence

obtained with the isotype-matched negative control. The

results presented in Fig. 3 show that DOX significantly

increased P-gp expression in a time- and concentration-

dependent manner. At 5, 10, 50 and 100 lM DOX, a sig-

nificant increase in P-gp expression as soon as 6 h was

observed (147, 186, 312 and 365% when compared to

control, respectively), whereas for 1 lM DOX similar

results were obtained only after 48 h (204% when com-

pared to control). At the 0.1 and 0.5 lM DOX concentra-

tions, no significant increase was observed during the time

course of the experiment.

The P-gp transport activity was also studied by flow

cytometry using 1 lM rhodamine 123 (RHO 123) as a P-gp

fluorescent substrate and 10 lM cyclosporine as a P-gp

inhibitor. No cytotoxic effects were observed for RHO 123

and cyclosporine at these concentrations after 30 min of

incubation (data not shown). Figure 4 represents the results

obtained for the evaluation of P-gp transport activity in

Caco-2 cells when exposed to 0–100 lM DOX at different

time points (6, 12, 24, 48, 72 and 96 h). A time- and

concentration-dependent significant increase in P-gp

transport activity was observed as soon as 6 h after DOX

exposure. For the 50 and 100 lM concentrations, after 6 h

of incubation, P-gp transport activity significantly

increased to values of 126 and 132% when compared to

control, respectively. Moreover, for the 0.5–10 lM DOX

concentration range, P-gp activity significantly increased

after 48 h (128–136% when compared to control). When

Caco-2 cells were exposed to the lower 0.1 lM DOX

concentration, no significant effect in P-gp transport

activity was observed during the time course of the

experiment.

Paraquat cytotoxicity assays

Paraquat cytotoxicity (0–1,000 lM) was evaluated by the

MTT assay over a time period of 96 h. Figure 5 represents

the results expressed as percent viability compared to

control. Cell death occurred in a concentration- and time-

dependent manner. For the 0–10 lM PQ concentration

range, no significant effect was observed in cell viability

during the time course of the experiment. For the higher

tested PQ concentrations (500 and 1,000 lM), a small but

significant decrease in cell viability was observed as soon

as 6 h (down to 94 and 90% when compared to control

values, respectively). At the end of the experiment, for

Arch Toxicol (2011) 85:315–326 319

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these two concentrations, a significant cytotoxic effect

could be observed (down to 27 and 9% cell viability when

compared to the control, respectively). For the 50 and

100 lM concentrations, a significant decrease in cell via-

bility was observed after 24 h of exposure (down to 89 and

80% when compared to control, respectively), which was

more pronounced after 96 h of incubation (87 and 73%

when compared to control, respectively).

To study the protective effects of the increase in P-gp

expression and activity, the cytotoxicity of PQ (0–5,000 lM)

Fig. 2 Doxorubicin (DOX) cytotoxicity in Caco-2 cells at different

time points. Results are presented as mean ± SEM from 4 indepen-

dent experiments (triplicates were performed in each experiment).

Statistical comparisons were made using the Kruskal–Wallis test

followed by the Dunn’s multiple comparison post hoc test (*p \ 0.05;

**p \ 0.01; ***p \ 0.001 vs control)

Fig. 3 P-glycoprotein expression levels in Caco-2 cells exposed to

doxorubicin (DOX) (0–100 lM) at different time points (6, 12, 24,

48, 72 and 96 h). X means that these concentrations were not tested.

Results are presented as mean ± SEM from 4 independent

experiments (triplicates were performed in each experiment). Statis-

tical comparisons were made using the Kruskal–Wallis test followed

by the Dunn’s multiple comparison post hoc test (*p \ 0.05;

**p \ 0.01; ***p \ 0.001 vs control)

320 Arch Toxicol (2011) 85:315–326

123

was further evaluated with or without DOX pre-incubation.

Doxorubicin concentration range (5–100 lM) and pre-

incubation time (24 h) were selected due to the previously

noted absence of relevant cytotoxicity and significant increase

in P-gp induction (protein expression and transport activity)

under these conditions.

Fig. 4 P-glycoprotein transport activity in Caco-2 cells exposed to

doxorubicin (DOX) (0–100 lM) at different time points (6, 12, 24,

48, 72 and 96 h). X means that these concentrations were not tested.

Results are presented as mean ± SEM from 4 independent

experiments (triplicates were performed in each experiment). Statis-

tical comparisons were made using the Kruskal–Wallis test followed

by the Dunn’s multiple comparison post hoc test (*p \ 0.05;

**p \ 0.01; ***p \ 0.001 vs control)

Fig. 5 Paraquat (PQ) cytotoxicity in Caco-2 cells at different time

points. Results are presented as mean ± SEM from 3 independent

experiments (triplicates were performed in each experiment).

Statistical comparisons were made using one-way analysis of variance

followed by the Bonferroni’s multiple comparison post hoc test

(*p \ 0.05; **p \ 0.01; ***p \ 0.001 vs control)

Arch Toxicol (2011) 85:315–326 321

123

Cytotoxicity was evaluated by the MTT assay 24 h after

PQ exposure. Figure 6 shows the concentration–response

curves obtained with only paraquat (PQ) and with DOX

pre-incubation (PQ ? DOX). Maximal cell death occurred

at the 5,000 lM PQ concentration both with and without

DOX pre-incubation (70% cell death when compared to

control values). However, significant differences were

observed for the EC50 values of both curves (representing

the half-maximum-effect concentrations from the fitted

curves) at all DOX tested concentrations. Significant

rightward shifts of the PQ concentration–response curves

(Fig. 6), accompanied by significant increases in the EC50

values, were observed for all the tested DOX concentra-

tions (Table 1). The observed increases in the EC50 values

were not concentration dependent, with similar EC50 val-

ues being found for all the tested DOX concentrations.

To correlate the observed EC50 increases with P-gp

expression and activity, this study was repeated in the

presence of a specific P-gp inhibitor (PQ ? UIC2 and

PQ ? UIC2 ? DOX curves, Fig. 7). Under P-gp inhibition

with the UIC2 antibody, an increase in the maximum

cytotoxic PQ effect was observed for all curves (Fig. 7)

when compared to the cytotoxicity curves obtained in the

absence of this specific P-gp inhibition (Fig. 6). A left-

wards shift in the PQ ? UIC2 ? DOX concentration–

response curves was observed with significant decreases in

the respective EC50 values (Table 2). The observed

decreases in the EC50 values were not concentration

dependent, with similar EC50 values being found for all

the tested DOX concentrations.

Discussion

P-glycoprotein (P-gp) has been viewed as a therapeutic

target for specific inhibition to overcome the well-known

problems of drug resistance in anticancer therapy. On the

other hand, its polarized expression is consistent with the

Fig. 6 Paraquat concentration–

response (cell death) curves

with (PQ ? DOX) or without

(PQ) previous exposure to

doxorubicin (5, 10, 50 and

100 lM). Three independent

experiments were performed

(triplicates were performed in

each experiment).

Concentration–response curves

were fitted using least squares as

the fitting method, and the

comparisons between

PQ ? DOX and control (PQ)

curves (bottom, top and LOG

EC50) were made using the

extra sum-of-squares F test. In

all cases, P values \ 0.05 were

considered statistically

significant

Table 1 EC50 values (half-maximum-effect concentrations) of the paraquat concentration–response fitted curves, with (PQ ? DOX) or without

(PQ) previous exposure of Caco-2 cells to doxorubicin

Doxorubicin

concentration (lM)

0 (PQ) 5 (PQ ? DOX5) 10 (PQ ? DOX10) 50 (PQ ? DOX50) 100 (PQ ? DOX100)

EC50 (lM) 1,047 1,825 1,899 1,853 1,806

EC50 P value (comparison

between EC50 values)

– 0.0016 0.0007 0.0025 0.0207

Curve P value (comparison

between the fitted curves)

– \0.0001 \0.0001 \0.0001 \0.0001

Concentration–response curves were fitted using least squares as the fitting method, and the comparisons between PQ ? DOX and control (PQ)

curves were made using extra sum-of-squares F test. In all cases, P values \ 0.05 were considered statistically significant

322 Arch Toxicol (2011) 85:315–326

123

proposed role of P-gp as a secretory protective system,

contributing to the gastrointestinal epithelial barrier in

limiting the bioavailability of its substrates (Hunter et al.

1993b). Thus, by using its efflux properties, a possible

antidotal pathway against the damage induced by xenobi-

otics that are substrates of this transporter could be pro-

posed. By increasing the expression and activity of this

important transport protein, a consequent increase in the

cellular efflux of such xenobiotics and a corresponding

decrease in their accumulation could therefore culminate in

an overall decrease in cytotoxicity.

Caco-2 cells have been reported to express P-gp

(Hidalgo and Jibin 1996; Hunter et al. 1993b; Shen et al.

2007; Watanabe et al. 2005), and the expression levels are

in good agreement with those of normal human jejunum

(Taipalensuu et al. 2001). Therefore, this in vitro model

could be used and validated for the screening and selection

of potent and safe P-gp inducers and also for the study of

the induction mechanism underlying their potential pro-

tective effects.

Several compounds were already reported to increase

P-gp expression in Caco-2 cells such as vinblastine

(Shirasaka et al. 2006b), venlafaxine (Ehret et al. 2007),

R-cetirizine (Shen et al. 2007), cadmium (Huynh-Delerme

et al. 2005) and benzo(e)pyrene (Sugihara et al. 2007).

Flavonoids, a subclass of dietary polyphenolic compounds

present in fruits, vegetables, and herbal plants, that are

thought to promote human health through their antioxidant,

antiviral and anticarcinogenic properties also induced P-gp

expression in Caco-2 cells (Lohner et al. 2007).

To validate the Caco-2 cells as a suitable in vitro model

to study and select safe, potent and specific P-gp inducers

Fig. 7 Paraquat concentration–

response (cell death) curves in

the presence of a specific

p-glycoprotein inhibitor (UIC2

antibody) with

(PQ ? UIC2 ? DOX) or

without (PQ ? UIC2) previous

exposure to doxorubicin (5, 10,

50 and 100 lM). Three

independent experiments were

performed (triplicates were

performed in each experiment).

Concentration–response curves

were fitted using least squares as

the fitting method, and the

comparisons between

PQ ? UIC2 ? DOX and

control (PQ ? UIC2) curves

(bottom, top and LOG EC50)

were made using the extra sum-

of-squares F test. In all cases,

P values \ 0.05 were

considered statistically

significant

Table 2 EC50 values (half-maximum-effect concentrations) of the

paraquat concentration–response fitted curves, in the presence of a

specific p-glycoprotein inhibitor (UIC2 antibody) with (PQ ? UIC2 ?

DOX) or without (PQ ? UIC2) previous exposure to doxorubicin (5, 10,

50 and 100 lM)

Doxorubicin

concentration (lM)

0 (PQ ? UIC2) 5 (PQ ? UIC2 ? DOX5) 10 (PQ ? UIC2 ? DOX10) 50 (PQ ? UIC2 ? DOX50) 100 (PQ ? UIC2 ? DOX100)

EC50 (lM) 1,933 1,034 927.7 1,246 1,187

EC50 P value

(comparison

between EC50

values)

– \0.0001 \0.0001 0.0024 0.0111

Curve P value

(comparison

between the

fitted curves)

– \0.0001 \0.0001 \0.0001 0.0012

Concentration–response curves were fitted using least squares as the fitting method, and the comparisons between PQ ? UIC2 ? DOX and control (PQ ? UIC2)

curves were made using extra sum-of-squares F test. In all cases, P values \ 0.05 were considered statistically significant

Arch Toxicol (2011) 85:315–326 323

123

as a tool for cell protection against xenobiotics, doxoru-

bicin, a known P-gp inducer (Zhou 2008), was used to

investigate changes in P-gp expression and activity and to

correlate this induction with cellular protection against

paraquat cytotoxicity.

Our results clearly indicated that doxorubicin is effec-

tive in increasing P-gp expression and activity. In fact,

when in the presence of this known P-gp inducer, P-gp

expression and transport activity increased in a concen-

tration- and time-dependent manner, with significant

results observed as soon as 6 h after incubation (Figs. 3

and 4). This rapid increase in P-gp expression was also

reported by Ehret et al. (2007), who showed that venla-

faxine increases the expression of MDR1 and MRP genes

in Caco-2 cells during an acute (1.5, 3 and 6 h) treatment

period. Similar results were observed for another known

P-gp inducer, rifampicine (Ehret et al. 2007), and for

several nonsteroidal anti-inflammatory drugs (NSAIDs)

including diclofenac, fenbufen, indomethacin and nimesu-

lide (Takara et al. 2009).

The observed remarkable increases in P-gp expression

levels induced by doxorubicin were not accompanied by

proportional increases in P-gp transport activity. For

example, the exposure of Caco-2 cells to 50 lM DOX for

24 h increased P-gp expression levels to approximately

530% of control values, although P-gp transport activity

increased only by 137%. This suggests that although P-gp

is being highly expressed and incorporated into the cell

membrane (since the monoclonal antibody recognizes an

external P-gp epitope), this transport efflux pump is not yet

fully functional. Noteworthy, our data suggest that, for the

screening of P-gp inducers, both P-gp expression and

activity should be investigated, since an increase in the first

may not be reflected in an increase in the second parameter.

Similarly, Takara et al. (2009) noted that P-gp transport

function remained unchanged in Caco-2 cells exposed to

several NSAIDs in spite of the observed increase in MDR1

mRNA (Takara et al. 2009).

One possible explanation for the differences noted

between P-gp expression and activity levels in these cells is

that Caco-2 full differentiation into enterocytes could be

needed to obtain fully functional P-gp. In fact, in a study

performed by Hosoya et al. (1996), based on the direc-

tionality of cyclosporine and verapamil transport, it was

observed that in spite of the observed early P-gp expres-

sion, the protein was only fully functional after the 17th

day in culture (Hosoya et al. 1996). Moreover, in that study

the rank order of P-gp expression levels was 4 week-

s & 1 week [ 3 weeks [ 2 weeks at equal loading of cell

proteins. These authors also observed, at the late stage of

culture (*27 days), an enhanced cyclosporine transport in

the basal-to-apical direction, which was due not only to an

increased level of P-gp expression in the apical cell

membrane but also to the full development of cell polarity,

which may be the most important factor in effecting efflux

pump function (Hosoya et al. 1996).

Paraquat dichloride (PQ) is an effective and widely used

herbicide as desiccant and defoliant in a variety of crops.

PQ is the third most extensively used herbicide in the

world, causing thousands of deaths due to accidental or

intentional ingestion (Dinis-Oliveira et al. 2006a,2006b,

2008). Studies performed by Nagao et al. 1993 suggested

that PQ is absorbed through a specialized mechanism

associated with the carrier-mediated transport system for

choline on the brush-border membrane (Nagao et al. 1993).

This carrier-mediated transport system for choline is

present in Caco-2 cells (Kamath et al. 2003), allowing PQ

to accumulate inside these cells. Noteworthy, a new ther-

apeutic approach for PQ poisonings, involving its intestinal

excretion, was already proposed by induction of de novo

synthesis of P-gp (Dinis-Oliveira et al. 2006b). Taking

these findings into account, PQ was used in the present

study as a model for a xenobiotic that is able to enter into

the Caco-2 cells and that is a known P-gp substrate.

The protective effects mediated by P-gp were studied

through the evaluation of PQ cytotoxic effects with or

without previous exposure to the P-gp inducer. We

observed that pre-exposure of these cells to DOX resulted

in a significant decrease in PQ cytotoxicity as shown by the

PQ ? DOX concentration–response curves shift to the

right (Fig. 6) and by the corresponding significant increa-

ses in EC50 values (Table 1). However, the protective

effects mediated by DOX were not concentration depen-

dent for the DOX tested concentrations since the EC50

values obtained with the different DOX concentrations

were very similar. In fact, the EC50 values increased from

1,047 lM in the absence of DOX to 1,825, 1,899, 1,853

and 1,806 lM when the cells were pre-exposed to 5, 10, 50

and 100 lM DOX, respectively (Table 1). This can be

explained because in spite of the significant differences

between P-gp expression levels for these DOX concentra-

tions, smaller differences in P-gp transport activity were

noted. In fact, P-gp expression levels were increased by

237, 294, 529 and 646% after 24 h incubation with 5, 10,

50 and 100 lM DOX, respectively (Fig. 3). However, the

corresponding increases in P-gp activity were only of 110,

111, 136 and 150% (Fig. 4). These data indicate that

although the P-gp expression levels increased in a con-

centration-dependent manner upon exposure to the tested

inducer, the magnitude of the expected protective effect

against a xenobiotic such as PQ did not increase in a

similar trend. Several monoclonal antibodies recognizing

discontinuous extracellular epitopes of P-gp have been

developed. UIC2, in particular, seems to inhibit

P-gp-mediated drug export in vitro (Chaudhary et al. 1992;

Mechetner 2007; Mechetner and Roninson 1992). Thus,

324 Arch Toxicol (2011) 85:315–326

123

this inhibitor was chosen to investigate whether there is a

link between P-gp induction and the reduction in PQ-

induced cytotoxicity. Under P-gp inhibition, the maximum

cytotoxic effect was increased for PQ (PQ ? UIC2) when

compared with PQ alone (Fig. 8). Similarly, for all

PQ ? UIC2 ? DOX curves it was also observed an

increase in the maximum cytotoxic effect when compared

with the PQ ? DOX curves (Figs. 6 and 7). This can be

explained by the higher intracellular PQ accumulation that

is expected when P-gp is inhibited. Given these differences

in the observed maximum cytotoxic effect, the EC50 val-

ues (defined as the concentration of PQ that causes 50% of

the observed maximum effect) can only be compared

among the same experimental group (either in the presence

or in the absence of the UIC2 antibody).

The analysis of concentration–response curves in the

presence of the UIC2 antibody revealed that DOX pro-

tective effect was completely abolished in the presence of

this P-gp specific inhibitor, with the PQ ? UIC2 ? DOX

curves leftwards shift and with the corresponding decrease

in the EC50 values (Table 2). In fact, when the cells were

pre-exposed to doxorubicin and afterward co-exposed to

PQ ? UIC2, the EC50 values decreased significantly from

1,933 lM in the absence of DOX to 1,034, 927.7, 1,246

and 1,187 lM when the cells were pre-exposed to 5, 10, 50

and 100 lM DOX, respectively (Table 2).

These results suggest that P-gp induction by DOX pro-

tected the cells from PQ cytotoxicity. Moreover, this

mechanistic study proved that P-gp induction can be an

extremely important cellular protection tool against xeno-

biotics toxicity and an important antidotal pathway to be

explored.

In conclusion, effective antidotal pathways can be

achieved by promoting the cellular efflux of deleterious

xenobiotics. As such, appropriate in vitro models

addressing P-gp induction are needed. Our results showed

that P-gp induction with doxorubicin was effective in

increasing P-gp expression and activity, indicating that the

present in vitro model could be useful for the screening of

potential P-gp inducers. However, it should be noted that

for the quantitative estimation of the P-gp-mediated drug

transport, higher differentiation of the Caco-2 cells for

maximal enterocytic differentiation may be required.

Moreover, it was possible to prove the involvement of P-gp

in the decrease in paraquat cytotoxicity.

Acknowledgments This work was supported by the Fundacao para

a Ciencia e Tecnologia (FCT) - project [PTDC/SAU-OSM/101437/

2008] - QREN initiative with EU/FEDER financing through COM-

PETE - 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 Rosario 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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