Download - P-glycoprotein–mediated transport of berberine across Caco-2 cell monolayers

P-Glycoprotein–Mediated Transport of Berberine acrossCaco-2 Cell Monolayers

HAN-JOO MAENG, HO-JUNG YOO, IN-WHA KIM, IM-SOOK SONG, SUK-JAE CHUNG, CHANG-KOO SHIM

Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul 151-742, Korea

Received 7 March 2002; revised 30 May 2002; accepted 6 July 2002

ABSTRACT: The objective of this study was to investigate the mechanisms by whichberberine is transported in the secretory and absorptive directions across Caco-2 cellmonolayers. The basolateral-to-apical (B-A) flux was 30-fold greater than the apical-to-basolateral flux and temperature dependent (i.e., drastic decrease at 48C compared with378C). The above results suggest the involvement of a carrier-mediated active transportmechanism for the B-A transport of berberine. However, no significant concentrationdependency for the permeability (Papp) of berberinewas observed forB-A transport over aconcentration range of 5–300mM, indicating that theKmvalue of berberine for the carriersystem is greater than 300 mM. Well-documented P-glycoprotein (P-gp) substrates suchas verapamil, daunomycin, and rhodamine123 inhibited the B-A flux of berberine,whereas tetraethylammonium and taurocholate did not, suggesting that P-gp is involv-ed in the transport. For the case of daunomycin, the B-A flux, but not the apical-to-basolateral flux, was significantly increased after pretreatment of the cell monolayerswith berberine. In addition, the uptake of 1 mM daunomycin into Caco-2 cells was de-creased as a result of this pretreatment. These results suggest that the repeatedadministration of berberinemay up-regulate P-gp functions in Caco-2 cells. If this occursin the gastrointestinal epithelial cells, the repeated administration of berberine mayreduce the gastrointestinal absorption of P-gp substrates including chemotherapeuticagents such as daunomycin. � 2002 Wiley-Liss, Inc. and the American Pharmaceutical

Association J Pharm Sci 91:2614–2621, 2002

Keywords: berberine; P-gp; Caco-2 cell monolayers; transport

INTRODUCTION

Berberine (Fig. 1) is an isoquinoline1 that has avaried pharmacology including anti-microbial,2

anti-motility,3 and anti-secretory activities.4–6 Ithas been used for more than 2000 years in tradi-tional Eastern medicine as an effective medi-cation for the treatment of gastro-enteritis andsecretory diarrhea.1 It is also effective in theprevention and treatment of diarrhea in animalmodels.7–9

It has recently been reported that berberineis readily extruded from microbial cells by the

multidrug resistance pump of cell membranes,which has a role in protecting the cells from anti-microbials.10 In vitro interactions of berberinewith P-glycoprotein (P-gp) have also been report-ed in hepatoma cells.11 These reports stronglysuggest that berberine is a substrate for P-gp,a 170 kDa membrane glycoprotein encoded bythe multidrug resistance gene (e.g., MDR1 inhumans).

P-gp confersmultidrug resistance by enhancingthe active efflux of chemotherapeutic agents fromcells,12 and is involved in the transport of a varietyof structurally and pharmacologically unrelatedhydrophobic drugs, including vinca alkaloids,anthracyclines, cyclosporin A, digoxin, and gluco-corticoids.13–16 It is expressed in normal tissuessuch as the brush-border membrane of renalproximal tubules, the bile canalicular membrane

2614 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 12, DECEMBER 2002

Correspondence to: Chang-Koo Shim (Telephone: 82-2-880-7873; Fax: 82-2-885-8429.; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 91, 2614–2621 (2002)� 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

of hepatocytes, intestinal epithelial cells, capillaryendothelial cells of brain and testis, adrenal glandand placental trophoblasts, aswell as inmultidrug-resistant tumor cells.17,18 Thus, it contributes tothe excretion of xenobiotics and endogenous com-pounds into the urine, bile, and intestinal lumen,as well as the formation of biological barriersagainst such compounds in various tissues ororgans including brain, placenta, and tumor cells.For example, intestinal P-gp is thought to limit theabsorption of drugs after oral administration.19–22

If berberine is a substrate of P-gp in the body, itwould be expected to influence the pharmacoki-netics of various substances that are transportedvia the P-gp system. The objective of this study,therefore, was to investigate the mechanisms bywhich berberine is transported in secretory andabsorptive directions across a biological mem-brane. Of particular interest was its possible in-teractionwith the P-gp system on cell membranes.Caco-2 cells were used as a representativemodel of biological membranes, on which P-gp isexpressed.20,23,24

MATERIALS AND METHODS

Materials

Berberine chloride (Sigma Chemical Co., St.Louis, MO), [14C]mannitol (50 mCi), [3H]taurocho-late (250 mCi), [3H]daunomycin (250 mCi) (all fromNew England Nuclear, Boston, MA), fetal bovineserum (Hyclone Laboratories, Logan, UT), tryp-sin-EDTA (Gibco Laboratories, Gaithersburg,MD), and Dulbecco’s modified Eagle’s medium,nonessential amino acid solution, penicillin-streptomycin, Hank’s balanced salt solution

(HBSS), HEPES (all from Sigma Chemical Co.)were used as purchased. All other reagents wereof analytical grade.

Cell Culture

The human colon adenocarcinoma cell line, Caco-2 (American Type Culture Collection, Rockville,MD), was grown in the form of monolayers inDulbecco’s modified Eagles’ medium, 10% fetalbovine serum, 1% nonessential amino acid solu-tion, 100 U/mL penicillin, and 0.1 mg/mL strepto-mycin at 378C in an atmosphere of 5% CO2 and90% relative humidity. Stock cultures were grownin 75-cm2 tissue culture flasks and were split 1:3at 80 to 90% confluency using 0.02% EDTA and0.05% trypsin. The Caco-2 cells from passagenumbers 46 to 55 were seeded on the permeablepolycarbonate inserts (1-cm2, 0.4-mm pore size;Corning Costar Co., Cambridge, MA) in 12 Trans-well plates (Corning Costar Co.) at a density of2.5� 105 to 3.0� 105 cells/cm2. The inserts werefed with the incubation medium at 2-day intervalsfor the first week and then at daily intervals,usually for 2 weeks, until they were used for thetransport experiments.

In experiments to investigate the effect of ber-berine pretreatment on the transport of dauno-mycin, the cell monolayer grown on the insert wasfurther fed with the incubation medium contain-ing berberine (30 mM) for specified time periods(i.e., 1, 2, 3, 5, 7, or 10 days).

The integrity of the cell monolayers wasevaluated by means of transepithelial electricalresistance (TEER) measurements using anEVOMTM epithelial volt/ohm-meter (World Preci-sion Instruments, Sarasota, FL). When the TEERvalue reached 300–700 Ocm2, the cell insertswere used for the transport experiments. Thetransport of [14C]mannitol (5.4 mM)was<0.25% ofthe dose/h, corresponding to an apparent per-meability (Papp) value of 5.6� 10�7cm/s under thegiven culture conditions.

Transport Study

Before the transport experiments, the cell mono-layers were washed three times with the trans-port medium (pH 7.4, HBSS containing 25 mMHEPES and 25 mM glucose). After each wash, theplates were incubated in the transport mediumfor 1 h at 378C, and the TEER value was thenmeasured. The transport medium on both sidesof the cell monolayers was then removed by

Figure 1. Chemical structure of berberine chloride.

BERBERINE TRANSPORT ACROSS CACO-2 MONOLAYERS 2615

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 12, DECEMBER 2002

aspiration.25 In each transport experiment, threeinserts were used. To measure the transport ofberberine from the apical-to-basolateral (A-B)side, 0.5 mL of transport medium (pH 7.4) con-taining 1% (v/v) dimethylsulfoxide and the drugwas added to the apical side, and 1.5 mL of thetransport medium without the drug to the basalside. The inserts were moved to wells containingfresh transport medium at 30-min intervals for2 h. A 300-mL aliquot of the medium, taken at eachtime point, was assayed for berberine by high-performance liquid chromatography (HPLC).

For measurement of the basolateral-to-apical(B-A) transport of berberine, 1.5 mL of the trans-port medium containing the drug was added to thebasolateral side, and 0.5 mL of the transportmedium without the drug to the apical side. Theinserts were then incubated at 378C, and a 300-mLaliquotwas removed from the apical side at 30-minintervals for 2 h and replaced with 300 mL of thefresh transport medium. A 300-mL aliquot of eachsample was assayed for berberine by HPLC.

In experiments to investigate the effect of vari-ous compounds on the B-A transport of 20 mM ber-berine, the inhibitors were added to the transportmedium on the basal side of the cell monolayers.

Berberine levels were determined by the HPLCprocedure described below, whereas [14C]manni-tol, [3H]taurocholate, and [3H]daunomycin weredetermined by liquid scintillation counting using aWallac model 1409 instrument (Wallac, Gaithers-burg, MD).

HPLC Assay of Berberine

Themethod of Chen and Chang26 was used for thedetermination of berberine, with minor modifica-tions. To summarize, the HPLC system consistedof a Hitachi L-7110 pump (Hitachi, Japan), aShimadzu SPD-6AV UV-Vis detector (Shimadzu,Japan), a Hitachi L-7200 autosampler (Hitachi),and a Shimadzu C-R6A integrator (Shimadzu).C18 column was used. The mobile phase wasprepared by mixing 60% acetonitrile in a 0.1%phosphoric acid solution and adjusting the pH to6.0 using concentrated ammonia solution. Theeluent resulted in sharp, well-resolved peaks. Thechromatogram was monitored by UV detection ata maximum wavelength of 267 nm. Calibrationcurves for berberine were linear over the con-centration range of 25� 1000 ng/mL with acorrelation coefficient of 0.999, but the limit ofdetection which exhibits <�20% variability was50 ng/mL.

Calculation

For each transport experiment, the mean trans-port rate was calculated from the linear portion(i.e., 30-, 60-, 90-, and 120-min time points) of aplot of the total amount of drug transported ver-sus time. The apparent permeability values, Papp,of drug across the Caco-2 cell monolayers, ex-pressed as cm/s, were calculated as DQ/Dt�1/60� 1/A� 1/Co, where DQ/Dt is the permeabilityrate (mmole/min), A the surface area of the mem-brane (cm2, 1 in the present study), and Co theinitial concentration of drug in the donor chamber(mmole/mL). The transport clearance (CLlinear, mL/min/cm2) was calculated as DQ/Dt/Co/A. All dataare expressed as the mean�SD of three experi-ments. The statistical significance of differencesbetween treatments was evaluated using unpair-ed Student’s t tests, and a value of p< 0.05 wasconsidered to be statistically significant.

Kinetic parameters according to the Michaelis-Menten equation were calculated by a nonlinearregression analysis27 of the transport rate versusconcentration profiles of daunomycin:V¼Vmax[S]/(Kmþ [S]), where V is the apparent linear initialrate, [S] the initial concentration, Vmax the maxi-mum uptake rate, and Km the Michaelis-Mentenconstant for daunomycin.

RESULTS AND DISCUSSION

Transepithelial Transport of Berberinein Caco-2 Cells

To examine the issue of whether carrier-mediatedtransport is involved in the transepithelial trans-port of berberine across Caco-2 cell monolayers,the directional transport of berberine was deter-mined as a function of time (Fig. 2). The polarizedtransport of berberine was observed to be muchfaster for the B-A direction compared with theA-B direction. The apparent permeability coeffi-cient of berberine in the B-A direction was signi-ficantly greater compared with the A-B directionfor 5, 10, 20, 50, 100, and 300 mM berberine (datanot shown except for 100 mM). For example, appro-ximately 30-fold greater permeability coefficientwas observed for the B-A flux (1.8� 10�5 cm/s)compared with the A-B flux (6.0� 10�7 cm/s) for100 mM berberine (Fig. 2), suggesting the involve-ment of a carrier-mediated system for the B-Atransport of berberine in Caco-2 cells. However,the flux of berberine in both directions increas-ed linearly with increasing concentration of

2616 MAENG ET AL.


berberine over the range of 5 � 300 mM (Fig. 3 forthe B-A transport, data not shown for the A-Btransport), exhibiting no significant concentra-tion dependency in the permeability (Papp) of thetransport in either direction. This indicates thatthe Km of berberine for the responsible carriersystem in Caco-2 cells is >300 mM. Because ofprobable cytotoxicity problems,26,28,29 the trans-port rates for concentrations >300 mM were notpursued. The transport was found to be tempera-ture dependent (i.e., drastic decrease in the flux at48C compared with 378C) for the B-A flux of

berberine (62.4� 1.6 versus 5.7� 0.4 pmole/min,mean�SD, n¼ 3). Collectively, the above resultssuggest the involvement of a carrier-mediatedactive transport mechanism in the B-A transportof berberine in Caco-2 cells.

Effect of Transport Inhibitorson Berberine Transport

It is well known that P-gp is expressed in theapical membrane of mucosal cells in the intestineand often pumps (i.e., secretion) a variety of xeno-biotics into the lumen, leading to a limited netintestinal absorption of such compounds. Thus,the relationship between the active transport ofberberine in the efflux (secretary) direction acrossthe Caco-2 cell monolayers and P-gp was thenexamined. For this purpose, the flux of berberine(20 mM) was examined in the absence and pre-sence of representative P-gp inhibitors such asverapamil (1 mM), daunomycin (1 mM), andrhodamine 123 (1 mM) on the basal side. Thepresence of the above P-gp inhibitors had nosignificant influence on the TEER values ofCaco-2 cell monolayers, but substantially inhib-ited the B-A flux of berberine (Fig. 4), suggestingthat berberine is transported across Caco-2 cellmonolayers via assistance from P-gp, and that acarrier-mediated active mechanism is operative.However, the presence of tetraethylammonium(1 mM) or taurocholate (1 mM) on the basal side

Figure 2. Time course for the apical-to-basolateral(*) and basolateral-to-apical (*) transport of 100 mMberberine (mean�SD, n¼ 3) across Caco-2 cell mono-layers in HBSS at 378C.

Figure 3. The basolateral-to-apical (B-A) flux ofberberine (mean�SD, n¼ 3) across Caco-2 cell mono-layers as a function of berberine concentration (5, 10, 20,50, 100, and 300 mM). Each point represents themean�SD for three experiments. The inset indicatesan Eadie-Hofstee plot of the transport rates (V) andberberine concentration (C).

Figure 4. Effect of P-gp inhibitors (verapamil, rhoda-mine123, and daunomycin, 1 mM each), an organicanion (taurocholate, 1 mM), and an organic cation(tetraethylammonium, TEA, 1 mM) in the basal sideon thebasolateral-to-apical (B-A)fluxof 20mMberberine(mean�SD, n¼ 3) across Caco-2 cell monolayers. BDL,below detection limit.



had no effect on the B-A transport of berberine(20 mM) (Fig. 4), suggesting that the transportsystem for berberine is independent of the othertransport systems that are involved in the trans-port of, for example, organic cations and bileacids.

If P-gp is responsible for the efflux of berberine,then the presence of P-gp inhibitors might in-crease the absorptive flux of berberine. However,the presence of 1 mM verapamil in the apical sidehad no effect on the A-B transport of 100 mMberberine, suggesting that the inhibitory effect ofverapamil on the A-B transport of berberine isoperative only in the presence of verapamil in thebasal side.

Effect of Berberine Pretreatment onDaunomycin Transport

A variety of factors including heat shock, differ-entiation inducers, carcinogens, and anticancerdrugs often induce the expression of P-gp in tumorcells through transcriptional regulation.30 A simi-lar induction of P-gp has been reported for variousP-gp substrates. Thus, the effect of berberinepretreatment on P-gp function in Caco-2 cellmonolayers was examined. The effect of the pre-treatment period on daunomycin transport wasinitially examined for 30 mM berberine. For thispurpose, Caco-2 cell monolayers were pretreatedwith 30 mM berberine for specified time periods(i.e., 1, 2, 3, 5, 7, or 10 days) as described inMethods. The monolayers were then washedthree times with 2 mL of the fresh transportmedium to remove residual berberine in the cells,and the transport of 1 mM [3H]daunomycin inboth directions was measured. As indicated byFigure 5, no change was observed in the A-B fluxfor daunomycin regardless of the pretreatmentperiod used, whereas a significant increase in B-A flux was observed within 3 days of pretreat-ments which approached a plateau thereafter for10 days. Thus, the pretreatment period was set at7 days in subsequent experiments.

The effect of berberine concentration (1, 10, and30 mM) on the directional transport of daunomycinwas then examined. As shown by Figure 6, the B-Aflux of 1 mM daunomycin was increased by the7-day pretreatment as the concentration of ber-berine in the pretreatment medium increased.This increase was clearly evident for the pretreat-ment, evenwith 1 mMberberine.However, such aneffect was not observed for the A-B flux of 1 mMdaunomycin (Fig. 6). To determine whether the

increase in the B-A flux of daunomycin as theresult of berberine pretreatment is associatedwithcell integrity, the effects of a 7-day pretreatmentwith berberine (1, 10, 30 mM) on mannitol leakage(A-B direction) and TEER values were measured(Fig. 7). No significant changes in these character-istics were detected (data not shown), consistentwith the previous study that reported the non-toxicity of berberine pretreatment in termsof HepG-2 cell viability.11 This indicates that theincrease in the transport of daunomycin is notassociated with the reduced integrity of the cellmonolayer.

Figure 5. Effect of the period of pretreatment withberberine (30 mM) on the vectorial transport of 1 mMdaunomycin (mean�SD,n¼ 3) acrossCaco-2 cellmono-layers. *, apical-to-basal; *, basal-to-apical.

Figure 6. Effect of berberine concentration in thepretreatmentmedium on the vectorial transport of 1 mMdaunomycin (mean�SD,n¼ 3) acrossCaco-2 cellmono-layers. The period of pretreatment was set at 7 days.*,apical-to-basolateral; *, basolateral-to-apical.

2618 MAENG ET AL.


The effect of berberine pretreatment was thenexamined to elucidate the issue of whether it isspecific to the B-A transport of daunomycin acrossCaco-2 cells. For this purpose, the effect of the7-day pretreatment with 10 mM berberine on theA-B transport of 1 mM taurocholate was alsoexamined. Results showed no significant changesin the A-B flux of taurocholate (data not shown),confirming that the effect of berberine pre-treatment is specific to the B-A transport ofdaunomycin.

The effect of a 7-day pretreatment with 30 mMberberine on the B-A transport characteristics ofdaunomycin (1–200 mM) was further examined.Again, an apparent increase in the flux of dauno-mycin was observed as a result of pretreatmentover the entire concentration range (data notshown). The results of an Eadie-Hofstee analysisare summarized in Table 1, in which a significant

increase (approximately 1.5-fold) as the result ofthe pretreatment was observed for Vmax, whereasno significant increase was observed for Km (themedium concentration at half the maximal trans-port rate) and CLlinear (the linear transport clear-ance). As a result, the intrinsic clearance ofdaunomycin for the carrier-mediated transport inthe B-A direction (i.e., efflux) was increased byapproximately 1.4-fold as the result of berberinepretreatment (i.e., 0.88 versus 1.26mL/min/cm2). Ithad been documented that the efflux of daunomy-cin is correlated well with P-gp expression. By thisrationale, it can be hypothesized that the signifi-cant increase in Vmax reflects the up-regulatedexpression or function of P-gp in berberine-treatedCaco-2 cells. This hypothesis is supported bythe reported up-regulation of P-gp expression byberberine in mammalian cells.31 To further exa-mine this hypothesis, the apical uptake of dauno-mycin (1 mM) into Caco-2 cellmonolayers grown oncollagen-coated 12-well plates (Corning CostarCo.) after berberine pretreatment (30 mM, for7 days) was determined by measuring the amountof daunomycin accumulated in the cell for 15-, 30-,and 45-min periods. Consistent again with thehypothesis, a 63% decrease in accumulation wasobserved as the result of berberine pretreatment(i.e., from 5.6� 1.2 to 2.1� 0.6 pmole/well/min,mean�SD, n¼ 3). The mechanism involved inthe elevated expression or function of P-gp byberberine is presently unclear. However, consider-ing the fact that berberine is frequently usedworldwide as an oral antidiarrheal medicine, andthat berberine is often administered over a re-latively long term, the up-regulation of P-gpfunction, during or after the administration ofberberine, seems to be probable. Thus, it can beconcluded that berberine may influence thepharmacokinetics of P-gp substrates, includingchemotherapeutic agents.

CONCLUSION

P-gp seems to be involved in the efflux of ber-berine across Caco-2 cell monolayers. The re-peated administration of berberine up-regulatedP-gp functions on the apical membrane of Caco-2cells. These results suggest that, if this occursin gastrointestinal epithelial cells, the repeatedadministration of berberine may lead to a de-creased gastrointestinal absorption of P-gp sub-strates including chemotherapeutic agents suchas daunomycin.

Table 1. Summary of the Effect of BerberinePretreatment (30 mM, 7 Days) on the Basolateral-to-Apical Transport of 1–200 mM Daunomycin(Mean�SD, n¼ 3) across Caco-2 Cell Monolayers

Control Pretreatment

Vmax (pmole/min/cm2) 29.38� 2.79 43.54� 2.70a

Km (mM) 33.43� 1.91 34.60� 2.23CLlinear (mL/min/cm2) 0.29� 0.02 0.30� 0.01

ap<0.05.

Figure 7. Effect of a 7-day pretreatment with 30 mMberberine on the basolateral-to-apical (B-A) flux ofdaunomycin (mean�SD,n¼ 3) acrossCaco-2 cellmono-layers as a function of daunomycin concentration. *,control; *, berberine pretreatment.



ACKNOWLEDGMENT

This study was supported by a grant (02-PJ2-PG1-CH12-0002) from The Ministry of Healthand Welfare of Korea.

REFERENCES

1. Chopra RN, Dikshit BB, Chowan JS. 1932. Phar-macological action of berberine. Indian J Med Res19:1193–1203.

2. Tang W, Eisenbrand G. 1992. Chinese drugsof plant origin. London: Springer-Verlag Press.pp 361–371.

3. Yamamoto K, Takase H, Abe K, Saito Y, Suzuki A.1993. Pharmacological studies on antidiarrhealeffects of a preparation containing berberine andgeranii herba. Nippon Yakurigaku Zasshi 101:169–175.

4. Tai YH, Feser JF, Marnane WG, Desjeux JF. 1981.Anti-secretory effects of berberine in rat ileum. AmJ Physiol 241:G253–G258.

5. Guandalini S, Fasano A, Migliavacca M,Marchesano G, Ferola A, Rubino A. 1987. Effectsof berberine on basal and secretagogue-modifiedion transport in the rabbit ileum in vitro. J PediatrGastroenterol Nutr 6:953–960.

6. Taylor CT, Baird AW. 1995. Berberine inhibitionof electrogenic ion transport in rat ileum. Br JPharmacol 116:2667–2672.

7. Dutta NK, Marker PH, Rao NR. 1972. Berberine intoxin induced experimental cholera. Br J Pharma-col 44:153–159.

8. Sabir M, Akhter MH, Bhide NK. 1977. Antagonismof cholera toxin by berberine in the gastrointestinaltract of adult rats. Indian J Med Res 65:305–313.

9. Sack RB, Froehlich JL. 1982. Berberine inhibitsintestinal secretory response of Vibrio cholerae andEscherichia coli enterotoxins. Infect Immun 35:471–475.

10. Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA,Lewis K. 2000. Synergy in medicinal plant: Anti-microbial action of berberine potentiated by 50-methoxyhydnocarpin, a multidrug pump inhibitor.Proc Natl Acad Sci USA 97:1433–1437.

11. Lin HL, Liu TY, Lui WY, Chi CW. 1999. Up-regulation of multidrug resistance transporterexpression by berberine in human and murinehepatoma cells. Cancer 85:1937–1942.

12. Gottesman MM, Pastan I. 1993. Biochemistry ofmultidrug resistance mediated by the multidrugtransporter. Annu Rev Biochem 62:385–427.

13. Tanigawara Y, Okamura N, Hirai M, YasuharaM, Ueda K, Kioka N, Komano T, Hori R. 1992.Transport of digoxin by human P-glycoproteinexpressed in a porcine kidney epithelial cell line(LLC-PK1). J Pharmacol Exp Ther 263:840–845.

14. Ueda K, Okamura N, Hirai M, Tanigawara Y,Saeki T, Kioka N, Komano T, Hori R. 1992. HumanP-glycoprotein transports cortisol, aldosterone, anddexamethasone, but not progesterone. J Biol Chem267:24248–24252.

15. Wacher VJ, Wu CY, Benet LZ. 1995. Overlappingsubstrate specificities and tissue distribution ofcytochrome P450 3A and P-glycoprotein: Implica-tions for drug delivery and activity in cancerchemotherapy. Mol Carcinog 13:129–134.

16. TanakaK, Hirai M, Tanigawara Y, UedaK, TakanoM, Hori R, Inui K. 1997. Relationship betweenexpression level of P-glycoprotein and daunorubi-cin transport in LLC-PK1 cells transfected withhuman MDR1 gene. Biochem Pharmacol 53:741–746.

17. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottes-man MM, Pastan I. 1987. Expression of a multi-drug-resistance gene in human tumors and tissues.Proc Natl Acad Sci USA 84:265–269.

18. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM,Pastan I, Willingham MC. 1987. Cellular locali-zation of the multidrug-resistance gene productP-glycoprotein in normal human tissues. Proc NatlAcad Sci USA 84:7735–7738.

19. Hsing S, Gatmaitan Z, Arias IM. 1992. The functionof Gp170, themultidrug-resistance gene product, inthe brush border of rat intestinal mucosa. Gastro-enterology 102:879–885.

20. Hunter J, Hirst BH, Simmons NL. 1993. Trans-epithelial secretion, cellular accumulation, andcytotoxicity of vinblastine in defined MDCK cellstrains. Biochim Biophys Acta 1179:1–10.

21. Terao T, Hisanaga E, Sai Y, Tamai I, Tsuji A. 1996.Active secretion of drugs from the small intestinalepithelium in rats by P-glycoprotein functioningas an absorption barrier. J Pharm Pharmacol 48:1083–1089.

22. Sparreboom A, van Asperen J, Mayer U, SchinkelAH, Smit JW, Meijer DK, Borst P, Nooijen WJ,Beijnen JH, van Tellingen O. 1997. Limited oralbioavailability and active epithelial excretion ofpaclitaxel (Taxol) caused by P-glycoprotein inthe intestine. Proc Natl Acad Sci USA 94:2031–2035.

23. Wils P, Phung-Ba V, Warnery A, LechardeurD, Raeissi S, Hidalgo IJ, Scherman D. 1994.Polarized transport of docetaxel and vinblastinemediated by P-glycoprotein in human intestinalepithelial cell monolayers. Biochem Pharmacol 48:1528–1530.

24. Yee S. 1997. In vitro permeability across Caco-2cells (colonic) can predict in vivo (small intestinal)absorption in man: Fact or myth. Pharm Res 14:763–766.

25. Augustijns PF, Bradshaw TP, Gan LS, HendrenRW, Thakker DR. 1993. Evidence for a polariz-ed efflux system in CACO-2 cells capable of

2620 MAENG ET AL.


modulating cyclosporin A transport. Biochem Bio-phys Res Commun 197:360–365.

26. Chen CM, Chang HC. 1995. Determination ofberberine in plasma, urine, and bile by high-performance liquid chromatography. J ChromatogrB Biomed Appl 665:117–123.

27. Yamaoka K, Tanigawara Y, Nakagawa T, Uno T.1981. A pharmacokinetic analysis program (multi)for microcomputer. J Pharmacobiodyn 4:879–885.

28. Ikekawa T, Ikeda Y. 1982. Antitumor activity of 13-methyl-berberine derivatives. J Pharmacobiodyn 5:469–474.

29. Zhang RX, Dougherty DV, Rosenblum ML. 1990.Laboratory studies of berberine used alone andin combination with 1,3-bis(2-chloroethyl)-1-nitro-sourea to treat malignant brain tumors. Chin MedJ 103:658–665.

30. Bellamy WT. 1996. P-glycoproteins and multidrugresistance. Annu Rev Pharmacol Toxicol 36:161–183.

31. Lin HL, Liu TY, Wu CW, Chi CW. 1999. Berberinemodulates expression of mdr1 gene product andresponse of digestive track cancer cells to pacli-taxel. Br J Cancer 81:416–422.



Download - P-glycoprotein–mediated transport of berberine across Caco-2 cell monolayers

Top Related