Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein

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Abstract

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [3H]vinblastine and [3H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [3H]vinblastine, [3H]vincristine and [3H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [3H]vinblastine, [3H]vincristine and [3H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [3H]vinblastine, [3H]vincristine and [3H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti-P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [3H]vinblastine, [3H]vincristine and [3H]digoxin into BeWo cells, the uptake of [3H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.

Introduction

Vinblastine and vincristine, antineoplastic vinca alkaloids, are commonly used for the treatment of breast cancer and Hodgkin's disease, malignancies that are often found in gravida. Their teratogenic potencies have been examined in a variety of animal models Joneja and Ungthavorn, 1969, Courtney and Valerio, 1968, Ferm, 1963, Tanaki et al., 1967. In hamsters, intravenous injection of vinblastine (0.25 mg/kg) or vincristine (0.1 mg/kg) was reported to induce malformations such as microphthalmia, anophthalmia, exencephaly and spina bifida (Ferm, 1963). Similar results were reported in mice (Joneja and Ungthavorn, 1969), rats (Tanaki et al., 1967) and rhesus monkeys (Courtney and Valerio, 1968). In humans, two cases of vinca alkaloid-induced teratogenicity were reported after maternal treatment with vinblastine or vincristine Thomas, 1976, Mannuti et al., 1975. Digoxin was also reported to be teratogenic (Johnny and Menachem, 1987). However, the permeability and transport mechanisms of these drugs across the placenta remain to be investigated.

Throughout gestation, the placenta plays important roles in regulating the exchange of various materials between the maternal and the fetal circulations (Stulc, 1997). Many investigators have examined the transport mechanisms of nutrients, such as amino acids, vitamins and glucose, across the blood–placental barrier, which consists of trophoblast cells (placental microvillous membrane epithelial cells) shown in Fig. 1 (Hay, 1994). Several transport systems for amino acids, including the Na+-dependent A or ASC system and the Na+-independent L system, were reported to be present in trophoblasts Furesz et al., 1993, Moe et al., 1994, Moe, 1995, Ramamoorthy et al., 1992. It was also reported that glucose transporter 1 (GLUT 1) was expressed both on the brush-border (maternal side) and the basal (fetal side) membranes, while glucose transporter 3 (GLUT 3) was expressed only on the brush-border membrane Bissonnette, 1981, Reid and Boyd, 1994, Hahn and Desoye, 1996. Thiamine (vitamin B1) is transported in exchange for H+ via a Na+- and membrane potential-dependent transport system (Grassl, 1998). Biotin, lipoate and pantothenate are cotransported with Na+ Hu et al., 1994, Schenker et al., 1992, Prasad et al., 1998. However, little is known about the transport of drugs or xenobiotics across the blood–placental barrier, though, several drugs are known to cross the placenta and reach the developing fetus when administered during pregnancy. The fetal blood concentration of drugs is not always equal to the maternal blood concentration Pacifici and Nottoli, 1995, Van der Aa et al., 1998, suggesting that there are active transport systems for drugs in the placenta. In several tissues such as kidney, adrenal gland, vessels at blood–brain barrier sites, liver, intestine and testis, P-glycoprotein was found to be expressed and to extrude a range of hydrophobic natural products and drugs against a concentration gradient Thiebaut et al., 1987, Tsuji et al., 1992, Cordon-Cardo et al., 1989, Sugawara et al., 1988, Sugawara et al., 1997. P-Glycoprotein is encoded by a multidrug resistance gene (MDR1), and can confer multidrug resistance by extruding a wide range of structurally unrelated, amphiphilic hydrophobic drugs from cells in an ATP-dependent manner Juliano and Ling, 1976, Kartner et al., 1983. In recent studies, P-glycoprotein was shown to be expressed in the placenta Sugawara et al., 1997, Nakamura et al., 1997. However, these reports were based on immunohistochemical techniques, and functional studies have not been carried out. Therefore, it is essential to investigate whether P-glycoprotein operates as a drug-efflux pump in the human placenta. Human placental choriocarcinoma epithelial cells (BeWo cells) are commonly used for studies of the blood–placental barrier, including transport mechanisms. BeWo cells have similar properties to normal trophoblasts in terms of morphology, biochemical markers and hormone secretion (Liu et al., 1997).

In the present study, we aimed to elucidate the role of P-glycoprotein in the placenta by means of immunoblotting studies and uptake and transcellular transport studies using BeWo cells. We employed vinblastine, vincristine, digoxin and progesterone as model drugs. Immunoblot analysis was carried out with cultured BeWo cells, isolated human placental trophoblast cells, human placental brush-border membrane vesicles and human placental basolateral membrane vesicles.

Section snippets

Materials and reagents

[3H]Vinblastine sulphate (15.5 Ci/mmol) and [3H]vincristine sulphate (5.70 Ci/mmol) were purchased from Amersham International (Buckinghamshire, UK). [3H]Digoxin (19.0 Ci/mmol) and [1, 2-3H]progesterone (52.0 Ci/mmol) were purchased from NEN Research Products (MA, USA). [1-14C]d-mannitol (53 Ci/mmol) was purchased from Moravek Biochemicals (CA, USA). Trypsin was purchased from GIBCO BRL Life Technologies (Rockville, MD, USA). Collagenase Type I was purchased from Worthington Biochemical

Purity of human placental brush-border membrane vesicles and human placental basolateral membrane vesicles

The purity of human placental brush-border membrane vesicles or human placental basolateral membrane vesicles was confirmed by examining the enzyme activities of alkaline phosphatase and γ-glutamyl transpeptidase or dihydroalprenolol binding activity, respectively. In the case of human placental brush-border membrane vesicles, the activities of alkaline phosphatase and γ-glutamyl transpeptidase for the vesicles and homogenate were 7.14±0.03 and 0.41±0.01 (pmol/mg protein/min) and 26.79±0.43 and

Discussion

P-Glycoprotein can confer multidrug resistance by extruding a wide range of structurally unrelated, amphiphilic hydrophobic drugs from cells in an ATP-dependent manner Juliano and Ling, 1976, Kartner et al., 1983. In recent studies, P-glycoprotein was reported to be expressed in trophoblast cells but not endothelial cells of the placenta Sugawara et al., 1997, Cordon-Cardo et al., 1989, Nakamura et al., 1997. Therefore, P-glycoprotein in the trophoblast cells is likely to be involved in the

References (53)

  • I Sugawara et al.

    Lung resistance protein (LRP) expression in human normal tissues in comparison with that of MDR1 and MRP

    Cancer Lett.

    (1997)
  • A Tsuji et al.

    P-Glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells

    Life Sci.

    (1992)
  • K Ueda et al.

    Human P-glycoprotein transports cortisol, aldosterone, and dexamethadone, but not progesterone

    J. Biol. Chem.

    (1992)
  • L.T Williams et al.

    Adipocyte β-adrenergic receptors

    J. Biol. Chem.

    (1976)
  • P Wils et al.

    Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers

    Biochem. Pharmacol.

    (1994)
  • M.K Barnes et al.

    Steroid transport, accumulation, and antagonism of P-glycoprotein in multidrug-resistant cells

    Biochemistry

    (1996)
  • J.M Bissonnette

    Placental transport of carbohydrates

    Mead Johnson Symp. Perinat. Dev. Med.

    (1981)
  • C Cordon-Cardo et al.

    Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood–brain barrier sites

    Proc. Natl. Acad. Sci. U. S. A.

    (1989)
  • K Courtney et al.

    Teratology in the Macaca mulatta

    Teratology

    (1968)
  • L.O Derewlany et al.

    The transport of digoxin across the perfused human placental lobule

    J. Pharmacol. Exp. Ther.

    (1991)
  • V Ferm

    Congenital malformation in hamster embryos after treatment with vinblastine and vincristine

    Science

    (1963)
  • T.C Furesz et al.

    ASC system activity is altered by development of cell polarity in trophoblast from human placenta

    Am. J. Physiol.

    (1993)
  • T Hahn et al.

    Ontogeny of glucose transport systems in the placenta and its progenitor tissues

    Early Pregnancy

    (1996)
  • H Hamada et al.

    Functional role of the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies

    Proc. Natl. Acad. Sci. U. S. A.

    (1986)
  • W.W Hay

    Placental transport of nutrients to the fetus

    Horm. Res.

    (1994)
  • Z.-Q Hu et al.

    Biotin uptake by basolateral membrane vesicles of human placenta: normal characteristics and role of ethanol

    Proc. Soc. Exp. Biol. Med.

    (1994)
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