Inhibition of P-Glycoprotein Activity and Chemosensitization of Multidrug-Resistant Ovarian Carcinoma 2780AD Cells by Hexanoylglucosylceramide Robert Jan Veldman, Hannie Sietsma, Karin Klappe, Dick Hoekstra, and Jan Willem Kok 1 Groningen Institute of Drug Studies, Department of Physiological Chemistry, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands Received November 11, 1999 In the present study we show that neutral hexanoyl- (glyco)sphingolipids inhibit P-glycoprotein (Pgp) ac- tivity in human ovarian 2780AD cells. By contrast, hex- anoylceramide and the gangliosides GM 3 and GM 2 had no effect on Pgp activity, whereas sphingosine had a stimulating effect. In the case of hexanoylglucosylcer- amide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid. Our results lead to the conclusion that a direct transmodu- lation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in re- duced resistance to the chemotherapeutic agent doxorubicin. © 1999 Academic Press Key Words: P-glycoprotein; sphingolipids; glucosyl- ceramide; cell survival. Clinically, MDR of tumor cells often results in failure of anticancer chemotherapy. An important cellular mechanism underlying this phenomenon, is the overexpression of drug transporter proteins such as Pgp [1]. Being a member of the ATP-binding cas- sette (ABC) superfamily of transporter proteins, this 170 kDa integral plasma membrane phosphoglyco- protein effectively prevents cellular accumulation of cytotoxic compounds, and hence increases tumor cell survival. From a chemical point of view, Pgp sub- strates are rather diverse by nature. However, the fact that most of its substrates are amphipatic ex- plains the observation that many drugs, once taken up by the cell, eventually gain access to the inner leaflet of the plasma membrane [2]. It has been pro- posed that Pgp removes drugs from the inner leaflet either by expelling them directly into the extracellu- lar space or by translocating the lipophilic com- pounds to the external leaflet of the membrane, from which they eventually diffuse [3]. Being an integral plasma membrane protein, Pgp is dependent on its lipid environment for optimal func- tioning [4, 5]. An even more intimate relation between lipids and ABC-transporters, such as Pgp, is indicated by observations that some phospholipids might serve as substrate for MDR proteins, thus mediating their translocation across membranes [6 – 8]. In addition, it has been reported that sphingolipids such as SM and GlcCer can be subjected to Pgp-mediated translocation [9, 10]. Therefore, in addition to removing xenotoxic compounds, Pgp might act as a flippase and hence, could be involved in the maintenance of the lipid asym- metry which exists between the leaflets of cellular plasma membranes. Other correlations between lipids and MDR have also been reported. For example, the phospholipid and ether lipid composition in Pgp-overexpressing cells dif- fers from that in wild type cells [11]. In addition, in- creased levels of GlcCer have been reported in MDR cells [12, 13]. The latter is particularly interesting since GlcCer is known for its role in cell growth regu- lation. High levels of this lipid have been implicated in proliferation activity [14, 15], whereas low levels in- duce differentiation of cells [16]. In addition to the overexpression of drug transporters such as Pgp, sev- eral other MDR mechanisms exist, e.g. the ability of some MDR cells to escape from apoptosis. Since Cer plays a major role in the regulation of apoptosis [17], this indicates an additional relationship between sphingolipids and MDR. Abbreviations used: ABC-protein, ATP-binding cassette protein; bSMase, Staphylococcus aureus sphingomyelinase; C 6, n-hexanoic acid/n-hexanoyl; Cer, ceramide; CSA, cyclosporin A; GalCer, galac- tosylceramide; GlcCer, glucosylceramide; HBSS, Hanks’ balanced salt solution; LacCer, lactosylceramide; MDR, multidrug resistance; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PDMP, DL-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propa- nol; Pgp, P-glycoprotein; Rh 123 , rhodamine 123; SM, sphingomyelin; So, sphingosine. 1 To whom correspondence should be addressed. Fax: 31-50- 3632728. E-mail: j.w.kok@med.rug.nl. Biochemical and Biophysical Research Communications 266, 492– 496 (1999) Article ID bbrc.1999.1850, available online at http://www.idealibrary.com on 492 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.