Lobeline, a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells
Introduction
Cancer cells can develop resistance to the anticancer drugs used in cancer therapy. Furthermore, the cells become resistant to other chemotherapeutic drugs whose structures and functions are unrelated. This phenomenon is called multidrug resistance (MDR) (Ambudkar et al., 1999; Gottesman, 2002). MDR is multifactorial, but P-gp, also known as P-glycoprotein, Pg-170, or P-170, plays an important role in MDR. Encoded by ABCB1 gene, it is a member of the ATP-binding cassette family of transmembrane transporters.
P-gp is composed of two transmembrane domains and two ATP binding domains. The substrate molecules bind to the transmembrane domains and are then exported to extracellular space driven by the energy of ATP hydrolysis. P-gp is a broad-spectrum transporter, which can expel different classes of cytotoxic agents, such as Vinca alkaloids, taxanes, anthracyclines, colchicine, etc. (Ambudkar et al., 1999; Gottesman, 2002; Schinkel and Jonker, 2003). Because P-gp can pump anti-cancer drugs out of tumor cells, and thus impairs the efficacy of chemotherapy, a new tentative strategy aims to find compounds that can reverse MDR. Several synthetic compounds, such as verapamil (Tsuruo et al., 1981; Salmon et al., 1991), PSC833 (Boesch et al., 1991; Twentyman and Bleehen, 1991), GF120918 (Hyafil et al., 1993), LY335979 (Dantzig et al., 2001), etc., and natural products, such as the quinone alkaloids cinchonine (Genne et al., 1992), quinine (Solary et al., 1990), etc., are known to at least partially overcome P-gp mediated MDR. Although these agents work successfully in some patients, most results of clinical trials were disappointing (Salmon et al., 1991; Langenbuch et al., 1990; Baer et al., 2002; Rubin et al., 2002; Solary et al., 2000, Solary et al., 2003). Some of these reversal agents did not work in vivo or some had too severe side effects.
Breast cancer resistance protein (BCRP) is another member of ABC superfamily. It is expressed in tumor cells as well. BCRP pumps out anti-cancer drugs, such as anthracyclines, camptothecin, and mitoxantrone from cells. BCRP confers MDR to tumor cells as well (Krishnamurthy and Schuetz, 2006).
In this study, several natural products, among them several alkaloids were screened as potential P-gp and BCRP substrates. Lobeline, a piperidine alkaloid, which is produced by Lobelia inflata (family Lobeliaceae) and several other Lobelia species, stimulates chemoreceptors in carotid and aortic bodies, and then exerts reflectory activation of respiratory centre (Fig. 1). Lobeline, which is also available synthetically (Felpin and Lebreton, 2003), has been used as a respiratory stimulant.
We could show for the first time in our studies that lobeline can moderately inhibit P-gp mediated efflux and reverse P-gp dependent resistance at non-toxic concentrations. It may be a promising lead compound for the development of new MDR reversal drugs.
Section snippets
Chemicals
RPMI 1640 medium, DMEM medium, glutamine and non-essential amino acid were purchased from GIBCO. Fetal bovine serum was from Biochrom AG. Lobeline, verapamil, doxorubicin, MTT, rhodamine 123, G418, mitoxantrone, and other chemicals were from Sigma Aldrich. Fumitremorgin C (FTC) was from Alexis.
Cell lines
A human colon carcinoma cell line, Caco-2 cells, was maintained in DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 1% non-essential amino acid. No antibiotics were added. The
Results
In order to determine the inhibition of P-gp activity, a rhodamine 123 efflux assay was employed. Caco-2 cells, which highly express P-gp on their apical membrane, were loaded with rhodamine 123 (a fluorescent substrate of P-gp) after confluence of the cell layer had formed. As shown in Fig. 2, the rhodamine fluorescence of Caco-2 cells is positively correlated with an increase of lobeline concentration. Thus, lobeline treated cells retained more rhodamine 123 than untreated control cells. This
Discussion
Multidrug resistance impairs the efficacy of some chemotherapeutics. Drug efflux from cells mediated by P-gp overexpression is a major reason of MDR. P-gp is known to export a wide variety of anticancer drugs, such as daunorubicin, doxorubicin, etoposide, vinblastine, vincristine, etc. P-gp activity decreases the accumulation of anticancer drugs in the cells and protects cancer cells from the cytotoxic effects (Ambudkar et al., 1999; Gottesman, 2002; Schinkel and Jonker, 2003). In order to
Acknowledgements
We thank the foundation Landesstiftung Baden-Wuerttemberg for providing part financial support.
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