- Email: [email protected]
Bicalutamide failure in prostate cancer treatment: Involvement of Multi Drug Resistance proteins
European Journal of Pharmacology 601 (2008) 38–42
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Bicalutamide failure in prostate cancer treatment: Involvement of Multi Drug Resistance proteins Nicola Antonio Colabufo a,⁎, Vincenzo Pagliarulo b,⁎, Francesco Berardi a, Marialessandra Contino a, Carmela Inglese a, Mauro Niso b, Patrizia Ancona b, Giancarlo Albo b, Arcangelo Pagliarulo b, Roberto Perrone a a b
Dipartimento Farmacochimico, Universita' degli Studi di Bari, via Orabona 4, 70125, Bari, Italy Dipartimento Emergenza e Trapianti d'Organo, Sezione di Urologia, Universita' degli Studi di Bari, Piazza G. Cesare 11, 70124 Bari, Italy
a r t i c l e
i n f o
Article history: Received 19 June 2008 Received in revised form 25 September 2008 Accepted 9 October 2008 Available online 29 October 2008 Keywords: Bicalutamide Calcein-AM P-glycoprotein BCRP MRP1
a b s t r a c t Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. As experimental models androgen-dependent (LnCap) and androgen-independent (PC-3) prostate cancer cell lines have been employed. Bicalutamide has been tested in the cell lines mentioned above in the absence and in the presence of MC18, our potent P-glycoprotein/BCRP/MRP1 inhibitor. The results displayed that bicalutamide antiproliferative effect at 72 h was ameliorated in LnCap cells (EC50 from 51.9 ± 6.1 µM to 17.8 ± 2.6 µM in the absence and in the presence of MC18, respectively) and restored in PC-3 cells (EC50 from 150 ± 2.4 µM to 60 ± 3.5 µM in the absence and in the presence of MC18, respectively). Moreover, we established the contribution of each transporter employing stable transfected cells (MDCK) overexpressing P-glycoprotein or BCRP or MRP1 pump. The results displayed that P-glycoprotein and BCRP were involved in bicalutamide efflux while MRP1 was unable to bind the antiandrogen drug. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The androgen receptors are involved in prostate cancer development and progression (Damber and Aus, 2008), and the treatment with androgen receptor antagonists (flutamide or bicalutamide) represented the standard systemic therapy (Moul, 2003; Gillatt, 2006; Iversen and Roder, 2008). Prolonged androgen receptor antagonist treatment caused evidence of pathology regression (Hsieh and Ryan, 2008) but relapse with a more aggressive form of prostate cancer has been observed (McLeod, 2004). This relapse has been termed hormone-refractory, castration-resistant, or androgenindependent prostate cancer (Shand and Gelmann, 2006; Kageyama et al., 2007; Schrijvers, 2007). Bicalutamide failed in androgen-independent prostate cancer although androgen receptor and androgen receptor-regulated genes were found highly expressed, since in these tumors androgen receptor transcriptional activity was reactivated and androgen receptors were potential therapeutic targets (Suzuki et al., 2003). A mechanism ⁎ Corresponding authors. Colabufo is to be contacted at Tel.: +39 080 5442727; fax: +39 080 5442231. Pagliarulo, Tel.: +39 080 5586499. E-mail addresses: [email protected] (N.A. Colabufo), [email protected] (V. Pagliarulo). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.038
explaining this failure involved androgen receptor mutation (Culig et al., 2001; Hara et al., 2003; Unni et al., 2004; Bohl et al., 2005) since the points suitable of this alteration have been identified (Hirawat et al., 2003; Elhaji et al., 2004; Lumbroso et al., 2004). In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. In order to verify our hypothesis we selected two cell lines: an androgen-dependent (LnCap) and an androgen-independent (PC-3) tumor prostate cell lines. LnCap cell line is derived from a metastatic lesion of the lymphnodes of a patient with prostate cancer (Veldscholte et al., 1990). The growth of LnCap cells is stimulated in vitro by androgens, estrogens, progestogens and several antiandrogens, indicating a widely responsive property of this cell line. PC-3 cells are an epithelial cell line derived from a human prostatic adenocarcinoma metastatic to bone (Kaighn et al., 1979). In these cell lines we verified bicalutamide antiproliferative effect either alone or in co-administration with an inhibitor of ABC transporters MC18 (6,7-dimethoxy-2-{3-[4-methoxy-3,4-dihydro-2Hnaphthalen-(1E)-ylidene]-propyl}-1,2,3,4-tetrahydro-isoquinoline) reported in Fig. 1 (Colabufo et al., 2008a). This inhibitor, previously
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
39
2.3. Permeability experiments
Fig. 1. Structure of MC18, a potent P-glycoprotein/BCRP/MRP1 inhibitor.
characterized (Colabufo et al., submitted for publication), displayed high pump inhibition activity towards P-glycoprotein (EC50 = 1.64 µM), BCRP (EC50 = 1.56 µM) and MRP1 (EC50 = 5.1 µM) measured in Madin–Darby Canine Kidney (MDCK) cell line overexpressing each human transporter by Calcein-AM assay (Colabufo et al., submitted for publication). Aims of this paper were: (i) to determine bicalutamide Apparent Permeability (Papp) so that the capability of this compound to permeate cell membranes may be established; (ii) to verify if MC18–bicalutamide co-administration in PC-3 cells restored anti-androgen activity of tested drug; (iii) to identify the target pump responsible for bicalutamide efflux. 2. Materials and methods 2.1. Drug and chemicals RPMI 1640 medium, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Phosphate Buffered Saline (PBS), Hank's Balanced Salt Solution (HBSS), trypsin–EDTA, penicillin (10,000 U/ml) streptomycin (10 mg/ml), L-glutamine solution (100×), and fetal calf serum, were purchased from Celbio s.r.l. Italy. Disposable culture flasks and Petri dishes were purchased from Corning, Glassworks (Corning, NY, USA). Millicell® Cell Culture Insert Plate was from Millipore (Carrigtwohill, Co Cork, Ireland). 3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide (MTT) was purchased from SigmaAldrich s.r.l. (Milan, Italy). Bicalutamide was purchased from Astra Zeneca (AstraZeneca S.p.A. Caponago, (Milan, Italy). Calcein-AM was purchased from Molecular Probes (Eugene, OR).
2.3.1. Preparation of Caco-2 monolayer This procedure has been previously reported by Colabufo et al. (2006). Briefly, Caco-2 cells were harvested with trypsin–EDTA and seeded onto Millicell® assay system at a density of 10,000 cells/well. The culture medium was replaced every 48 h for the first 6 days and every 24 h thereafter, and after 21 days in culture, the Caco-2 monolayer was utilized for the permeability experiments. The Trans Epithelial Electrical Resistance (TEER) of the monolayers was measured daily before and after the experiment using an epithelial voltohometer (Millicell®-ERS; Millipore, Billerica, MA). Generally, TEER values obtained are greater than 1000 Ω for a 21 day culture. 2.3.2. Drug transport experiment Apical to basolateral (Papp A→B) and basolateral to apical (Papp B→A) permeabilities of bicalutamide were measured at 120 min and at various bicalutamide concentrations (1–100 µM) (Polli et al., 2001). Drugs were dissolved in Hank's balanced salt solution (HBSS, pH 7.4) and sterile filtered. After 21 days of cell growth, the medium was removed from filter wells and from the receiver plate. The filter wells were filled with 75 µl of fresh HBSS buffer and the receiver plate with 250 µl/well of the same buffer. This procedure was repeated twice, and the plates were incubated at 37 °C for 30 min. After incubation time, the HBSS buffer was removed and drug solutions added to the filter well (75 µl). HBSS without bicalutamide was added to the receiver plate (250 µl). The plates were incubated at 37 °C for 120 min. After incubation time, samples were removed from the apical (filter well) and basolateral (receiver plate) sides of the monolayer and then were stored in a freezer (−20 °C) pending analysis. The Apparent Permeability (Papp), in units of nm/s, was calculated using the following equation: Papp =
½drugacceptor VA × Area×time ½druginitial
where VA is the volume (in ml) in the acceptor well; Area is the surface area of the membrane (0.11 cm2 of the well); time is the total transport time in seconds (7200 s); [drug]acceptor is the concentration of the drug measured by ESI-MS analyses or U.V. spectroscopy; [drug]initial is the initial drug concentration (1 × 10− 4 M) in the apical or basolateral wells. 2.4. Cell antiproliferative effect
2.2. Cell lines LnCap and PC-3 prostate cancer cell lines were obtained from Interlab Cell Line Collection (ICLC, Genova). Caco-2 cells were obtained from IRCCS “S. De Bellis”, Castellana Grotte (Bari, Italy). The prostate cancer cell lines of human origin, LnCap and PC-3, were routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37 °C with a 5% CO2 atmosphere. Caco-2 cells were grown in DMEM medium with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. MDCK-MRP1 (gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam, Nederland) and MDCK-BCRP cells (gift of Dr. A. Schinkel, NKI-AVL Institute, Amsterdam, Nederland) were grown in DMEM high glucose supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, in a humidified incubator at 37 °C with a 5% CO2 atmosphere. The cells were trypsinized twice a week with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%/0.02%) and the medium was changed twice a week.
The antiproliferative effect was evaluated using the 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay as reported by Colabufo et al. (2004) with minor modifications. The cells were seeded into 96-well plates in the absence and presence of known concentrations of test compound for 48 h. In each well 10 µl of MTT solution (5 mg/ml) freshly prepared was added and the plate was incubated in a humidified atmosphere 5% CO2 at 37 °C for 3–4 h. MTT solution was removed and 200 µl of EtOH/DMSO (1:1) was added to each well to dissolve the blue formazan solid crystals. The optical density was measured at 570 nm and 650 nm wavelengths using Victor3, from PerkinElmer Life Sciences. 2.5. Calcein-AM experiment This experiment was carried out as described by Feng et al. (2008) with minor modifications. Each cell line (50,000 cells/well) was seeded into black CulturePlate 96/wells plate with 100 µl medium and allowed to become confluent overnight. Test compounds were solubilized in 100 µl of culture medium and added to monolayers. 96/wells plate was incubated at 37 °C for 30 min. Calcein-AM was
40
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
3.2. Bicalutamide antiproliferative effect in LnCap and PC-3 prostate cancer cells
Fig. 2. Bicalutamide antiproliferative effect in the absence and in the presence of MC18 at 48 h (A) and 72 h (B) in LnCap cell line.
Bicalutamide antiproliferative effect in LnCap (Fig. 2) and PC-3 (Fig. 3) cells was performed at 48 h (Fig. 2A) and 72 h (Fig. 3A). The data at 24 h are not reported because the effect was unappreciable considering the slow cell growth of each line. In LnCap cells bicalutamide, tested from 1.0 µM to 200 µM, at 48 h and 72 h displayed moderate antiproliferative effect (EC50 = 72.6 ± 2.6 µM and 51.9 ± 6.1 µM, respectively). In the same experimental conditions bicalutamide displayed antiproliferative effect in PC-3 cells (EC50 = 100 ± 5.2 µM and 150 ± 8.6 µM, respectively) lower than that found in LnCap cells. These results reflected bicalutamide different sensitivity of androgen-dependent (LnCap) and androgen-independent (PC-3) cell lines. In order to verify our hypothesis, we employed 10 µM MC18, a potent non selective P-glycoprotein/BCRP/MRP1 inhibitor, in bicalutamide co-administration. Our inhibitor at 10 µM did not display antiproliferative effect in each cell line (data not shown) and at this concentration it blocked the above mentioned ABC transporters. In LnCap cells bicalutamide–MC18 co-administration at 48 h (Fig. 2A) ameliorated bicalutamide antiproliferative effect about 2-fold (EC50 from 72.6 ± 2.6 µM to 42.7 ± 2.4 µM). At 72 h the co-administration (Fig. 2B) ameliorated about 3-fold bicalutamide antiproliferative effect (EC50 from 51.9 ± 6.1 µM to 17.8 ± 2.6 µM). By contrast, in PC-3 cell line at 48 h bicalutamide–MC18 co-administration (Fig. 3A) did not improve antiproliferative effect of the anti-androgen agent. Conversely at 72 h (Fig. 3B) the co-administration increased about 3-fold bicalutamide antiproliferative effect (EC50 from 150 ± 2.4 µM to 60 ± 3.5 µM). This result (EC50 = 60 ± 3.5 µM) is comparable to the effect found in LnCap cells at 48 h and at 72 h. This finding suggested that bicalutamide failure in PC-3 could be due to the presence of one of the three studied pumps.
added in 100 µl of PBS to yield a final concentration of 2.5 µM and plate was incubated for 30 min. Each well was washed 3 times with ice cold PBS. Saline buffer was added to each well and the plate was read to Victor3 at excitation and emission wavelengths of 485 nm and 535 nm, respectively. In these experimental conditions Calcein cell accumulation in the absence and in the presence of tested compounds was evaluated and fluorescence basal level was estimated by untreated cells. In treated wells the increase of fluorescence with respect to basal level was measured. The IC50 values were determined by fitting the fluorescence increase percentage versus log[dose]. 3. Results 3.1. Bicalutamide Apparent Permeability (Papp) In Caco-2 cells experimental model, bicalutamide Apparent Permeability (Papp) has been determined evaluating both the basolateral–apical flux (B→A) and apical–basolateral flux (A→B). In this assay 100 µM bicalutamide was added to apical compartment and, at equilibrium, the drug concentration in basolateral compartment was determined (Papp A→B). In the same experimental conditions bicalutamide was added to basolateral compartment and the drug concentration, at the equilibrium in the apical compartment was measured (Papp B→A). A BA/AB ratio N2 is an indication that the compound could be linked and flipped to extra cellular compartment by P-glycoprotein and/or BCRP and/or MRP1, transmembrane ATPase pumps overexpressed in cancer cells. Bicalutamide displayed Papp A→B = 221 nm/s, Papp B→A = 1193 nm/s and BA/AB ratio was 5.4.
Fig. 3. Bicalutamide antiproliferative effect in the absence and in the presence of MC18 at 48 h (A) and 72 h (B) in PC-3 cell line.
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
Fig. 4. Calcein-AM assay in MDCK-P-gp, MDCK-BCRP and MDCK-MRP1 cell lines.
3.3. Bicalutamide Calcein-AM assay A biological assay to study Multi Drug Resistance proteins activity is monitoring Calcein accumulation in transfected MDCK cells. These cells are incubated with a lipophilic P-glycoprotein/BCRP/MRP1 substrate, AcetoxyMethyl (AM) esters of Calcein (Calcein-AM). This non fluorescent dye diffuses across the plasma membrane into the cell and is hydrolyzed by endogenous cytoplasmic esterases into highly fluorescent Calcein (Fig. 4). This compound is not a Pglycoprotein/BCRP/MRP1 pump substrate and cannot cross the cell membrane via passive diffusion because of its hydrophilicity. Therefore, a rapid fluorescence increase can be monitored. P-glycoprotein/ BCRP/MRP1 pumps, present in the plasma membrane, rapidly efflux Calcein-AM so that it cannot enter the cytosol determining a reduction in the fluorescent signal due to Calcein accumulation. In each transfected cell line, bicalutamide has been tested in order to verify its ability to compete with Calcein-AM for the same binding site. In MDCK-P-glycoprotein cells, as depicted in Fig. 5A (fluorescence decrease versus log[bicalutamide]), bicalutamide influenced Calcein-AM transport (EC50 = 73.5 ± 4.6 µM). Similarly, as reported in Fig. 5B, bicalutamide displayed high affinity towards BCRP CalceinAM site resulting in appreciable fluorescent decrease (EC50 = 31.0 ± 1.6 µM). Conversely, bicalutamide at 200 µM failed to interfere with MRP1 Calcein-AM site.
41
we established the ability of this drug to permeate cell membrane in Caco-2 cell model where P-glycoprotein was highly expressed. The results demonstrated that bicalutamide was transported by Pglycoprotein and this finding encouraged us to deepen the potential Multi Drug Resistance involvement in bicalutamide failure both in LnCap and PC-3 cell lines. For this purpose we verified bicalutamide antiproliferative effect in each cell line employing in bicalutamide coadministration our potent Multi Drug Resistance pumps inhibitor, MC18. The results demonstrated that MC18 restored bicalutamide cell permeation in PC-3 and ameliorated the effect in LnCap with respect to bicalutamide alone. Since MC18 inhibited in the same rank order P-glycoprotein, BCRP and MRP1, we employed cell lines overexpressing each of the above mentioned pumps. The results displayed that bicalutamide is effluxed by P-glycoprotein and BCRP while MRP1 was unable to efflux this ligand. This finding disagreed with the hypothesis that MRP1 was the predominant pump involved in prostate cancer resistance (Sullivan et al., 2000; Zalcberg et al., 2000). Moreover, Ho et al. (2008) demonstrated that ABCC4/MRP4, another pump highly expressed in prostate (Belinsky et al., 1998; Kool et al., 1997) and able to transport typical MRP1 substrates (Chen et al., 2001), was downregulated by bicalutamide treatment in vitro in LnCap cells. By contrast, the results seem to confirm the hypothesis of BCRP involvement in animal model. On the other hand also Pglycoprotein, although reported as undetectable in the studied cell lines, seems to be involved in bicalutamide failure. In conclusion, our results demonstrated that a possible strategy to restore bicalutamide activity in prostate cancer could be its co-administration with BCRP/ P-glycoprotein inhibitors.
4. Discussion In the present study we reported several biological assays in order to support the hypothesis that bicalutamide failure in prostate cancer, after prolonged treatment, is due to Multi Drug Resistance proteins involvement. In the last years, several authors reported that P-glycoprotein both in LnCap and PC-3 was below detection level (Theyer et al., 1993; van Brussel et al., 1999) while MRP1 overexpression could be considered the predominant mechanism of resistance in PC-3 (Sullivan et al., 2000; Zalcberg et al., 2000). Moreover, the effect of BCRP inhibition on the progression of prostate cancer and on the transition to androgen-insensitive prostate cancer has been demonstrated in animal model (Lee et al., 2007). Finally, it was reported that resistance of prostate cancer cells to bicalutamide was determined by Multi Drug Resistance mechanism involving some ABC transporters such as P-glycoprotein, MRP1 and BCRP (van Brussel et al., 1999). In order to better clarify if bicalutamide failure in prostate cancer treatment was due to Multi Drug Resistance, firstly
Fig. 5. Bicalutamide effect on Calcein cell accumulation in MDCK-P-glycoprotein (A) and MDCK-BCRP (B) cell lines.
42
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
References Belinsky, M.G., Bain, L.J., Balsara, B.B., Testa, J.R., Kruh, G.D., 1998. Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins. J. Natl. Cancer Inst. 90, 1735–1741. Bohl, C.E., Gao, W., Miller, D.D., Bell, C.E., Dalton, J.T., 2005. Structural basis for antagonism and resistance of bicalutamide in prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 6201–6206. Chen, Z.S., Lee, K., Kruh, G.D., 2001. Transport of cyclic nucleotides and estradiol 17-β-Dglucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J. Biol. Chem. 276, 33747–33754. Colabufo, N.A., Berardi, F., Contino, M., Niso, M., Abate, C., Perrone, R., Tortorella, V., 2004. Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumour cell lines. Naunyn-Schmiedeberg's Arch. Pharmacol. 370, 106–113. Colabufo, N.A., Berardi, F., Perrone, R., Rapposelli, S., Digiacomo, M., Balsamo, A., 2006. Arylmethyloxyphenyl derivatives: small molecules displaying P-glycoprotein inhibition. J. Med. Chem. 49, 6607–6613. Colabufo, N.A., Berardi, F., Cantore, M., Perrone, M.G., Contino, M., Inglese, C., Niso, M., Perrone, R., Azzariti, A., Simone, G.M., Porcelli, L., Paradiso, A., 2008a. Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives. Bioorg. Med. Chem. 16, 362–373. Colabufo, N.A., Cantore, M., Contino, M., Inglese, C., Niso, M., Berardi, F., Perrone, R., Cassano, G., Gasparre, G., Azzariti, A., Paradiso, A., submitted for publication. Biological characterization of PB28 and 6,7-dimethoxytetrahydroisoquinoline derivatives, MC18 and MC70, as potent inhibitors of ABC transporters involved in MultiDrug Resistance. Eur. J. Pharmacol. Culig, Z., Klocker, H., Bartsch, G., Hobisch, A., 2001. Androgen receptor mutations in carcinoma of the prostate: significance for endocrine therapy. Am. J. Pharmacogenomics 1, 241–249. Damber, J.E., Aus, G., 2008. Prostate Cancer. Lancet 17, 1710–1721. Elhaji, Y.A., Wu, J.H., Gottlieb, B., Betel, L.K., Alvarado, C., Batist, G., Trifido, M.A., 2004. An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes. Mol. Endocrinol. 18, 1876–1886. Feng, B., Mills, J.B., Davidson, R.E., Mireles, R.J., Janiszewski, J.S., Troutman, M.D., de Morais, S.M., 2008. In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system. Drug Metab. Dispos. 36, 268–275. Gillatt, D., 2006. Antiandrogen treatments in locally advanced prostate cancer: are they all the same? J. Cancer Res. Clin. Oncol. 132, S17–26. Hara, T., Miyazaki, J., Araki, H., Yamaoka, M., Kanzaki, N., Kusaka, M., Miyamoto, M., 2003. Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res. 63, 149–153. Hirawat, S., Budman, D.R., Kreis, W., 2003. The androgen receptor: structure, mutations, and antiandrogens. Cancer Investig. 21, 400–417. Ho, L.L., Kench, J.G., Handelsman, D.J., Scheffer, G.L., Stricker, P.D., Grygiel, J.G., Sutherland, R.L., Henshall, S.M., Allen, J.D., Horvath, L.G., 2008. Androgen regulation of multidrug resistance-associated protein 4 (MRP4/ABCC4) in prostate cancer. Prostate 68, 1421–1429. Hsieh, A.C., Ryan, C.J., 2008. Novel concepts in androgen receptor blockade. Cancer J. 14, 11–14. Iversen, P., Roder, M.A., 2008. The Early Prostate Cancer program: bicalutamide in nonmetastatic prostate cancer. Expert Rev. Anticancer Ther. 8, 361–369.
Kageyama, Y., Hyochi, N., Kihara, K., Sugiyama, H., 2007. The androgen receptor as putative therapeutic target in hormone-refractory prostate cancer. Recent Patents Anticancer Drug Discov. 2, 203–211. Kaighn, M.E., Narayan, K.S., Ohnuki, Y., Lechner, J.F., Jones, L.W., 1979. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest. Urol. 17, 16–23. Kool, M., de Haas, M., Scheffer, G.L., Scheper, R.J., van Eijk, M.J., Juijn, J.A., Baas, F., Borst, P., 1997. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 57, 3537–3547. Lee, D.I., Sumbilla, C., Lee, M., Natesavelalar, C., Klein, M.G., Ross, D.D., Inesi, G., Hussain, A., 2007. Mechanisms of resistance and adaptation to thapsigargin in androgenindependent prostate cancer PC3 and DU145 cells. Arch. Biochem. Biophys. 464, 19–27. Lumbroso, S., Wagschal, A., Bourguet, W., Georget, V., Mazen, I., Servant, N., Audran, F., Sultan, C., Auzou, G., 2004. A new mutation of the androgen receptor, P817A, causing partial androgen insensitivity syndrome: in vitro and structural analysis. J. Mol. Endocrinol. 32, 679–687. McLeod, D.G., 2004. Success and failure of single-modality treatment for early prostate cancer. Rev. Urol. 6, S13–19. Moul, J.W, 2003. Biochemical recurrence of prostate cancer. Curr. Probl. Cancer 27, 243–272. Polli, J.W., Wring, S.A., Humphreys, J.E., Huang, L., Morgan, J.B., Webster, L.O., SerabjitSingh, C.S., 2001. Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 299, 620–628. Schrijvers, D., 2007. Androgen-independent prostate cancer. Recent Results Cancer Res. 175, 239–249. Shand, R.L., Gelmann, E.P., 2006. Molecular biology of prostate-cancer pathogenesis. Curr. Opin. Urol. 16, 123–131. Sullivan, G.F., Yang, J.M., Vassil, A., Yang, J., Bash-Babula, J., Hait, W.N., 2000. Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells. J. Clin. Invest. 105, 1261–1267. Suzuki, H., Ueda, T., Ichikawa, T., Ito, H., 2003. Androgen receptor involvement in the progression of prostate cancer. Endocr. Relat. Cancer 10, 209–216. Theyer, G., Schirmbock, M., Thalhammer, T., Sherwood, E.R., Baumgartner, G., Hamilton, G., 1993. Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines. J. Urol. 150, 1544–1547. Unni, E., Sun, S., Nan, B., McPhaul, M.J., Cheskis, B., Mancini, M.A., Marcelli, M., 2004. Changes in androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to androgen independence. Cancer Res. 1, 7156–7168. van Brussel, J.P., van Steenbrugge, G.J., Romijn, J.C., Schröder, F.H., Mickisch, G.H.J., 1999. Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins. Eur. J. Cancer 35, 664–671. Veldscholte, J., Voorhorst-Ogink, M.M., Bolt-de Vries, J., van Rooij, H.C., Trapman, J., Mulder, E., 1990. Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids. Biochim. Biophys. Acta 1052, 187–194. Zalcberg, J., Hu, X.F., Slater, A., Parisot, J., El-Osta, S., Kantharidis, P., Chou, S.T., Parkin, J.D., 2000. MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines. Prostate Cancer Prostatic Dis. 3, 66–75.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Bicalutamide failure in prostate cancer treatment: Involvement of Multi Drug Resistance proteins Nicola Antonio Colabufo a,⁎, Vincenzo Pagliarulo b,⁎, Francesco Berardi a, Marialessandra Contino a, Carmela Inglese a, Mauro Niso b, Patrizia Ancona b, Giancarlo Albo b, Arcangelo Pagliarulo b, Roberto Perrone a a b
Dipartimento Farmacochimico, Universita' degli Studi di Bari, via Orabona 4, 70125, Bari, Italy Dipartimento Emergenza e Trapianti d'Organo, Sezione di Urologia, Universita' degli Studi di Bari, Piazza G. Cesare 11, 70124 Bari, Italy
a r t i c l e
i n f o
Article history: Received 19 June 2008 Received in revised form 25 September 2008 Accepted 9 October 2008 Available online 29 October 2008 Keywords: Bicalutamide Calcein-AM P-glycoprotein BCRP MRP1
a b s t r a c t Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. As experimental models androgen-dependent (LnCap) and androgen-independent (PC-3) prostate cancer cell lines have been employed. Bicalutamide has been tested in the cell lines mentioned above in the absence and in the presence of MC18, our potent P-glycoprotein/BCRP/MRP1 inhibitor. The results displayed that bicalutamide antiproliferative effect at 72 h was ameliorated in LnCap cells (EC50 from 51.9 ± 6.1 µM to 17.8 ± 2.6 µM in the absence and in the presence of MC18, respectively) and restored in PC-3 cells (EC50 from 150 ± 2.4 µM to 60 ± 3.5 µM in the absence and in the presence of MC18, respectively). Moreover, we established the contribution of each transporter employing stable transfected cells (MDCK) overexpressing P-glycoprotein or BCRP or MRP1 pump. The results displayed that P-glycoprotein and BCRP were involved in bicalutamide efflux while MRP1 was unable to bind the antiandrogen drug. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The androgen receptors are involved in prostate cancer development and progression (Damber and Aus, 2008), and the treatment with androgen receptor antagonists (flutamide or bicalutamide) represented the standard systemic therapy (Moul, 2003; Gillatt, 2006; Iversen and Roder, 2008). Prolonged androgen receptor antagonist treatment caused evidence of pathology regression (Hsieh and Ryan, 2008) but relapse with a more aggressive form of prostate cancer has been observed (McLeod, 2004). This relapse has been termed hormone-refractory, castration-resistant, or androgenindependent prostate cancer (Shand and Gelmann, 2006; Kageyama et al., 2007; Schrijvers, 2007). Bicalutamide failed in androgen-independent prostate cancer although androgen receptor and androgen receptor-regulated genes were found highly expressed, since in these tumors androgen receptor transcriptional activity was reactivated and androgen receptors were potential therapeutic targets (Suzuki et al., 2003). A mechanism ⁎ Corresponding authors. Colabufo is to be contacted at Tel.: +39 080 5442727; fax: +39 080 5442231. Pagliarulo, Tel.: +39 080 5586499. E-mail addresses: [email protected] (N.A. Colabufo), [email protected] (V. Pagliarulo). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.038
explaining this failure involved androgen receptor mutation (Culig et al., 2001; Hara et al., 2003; Unni et al., 2004; Bohl et al., 2005) since the points suitable of this alteration have been identified (Hirawat et al., 2003; Elhaji et al., 2004; Lumbroso et al., 2004). In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. In order to verify our hypothesis we selected two cell lines: an androgen-dependent (LnCap) and an androgen-independent (PC-3) tumor prostate cell lines. LnCap cell line is derived from a metastatic lesion of the lymphnodes of a patient with prostate cancer (Veldscholte et al., 1990). The growth of LnCap cells is stimulated in vitro by androgens, estrogens, progestogens and several antiandrogens, indicating a widely responsive property of this cell line. PC-3 cells are an epithelial cell line derived from a human prostatic adenocarcinoma metastatic to bone (Kaighn et al., 1979). In these cell lines we verified bicalutamide antiproliferative effect either alone or in co-administration with an inhibitor of ABC transporters MC18 (6,7-dimethoxy-2-{3-[4-methoxy-3,4-dihydro-2Hnaphthalen-(1E)-ylidene]-propyl}-1,2,3,4-tetrahydro-isoquinoline) reported in Fig. 1 (Colabufo et al., 2008a). This inhibitor, previously
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
39
2.3. Permeability experiments
Fig. 1. Structure of MC18, a potent P-glycoprotein/BCRP/MRP1 inhibitor.
characterized (Colabufo et al., submitted for publication), displayed high pump inhibition activity towards P-glycoprotein (EC50 = 1.64 µM), BCRP (EC50 = 1.56 µM) and MRP1 (EC50 = 5.1 µM) measured in Madin–Darby Canine Kidney (MDCK) cell line overexpressing each human transporter by Calcein-AM assay (Colabufo et al., submitted for publication). Aims of this paper were: (i) to determine bicalutamide Apparent Permeability (Papp) so that the capability of this compound to permeate cell membranes may be established; (ii) to verify if MC18–bicalutamide co-administration in PC-3 cells restored anti-androgen activity of tested drug; (iii) to identify the target pump responsible for bicalutamide efflux. 2. Materials and methods 2.1. Drug and chemicals RPMI 1640 medium, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Phosphate Buffered Saline (PBS), Hank's Balanced Salt Solution (HBSS), trypsin–EDTA, penicillin (10,000 U/ml) streptomycin (10 mg/ml), L-glutamine solution (100×), and fetal calf serum, were purchased from Celbio s.r.l. Italy. Disposable culture flasks and Petri dishes were purchased from Corning, Glassworks (Corning, NY, USA). Millicell® Cell Culture Insert Plate was from Millipore (Carrigtwohill, Co Cork, Ireland). 3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide (MTT) was purchased from SigmaAldrich s.r.l. (Milan, Italy). Bicalutamide was purchased from Astra Zeneca (AstraZeneca S.p.A. Caponago, (Milan, Italy). Calcein-AM was purchased from Molecular Probes (Eugene, OR).
2.3.1. Preparation of Caco-2 monolayer This procedure has been previously reported by Colabufo et al. (2006). Briefly, Caco-2 cells were harvested with trypsin–EDTA and seeded onto Millicell® assay system at a density of 10,000 cells/well. The culture medium was replaced every 48 h for the first 6 days and every 24 h thereafter, and after 21 days in culture, the Caco-2 monolayer was utilized for the permeability experiments. The Trans Epithelial Electrical Resistance (TEER) of the monolayers was measured daily before and after the experiment using an epithelial voltohometer (Millicell®-ERS; Millipore, Billerica, MA). Generally, TEER values obtained are greater than 1000 Ω for a 21 day culture. 2.3.2. Drug transport experiment Apical to basolateral (Papp A→B) and basolateral to apical (Papp B→A) permeabilities of bicalutamide were measured at 120 min and at various bicalutamide concentrations (1–100 µM) (Polli et al., 2001). Drugs were dissolved in Hank's balanced salt solution (HBSS, pH 7.4) and sterile filtered. After 21 days of cell growth, the medium was removed from filter wells and from the receiver plate. The filter wells were filled with 75 µl of fresh HBSS buffer and the receiver plate with 250 µl/well of the same buffer. This procedure was repeated twice, and the plates were incubated at 37 °C for 30 min. After incubation time, the HBSS buffer was removed and drug solutions added to the filter well (75 µl). HBSS without bicalutamide was added to the receiver plate (250 µl). The plates were incubated at 37 °C for 120 min. After incubation time, samples were removed from the apical (filter well) and basolateral (receiver plate) sides of the monolayer and then were stored in a freezer (−20 °C) pending analysis. The Apparent Permeability (Papp), in units of nm/s, was calculated using the following equation: Papp =
½drugacceptor VA × Area×time ½druginitial
where VA is the volume (in ml) in the acceptor well; Area is the surface area of the membrane (0.11 cm2 of the well); time is the total transport time in seconds (7200 s); [drug]acceptor is the concentration of the drug measured by ESI-MS analyses or U.V. spectroscopy; [drug]initial is the initial drug concentration (1 × 10− 4 M) in the apical or basolateral wells. 2.4. Cell antiproliferative effect
2.2. Cell lines LnCap and PC-3 prostate cancer cell lines were obtained from Interlab Cell Line Collection (ICLC, Genova). Caco-2 cells were obtained from IRCCS “S. De Bellis”, Castellana Grotte (Bari, Italy). The prostate cancer cell lines of human origin, LnCap and PC-3, were routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37 °C with a 5% CO2 atmosphere. Caco-2 cells were grown in DMEM medium with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. MDCK-MRP1 (gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam, Nederland) and MDCK-BCRP cells (gift of Dr. A. Schinkel, NKI-AVL Institute, Amsterdam, Nederland) were grown in DMEM high glucose supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, in a humidified incubator at 37 °C with a 5% CO2 atmosphere. The cells were trypsinized twice a week with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%/0.02%) and the medium was changed twice a week.
The antiproliferative effect was evaluated using the 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay as reported by Colabufo et al. (2004) with minor modifications. The cells were seeded into 96-well plates in the absence and presence of known concentrations of test compound for 48 h. In each well 10 µl of MTT solution (5 mg/ml) freshly prepared was added and the plate was incubated in a humidified atmosphere 5% CO2 at 37 °C for 3–4 h. MTT solution was removed and 200 µl of EtOH/DMSO (1:1) was added to each well to dissolve the blue formazan solid crystals. The optical density was measured at 570 nm and 650 nm wavelengths using Victor3, from PerkinElmer Life Sciences. 2.5. Calcein-AM experiment This experiment was carried out as described by Feng et al. (2008) with minor modifications. Each cell line (50,000 cells/well) was seeded into black CulturePlate 96/wells plate with 100 µl medium and allowed to become confluent overnight. Test compounds were solubilized in 100 µl of culture medium and added to monolayers. 96/wells plate was incubated at 37 °C for 30 min. Calcein-AM was
40
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
3.2. Bicalutamide antiproliferative effect in LnCap and PC-3 prostate cancer cells
Fig. 2. Bicalutamide antiproliferative effect in the absence and in the presence of MC18 at 48 h (A) and 72 h (B) in LnCap cell line.
Bicalutamide antiproliferative effect in LnCap (Fig. 2) and PC-3 (Fig. 3) cells was performed at 48 h (Fig. 2A) and 72 h (Fig. 3A). The data at 24 h are not reported because the effect was unappreciable considering the slow cell growth of each line. In LnCap cells bicalutamide, tested from 1.0 µM to 200 µM, at 48 h and 72 h displayed moderate antiproliferative effect (EC50 = 72.6 ± 2.6 µM and 51.9 ± 6.1 µM, respectively). In the same experimental conditions bicalutamide displayed antiproliferative effect in PC-3 cells (EC50 = 100 ± 5.2 µM and 150 ± 8.6 µM, respectively) lower than that found in LnCap cells. These results reflected bicalutamide different sensitivity of androgen-dependent (LnCap) and androgen-independent (PC-3) cell lines. In order to verify our hypothesis, we employed 10 µM MC18, a potent non selective P-glycoprotein/BCRP/MRP1 inhibitor, in bicalutamide co-administration. Our inhibitor at 10 µM did not display antiproliferative effect in each cell line (data not shown) and at this concentration it blocked the above mentioned ABC transporters. In LnCap cells bicalutamide–MC18 co-administration at 48 h (Fig. 2A) ameliorated bicalutamide antiproliferative effect about 2-fold (EC50 from 72.6 ± 2.6 µM to 42.7 ± 2.4 µM). At 72 h the co-administration (Fig. 2B) ameliorated about 3-fold bicalutamide antiproliferative effect (EC50 from 51.9 ± 6.1 µM to 17.8 ± 2.6 µM). By contrast, in PC-3 cell line at 48 h bicalutamide–MC18 co-administration (Fig. 3A) did not improve antiproliferative effect of the anti-androgen agent. Conversely at 72 h (Fig. 3B) the co-administration increased about 3-fold bicalutamide antiproliferative effect (EC50 from 150 ± 2.4 µM to 60 ± 3.5 µM). This result (EC50 = 60 ± 3.5 µM) is comparable to the effect found in LnCap cells at 48 h and at 72 h. This finding suggested that bicalutamide failure in PC-3 could be due to the presence of one of the three studied pumps.
added in 100 µl of PBS to yield a final concentration of 2.5 µM and plate was incubated for 30 min. Each well was washed 3 times with ice cold PBS. Saline buffer was added to each well and the plate was read to Victor3 at excitation and emission wavelengths of 485 nm and 535 nm, respectively. In these experimental conditions Calcein cell accumulation in the absence and in the presence of tested compounds was evaluated and fluorescence basal level was estimated by untreated cells. In treated wells the increase of fluorescence with respect to basal level was measured. The IC50 values were determined by fitting the fluorescence increase percentage versus log[dose]. 3. Results 3.1. Bicalutamide Apparent Permeability (Papp) In Caco-2 cells experimental model, bicalutamide Apparent Permeability (Papp) has been determined evaluating both the basolateral–apical flux (B→A) and apical–basolateral flux (A→B). In this assay 100 µM bicalutamide was added to apical compartment and, at equilibrium, the drug concentration in basolateral compartment was determined (Papp A→B). In the same experimental conditions bicalutamide was added to basolateral compartment and the drug concentration, at the equilibrium in the apical compartment was measured (Papp B→A). A BA/AB ratio N2 is an indication that the compound could be linked and flipped to extra cellular compartment by P-glycoprotein and/or BCRP and/or MRP1, transmembrane ATPase pumps overexpressed in cancer cells. Bicalutamide displayed Papp A→B = 221 nm/s, Papp B→A = 1193 nm/s and BA/AB ratio was 5.4.
Fig. 3. Bicalutamide antiproliferative effect in the absence and in the presence of MC18 at 48 h (A) and 72 h (B) in PC-3 cell line.
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
Fig. 4. Calcein-AM assay in MDCK-P-gp, MDCK-BCRP and MDCK-MRP1 cell lines.
3.3. Bicalutamide Calcein-AM assay A biological assay to study Multi Drug Resistance proteins activity is monitoring Calcein accumulation in transfected MDCK cells. These cells are incubated with a lipophilic P-glycoprotein/BCRP/MRP1 substrate, AcetoxyMethyl (AM) esters of Calcein (Calcein-AM). This non fluorescent dye diffuses across the plasma membrane into the cell and is hydrolyzed by endogenous cytoplasmic esterases into highly fluorescent Calcein (Fig. 4). This compound is not a Pglycoprotein/BCRP/MRP1 pump substrate and cannot cross the cell membrane via passive diffusion because of its hydrophilicity. Therefore, a rapid fluorescence increase can be monitored. P-glycoprotein/ BCRP/MRP1 pumps, present in the plasma membrane, rapidly efflux Calcein-AM so that it cannot enter the cytosol determining a reduction in the fluorescent signal due to Calcein accumulation. In each transfected cell line, bicalutamide has been tested in order to verify its ability to compete with Calcein-AM for the same binding site. In MDCK-P-glycoprotein cells, as depicted in Fig. 5A (fluorescence decrease versus log[bicalutamide]), bicalutamide influenced Calcein-AM transport (EC50 = 73.5 ± 4.6 µM). Similarly, as reported in Fig. 5B, bicalutamide displayed high affinity towards BCRP CalceinAM site resulting in appreciable fluorescent decrease (EC50 = 31.0 ± 1.6 µM). Conversely, bicalutamide at 200 µM failed to interfere with MRP1 Calcein-AM site.
41
we established the ability of this drug to permeate cell membrane in Caco-2 cell model where P-glycoprotein was highly expressed. The results demonstrated that bicalutamide was transported by Pglycoprotein and this finding encouraged us to deepen the potential Multi Drug Resistance involvement in bicalutamide failure both in LnCap and PC-3 cell lines. For this purpose we verified bicalutamide antiproliferative effect in each cell line employing in bicalutamide coadministration our potent Multi Drug Resistance pumps inhibitor, MC18. The results demonstrated that MC18 restored bicalutamide cell permeation in PC-3 and ameliorated the effect in LnCap with respect to bicalutamide alone. Since MC18 inhibited in the same rank order P-glycoprotein, BCRP and MRP1, we employed cell lines overexpressing each of the above mentioned pumps. The results displayed that bicalutamide is effluxed by P-glycoprotein and BCRP while MRP1 was unable to efflux this ligand. This finding disagreed with the hypothesis that MRP1 was the predominant pump involved in prostate cancer resistance (Sullivan et al., 2000; Zalcberg et al., 2000). Moreover, Ho et al. (2008) demonstrated that ABCC4/MRP4, another pump highly expressed in prostate (Belinsky et al., 1998; Kool et al., 1997) and able to transport typical MRP1 substrates (Chen et al., 2001), was downregulated by bicalutamide treatment in vitro in LnCap cells. By contrast, the results seem to confirm the hypothesis of BCRP involvement in animal model. On the other hand also Pglycoprotein, although reported as undetectable in the studied cell lines, seems to be involved in bicalutamide failure. In conclusion, our results demonstrated that a possible strategy to restore bicalutamide activity in prostate cancer could be its co-administration with BCRP/ P-glycoprotein inhibitors.
4. Discussion In the present study we reported several biological assays in order to support the hypothesis that bicalutamide failure in prostate cancer, after prolonged treatment, is due to Multi Drug Resistance proteins involvement. In the last years, several authors reported that P-glycoprotein both in LnCap and PC-3 was below detection level (Theyer et al., 1993; van Brussel et al., 1999) while MRP1 overexpression could be considered the predominant mechanism of resistance in PC-3 (Sullivan et al., 2000; Zalcberg et al., 2000). Moreover, the effect of BCRP inhibition on the progression of prostate cancer and on the transition to androgen-insensitive prostate cancer has been demonstrated in animal model (Lee et al., 2007). Finally, it was reported that resistance of prostate cancer cells to bicalutamide was determined by Multi Drug Resistance mechanism involving some ABC transporters such as P-glycoprotein, MRP1 and BCRP (van Brussel et al., 1999). In order to better clarify if bicalutamide failure in prostate cancer treatment was due to Multi Drug Resistance, firstly
Fig. 5. Bicalutamide effect on Calcein cell accumulation in MDCK-P-glycoprotein (A) and MDCK-BCRP (B) cell lines.
42
N.A. Colabufo et al. / European Journal of Pharmacology 601 (2008) 38–42
References Belinsky, M.G., Bain, L.J., Balsara, B.B., Testa, J.R., Kruh, G.D., 1998. Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins. J. Natl. Cancer Inst. 90, 1735–1741. Bohl, C.E., Gao, W., Miller, D.D., Bell, C.E., Dalton, J.T., 2005. Structural basis for antagonism and resistance of bicalutamide in prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 6201–6206. Chen, Z.S., Lee, K., Kruh, G.D., 2001. Transport of cyclic nucleotides and estradiol 17-β-Dglucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J. Biol. Chem. 276, 33747–33754. Colabufo, N.A., Berardi, F., Contino, M., Niso, M., Abate, C., Perrone, R., Tortorella, V., 2004. Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumour cell lines. Naunyn-Schmiedeberg's Arch. Pharmacol. 370, 106–113. Colabufo, N.A., Berardi, F., Perrone, R., Rapposelli, S., Digiacomo, M., Balsamo, A., 2006. Arylmethyloxyphenyl derivatives: small molecules displaying P-glycoprotein inhibition. J. Med. Chem. 49, 6607–6613. Colabufo, N.A., Berardi, F., Cantore, M., Perrone, M.G., Contino, M., Inglese, C., Niso, M., Perrone, R., Azzariti, A., Simone, G.M., Porcelli, L., Paradiso, A., 2008a. Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives. Bioorg. Med. Chem. 16, 362–373. Colabufo, N.A., Cantore, M., Contino, M., Inglese, C., Niso, M., Berardi, F., Perrone, R., Cassano, G., Gasparre, G., Azzariti, A., Paradiso, A., submitted for publication. Biological characterization of PB28 and 6,7-dimethoxytetrahydroisoquinoline derivatives, MC18 and MC70, as potent inhibitors of ABC transporters involved in MultiDrug Resistance. Eur. J. Pharmacol. Culig, Z., Klocker, H., Bartsch, G., Hobisch, A., 2001. Androgen receptor mutations in carcinoma of the prostate: significance for endocrine therapy. Am. J. Pharmacogenomics 1, 241–249. Damber, J.E., Aus, G., 2008. Prostate Cancer. Lancet 17, 1710–1721. Elhaji, Y.A., Wu, J.H., Gottlieb, B., Betel, L.K., Alvarado, C., Batist, G., Trifido, M.A., 2004. An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes. Mol. Endocrinol. 18, 1876–1886. Feng, B., Mills, J.B., Davidson, R.E., Mireles, R.J., Janiszewski, J.S., Troutman, M.D., de Morais, S.M., 2008. In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system. Drug Metab. Dispos. 36, 268–275. Gillatt, D., 2006. Antiandrogen treatments in locally advanced prostate cancer: are they all the same? J. Cancer Res. Clin. Oncol. 132, S17–26. Hara, T., Miyazaki, J., Araki, H., Yamaoka, M., Kanzaki, N., Kusaka, M., Miyamoto, M., 2003. Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res. 63, 149–153. Hirawat, S., Budman, D.R., Kreis, W., 2003. The androgen receptor: structure, mutations, and antiandrogens. Cancer Investig. 21, 400–417. Ho, L.L., Kench, J.G., Handelsman, D.J., Scheffer, G.L., Stricker, P.D., Grygiel, J.G., Sutherland, R.L., Henshall, S.M., Allen, J.D., Horvath, L.G., 2008. Androgen regulation of multidrug resistance-associated protein 4 (MRP4/ABCC4) in prostate cancer. Prostate 68, 1421–1429. Hsieh, A.C., Ryan, C.J., 2008. Novel concepts in androgen receptor blockade. Cancer J. 14, 11–14. Iversen, P., Roder, M.A., 2008. The Early Prostate Cancer program: bicalutamide in nonmetastatic prostate cancer. Expert Rev. Anticancer Ther. 8, 361–369.
Kageyama, Y., Hyochi, N., Kihara, K., Sugiyama, H., 2007. The androgen receptor as putative therapeutic target in hormone-refractory prostate cancer. Recent Patents Anticancer Drug Discov. 2, 203–211. Kaighn, M.E., Narayan, K.S., Ohnuki, Y., Lechner, J.F., Jones, L.W., 1979. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest. Urol. 17, 16–23. Kool, M., de Haas, M., Scheffer, G.L., Scheper, R.J., van Eijk, M.J., Juijn, J.A., Baas, F., Borst, P., 1997. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 57, 3537–3547. Lee, D.I., Sumbilla, C., Lee, M., Natesavelalar, C., Klein, M.G., Ross, D.D., Inesi, G., Hussain, A., 2007. Mechanisms of resistance and adaptation to thapsigargin in androgenindependent prostate cancer PC3 and DU145 cells. Arch. Biochem. Biophys. 464, 19–27. Lumbroso, S., Wagschal, A., Bourguet, W., Georget, V., Mazen, I., Servant, N., Audran, F., Sultan, C., Auzou, G., 2004. A new mutation of the androgen receptor, P817A, causing partial androgen insensitivity syndrome: in vitro and structural analysis. J. Mol. Endocrinol. 32, 679–687. McLeod, D.G., 2004. Success and failure of single-modality treatment for early prostate cancer. Rev. Urol. 6, S13–19. Moul, J.W, 2003. Biochemical recurrence of prostate cancer. Curr. Probl. Cancer 27, 243–272. Polli, J.W., Wring, S.A., Humphreys, J.E., Huang, L., Morgan, J.B., Webster, L.O., SerabjitSingh, C.S., 2001. Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 299, 620–628. Schrijvers, D., 2007. Androgen-independent prostate cancer. Recent Results Cancer Res. 175, 239–249. Shand, R.L., Gelmann, E.P., 2006. Molecular biology of prostate-cancer pathogenesis. Curr. Opin. Urol. 16, 123–131. Sullivan, G.F., Yang, J.M., Vassil, A., Yang, J., Bash-Babula, J., Hait, W.N., 2000. Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells. J. Clin. Invest. 105, 1261–1267. Suzuki, H., Ueda, T., Ichikawa, T., Ito, H., 2003. Androgen receptor involvement in the progression of prostate cancer. Endocr. Relat. Cancer 10, 209–216. Theyer, G., Schirmbock, M., Thalhammer, T., Sherwood, E.R., Baumgartner, G., Hamilton, G., 1993. Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines. J. Urol. 150, 1544–1547. Unni, E., Sun, S., Nan, B., McPhaul, M.J., Cheskis, B., Mancini, M.A., Marcelli, M., 2004. Changes in androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to androgen independence. Cancer Res. 1, 7156–7168. van Brussel, J.P., van Steenbrugge, G.J., Romijn, J.C., Schröder, F.H., Mickisch, G.H.J., 1999. Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins. Eur. J. Cancer 35, 664–671. Veldscholte, J., Voorhorst-Ogink, M.M., Bolt-de Vries, J., van Rooij, H.C., Trapman, J., Mulder, E., 1990. Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids. Biochim. Biophys. Acta 1052, 187–194. Zalcberg, J., Hu, X.F., Slater, A., Parisot, J., El-Osta, S., Kantharidis, P., Chou, S.T., Parkin, J.D., 2000. MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines. Prostate Cancer Prostatic Dis. 3, 66–75.