Therese Thalhammer

Role of the MDR-1-Encoded Multiple Drug Resistance Phenotype in Prostate Cancer Cell Lines

The treatment of advanced metastatic prostate cancer by hormone manipulation or orchiectomy is frequently followed by the appearance of hormone-insensitive and highly chemoresistant tumor cells. In this study we have investigated the contribution of the P-glycoprotein-mediated drug efflux (multidrug-resistance; MDR) to the cellular resistance of prostate carcinoma-derived cell lines to diverse cytotoxic drugs by detection of P-glycoprotein (P-gp) measurement of P-gp-mediated drug transport and reversal of MDR by chemosensitizers. The in vitro chemosensitivity of three prostate cancer cell lines (PC-3, DU-145 and LNCaP) to doxorubicin was measured in a thymidine incorporation proliferation assay. Growth of the partially hormone-sensitive cell line LNCaP is inhibited by low doses of doxorubicin (IC50:27 ng./ml.), but PC-3 and DU-145 are highly resistant to the drug, with IC50 values of 10 micrograms./ml. and 7.5 micrograms./ml., respectively. The chemosensitivity of the PC-3 and DU-145 cells is increased in response to 1 microM. verapamil, 1 micrograms./ml. cyclosporine A and 2 microM. tamoxifen, which are known to partially reverse the MDR phenotype in other resistant tumors. A verapamil-sensitive drug efflux has been demonstrated for the PC-3 and Du-145, but not for the LNCaP, cell lines, using flow cytometric measurements of the P-gp substrate rhodamine 123 efflux from preloaded cells. In agreement with the functional measurements, the expression of the P-glycoprotein was detected in the PC-3 and Du-145 cell lines in Western blots using the monoclonal C 219 antibody. In conclusion, the chemoresistant and hormone-insensitive PC-3 and Du-145 cell lines express P-gp and exhibit verapamil-sensitive drug efflux, indicative of MDR. However, the low MDR-reversal rates observed in these cell lines in response to chemosensitizers in clinically achievable concentrations (approximately 2- to 3-fold reversal), point to non-MDR-associated cellular mechanisms as dominant factors of chemoresistance in prostate cancer.

METABOLISM OF THE ANTICANCER DRUG FLAVOPIRIDOL, A NEW INHIBITOR OF CYCLIN DEPENDENT KINASES, IN RAT LIVER

Flavopiridol (FLAP) is a promising novel chemotherapeutic agent currently undergoing clinical phase I trials. To examine hepatic metabolism and biliary disposition of FLAP we applied the isolated perfused rat liver system. Besides FLAP two metabolites were detected by high performance liquid chromatography in bile and perfusate. Twenty-five min after FLAP (30 microM) addition to the perfusion medium, biliary secretion of metabolite 1 and 2 reached a maximum of 1.04 +/- 0.52 and 11.34 +/- 4.72 nmol/g.liver.min, respectively. Biliary excretion of parent FLAP, however, continuously increased for 60 min up to 406 +/- 134 pmol/g liver.min. In the perfusate, metabolite 1 was below detection limit and release of metabolite 2 was low (2.8 +/- 0.7 pmol/g liver.min after 60 min). Enzymatic hydrolysis with beta-glucuronidase, mass spectroscopy and electron absorption spectroscopy revealed that both metabolites are monoglucuronides with the glucuronide in position 5 and 7 of the flavonoid core, respectively. The amount of FLAP, metabolite 1 and metabolite 2 excreted into bile during the 60 min of perfusion was 1.94 +/- 0.91, 5.15 +/- 1.95 and 57.29 +/- 23.60% of FLAP cleared from the perfusate during 60 min, respectively. In contrast to the structurally similar flavonoids genistein and daidzein, no inhibition of UDP-glucuronyltransferase with methylumbelliferone as a substrate was observed indicating that different UDP-glucuronyltransferase isoforms are involved in FLAP metabolism. In conclusion, we find that glucuronidation is the major mechanism of hepatic FLAP biotransformation. Metabolites are mainly excreted into bile but also released into systemic circulation. The pharmacological and toxicological effects of these metabolites remain to be elucidated.

Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R>1,400 Ω cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitisers inhibited drug flux (137 pmol h−1 cm−2; range, 133–142 pmol h−1 cm−2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 μg/ml (0.9 μM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 μM; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhdamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of r-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation. Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly. As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.

Angiogenesis stimulation in explanted hearts from patients pre-treated with intravenous prostaglandin E1

BACKGROUND Prostaglandin E(1) (PGE(1)) is a potent vasodilator and induces angiogenesis in animal tissues. Previous clinical studies demonstrated that PGE(1) improves hemodynamic parameters in patients with heart failure listed for heart transplantation (HTX). Therefore, we designed a retrospective immunohistochemistry study to investigate various markers of angiogenesis using hearts explanted from PGE(1)-treated patients with idiopathic dilated cardiomyopathy (IDCM). METHODS AND RESULTS We investigated neovascularization in 18 hearts explanted from patients with IDCM: 9 patients received treatment with chronic infusions of PGE(1) for end-stage heart failure before HTX, whereas the remaining patients with IDCM did not receive PGE(1) and served as controls. We used immunoreactivity against CD34, von Willebrand factor (vWf), vascular endothelial growth factor (VEGF), and MIB-1 (Ki-67) to quantify angiogenesis, and used sirius red staining to determine the degree of fibrosis. Compared with the control group, PGE(1)-treated patients had significantly more CD34-, vWf- and MIB-1-positive cells in the sub-endocardium, myocardium and sub-epicardium (p < 0.01). The degree of fibrosis in the hearts of PGE(1)-treated patients was significantly lower than in control patients (p < 0.05), but we did not see any difference in the percentage of muscle mass. Finally, throughout the ventricles, we found significantly more VEGF-positive capillaries in the PGE(1) group (p < 0.0001). CONCLUSIONS The data suggest that PGE(1) could be a potent inducer of angiogenesis and the angiogenic factor VEGF, and could cause reduced fibrosis in the failing human heart.

Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport

This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

Metabolism and disposition of the novel antileukaemic drug, benzamide riboside, in the isolated perfused rat liver.

Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cells, however, little is known about its biotransformation. To analyze for BR and its metabolites, livers of Wistar and mutant TR- rats were perfused with BR in a single pass system. In bile, native BR and its deamination product, benzene carboxylic acid riboside (BR-COOH) was quantified by HPLC. Total excretion of BR and BR-COOH into bile of Wistar rats was low (< 0.2%) whereas cumulative efflux of BR and its metabolite BR-COOH was high, representing 79% and 1.6% of infused BR, respectively. Biliary excretion of BR and BR-COOH in TR- rats, deficient in canalicular multispecific organic anion transporter, a membrane protein identical to MRP2 in tumor cells, was only slightly lower than in Wistar rats, indicating that BR and BR-COOH are non-substrates of MRP2. Experiments using rat hepatocytes incubated with BR did show a linear uptake of BR and a subsequent metabolism to BR-COOH that was largely excreted into the cellular supernatant. Examination of the cytotoxic activity against the human HL60 and K562 leukemia cells in a clonogenic assay demonstrated an IC50 of 619 microM and 1013 microM for BR-COOH compared to the IC50 of 0.21 microM and 0.46 microM for BR, suggesting the inertness of the metabolite. In summary, we found that deamination of BR to BR-COOH is the main metabolic pathway in rat liver. BR-COOH formation should also be considered in human liver during cancer therapy.

Metabolism of roxithromycin in the isolated perfused rat liver.

Roxithromycin is a macrolide antibiotic with high clinical potency. N‐Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver.

Die Hemmung des P-Glykoprotein-mediierten, transepithelialen Zytostatikatransportes durch R-Verapamil, Cyclosporin SDZ PSC-833 und Tamoxifen in einem Adenokarzinom-Monolayer-Modell

ZusammenfassungGrundlagenDie Multidrug-Resistenz (MDR) gilt als wichtiger Chemoresistenzmechanismus besonders bei hämatologischen, aber auch bei soliden Malignomen. Inhibitoren des MDR-Effektors P-Glycoprotein (P-GP), Chemomodulatoren genannt, zeigten für den Fall solider Tumoren aufgrund nebenwirkungsbedingter Einschränkungen der erreichbaren Konzentrationen bisher noch keine klinischen Erfolge. Mit der Fragestellung, ob und wie weit Chemomodulatoren den P-Gpmediierten Zytostatikatransport in intestinalen Karzinomzellen hemmen, wurden neben dem Antiöstrogen Tamoxifen (TMX) die speziell für die MDR-Reversion entwickelten Chemomodulatoren, nämlich das weniger kardiotoxische R-Enantiomer von Verapamil (R-VPL) und das nichtimmunsuppressive Cyclosporin SDZ PSC-833 (PSC), in vitro getestet.MethodikUnter Verwendung von HCT-8 intestinalen Adenokarzinomzell-Monolayem untersuchten wir den P-Gp-mediierten, transmembranen, sekretorischen Transport von Vinblastin, einem P-Gp-Substrat, sowie den zellassoziierten Vinblastingehalt in An- oder Abwesenheit obengenannter Chemomodulatoren unter verschiedenen extrazellulären pH-(pHo-)Bedingungen, wie sie in soliden Tumoren vorkommen. Zusätzlich führten wir durchflußzytometrische Effluxmessungen mit dem P-Gp-Substrat Rhodamin 123 unter denselben extrazellulären Bedingungen durch. Das Ausmaß der durch die Chemomodulatoren bedingten Resistenzreversion wurde anhand von Chemosensitivitätsassays (Inkorporation von3H-Thymidin) mit HCT-8 Einzelzellsuspensionen gezeigt.ErgebnisseDie Modulatoren R-VPL, PSC und TMX inhibierten den transepithelialen VIN-Transport in einem Ausmaß von bis zu 50,55 bzw. 30%, der zellassoziierte Vinblastingehalt war nach 5h im Vergleich zur Kontrolle 3,7-, 2,3- bzw. 1.7mal größer. Im Gegensatz zu PSC und TMX stellten wir bei Verwendung von R-VPL einen 32-bzw. 47%igen Verlust der modulierenden Wirkung bei extrazellulärer Ansäuerung (pHo 7,0 bzw. 6,8) fest. Damit übereinstimmende Ergebnisse erzielten wir auch bei den durchflußzytometrischen Studien unter gleichen extrazellulären Bedingungen. Die Chemosensitivitätsassays ergaben einen 8-, 5.3- und 10,7fachen Anstieg der VIN-Toxizität durch R-VPL (10 μM), PSC (100 ng/ml) bzw. TMX (10 μM).SchlußfolgerungenMit TMX konnte trotz schwacher P-Gp-Inhibition eine deutliche Resistenzreversion bewirkt werden, was darauf hindeutet, daß zusätzliche Mechanismen an der Resistenz der Zellinie beteiligt sind, die durch TMX, unabhängig von P-Gp-Interaktionen, beeinflußbar sind. Der festgestellte Wirkungsverlust von R-VPL unter sauren Bedingungen könnte als Erklärung der vorherrschend negativen Ergebnisse klinischer Studien mit Verapamil bei Trägern solider Tumoren dienen. Sofern MDR wesentlich zum fehlenden Ansprechen solider Tumoren auf Chemotherapie beiträgt, würden wir klinische Erfolge mit PSC erwarten, das sich bei geringer klinischer Toxizität durch ein hohes vom pHo weitgehend unabhängiges P-Gp-Inhibitions-potential auszeichnet.SummaryBackgroundMultidrug resistance (MDR) is known to be an important mechanism of chemoresistance especially in hematologic and presumably in solid malignoma. Inhibitors of MDR-mediating P-glycoprotein (P-Gp) called chemomodulators. however, did not cause significant reversal of resistance in clinical trials so far. Main problems consisted in achieving sufficient concentrations and adverse side effects of chemomodulators. We tested chemomodulators especially developed for MDR reversal, namely the less cardiotoxic R-enantiomer of verapamil (R-VPL) and the non-immunosuppressive cyclosporine SDZPSC-833 (PSC), and further the antiestrogen tamoxifen (TMX) with respect to the inhibition of P-Gp-mediated transport of cytostatics in polarized intestinal carcinoma cells in vitro.MethodsUsing the HCT-8 intestinal adenocarcinoma cell monolayer model we assessed P-Gp-mediated, transmembrane, secretory transport of vinblastine (3H-VIN), a P-Gp substrate, as well as the cell-associated VIN-content in presence/absence of above chemomodulators under different extracellular pH (pHo) conditions known to prevail in carcinoma. Additionally we performed flow cytometric efflux experiments with the P-Gp substrate rhodamine 123 under same extracellular conditions. Reversion of resistance due to chemomodulators is shown by chemosensitivity assays (incorporation of3H-thymidine) of HCT-8 suspension cells.ResultsCompared to controls R-VPL (10 μM), PSC (100 ng/ml) and TMX (10 μM) inhibited3H-VIN transport up to 50, 55 and 30%, respectively and cell associated drug content was increased about 3.7, 2.3 and 1.7 times, respectively. In contrast to PSC and TMX effect we observed a significant 32 and 47% loss of chemomodulating capacity of R-VPL at pHo 7.0 and 6.8 when compared with the inhibition at pHo 7.5. Corresponding results were gathered from flow cytometry. Chemosensitivity assays showed an 8-, 5.3- and 10.7-fold increase of VIN-cytotoxity due to R-VPL, PSC and TMX, respectively.ConclusionsTMX displayed high overall resistance reversion, despite of low specific inhibition of P-Gp-mediated fluxes indicating further resistance mechanisms in HCT-8 cells revertable by TMX independent of its interaction with P-Gp. Our results could serve possible explanation for the predominately negative results of clinical studies with verapamil as MDR revertant in patients with resistant solid tumors because of only partial P-Gp inhibition at clinically achievable concentrations and significant loss of efficacy under acidic pHo-conditions. Because of high and pHo-independent capacity of P-Gp-inhibition PSC seems to be the most suitable chemomodulator as far as MDR contributes to the overall unresponsiveness to chemotherapy in solid tumours.

Hepatobiliary transport of the anionic organomercury compound (mersalyl) is carrier mediated

The hepatobiliary excretion of the anionic organic mercury compound (mersalyl) was studied in the isolated perfused rat liver and in isolated rat liver plasma membrane vesicles. In the isolated perfused liver, mersalyl is immediately taken up from the perfusion medium and concentratively excreted into bile. Uptake is characterized by saturation kinetics (S)0.5 = 20 microM, Vmax = 117 nmoles/min/g liver, cooperatively of mersalyl binding sites, stimulation by extracellular sodium and temperature dependence. Uptake of mersalyl into basolateral membrane vesicles also exhibits characteristics of a carrier mediated transport: saturation kinetics (S)0.5 = 28 microM, Vmax = 1.6 nmoles/min/mg protein, dependence on extravesicular sodium, cooperativity of mersalyl binding sites, temperature dependence and transstimulation by intravesicular non-radioactive mersalyl. Uptake was inhibited by alpha-naphthylacetic acid and mercapto group reagents, indicating involvement of mercapto groups on the carrier and a binding site for carboxylic anions. Data from the isolated perfused liver and from isolated basolateral vesicles indicate that mersalyl uptake into the liver is carrier mediated. Uptake mechanism and driving forces appear analogous to those for the uptake of chemically related compounds such as taurocholic acid. Therefore it is speculated that mersalyl may be transported by carrier molecules which apparently accept numerous chemically unrelated compounds.

Simultaneous determination of the new anticancer agent amidox and its metabolites in rat bile and plasma by high-performance liquid chromatography

A new reversed-phase ion-pair high-performance liquid chromatography method was developed to study the first-pass hepatic metabolism of the anti cancer drug amidox in bile. Separation of the metabolites was achieved on a Spherisorb C18 column after liquid-liquid extraction using a linear gradient system of heptanesulfonic acid in potassium phosphate monobasic (pH 4.0) with increasing amounts of methanol (0-40%). The method was further applied to a pharmacokinetic study of amidox in rats after 200 mg kg-1 intraperitoneal administration. Using 50 microliters of rat bile and 300 microliters of rat plasma the limit of detection for amidox was 60 ng and 85 ng, respectively, and the assay was linear from 0.1 to 150 micrograms ml-1. This method appears to be sensitive enough to be used in further pharmacokinetic studies of amidox in human volunteers.