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Grundlagen Die Multidrug-Resistenz (MDR) gilt als wichtiger Chemoresistenzmechanismus besonders bei hamatologischen, aber auch bei soliden Malignomen. Inhibitoren des MDR-Effektors P-Glycoprotein (P-GP), Chemomodulatoren genannt, zeigten fur den Fall solider Tumoren aufgrund nebenwirkungsbedingter Einschrankungen 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 Antiostrogen Tamoxifen (TMX) die speziell fur die MDR-Reversion entwickelten Chemomodulatoren, namlich das weniger kardiotoxische R-Enantiomer von Verapamil (R-VPL) und das nichtimmunsuppressive Cyclosporin SDZ PSC-833 (PSC), in vitro getestet. Methodik Unter 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 extrazellularen pH-(pHo-)Bedingungen, wie sie in soliden Tumoren vorkommen. Zusatzlich fuhrten wir durchfluszytometrische Effluxmessungen mit dem P-Gp-Substrat Rhodamin 123 unter denselben extrazellularen Bedingungen durch. Das Ausmas der durch die Chemomodulatoren bedingten Resistenzreversion wurde anhand von Chemosensitivitatsassays (Inkorporation von3H-Thymidin) mit HCT-8 Einzelzellsuspensionen gezeigt. Ergebnisse Die Modulatoren R-VPL, PSC und TMX inhibierten den transepithelialen VIN-Transport in einem Ausmas von bis zu 50,55 bzw. 30%, der zellassoziierte Vinblastingehalt war nach 5h im Vergleich zur Kontrolle 3,7-, 2,3- bzw. 1.7mal groser. Im Gegensatz zu PSC und TMX stellten wir bei Verwendung von R-VPL einen 32-bzw. 47%igen Verlust der modulierenden Wirkung bei extrazellularer Ansauerung (pHo 7,0 bzw. 6,8) fest. Damit ubereinstimmende Ergebnisse erzielten wir auch bei den durchfluszytometrischen Studien unter gleichen extrazellularen Bedingungen. Die Chemosensitivitatsassays ergaben einen 8-, 5.3- und 10,7fachen Anstieg der VIN-Toxizitat durch R-VPL (10 μM), PSC (100 ng/ml) bzw. TMX (10 μM). Schlusfolgerungen Mit TMX konnte trotz schwacher P-Gp-Inhibition eine deutliche Resistenzreversion bewirkt werden, was darauf hindeutet, das zusatzliche Mechanismen an der Resistenz der Zellinie beteiligt sind, die durch TMX, unabhangig von P-Gp-Interaktionen, beeinflusbar sind. Der festgestellte Wirkungsverlust von R-VPL unter sauren Bedingungen konnte als Erklarung der vorherrschend negativen Ergebnisse klinischer Studien mit Verapamil bei Tragern solider Tumoren dienen. Sofern MDR wesentlich zum fehlenden Ansprechen solider Tumoren auf Chemotherapie beitragt, wurden wir klinische Erfolge mit PSC erwarten, das sich bei geringer klinischer Toxizitat durch ein hohes vom pHo weitgehend unabhangiges P-Gp-Inhibitions-potential auszeichnet.

Cytokeratin 18 (CK18) and Caspase-Cleaved CK18 (ccCK18) as Response Markers in Anticancer Therapy

Grundlagen: Resistenzmechanismen auf Tumorebene und zellularer Art sind fur die niedrigen Ansprechraten und Uberlebenszeiten nach Chemotherapie, insbesondere bei den meisten soliden Tumoren, hauptverantwortlich. Die sogenannte Multidrug-Resistenz (MDR) wird durch ein Transmembranprotein (P-Gp) bewirkt, das klinisch wichtige Zytostatika aus dem Zellinneren transportiert und in hamatologischen und soliden Tumoren nachgewiesen wurde. P-Gp wird im Normalgewebe in Epithelien der Nierentubuli, der Leber, der Nebenniere, der Blut-Hirnschranke und andernorts exprimiert. Es war das Ziel der vorliegenden Arbeit, die Funktion des P-Gp und seine Inhibierbarkeit durch Chemomodulatoren, im Gegensatz zu den normalerweise untersuchten Suspensionszellen, in polarisierter, apikaler Expression eines Epithelmodells zu erfassen. Als Zellsystem wurden Monolayerkulturen der humanen HCT-8-Adenokarzinomlinie auf porosen Gewebekulturinserts etabliert.

Hintergrund: Solide Tumoren weisen azidotische Regionen mit durchschnittlichen extrazellularen pH Werten (pHo) von 6,9 bis 7,0 (Normalgewebe: 7,4 bis 7,5) auf. In humanen SW620-Kolonkarzinomzellen konnten in einer fruheren Arbeit ein Na/H-Austauscher sowie ein Na/HCO3-Kotransporter als Saureextrusoren nachgewiesen werden, ein Cl/HCO3-Austauscher wurde im Gegensatz zu anderen Tumorlinien nicht gefunden. Diese Studie untersucht die Auswirkungen unterschiedlicher extrazellularer pH-Bedingungen auf die intrazellulare pH-Regulation am Modell der SW620-Kolonkarzinomzellen, durch Variation des CO2-Partialdrucks und der HCO3-Konzentration der Inkubationslosungen.

In order to investigate the regulation of intracellular pH (pHi) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37 degrees C). In N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic-acid-(HEPES)-buffered Ringer solution (HBS) a baseline pHi of 7.35 +/- 0.03 (mean +/- SD; n = 42) was found for human colonocytes and in HBS, NH4Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na+/H+ exchanger. Switching from HBS to HCO(3-)-buffered solution (BBS) led to a transient intracellular acification (7.29 +/- 0.09), followed by a recovery to a final resting pHi of 7.43 +/- 0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (H2DIDS)-sensitive HCO(3-)-dependent mechanisms. The functional activity of an acid-extruding Na+/HCO3- cotransporter in human colonocytes was observed in response to the reintroduction of Na+ into amiloride-containing Na+/Cl(-)-free BBS. In addition, the mechanism leading to alkalinization (7.56 +/- 0.05) in Cl(-)-free BBS was identified as Na(+)-dependent Cl-/HCO3- exchange, by its H2DIDS sensitivity and the specific requirement for Cl- and Na+. The intrinsic buffering capacity (beta i) of the human colonocytes was calculated from pH changes induced by sequential NH4Cl-loading steps during blockage of acid/base transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (R1,400 omega cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitizers 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 microgram/ml (0.9 microM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 microM; 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 rhodamine 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.

Prostate cancer is among the most common types of malignancies and causes of cancerrelated deaths in men worldwide (Fitzpatrick, et al., 2009). Primary tumor involvement outside the prostatic capsule or relapse following radical prostatectomy results generally in incurability (Lassi & Dawson, 2009). Androgen deprivation therapy has progressed since attempts in 1941, when surgical castration had been shown to improve outcomes for the first time. Palliative treatment consists of hormonal manipulation to deprive the cancer cells of androgenic stimulation by orchidectomy or use of LHRH analogs and steroidal or nonsteroidal antiandrogens (Kollmeier & Zelefsky, 2008). Although continuous androgen suppression therapy (CAS) has been a cornerstone of the management of prostate cancer for more than 50 years, controversy remains regarding its optimum application. Generally, androgen suppression (AS) is performed as continuous treatment, resulting in apoptotic regression of the tumor cells in a high percentage of cases. However, surgical or medical castration results in median progression-free survival of only 2–3 years, with no other effective treatment left (Mellado, et al., 2009). Responses to cytotoxic therapy are low and only recently several studies revealed a possible benefit of incorporating chemotherapeutic agents in treatment regimen for prostate cancer (Chang & Kibel, 2009). Taxanes like docetaxel and cabazitaxel, therapeutic cancer vaccines and newly developed agents targeting androgen receptor signaling are expected to improve therapy (Madan et al., 2011). Following experimental research using animal models, intermittent androgen suppression (IAS) was introduced as new clinical concept, assuming that during limited regrowth in the treatment cessation periods tumorigenic cells are residing in an androgen-responsive state (Goldenberg, et al., 1995). Since induction of androgen independence may occur early after treatment initiation, cessation of antiandrogen therapy prior to this switch is expected to maintain the apoptotic potential of the tumor cells and keep them sensitive to retreatment. Providing an easy method for selection of the type of treatment and early assessment of tumor growth during the off-periods serial serum PSA determinations made IAS feasible. In detail, IAS consists of an initial androgen suppression period of up to nine months combining LHRH antagonists and antiandrogens, which is followed by treatment cessation

1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3-bis{3-[1(4-butylpiperazinyl)]-propyl}-disiloxan-tetrahydrochlorid (SILA 421) is a compound that was developed as modulator of the ABC cassette transporter P-glycoprotein. Furthermore, it exerted antimicrobial toxicity, vascular effects, downregulation of chaperone induction and plasmid curing in bacterial cells. Here, this drug was found to possess cytotoxic activity against a panel of human cancer cell lines that do not overexpress P-gp, with 50% inhibitory concentrations ranging between 1.75±0.38 μM for GLC14 small cell lung cancer and 34.00±4.75 μM for PC-3 prostate cancer cells. HL-60 leukemia and MDA-MB-435 breast cancer cells exhibited cell cycle arrest and apoptotic cell death in response to SILA 421. Assessment of global gene expression of SILA 421-treated HL-60 cells was employed to identify cellular pathways affected by the compound and revealed disturbance of DNA replication, transcription and production of apparently misfolded proteins. Endoplasmatic reticulum stress and downregulation of cell cycle, cellular repair mechanisms and growth factor-related signaling cascades eventually resulted in induction of apoptosis in this cell line. In addition to the well established P-gp inhibitory effect of SILA compounds, reversal of resistance to taxanes, which had been reported for SILA 421 and the related molecule SILA 409, may be linked to downregulation of gene expression of kinesins. Interference with DNA replication and transcription seems to be the common denominator of antimicrobial activity and plasmid curing, as well as anticancer toxicity in human cell lines. Thus, in consideration of the full range of putative cellular targets found in the present work, the application of these SILA compounds for treatment of tumors should be further evaluated.

Ulrike Olszewski, Ernst Ulsperger, Klaus Geissler, Gerhard HamiltonLudwig Boltzmann Institute of Clinical Oncology and Photodynamic Therapy, Ludwig Boltzmann Cluster of Translational Oncology, Vienna, AustriaAbstract: Platinum(IV) coordination complexes like oxoplatin (cis,cis,trans-diammine-dichlorido-dihydroxido-platinum[IV]) show high stability and therefore can be utilized orally for outpatient care. Although oxoplatin is capable of binding directly to DNA after prolonged incubation, platinum(IV) agents are considered to be largely inert prodrugs that are converted to highly cytotoxic platinum(II) compounds by reducing substances, enzymes, or microenvironmental conditions. Reaction of oxoplatin with 0.1 M hydrogen chloride mimicking gastric acid yields cis-diammine-tetrachlorido-platinum(IV) (DATCP[IV]), which exhibits two-fold increased activity. The presence of chlorides as ligands in the axial position results in a high reduction potential that favors transformation to platinum(II) complexes. In this study, the intracellular effect of the highly reactive tetrachlorido derivative was investigated in comparison with an equipotent dose of cisplatin. Genome-wide expression profiling of NCI-H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes and concerned cellular pathways between DATCP(IV) and cisplatin. Application of DATCP(IV) resulted in extensive downregulation of protein and ATP synthesis, cell cycle regulation, and glycolysis, in contrast to cisplatin, which preferentially targeted glutathione conjugation, pyruvate metabolism, citric acid cycle, and the metabolism of amino acids and a range of carbohydrates. Thus, the oxoplatin metabolite DATCP(IV) constitutes a potent cytotoxic derivative that may be produced by gastric acid or acidic areas prevailing in larger solid tumors, depending on the respective pharmaceutical formulation of oxoplatin. Furthermore, DATCP(IV) exhibits intracellular effects that are clearly different from the expected reduced product cisplatin(II). In conclusion, activation of the platinum(IV) complex oxoplatin seems to involve the generation of a cytotoxic six-coordinate species, dependent on prevailing conditions, and its effects need to be considered in addition to the effects of the potential final platinum(II) product.Keywords: platinum, oxoplatin, metabolites, small cell lung cancer, cell line, gene expression, microarray

Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients.

Chemotherapy for advanced well-differentiated carcinoids is characterised by low response rates and short duration of responses. The present study aimed to assess the in vitro activity of novel platinum-based chemotherapeutic drugs in combination with dichloroacetate (DCA), a sensitiser to apoptosis, against lung carcinoid cell lines. Three permanent cell lines (UMC-11, H727 and H835) were exposed to 14 different established cytotoxic drugs and the novel platinum-based compounds as satraplatin, JM118 and picoplatin in combination with DCA, and viability of the cells was measured using a tetrazoliumbased dye assay. With exception of the highly chemoresistant UMC-11 line, the carcinoid cell lines (H727, H835) were sensitive to the majority of chemotherapeutics in vitro. Among the platinum-based drugs, carboplatin and oxaliplatin showed highest efficacy. H835 cells growing as multicellular spheroids were 2.7–8.7-fold more resistant to picoplatin, satraplatin and its metabolite compared to single cell suspensions. DCA (10 mM) inhibited the growth of UMC-11 cells by 22% and sensitised these highly resistant cells to carboplatin, satraplatin and JM118 1.4–2.4-fold. The highly resistant UMC-11 lung carcinoid cells are sensitive to carboplatin, oxaliplatin and the satraplatin metabolite JM118, but multicellular spheroidal growth, as observed in the H835 cell line and pulmonary tumourlets, seems to increase chemoresistance markedly. The activity of carboplatin and JM118 is significantly and specifically increased in combination with the apoptosis sensitiser DCA that promotes mitochondrial respiration over aerobic glycolysis. In summary, among the novel platinum drugs satraplatin has the potential for treatment of lung carcinoids and DCA potentiates the cytotoxicity of selected platinum drugs.

Pancreatic adenocarcinoma is a devastating disease characterized by early dissemination and poor prognosis. These solid tumors express receptors for neuropeptides like neurotensin (NT) or epidermal growth factor (EGF) and exhibit acidic regions when grown beyond a certain size. We previously demonstrated increases in intracellular Ca2+ levels, intracellular pH and interleukin-8 (IL-8) secretion in BxPC-3 and PANC-1 pancreatic cancer cells in response to a stable NT analog. The present study aimed at investigation of the dependence of the relative expression of NT receptor 1 (NTR1) and EGFR in BxPC-3 and MIA PaCa-2 cells on cell density and extracellular pH (pHe). MTT assays revealed the NTR1 inhibitor SR 142948-sensitive Lys8-ψ-Lys9NT (8–13)-induced proliferation in BxPC-3 and PANC-1 cells. Confluent cultures of BxPC3 and HT-29 lines exhibited highest expression of NTR1 and lowest of EGFR and expression of NTR1 was maximal at slightly acidic pHe. IL-8 production was stimulated by Lys8-ψ-Lys9NT (8–13) and even enhanced at both acidic and alkaline pHe in BxPC-3 and PANC-1 cells. In conclusion, our in vitro study suggests that one contributing factor to the minor responses obtained with EGFR-directed therapy may be downregulation of this receptor in tumor cell aggregates, possibly resulting in acquisition of a more aggressive phenotype via other growth factor receptors like NTR1.

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.

In human orthotopic liver transplantation (LTX) intraoperative elevations of TNF-α (> 100 pg/ml) and IL-6 (>800 pg/ml) have been found to correlate with early post-operative rejections and infections respectively. In this study the possible mechanism responsible for the induction of these cytokines has been investigated during liver allografting in 38 recipients. Intraoperative elevations of TNF-α (> 100 pg/ml) were detected in the majority of pre-transplant endotoxin positive recipients (8/12, > 10 endotoxin units/ml), the patients turning endotoxin positive until the end of grafting (3/5), and in a subgroup (6/21 patients), apparently endotoxin negative for the whole operation. Therefore endotoxin (ET) seems to stimulate release of TNF-α in approximately 50% of the patients, whereas sensitized Kupffer graft cells or immediate allograft reactivity of the host are likely to account for the remaining TNF-α positive cases. Elevations of IL-6 > 800 pg/ml) were found in approximately 50% of the TNF-α positive cases, indicating partially independent regulatory pathways for IL-6 induction in the TNF-α negative patients. In agreement with a previous study, 11/13 (85%) of the intraoperative TNF-α positive recipients rejected their grafts within the first 10 days post-operatively. These data demonstrate that ET/infection associated as well as ET independent/reperfusion associated intraoperative TNF-α elevations, promote the initiation of allograft rejection in human liver transplantation. The transient and low endotoxaemia caused by the liver grafting procedure performed without veno-venous bypass seems to be of minor importance in the intraoperative induction of TNF-α.

Acidosis commonly observed in solid tumors like pancreatic cancer promotes genetic instability and selection of a more malignant phenotype of cancer cells. Overexpression or activation of integral membrane proteins mediating H+ efflux may contribute to extracellular acidification. Neurotensin (NT) induces intracellular alkalinization and stimulates interleukin-8 production in pancreatic cancer cells and, as demonstrated here, the stable NT analog Lys(8)-psi-Lys(9)NT(8-13) enhances the amiloride-sensitive, Na+-dependent transmembrane H+ flux by a factor of 2.05+/-0.28 and 2.69+/-0.07 in BxPC-3 and PANC-1 pancreatic cancer cells, respectively, by phosphorylation of the Na+/H+ exchanger 1 (NHE1). Human genome-wide gene expression analysis was performed to detect effects of Lys(8)-psi-Lys(9)NT(8-13) on BxPC-3 cells. Results indicated upregulation of genes involved in regulation of NHE1, hypoxic response and glycolysis in response to Lys(8)-psi-Lys(9)NT(8-13) even under normoxic conditions. Therefore, our findings suggest that growth factors like NT may be implicated in the early progression of pancreatic cancer by localized acidification and induction of an aerobic glycolytic phenotype with higher metastatic potential in small cell aggregates.

IMPORTANCE IN THE FIELD: Primary bone tumors are considered as (sex steroid) hormone-dependent tumors. Osteosarcoma, osteoblastoma and bone cysts are preferentially found in males, while giant cell tumors are more common in females. Indeed, bone tumor development and progression are influenced by sex steroid hormones derived from in situ synthesis in bone cells.This review describes intracrine mechanisms for local formation of the biologically most active estrogen, 17beta-estradiol (E2), from circulating steroid precursors through the 'aromatase' (aromatization of androgens) and the 'sulfatase' (conversion of inactive estrone-sulfate) pathway.The reader gains knowledge on both pathways and the enzymes, which contribute to the in situ availability of active hormones, namely 3beta-hydroxysteroid dehydrogenases, 17beta-hydroxysteroid dehydrogenases, aromatase, steroid sulfatases and sulfotransferases. An overview is given and the expression and function of these enzymes in bone tumors are discussed.Knowledge on pathways for the in situ formation of E2 in bone cells may allow the identification of potential targets for i) novel endocrine therapeutic options in primary bone tumors and ii) future preventive interventions.

In attempts to overcome the drawbacks of cisplatin – severe toxicity, drug resistance and poor oral bioavailability – the development of platinum-based drugs has progressed from carboplatin and oxaliplatin to the newest generation of drugs, such as satraplatin, picoplatin and the multinuclear platinum complex BBR3464 (triplatin). Despite encouraging preclinical in vitro and in vivo results, outcomes of clinical trials of these coordination complexes remained below expectations. Biased rationale underlying the drug design along with in vitro and in vivo preclinical tests with inadequate predictive power seem to have eventually resulted in the selection of drug candidates of limited clinical activity. The nature of the active species generated in vivo, uptake, efflux, intracellular trafficking and detailed mechanisms involved in chemoresistance of platinum drugs in vivo are topics that need further investigation to provide clues for the rational formulation of new drugs. Insufficient diffusion in tumor tissues is likely to constitute an important limiting step in the treatment of solid tumors with platinum compounds. Preclinical assays with improved predictive power for the clinical outcome of the compounds should be based on more representative tumor models, such as resistant primary cancer cell lines, spheroids and orthotopic xenograft models, respectively. Finally, use of new pharmaceutical formulations and bifunctional complexes, as well as their selection by decisive preclinical tests, are expected to result in the generation of platinum-based anticancer drugs with the potential to achieve clinical activity even in multidrug-resistant tumors.

Im Verlauf einer gramnegativen abdominellen Sepsis wurden prospektiv bei 18 Patienten Endotoxin, TNFa und IL-6 im Plasma gemessen und mit dem Uberleben sowie der Lungen- und Leberfunktion korreliert. Wahrend Endotoxin (median 12 vs. 9 EU/ml) und TNFa (12 vs. 21 pg/ml) bei Uberlebenden und Toten nicht unterschiedlich waren, fand sich eine signifikante Erhohung von IL-6 bei letalem Verlauf (82 vs. 171 pg/ml, p=0,02). An Tagen mit Lungenversagen war IL-6 signifikant erhoht (70 vs. 159 pg/ml, p=0,009). Endotoxin (11 vs. 13 EU/ml) und TNFa (12 vs. 15 pg/ml) waren bei Patienten mit und ohne respiratorischer Insuffizienz nicht different. Ebenso war IL-6 bei Leberversagen signifikant hoher (349 vs. 69 pg/ml, p=0,0000), wahrend fur Endotoxin (11 vs. 13 EU/ml) und TNFa (33 vs. 17 pg/ml) kein Unterschied nachgewiesen werden konnte. Bei gramnegativer abdomineller Sepsis fand sich eine Korrelation von IL-6 mit Uberleben, Lungen- und Leberfunktion, wahrend Endotoxin und TNFa in diesen Parametern nicht signifikant waren.

The correlation of endotoxin (ET), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and cellular immune parameters with multiple organ failure and lethal outcome in intraabdominal infections was studied in a group of 18 patients with peritonitis, abscess or pancreatitis. Of these patients, 7 developed respiratory failure and 5 died due to multiple septic organ failure. The peak levels of ET (2.7 +/- 1.3 ng/ml) in the course of the disease were followed by moderate increases of TNF-alpha (mean 147 +/- 41 pg/ml) and IL-6 (170 +/- 61 pg/ml) within 2 days. Analysis of the parameters for the last 12 days prior to death or discharge showed, that the patient group with lethal outcome was characterized by significant lower mean plasma levels of TNF-alpha (less than 75 pg/ml versus greater than 160 pg/ml) and IL-6 (less than 130 pg/ml versus greater than 270 pg/ml), as well as high rates of unstimulated thymidine uptake into peripheral mononuclear blood cells (greater than 44000 cpm/8 x 10(6) PMBC/18 h versus less than 24000 cmp), T-lymphocyte depression (CD3; approximately greater than 40% reduction) with lower T-helper/inducer subset cell numbers (mean CD:CD8 ratio 1.0 +/- 0.55 versus 1.8 +/- 0.2) and lower lectin (PHA) stimulation values (1.9 +/- 1.4 versus 4.1 +/- 1.0). These data demonstrate an anergic immune status with low mediator levels and depressed T-lymphocyte function in patients with poor prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

In cancer patients detection of systemic disease is of great importance to obtain prognostic information and to guide therapy. Bone marrow (BM) seems to be a common homing tissue for the early spread of tumor cells from various epithelial tumors; however, verification of the prognostic significance of BM-disseminated tumor cells (BM-DTCs), is restricted to breast cancer so far. These cells may be dormant for a long time, and signals triggering their activation leading to recurrence remain to be characterized. A recent study involving metastatic breast cancer patients reported that the shortest disease-free survival is correlated with cytokeratin (CK)-negative BM aspirates and that CK-positive BM-DTCs correspond to dormant tumor cells. Soluble CK fragments in serum including CK18 and 19 (measured as TPS and CYFRA 21-1, respectively) and caspase-cleaved CK18 are widely used to monitor tumor progression and response to therapy, actually indicating proliferation and/or necrotic/apoptotic cell death. In order to assess the source of the CK fragments, we used determinations of CK18 and caspase-cleaved CK18 fragments in serum samples before and after radical tumor surgery in colon cancer patients. Elevated serum concentrations of CK18 were found to persist in patients with a high incidence of BM-DTCs, and high perioperative levels of caspase-cleaved CK18 fragments were detected in patients with early relapses, respectively. These results indicate that in some patients at increased risk of recurrence disseminated cell populations exist that are responsible for the release of the bulk of CK fragments after removal of the apparently nonmetastatic tumor. In good agreement with the results in metastatic breast cancer patients, release of CK18 or 19 fragments by BM-DTCs seem to indicate disseminated tumor cells mainly in a dormant state, whereas caspase-cleaved CK18 may indicate skipping of this latent phase and early progression. Therefore, caspase cleavage of CKs in intact tumor cells seems to accompany or is involved in the differentiation leading from dormant to progressively active disseminated tumor cells. Release of respective CK fragments would result in an apparent clearing of CK-positive cells in BM, leaving malignant cells that have possibly undergone an epithelial-mesenchymal transition. Micrometastatic cancer cell lines derived from breast cancer patients were found to display loss of epithelial CK8, 18 and 19 as well as ectopic expression of vimentin as in mesenchymal cells. In conclusion, degradation of CKs may represent a marker indicating reactivation of dormant tumor cells in BM.

Platinum (Pt)(IV) complexes are thought to function as prodrugs for anticancer Pt(II) drugs. We studied two pairs of Pt(II)/Pt(IV) complexes to explore whether there were differences in their cytotoxic activities, their abilities to cause acquired resistance and their gene expression profiles in the resistant lines. Microtiter methods were used to evaluate the antiproliferative activity of cisplatin, oxoplatin, [trans-d,l-(1,2-diaminocyclo-hexane)]dichloroplatinum(II) [DACH-Pt(II)] and cis,trans-[trans-d,l-(1,2-diaminocyclo-hexane)]-dichlorodihydroxoplatinum(IV) [DACH-Pt(IV)] in a panel of 14 human cancer cell lines. Cisplatin and oxoplatin showed significant similar spectra of cytotoxicity, whereas DACH-Pt(II) and DACH-Pt(IV) did not. DACH-Pt(IV) required more than 24 h to reach full potency, whereas the other three Pt complexes achieved maximal activity in less than 24 h. The SISO cervical cell line was made four- to six-fold resistant to the four Pt complexes by weekly exposure to the respective agent. Glutathione (GSH) levels increased in all resistant lines except for the DACH-Pt(IV) resistant line. The catalytic concentrations of various redox enzymes (GSH transferase, GSH peroxidase, GSH reductase, catalase) were all unchanged in the resistant lines relative to the native line. Multidrug resistance protein 2 expression was detected in the cisplatin-resistant and oxoplatin-resistant cell lines but not in the native line. The transcription of 29,000 genes in the SISO lines resistant to either cisplatin or oxoplatin was studied by DNA-microarray methods and compared with the native line. Overall changes in gene transcription were very different between the cisplatin-resistant and oxoplatin-resistant cell lines. Thus, Pt(IV) complexes seem to have biological actions that distinguish them from their Pt(II) counterparts, even when they show cross-resistance.

Platinum(IV) compounds like oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)) show increased stability and therefore can be applied orally. In a panel of 38 human cancer cell lines this drug induced S-phase arrest and cell death withIC50values 2.5-fold higher than cisplatin. Oxoplatin may be converted to cisplatin by intracellular reducing agents, however, exposure to 0.1 M HCl mimicking gastric acid yielded cis-diammine-tetrachlorido-platinum(IV) exhibiting twofold increased activity. Similar results were obtained for another platinum(IV) compound, JM 149 (ammine-dichlorido-(cyclohexylamine)-dihydroxido-platinum(IV)), but not for its parent drug JM 216/satraplatin. Genome-wide expression profiling of H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes between oxoplatin and cisplatin. In conclusion, oxoplatin constitutes a potent oral agent that is either reduced or converted to distinct active compounds, for example, by gastric acid or acidic areas prevailing in solid tumors, in dependence of the respective pharmaceutical formulation.

Publisher Summary Hyaluronidase has been in clinical use involving chemotherapy for the treatment of extravasation in order to prevent tissue necrosis. A key event in alerting to the possible additional uses of hyaluronidase was the case of one myeloma patient who received a large dose of hyaluronidase locally to resolve extravasation of a highly toxic melphalan-containing drug. Hyaluronidase as an adjunct to chemotherapy increases the response of tumors to chemotherapeutic drugs in experimental animal models and in the clinical trials. Expression of hyaluronidase was detected in various tumors and at the time was regarded as an important factor supporting tumor growth and dissemination. Since expression of hyaluronan and hyaluronidase seems to have different effects in different tumors, hyaluronidase in combination with chemotherapy may be helpful only in specific tumor entities, however, in a wide dose range. The chapter presents a pilot study that yielded promising results, and further attempts are being made to improve and expand clinical applications of hyaluronidase by using more enriched preparations with higher specific activity. The clinical studies performed more than twenty years ago need to be repeated and evaluated using human recombinant hyaluronidase, in order to exclude side activities of impurities of the bovine preparations. Additionally, investigations into the mechanisms by which hyaluronidase improves the efficacy of specific anti-cancer drugs should be conducted.

Pancreatic adenocarcinomas express neurotensin receptors in up to 90% of cases, however, their role in tumor biology and as a drug target is not clear. In the present study, a stable neurotensin (NT) analog induced intracellular calcium release and intracellular alkalinization in BxPC-3 and PANC-1 pancreatic cancer cells that was abolished by inhibitors of NT receptor (NTR) and sodium-proton exchanger 1 (NHE1), amiloride and SR 142948, respectively. Activation of NHE1 involved increased phosphorylation of dimethylfumarate-sensitive mitogen- and stress-activated kinase 1/2 (MSK1/2). NTR signaling appears to promote a metastatic phenotype in pancreatic cancer cells by induction of localized extracellular acidification in normoxic cells, preceeding acidosis induced by hypoxia and switch to glycolysis in addition to increased expression of interleukin-8 (IL-8).

Background Soluble cytokeratin 18 (CK18; M65) and a caspase-cleaved fragment of CK18 (M30) have been used as biomarkers, corresponding to tumor cell death and apoptosis, respectively. Methods In the present study, M30 was quantified for the first time in serum samples of colon cancer patients pre- and postoperatively as well as during chemotherapy. Minimal residual disease (MRD) was assessed preoperatively by detection of pan-cytokeratin antibody A45-B/B3-positive cells in bone marrow aspirates. Results Out of 46 patients, those with colon tumors of stages I and IV had significantly elevated M30 serum concentrations compared to controls (n = 23). In 31 colon cancer patients, M30 determinations were performed prior to and seven days after tumor surgery. A group of 24 patients exhibited a significant decrease of M30 in response to tumor removal, in contrast to seven patients who revealed either persistent or higher M30 levels postoperatively. The frequency of MRD was not significantly different for patients with decreasing (4/24) and persisting (3/7) M30. However, M30 correlated significantly with the increased number of recurrences within 36 months in the group with persisting M30 (4/7 versus 2/24, p = 0.032; hazard ratio 8.3, p = 0.016). In a group of patients (n = 10) receiving capecitabine/oxaliplatin chemotherapy (CapOx), transient increases in M30 did not correlate with responses. Conclusion The data obtained within the present limited pilot study in colon cancer patients demonstrate that perioperative changes of M30 may indicate systemic residual tumor load and increased risk of recurrence warranting further evaluation of this marker of apoptosis in a larger prospective clinical trial.

Fourteen patients with metastatic pancreatic adenocarcinoma were treated with the long-acting somatostatin (SST) analogue lanreotide. No objective response was obtained, and the median survival was 4 months (range 1.8–7 months). Pancreatic cancer could not be visualized by means of SST-receptor (R) scintigraphy in our patients. In vitro data also demonstrated absence of SSTR2 expression, suggesting pancreatic cancer not to be a potential target for treatment with SST analogues. © 1999 Cancer Research Campaign

This work describes the syntheses and antitumoral properties of five prenyl compounds from which antiproliferative activities were predicted by using the TOPS-MODE approach, a computational method for drug design. The syntheses of 2-(3-methylbut-2-enyloxy)acetophenone ( 2 ), 2-hydroxy-5-(3-methylbut-2-enyl)acetophenone ( 3 ), 2-hydroxy-3-(1,1-dimethylallyl)acetophenone ( 4 ), and 5-(3-methylbut-2-enyl)-2-(3-methylbut-2-enyloxy)acetophenone ( 5 ) were realized by O -prenylation of phenolic compounds with prenyl bromide and by Claisen rearrangement, respectively. Reaction of 2-hydroxy-5-(3-methylbut-2-enyl)acetophenone 3 under Vilsmeier–Haack conditions with phosphoryl chloride and N , N -dimethylformamide yielded 6-(3-methylbut-2-enyl)chromone-3-carbaldehyde ( 6 ). The compounds were tested for their cytotoxicity toward a diverse panel of cultured human tumor cell lines. Compound 3 showed significant selective cytotoxic activity (IC 50

The overexpression of somatostatin receptors (SST-Rs) on various tumour cells provides the molecular basis for the successful use of radiolabelled SST analogues in clinical oncology. The objective of the study was to evaluate the tumour binding of In-1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetic acid-lanreotide (In-DOTA-LAN) and In-DOTA-tyrosine-octreotide (In-DOTA-Tyr-OCT) in patients with stage IV melanoma. In addition, we evaluated the potential antiproliferative effect of SST analogues, together with an assessment of the functionality of SST-Rs, on four melanoma cell lines. Twenty-three patients with advanced metastatic melanoma underwent scintigraphy. Thirty-eight of 61 lesions (62%) were positively imaged with In-DOTA-LAN, whereas 23 (37%) were negative. With In-DOTA-Tyr-OCT, 10 of the 23 documented lesions (43%) were positive and 13 (56%) were negative. In vitro, cell lines showed no growth inhibition in the presence of SST analogues and no influence on cell cycle distribution was found with the addition of SST analogues to cultured cells. In addition, no functional surface SST-Rs could be demonstrated on these cell lines. Taken together, our results demonstrate the visualization of metastatic melanoma in a high percentage of patients, probably due to binding of SST analogues to SST-Rs on tumour vessels or infiltrating immune cells. Judging from our data, however, there is no evidence of functional SST-R expression on melanoma cells.

Prostate carcinoma-derived factors induce a proliferative response in osteoblasts. The present study investigated the involvement of MAP kinase in the osteoblastic reaction of osteocytes and the response of 1alpha,25-hydroxy-vitamin D3 (1,25-vitD3)-pretreated osteoblasts. Conditioned media (CM) from prostate, colon, pancreatic, renal cell and breast cancer cell lines were tested on their proliferative activity using murine osteoblast-like MC3T3-E1 cells, MG63 human osteosarcoma cells and immortalized human osteoblasts (AHTO-7). Changes in osteoblastic activities of the supernantants were measured in the presence of MAP kinase inhibitors and following 1,25-vitD3-induced differentiation of the target osteoblasts. Supernatants of prostate cancer cells stimulated proliferation of osteoblasts in all three indicator cell lines, with AHTO-7 exhibiting the most significant correlation to human primary osteoblast cultures. 1,25-vitD3 induced the differentiation marker alkaline phosphatase (ALP) in MC3T3-E1 and AHTO-7, but only to a minor degree in MG63 cells. 1,25-vitD3-induced differentiation reduced the proliferative response to CM from several cell lines in MC3T3-E1 and MG63 to a minor degree, whereas in AHTO-7 cells the osteoblastic reaction was reduced for 2/4 pancreatic, 3/3 colon and 1/1 renal cancer CMs, however not for 3/3 prostate cancer CMs. Stimulation of AHTO-7 cells by CM from prostate cancer lines is inhibited significantly by MEK1 kinase inhibitor PD 98059 in contrast to CMs derived from other carcinomas, except ACHN renal cancer cells. The findings in the present study demonstrate that human AHTO-7 cells seem to represent a valid human system to monitor osteoblastic activity, especially in respect to 1,25-vitD3-induced differentiation. Vitamin D3-induced differentiation has no direct effect on prostate cancer-derived osteoblastic activity in the same cell line in vitro, which however, could be reversed by disruption of the signal transduction at the MAP kinase level, revealing a new target for the inhibition of prostate cancer-associated bone formation.

Prostate carcinoma-derived factors induce a proliferative response in osteoblasts. The present study investigated the involvement of MAP kinase in the osteoblastic reaction of osteocytes and the response of 1alpha,25-hydroxy-vitamin D3 (1,25-vitD3)-pretreated osteoblasts. Conditioned media (CM) from prostate, colon, pancreatic, renal cell and breast cancer cell lines were tested on their proliferative activity using murine osteoblast-like MC3T3-E1 cells, MG63 human osteosarcoma cells and immortalized human osteoblasts (AHTO-7). Changes in osteoblastic activities of the supernantants were measured in the presence of MAP kinase inhibitors and following 1,25-vitD3-induced differentiation of the target osteoblasts. Supernatants of prostate cancer cells stimulated proliferation of osteoblasts in all three indicator cell lines, with AHTO-7 exhibiting the most significant correlation to human primary osteoblast cultures. 1,25-vitD3 induced the differentiation marker alkaline phosphatase (ALP) in MC3T3-E1 and AHTO-7, but only to a minor degree in MG63 cells. 1,25-vitD3-induced differentiation reduced the proliferative response to CM from several cell lines in MC3T3-E1 and MG63 to a minor degree, whereas in AHTO-7 cells the osteoblastic reaction was reduced for 2/4 pancreatic, 3/3 colon and 1/1 renal cancer CMs, however not for 3/3 prostate cancer CMs. Stimulation of AHTO-7 cells by CM from prostate cancer lines is inhibited significantly by MEK1 kinase inhibitor PD 98059 in contrast to CMs derived from other carcinomas, except ACHN renal cancer cells. The findings in the present study demonstrate that human AHTO-7 cells seem to represent a valid human system to monitor osteoblastic activity, especially in respect to 1,25-vitD3-induced differentiation. Vitamin D3-induced differentiation has no direct effect on prostate cancer-derived osteoblastic activity in the same cell line in vitro, which however, could be reversed by disruption of the signal transduction at the MAP kinase level, revealing a new target for the inhibition of prostate cancer-associated bone formation.

BACKGROUND Recent studies have shown that vascular endothelial growth factor (VEGF) receptor is overexpressed in vascular endothelial cells of various human tumours as well as in human tumour cells. The aim of this study was to evaluate the usefulness of scanning with VEGF(165) labeled with (123)I for tumor localisation in patients with gastrointestinal tumours. PATIENTS AND METHODS Human recombinant VEGF(165) was radiolabelled with (123)I by electrophilic radioiodination using the chloramine T method. [(123)I]VEGF(165) was administered intravenously [mean dose 184 +/- 18 MBq (

10-hydroxycamptothecin (HCPT), a natural analog of the alkaloid camptothecin (CPT), is a promising anticancer agent currently undergoing preclinical trials. Though HCPT is less toxic and more active in various human cancer cell lines and in animal tumor models than the clinically approved CPT-analog topotecan, little is known about its biotransformation products and their route of elimination. To investigate the metabolism and biliary excretion, livers of male Wistar rats were perfused with HCPT (5 microM). Bile and perfusate samples were collected for 60 min and quantified by reversed-phase high-performance liquid chromatography (HPLC). Besides HCPT, three metabolites, namely HCPT glucuronide (M1), hydroxyHCPT glucuronide (M2), and hydroxyHCPT (M3) could be identified by enzymatic hydrolysis with beta-glucuronidase and mass spectroscopy. Biliary secretion of HCPT and M1-M3 reached a peak secretion of 1532+/-124, 75+/-16, 5.8+/-1.6 and 2.1+/-0.5 pmoles/g liver.min, respectively, after 25 min. The total amount of HCPT and M1-M3 excreted into bile during the time of perfusion (60 min) was low and represented a mean of 9.9+/-3.2%, 0.44+/-0.17%, 0.041+/-0.010%, and 0.022+/-0.004% of the initial HCPT dose, respectively. In the perfusate, besides HCPT M1 and M2 but not M3 could be detected (maximum concentrations after about 20 min: 3248+/-210, 16.8+/-2.8 and 1.0+/-0.4 pmoles/g liver.min, respectively). The cumulative efflux of HCPT and M1 and M2 into the perfusate was 21.1+/-3.9%, 0.145+/-0.036% and 0.018+/-0.004% of the initial dose, respectively, indicating a preferable non-biliary secretion for HCPT and a predominant biliary elimination for conjugated HCPT biotransformation products. In conclusion, HCPT is biotransformed in a rat liver model to three metabolites, mainly excreted into bile, which may be of clinical relevance during cancer therapy.

Aromatic fatty acids such as phenylbutyrate (PB) and its metabolite phenylacetate (PA) induce growth arrest, differentiation and apoptosis in solid tumor cells. Despite their antiproliferative action they were reported to exhibit a synergistic effect in combination with cytotoxic drugs like topotecan, and others. Since the activity of the camptothecines (CPTs) depends on local pH conditions, we investigated, whether PB/PA modulate CPT effects indirectly by affecting intracellular pH in SW620 and SW480 colon cancer cells. The results for the colon carcinoma cells show an antagonistic interaction for the combination of CPT and 0.25-5 mM PA in viability assays, resulting in an approximately 3-fold increase in IC50 (control: 20+/-7 nM). A synergistic effect with significantly increased numbers of late apoptotic/necrotic cancer cells (difference +21+/-4%) and 1.4-fold sensitization were detected upon inclusion of 2.5 mM PA during a 4-h CPT (10 micro;M) loading phase. In response to 0.25-1 mM PA/PB the cells exhibit a reversible decrease of pHi (0.1-0.31 pH units) in HEPES- or bicarbonate-buffered media. Dose-dependent acidification and pHi-recovery occurred following addition of PA and PB after an acid load and inhibition of the Na+/H+-antiporter and bicarbonate exchangers, pointing to a possible intracellular mechanism of cytoplasmic acidification. It is concluded that the synergistic modulation of CPT toxicity by short-term PA/PB treatment in colon carcinoma cells is caused by changes in intracellular pH, possibly affecting quantity and localization of the active closed lactone form of this drug.

Based on the fact that somatostatin (SST) analogs have given promising results for treatment of hepatocellular cancer, we performed both in vitro and in vivo investigations to define the role of a depot formulation of the long acting SST-analog lanreotide (LAN). A decrease of cells in the S-phase as compared to controls (p

Resveratrol, a natural phytoestrogen, has been reported to promote differentiation of murine MC3T3-E1 osteoblasts and to inhibit proliferation of prostate cancer cell lines. In the present study we tested the effects of resveratrol on the increased proliferation of human AHTO-7 osteoblastic cell line induced by conditioned media (CM) from a panel of carcinoma cell lines. This compound was found to modulate AHTO-7 proliferation in a tamoxifen-sensitive mechanism at lower concentrations, but failed to induce the osteoblast differentiation marker alkaline phosphatase (ALP) in contrast to vitamin D3. The proliferative response of AHTO-7 cells to conditioned media from carcinoma cell lines was diminished (30-71.4% inhibition) upon pretreatment with 0.5 microM resveratrol. Highest inhibition was demonstrated for pancreas (BxPC3, Panc-1), breast (ZR75-1) and renal (ACHN) carcinoma cell line supernatants whereas the effect on colon carcinoma (SW620, Colo320DM) cell CM and prostate cancer (PC3, DU145 and LNCaP) CM was less pronounced. Direct addition of resveratrol affected only supernatants of cell lines (

Treatment of advanced prostate cancer by continuous androgen suppression results in excellent short-term response but poor long-term survival. Intermittent androgen suppression (IAS) aims to maintain androgen responsiveness of tumor cells by regular cycles of treatment cessations and tumor regrowth to specific prostate-specific antigen limits. First clinical trials demonstrate consistent responses and improved quality of life in most patients on androgen suppression retreatment for up to five cycles, with mean off-treatment periods of approximately 5 to 16 months. Most patients with metastatic disease exhibit early disease progression or androgen independency under IAS, but a subgroup including patients with metastatic disease respond to a single androgen suppression cycle with off-treatment times for up to 48 months. In conclusion, IAS improves the quality of life in patients with primarily hormone-dependent tumors without adverse effects and seems to be most effective in patients with prostate cancer with asymptomatic biochemical progression and low tumor burden. Patients should be treated within the framework of randomized trials and characterized for survival and prognostic factors associated with response to IAS treatment.

The effects of pretreatment with the multidrug resistance (MDR) modulators verapamil (VPM), tamoxifen (TMX), cyclosporin A (CsA), and SDZ PSC833 (PSC) on drug sensitivity of the P-glycoprotein (Pgp) expressing human ileocecal carcinoma cell line HCT-8 is described. Following pretreatment of 2, 16 and 48 h with the individual modulators, rhodamine 123 efflux (RHO), transepithelial vinblastine transport (VIN) across treated HCT-8 monolayers, and chemosensitivity to doxorubicin (DOX) were determined and compared to Pgp protein expression and phosphorylation. After 2 h, VPM, TMX, CsA and PSC inhibited RHO efflux and VIN transport and increased the chemosensitivity of HCT-8 to DOX significantly. Prolonged exposure failed to further increase inhibition of Pgp-mediated transport, but in contrast maximized phosphorylation of Pgp (16 h) and Pgp protein expression (48 h), respectively.

430 Background: Organic anions transporters (OATPs) are important for tumor progression and therapeutic response by regulating cellular levels of hormones, second messenger proteins and drugs. OATP4A1 is a transporter of pro-inflammatory prostaglandin E2 and may contribute to cancer progression. Data on the expression of OATP4A1 and its clinical impact in primary colorectal cancer (CRC) is rare. Our study was designed to proof the overexpression of OATP4A1 in primary CRC. Methods: Frozen samples from 20 unselected CRC patients (pat) and five CRC cell lines were analyzed for OATP4A1 mRNA expression by real time PCR (mean level normalized to the calibrator, MNE). Immunohistochemistry was performed on paraffin- embedded tumor sections from 50 CRC pat., UICC 0-II (25/50 with subsequent relapse). An automatic quantitative image analysis program was applied to quantify OATP4A1 expression. Expression and intensity was correlated with clinical parameters and relapses. Results: Significant (p>0.05) higher levels of OATP4A1 mRNA were observed in 20 cancer samples as compared to adjacent non-cancerous tissue (2.44 vs. 0.46 MNE). The highest expression (9.85 MNE) was observed in a well-differentiated tumor sample. Similar high levels were observed in the COGA1A cell line, expression in the other cell lines ranged between 1.83 and 0.28 MNE. Immunoreactive staining for OATP4A1 was located in the membrane and occasionally in the cytosol of tumor cells, it was exclusively membrane located in the adjacent non-cancerous epithelial cells. The staining intensity was significantly higher in cancer cells compared to non-cancerous areas (1528±326 vs.376±218) while staining of stroma cells was only occasionally detectable. Surprisingly, the highest OATP4A1 levels were observed in immune cells (2839±381 vs.298±56). Data on the clinical impact of OATP4A1 in the early stage CRC pat. will be presented at the meeting. Conclusions: The profound expression of OATP4A1 in CRC cells and in the inflammatory infiltrates supports its implication on cancer progression. Suitability of OATP4A1 as a potential prognostic marker has to be established on a larger patient collective.

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5-bromonicotinic acid and 1-methyl-10 alpha-methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond-generating methods (mixed anhydride-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, and dicyclohexylcarbodiimide/N-hydroxysuccinimide methods) were used in bovine serum albumin (BSA)-coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23 mol of 1-methyl-10 alpha-methoxydihydrolysergol (MMD) or 41 mol of 5-bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten-inhibition ELISA using antigen-coated wells indicate that the supernatant of MMD-specific hybridoma exhibited 50% inhibition of antibody binding at 17 +/- 2 micrograms of MMD and at 24.5 +/- 2 micrograms of nicergoline, and the BNA-specific hybridoma exhibited similar inhibition at 147 +/- 6 micrograms of BNA and 500 +/- 30 micrograms of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten-inhibition ELISA.

Serum-free growth of Ewing's sarcoma (ES) and primitive peripheral neuroectodermal tumour (pPNET) cell lines was achieved by supplementing a basal medium with insulin-like growth factor-I (IGF-I). These cultures were used to investigate the sensitivity of 3 ES (EW-2, RD-ES, SK-ES-1) and 3 pPNET (SIM-1, KAL, SAL) cell lines to a panel of anti-tumour agents in short-term (48-h) proliferation assays. Of the four cytostatic drugs included in the currently used multi-drug regimens, cyclophosphamide, doxorubicin and actinomycin-D inhibit the proliferation of the cell lines with high efficacy, whereas the vinca alkaloids were less effective. Cisplatin, etoposide, mitomycin-C and mitoxanthrone were also found to have a high inhibitory activity in this in vitro ES/pPNET system. The most remarkable effect was observed for cytosine arabinoside (ARA-C), which gave a half-maximal inhibition at drug concentrations approximately 5000 times below the clinical peak plasma concentrations (250 μg/ml). The ARA-C sensitivity of ES and pPNET cell lines is comparable with the established ARA-C sensitivities of leukaemia-derived cells. The different ES and pPNET cell lines showed a rather uniform response to the different cytostatic drugs with decreased sensitivity of individual pPNET cell lines to vinblastin, ARA-C and mitoxanthrone. Modulation of the IGF-I/IGF-I receptor/IGF-I binding protein system, which seems to constitute an important stimulator of cell growth in neuroectoderm-derived or -related tumours, can be used to enhance the drug sensitivity of the tumour cells in vivo or in in vitro therapeutic procedures. According to our results, serum-free conditions for autologous bone marrow purification are expected to result in significantly increased chemosensitivity of ES and pPNET cells in response to anthracyclines and cisplatin.