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Cancer cell spheroids are used for drug screening as these three-dimensional (3D) assemblies recapitulate tumors more realistic than the widely employed 2D

Historically disseminated tumor cells in solid cancers were detected by immunohistochemistry of bone marrow aspirates. Blood samples are more easily and frequently accessible and are therefore employed to detect circulating tumor cells (CTCs), a procedure termed “liquid biopsy”. CTCs are shed from tumors and a fraction of these cells is capable of establishing distal lesions. Since patients die from metastatic disease, CTCs hold potential to have prognostic significance and to indicate the success or failure of therapeutic interventions. With few exceptions, CTCs are rare cells against a large background of blood cells and need to be enriched for detection and characterization. The FDA-approved CellSearch© system relies on the selection of epithelial cell adhesion molecule (EpCAM)-positive tumor cells and is incorporated in numerous clinical studies for risk stratification and assessment of therapy. However, CTCs may have undergone (partial) epithelial-mesenchymal transition and are therefore missed by methods dependent on epithelial markers. A host of other techniques to enrich/isolate CTCs which are based on physical and/or distinct biological properties are under evaluation. Although single CTCs were linked to an increased risk of tumor progression, the actual phenotype and fraction of these cells capable of forming metastases are not known, as well as the mechanisms and kinetics of tumor cell shedding and evasion. Despite considerable progress, CTC detection methods still need to be validated and their clinical significance confirmed in larger multicenter trials before entering clinical routine practice.

Small cell lung cancer (SCLC) is a highly malignant disease with dismal prognosis. Although great progress has been made in investigating genetic aberrations and putative drivers of this tumor entity, the mechanisms of rapid dissemination and acquisition of drug resistance are not clear. The majority of SCLC cases are characterized by inactivation of the tumor suppressors p53 and retinoblastoma (Rb) and, therefore, interchangeable drivers will be difficult to target successfully. Access to pure cultures of SCLC circulating tumor cells (CTCs) and study of their tumor biology has revealed a number of new potential targets. Most important, expression of chitinase-3-like-1/YKL-40 (CHI3L1) which controls expression of vascular epithelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) was newly described in these cells. The process switching CHI3L1-negative SCLC cells to CHI3L1-positive CTCs seems to be associated with cytokines released by inflammatory immune cells. Furthermore, these CTCs were found to promote monocyte-macrophage differentiation, most likely of the M2 tumor-promoting type, recently described to express PD-1 immune checkpoint antigen in SCLC. In conclusion, dissemination of SCLC seems to be linked to conversion of regular tumor cells to highly invasive CHI3L1-positive CTCs, which are protected by immune system suppression. Besides the classical targets VEGF, MMP-9 and PD-1, CHI3L1 constitutes a new possibly drugable molecule to retard down dissemination of SCLC cells, which may be similarly relevant for glioblastoma and other tumor entities.

Small cell lung cancer (SCLC) accounts for 15 % of all lung tumors and represents an invasive neuroendocrine malignancy with poor survival rates. This cancer is highly prevalent in smokers and characterized by inactivation of p53 and retinoblastoma. First in vitro expansion of circulating tumor cells (CTCs) of SCLC patients allowed for investigation of the cell biology of tumor dissemination. In the suggested CTC SCLC model, the primary tumor attracts and educates tumor-promoting and immunosuppressive macrophages which in turn arm CTCs to spread and generate distal lesions. Preexisting inflammatory processes associated with chronic obstructive pulmonary disease (COPD) seem to potentiate the subsequent activity of tumor-associated macrophages (TAM). Activation of signal transducer and activator of transcription 3 (STAT3) and expression of chitinase-3-like 1/YKL-40 in SCLC CTCs seems to be associated with drug resistance. In conclusion, inflammation-associated generation of invasive and chemoresistant CTCs most likely explains the characteristic features of SCLC, namely early dissemination and rapid failure of chemotherapy.

Small cell lung cancer (SCLC) has a poor prognosis and is found disseminated at first presentation in the majority of cases. The cell biological mechanisms underlying metastasis and drug resistance are not clear. SCLC is characterized by high numbers of circulating tumor cells (CTCs) and we were able to expand several CTC lines ex vivo and to relate chitinase-3-like-1/YKL-40 (CHI3L1) as marker. Availability of expanded SCLC CTC cells allowed for a screening of receptor tyrosine kinases (RTKs) expressed. The metastatic CHI3L1-negative SCLC cell line SCLC26A, established from a pleural effusion was used for comparison. The CTC cell line BHGc10 was found to exhibit increased expression of RYK, AXL, Tie-1, Dtk, ROR1/2, several ephrins (Eph) and FGF/EGF receptors compared to SCLC26A. All of these RTKs have been associated with cell motility, invasion and poor prognosis in diverse cancer entities without knowledge of their association with CTCs. The identification of RYK, AXL and ROR1/2 as pseudokinases, lacking activity, seems to be related to the observed failure of RTK inhibitors in SCLC. These kinases are involved in the noncanonical WNT pathway and their expression in SCLC CTCs represents a cancer stem cell-like phenotype.

Cancer cell lines are good in vitro models to study molecular mechanisms underlying chemoresistance and cancer recurrence. Recent works have demonstrated that most of the available ovarian cancer cell lines are most unlikely high grade serous (HGSOC), the major type of epithelial ovarian cancer. We aimed at establishing well characterized HGSOC cell lines, which can be used as optimal models for ovarian cancer research. We successfully established seven cell lines from HGSOC and provided the major genomic alterations and the transcriptomic landscapes of them. They exhibited different gene expression patterns in the key pathways involved in cancer resistance. Each cell line harbored a unique TP53 mutation as their corresponding tumors and expressed cytokeratins 8/18/19 and EpCAM. Two matched lines were established from the same patient, one at diagnosis and being sensitive to carboplatin and the other during chemotherapy and being resistant. Two cell lines presented respective BRCA1 and BRCA2 mutations. To conclude, we have established seven cell lines and well characterized them at genomic and transcriptomic levels. They are optimal models to investigate the molecular mechanisms underlying the progression, chemo resistance and recurrence of HGSOC. publisher: Elsevier articletitle: Molecular characterization of 7 new established cell lines from high grade serous ovarian cancer journaltitle: Cancer Letters articlelink: http://dx.doi.org/10.1016/j.canlet.2015.03.040 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd. ispartof: Cancer Letters vol:362 issue:2 pages:218-28 ispartof: location:Ireland status: published

Cancer and metastasis are closely associated with inflammation. Macrophages are important effector cells in enhancing tumor proliferation, invasion and providing protection against the immune system. Despite advanced knowledge of tumor-macrophage interactions, the role of macrophages in emergence and invasion of circulating tumor cells (CTCs) is not known. A series of six CTC cell lines have been derived from blood of patients with extensive disease small cell lung cancer (ED-SCLC) in our lab, most likely representing a homogenous cell population of the actual metastasis-initiating cells (MIC) of CTCs. SCLC has an unfavorable prognosis due to rapid dissemination and early chemoresistant relapses. SCLC CTCs recruit macrophages and elicit secretion of various cytokines and the six CTC lines express chitinase-3-like-1 (CHI3L1), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) in abundance. CHI3L1 is cytokine/growth factor expressed in inflammation and cancer and found to be correlated to metastasis and a dismal prognosis. In conclusion, SCLC CTCs have acquired the essential means for aggressiveness and invasion in a tumor microenvironment specifically shaped by macrophages and inflammation.

Cancer patients die of metastatic disease but knowledge regarding individual steps of this complex process of intravasation, spread and extravasation leading to secondary lesions is incomplete. Subpopulations of tumor cells are supposed to undergo an epithelial-mesenchymal transition (EMT), to enter the bloodstream and eventually establish metastases in a reverse process termed mesenchymal-epithelial transition (MET). Small cell lung cancer (SCLC) represents a unique model to study metastatic spread due to early dissemination and relapse, as well as availability of a panel of circulating cancer cell (CTC) lines recently. Additionally, chemosensitive SCLC tumor cells switch to a completely resistant phenotype during cancer recurrence. In advanced disease, SCLC patients display extremely high blood counts of CTCs in contrast to other tumors, like breast, prostate and colon cancer. Local inflammatory conditions at the primary tumor site and recruitment of macrophages seem to increase the shedding of tumor cells into the circulation in processes which may proceed independently of EMT. Since millions of cells are released by tumors into the circulation per day, analysis of a limited number of CTCs at specific time points are difficult to be related to the development of metastatic lesions which may occur approximately one year later. We have obtained a panel of SCLC CTC cell line from patients with relapsing disease, which share characteristic markers of this malignancy and a primarily epithelial phenotype with unique formation of large tumorospheres, containing quiescent and hypoxic cells. Although smoking and inflammation promote EMT, partial expression of vimentin indicates a transitional state with partial EMT in these cell lines at most. The CTC lines exhibit high expression of EpCAM , absent phosphorylation of β-catenin and background levels of Snail. Provided that these tumor cells had ever undergone EMT, here in advanced disease MET seem to have occurred already in the peripheral circulation. Alternative explanations for the expression of mesenchymal markers of the CTC lines are the heterogeneity of SCLC cells, cooperative migration or altered gene expression in response to the inflammatory tumor microenvironment allowing for tumor spread without EMT/MET.

Immune checkpoint inhibition holds great promise for selected tumors. The human monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect antibody-dependent cytotoxicity (ADCC) lysis of tumor cells. Areas covered: This article presents an overview of the current means to activate the antitumor immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated tumor cell elimination. Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1 which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown by targeting PD-1 and the upregulation of TIM-3.

In attempts to develop an orally applicable platinum-based drug, platinum(IV) drugs which exhibit higher in vivo stability compared to the platinum(II) drug cisplatin were formulated. The first such chemotherapeutic agent, namely satraplatin, failed to receive approval. In the present work, we checked the initial cellular stress response of the chemosensitive NCI-H526 small cell lung cancer (SCLC) cells by determination of the relative phosphorylation of 46 specific phosphorylation sites of 38 selected proteins in a six hours response to cisplatin (platinum(II)) or oxoplatin (platinum(IV)), respectively. Oxoplatin is considered as prodrug of cisplatin, although several findings point to differences in intracellular effects. Cisplatin induced hyperphosphorylation of p38α MAPK and AMPKα1, whereas oxoplatin treatment resulted in increased phosphorylation of a large number of signaling proteins involved in stress response/drug resistance, including JNK, GSK-3α, AMPKα1, src kinases, STATs, CHK-2 and especially focal adhesion kinase (FAK). Cisplatin exerts markedly higher cytotoxicity upon four hours short-term exposure in comparison to oxoplatin and, correspondingly, the extended initial stress response to the platinum(IV) drug oxoplatin thus is expected to increase clinical drug resistance. Induction of a substantial stress response to any prodrug of a platinum-based compound may likewise limit the effectivity of its active metabolite(s), such contributing to the failure of selected derivatized platinum complexes.

Anaplastic lymphoma kinase (ALK)-rearranged kinase drives non-small cell lung cancer (NSCLC) in 3–7% of patients and treatment consists of a broad range of approved inhibitors which are administered sequentially in case of resistance developing invariably after approximately 1 year of therapy. Besides the former standard tyrosine kinase inhibitor (TKI) crizotinib, the second-line ALK inhibitors alectinib, ceritinib, brigatinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find individual schemes of ALK inhibitors therapy which provide the best benefit for the patients. A host of ALK fusion partners, rearrangement variants and ALK mutations prevent a direct correlation of ALK alterations and sensitivity to specific TKIs within the framework of genomic precision medicine. However, recurrent ALK-positive NSCLC is an aggressive disease resulting in accumulation of tumor cells in pleural effusions which may be collected for in vitro sensitivity testing in a manner designated as functional precision medicine. Provided that these cells are present in sufficient numbers, they can be exposed to all possible drug candidates and their chemosensitivity tested in short-term proliferation assays. In addition to ALK-directed TKIs, cytotoxic drugs and inhibitors of non-target bypassing pathways may be included in the tests. In contrast to other trials in genome-guided precision medicine, a range of suitable therapeutics is available for modified ALK proteins. In summary, ALK-positive NSCLC with pleural effusions offer a unique model to develop, test and validate concepts of functional precision medicine.

Picoplatin was developed as platinum coordination complex to overcome development of resistance, through conjugation to thioles, by the introduction of a methyl-pyridine moiety into the cisplatin parent structure. Pharmacokinetic parameters of the drug, after intravenous and oral application, were studied in solid tumors and clinical Phase I - III trials performed, in particular in NSCLC and small cell lung cancer (SCLC). Results showed low clinical activity of picoplatin.This article presents an overview of the pharmacokinetic assessments of picoplatin in lung cancer. Specifically, the authors address the relationship between disposition and clinical activity of the drug.Picoplatin failed to overcome resistance to platinum compounds in lung cancer to achieve significant improved survival of most patients. Even highest doses of the drug reaching 150 m/m² given intravenously every 3 weeks were not sufficient to achieve better response than existing chemotherapeutics and the oral bioavailability of a dose of 200 - 400 mg corresponded only to 80 mg/m² iv. Picoplatin therefore seem to be quite ineffective. Picoplatin is expected to overcome tumor resistance in cases which overexpress thiol-conjugating pathways; however, this was not proved in clinical trials. To conclude, this blocked platinum complex is not able to reverse cisplatin resistance to a significant extent in vivo and its mechanisms and kinetics and of DNA damage failed to produce significant clinical results compared to second-line standard therapy for lung cancer.

Ulrike Olszewski, Robert Zeillinger, Klaus Geissler, Gerhard HamiltonLudwig Boltzmann Cluster of Translational Oncology, Ludwig Boltzmann Society, Vienna, AustriaPurpose: Carcinoids are highly chemoresistant tumors associated with a dismal prognosis. This study involved a comparison of the genome-wide gene expression pattern of a chemoresistant and a chemosensitive pulmonary carcinoid cell line to reveal factors that contribute to the resistant phenotype.Materials and methods: Gene expression of UMC-11 chemoresistant carcinoid cells as assessed by 32 K microarray was compared with H835 chemosensitive carcinoid cells, and the genes that were differentially expressed and expected to be related to chemoresistance were selected.Results: Drug-resistant UMC-11 cells exhibited increased expression of transcripts known to confer resistance to different cytostatics such as P-glycoprotein, multidrug resistance-associated proteins 2 and 3, effectors of the glutathione detoxification and xenobiotics degradation pathways, and ion transporters including Na+/K+-adenosine triphosphatase. In addition, enhanced transcription of several S100 proteins, capable of suppressing apoptosis, regulation of topoisomerase I (topo I) expression by antisense transcripts from TOPO1 pseudogenes, and alterations of the cytoskeleton seem to contribute to the multidrug-resistant phenotype. A multitude of epidermal growth factor (EGF)-related and neuropeptide growth factors, overexpression of proteases, and appearance of aerobic glycolytic metabolism complement the malignant phenotype of the UMC-11 cells.Conclusion: The multidrug-resistant phenotype of the UMC-11 pulmonary carcinoid cell line seems to be mediated by classical efflux pumps, drug metabolization or conjugation systems, and, possibly, modulation of apoptotic cell death by S100 proteins and topo I expression by pseudogene transcripts. Autocrine or paracrine stimulation by a host of EGF-related and neuropeptide growth factors, as well as high metastatic potency indicated by increased expression of components of aerobic glycolysis and proteolytic enzymes, may furthermore account for the failure of therapeutic interventions.Keywords: neuroendocrine tumor, drug resistance, microarray, drug transporter, apoptosis

Cancer patients die of metastatic disease but knowledge regarding individual steps of this complex process of intravasation, spread and extravasation leading to secondary lesions is incomplete. Subpopulations of tumor cells are supposed to undergo an epithelial-mesenchymal transition (EMT), to enter the bloodstream and eventually establish metastases in a reverse process termed mesenchymal-epithelial transition (MET). Small cell lung cancer (SCLC) represents a unique model to study metastatic spread due to early dissemination and relapse, as well as availability of a panel of circulating cancer cell (CTC) lines recently. Additionally, chemosensitive SCLC tumor cells switch to a completely resistant phenotype during cancer recurrence. In advanced disease, SCLC patients display extremely high blood counts of CTCs in contrast to other tumors, like breast, prostate and colon cancer. Local inflammatory conditions at the primary tumor site and recruitment of macrophages seem to increase the shedding of tumor cells into the circulation in processes which may proceed independently of EMT. Since millions of cells are released by tumors into the circulation per day, analysis of a limited number of CTCs at specific time points are difficult to be related to the development of metastatic lesions which may occur approximately one year later. We have obtained a panel of SCLC CTC cell line from patients with relapsing disease, which share characteristic markers of this malignancy and a primarily epithelial phenotype with unique formation of large tumorospheres, containing quiescent and hypoxic cells. Although smoking and inflammation promote EMT, partial expression of vimentin indicates a transitional state with partial EMT in these cell lines at most. The CTC lines exhibit high expression of EpCAM , absent phosphorylation of β-catenin and background levels of Snail. Provided that these tumor cells had ever undergone EMT, here in advanced disease MET seem to have occurred already in the peripheral circulation. Alternative explanations for the expression of mesenchymal markers of the CTC lines are the heterogeneity of SCLC cells, cooperative migration or altered gene expression in response to the inflammatory tumor microenvironment allowing for tumor spread without EMT/MET.

Extended-stage small cell lung cancer (SCLC) responds to platinum/vepeside-based first-line chemotherapy but relapses rapidly as drug-resistant tumor. Topotecan (TPT) is the single chemotherapeutic agent approved for second-line treatment of SCLC. However, the response to TPT is short-lived and novel treatment modalities need to be developed. Sequential treatment of cytotoxic drugs and inhibitors of cyclin-dependent kinases (CDKs) showed promising preclinical anticancer activity and, in the present work, combinations of TPT with CDK inhibitors olomoucine, roscovitine and CDK4I are shown to exhibit synergistic cytotoxic activity against SCLC cell lines. Highest activity was found against TPT-resistant NCI-H417 and DMS153 cell lines and moderate chemosensitizing effects against a primary SCLC cell line and sensitive GLC19 cells at levels of CDK inhibitors which exerted low toxicity. A combination of 0.6 μM TPT with 0.6 μM roscovitine, exhibiting no significant cytotoxicity as single agents, reduced viability of the TPT-resistant NCI-H417 line (IC50 > 10 μM) by 50%. In the TPT resistant cell lines olomoucine and roscovitine, targeting CDK1,2,5,7, were highly effective, whereas in the more sensitive cell lines CDK4I, inhibiting mainly CDK4/6, showed activity. In NCI-417 cells, preincubation with roscovitine for one day proved synergistic with TPT. Thus, in good accordance with previous findings, CDK inhibitors are able to convert SCLC cancer cells which are cell-cycle arrested by a blockade of topoisomerase I by TPT to apoptotic cells. Since nowadays several CDK inhibitors are at various phases of clinical testing their combination with TPT seems to constitute a promising approach to improve second-line chemotherapy in SCLC.

Extended stage small cell lung cancer (SCLC) responds to platinum/vepeside-based first-line chemotherapy but relapses early as drug-resistant tumor associated with a dismal prognosis. A pair of SCLC cell lines obtained from a single patient at different time points during treatment allows for the investigation of the changes in gene expression prior to (GLC14) and following cycles of chemotherapy and irradiation (GLC19). GLC19 cells were reported to reveal an increased doubling time and exhibit increased chemoresistance to doxorubicin, etoposide, melphalan and vinblastine. Upregulated transcripts in GLC19, as assessed by microarray analysis, comprised proteins involved in regulation of cellular growth (NGFRAP1/BEX3), adhesion, glutathione metabolism and, in particular, WNT/Notch pathways and the putative cancer stem cell phenotype (CD44, ALDH1A1, and AKR1C1/13). Metallothioneins, tubulins TUBA3/4 and tumor protein p53 inducible protein 11 (TP53IP11) were downregulated in this cell line compared to GLC14. Except increased expression of glutathione transferases no classical markers of chemoresistance were found, pointing to a role of altered growth control/differentiation and reduced accessibility of this SCLC tumor cells growing as multicellular spheroids. In conclusion, treatment of this single SCLC with cyclophosphamide, doxorubicin and etoposide (CDE) followed by radiotherapy ultimately resulted in an enrichment of tumor cells displaying the typical signature of tumor-initiating or cancer stem cells (CIC/CSC).

Objective: Vascular endothelial growth factor (VEGF) is an important angiogenic factor, and its receptors have been shown to be overexpressed in various human carcinomas. In this study, we investigated the role of scanning with iodine-123 (123I)-labelled VEGF165 in patients with highly malignant osteosarcoma. Methods: Two patients (a 15-year-old female and a 14-year-old male) with osteosarcoma were injected with 140 MBq [165 per patient] of 123I-VEGF165. Dynamic acquisition was initiated immediately after administration and carried out until 30 min after injection. Whole-body images were done in anterior and posterior views at various time points. All patients underwent single-photon emission tomography imaging. Results:123I-VEGF165 scans were positive in these patients. Sequential images clearly showed increased 123I-VEGF165 activity in osteosarcoma lesions. The tumour lesions were still visualized in whole-body images and single-photon emission tomography examinations 2 h after injection. Intravenous injection of 123I-VEGF165 did not cause any side effects. Conclusion: Our results suggest that 123I-VEGF165 receptor scintigraphy may be useful for the visualization of highly malignant osteosarcoma and/or metastasis and the angiogenic activity of the tumour.

Background : The histological origin of the Ewing's family of tumors (EFT) is still not clear. Since these small cell bone tumors may originate from osteogenic stem cells, the presence of the CD99/MIC2 antigen, known to be overexpressed in EFT, was studied in a human osteoblast cell line in response to differentiation inducers. Methods : The HBA-71 monoclonal antibody directed to the CD99/MIC2 antigen was used to stain a human osteoblast cell line as well as the two EFT cell lines KAL and EW-2 after pretreatment of the cells with the differentiation inducers calcitriol and the histone deacetylase (HDAC) inhibitors sodium butyrate (NaB), sodium phenylacetate (NaPA) as well as N, N'-hexamethylen-bis-acetamide (HMBA) . Alkaline phosphatase (ALP) levels were determined as cellular differentiation marker. Results : Significant expression of the CD99/MIC2 antigen, yielding a molecular weight of 32 kD in Western blotting, was found in the human osteoblast cell line. Pretreatment of the osteoblasts with calcitriol and HMBA increased ALP content and suppressed the CD99/MIC2 antigen. Calcitriol had no major effect on CD99/MIC2 expression of both EFT cell lines, but HMBA enhanced ALP activity in KAL cells and downregulated CD99/MIC2. EW-2 cells exhibited reduced levels of both CD99/MIC2 and ALP. Conclusions : This study supports the role of CD99/MIC2 as differentiation antigen of osteoblasts and a Ewing's sarcoma cell line with neuroectodermal phenotype. Response to calcitriol is absent or low in the two EFT cell lines tested. doi:10.4021/wjon415w

Intermittent androgen suppression (IAS) therapy for prostate cancer patients attempts to maintain the hormone dependence of the tumor cells by cycles alternating between androgen suppression (AS) and treatment cessation till a certain prostate-specific antigen (PSA) threshold is reached. Side effects are expected to be reduced, compared to standard continuous androgen suppression (CAS) therapy. The present study examined the effect of IAS on bone metabolism by determinations of serum procollagen I N-terminal peptide (PINP), a biochemical marker of collagen synthesis. A total of 105 treatment cycles of 58 patients with prostate cancer stages ≥pT2 was studied assessing testosterone, PSA and PINP levels at monthly intervals. During phases of AS lasting for up to nine months PSA levels were reversibly reduced, indicating apoptotic regression of the prostatic tumors. Within the first cycle PINP increased at the end of the AS period and peaked in the treatment cessation phase. During the following two cycles a similar pattern was observed for PINP, except a break in collagen synthesis as indicated by low PINP levels in the first months off treatment. Therefore, measurements of the serum PINP concentration indicated increased bone matrix synthesis in response to >6 months of AS, which uninterruptedly continued into the first treatment cessation phase, with a break into each of the following two pauses. In summary, synthesis of bone matrix collagen increases while degradation decreases during off-treatment phases in patients undergoing IAS. Although a direct relationship between bone matrix turnover and risk of fractures is difficult to establish, IAS for treatment of biochemical progression of prostate tumors is expected to reduce osteoporosis in elderly men often at high risk for bone fractures representing a highly suitable patient population for this kind of therapy.

Background The role of organic anion transporting polypeptide 5A1 (OATP5A1) a member of a family of drug transporters that mediate cellular uptake of drugs has not been characterized so far. Methods Gene expression levels of OATP5A1 in small cell lung cancer (SCLC) cell lines were determined by real-time qPCR and chemosensitivity of HEK-293- SLCO5A1-transfected cells to satraplatin in MTT assays. Results Significant expression of this transporter was found at the mRNA level, primarily in drug-resistant SCLC cells, and SLCO5A1-transfected HEK-293 cells showed higher resistance to satraplatin. OATP5A1 is found preferentially in cytoplasmic membranes of tumor cells, including SCLC. Conclusions OATP5A1 seems to effect intracellular transport of drugs and may participate in chemoresistance of SCLC by sequestration, rather than mediating cellular uptake. Since satraplatin failed to improve survival in SCLC patients, the relation of OATP5A1 expression to clinical drug resistance and its use as marker of chemoresistance should be further investigated.

Titanocenes constitute a class of metal-based anticancer agents that seem to display a mode of action distinct from that of platinum complexes and to be more tolerable with a differing spectrum of activity. In the present study, titanocene C (bis-(N,N-dimethylamino-2(N-methylpyrrolyl)-methyl-cyclopentadienyl) titanium(IV) dichloride) was shown to exhibit antiproliferative activity against human tumor cell lines with a mean IC₅₀ value of 48.3 ± 32.5 µM. In particular, high activity was found against small cell lung cancer (SCLC) cell lines with a profile different from cisplatin. Titanocene C induced cell cycle arrest at the G1/0-S interphase. Cross-resistance to either cisplatin or oxoplatin, respectively, was low for titanocene C and absent for titanocene Y in variant HL-60 cell lines. Alterations in gene expression of NCI-H526 SCLC cells induced by titanocene C were investigated using genome-wide expression arrays. Downregulation was found for genes coding for topoisomerases I and IIα, histones of the HIST1H4 cluster, enzymes involved in glycolysis, components of the cytoskeleton and vesicular transport, among others. In contrast, expression of genes involved in apoptosis, stress response, particularly members of the metallothionein gene cluster 1, DNA damage and growth factors was upregulated following exposure to titanocene C. Approximately 50% of those genes downregulated by titanocene C and cisplatin were concordant, including the previously identified markers of cisplatin-sensitivity, tubulin and stathmin, indicating partial overlap of the pathways affected by these metal complexes. The present findings point helicases/topoisomerases and HIST1H4 core histones out as targets of titanocene C and metallothioneins as putative main effectors of drug resistance.

Soluble cytokeratin 18 fragments (M30, M65) are released from human cancer cells during cell death and hold potential as biomarkers in colorectal cancer characterized by frequent metastatic spread. A total of 62 colorectal cancer and 27 control patients were included in the study. M65 (necrosis and apoptosis) and M30 (apoptosis) were quantified preoperatively (n = 62) and postoperatively (n = 31) using specific enzyme-linked immunosorbent assays. Presence of disseminated tumor cells (DTC) in the bone marrow was assessed by staining of A45-B/B3-positive cells in aspirates. M65 was significantly elevated in patients with International Union against Cancer stage I and IIA tumors compared to controls. A subgroup (19/31) exhibited a significant (p0.05) decrease of M65 after tumor surgery (503.9 +/- 230.7 to 342.6 + 94.8 U/l; -32.0 +/- 16.5%), in contrast to 12 patients who revealed higher M65 levels postoperatively (386.5 +/- 128.5 to 519.1 +/- 151 U/l; +37.4 +/- 32.3%). DTC in bone marrow were found in 10% (2/19) of patients with decreasing and 50% (6/12) of the patients with increasing M65 serum concentrations after surgery (p = 0.028). In conclusion, M65 as marker is likely to be valuable to identify patients with a high incidence of systemic disease.

Tumor-associated macrophages (TAMs) play an important role in tumor progression, suppression of antitumor immunity and dissemination. Blood monocytes infiltrate the tumor region and are primed by local microenvironmental conditions to promote tumor growth and invasion. Although many of the interacting cytokines and factors are known for the tumor-macrophage interactions, the putative contribution of circulating tumor cells (CTCs) is not known so far. These specialized cells are characterized by increased mobility, ability to degrade the extracellular matrix (ECM) and to enter the blood stream and generate secondary lesions which is a leading cause of death for the majority of tumor patients. The first establishment of two permanent CTC lines, namely BHGc7 and 10, from blood samples of advanced stage small cell lung cancer (SCLC) patients allowed us to investigate the CTC-immune cell interaction. Cocultures of peripheral blood mononuclear cells (PBMNCs) with CTCs or addition of CTC-conditioned medium (CTC-CM) in vitro resulted in monocyte-macrophage differentiation and appearance of CD14+, CD163weak and CD68+ macrophages expressing markers of TAMs. Furthermore, we screened the supernatants of CTC-primed macrophages for presence of approximately 100 cytokines and compared the expression with those induced by the local metastatic SCLC26A cell line. Macrophages recruited by SCLC26A-CM showed expression of osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), IL-8, chitinase3-like 1 (CHI3L1), platelet factor (Pf4), IL-1ra and matrix metalloproteinase-9 (MMP-9) among other minor cytokines/chemokines. In contrast, BHGc7-CM induced marked overexpression of complement factor D (CFD)/adipsin and vitamin D-BP (VDBP), as well as increased secretion of OPN, lipocalin-2 (LCN2), CHI3L1, uPAR, MIP-1 and GDF-15/MIC-1. BHGc10, derived independently from relapsed SCLC, revealed an almost identical pattern with added expression of ENA-78/CXCL5. CMs of the non-tumor HEK293 cell line revealed no induction of macrophages, whereas incubation of PBMNCs with recombinant CHI3L1 gave positive results. Thus, the specific contributions of CTCs in SCLC affect CFD/adipsin, possibly involved in immunity/cachexia, VDBP which gives rise to group-specific component protein-derived macrophage-activating factor (GcMAF), GDF-15/MIC-1 which enhances the malignant phenotype of tumor cells and ENA-78/CXCL5 which attracts angiogenic neutrophils. In conclusion, CTCs are competent to specifically manipulate TAMs to increase invasiveness, angiogenesis, immunosuppression and possibly lipid catabolism.

Lung cancer is the leading cause of cancer-related deaths worldwide, and the small cell lung cancer (SCLC) histotype is its most aggressive variant. Yu et al. recently reviewed the detection methods and clinical applications of circulating tumor cells (CTCs) in lung cancer in detail [1]. CTCs are shed from primary tumors or metastatic sites into the peripheral blood and may serve as liquid tumor biopsies for early diagnosis, risk evaluation, and monitoring of therapy [1, 2]. As CTCs are normally rare, detection methods comprise enrichment steps followed by identification with selective markers. The Cell-Search system is the only diagnostic assay approved by the US Food and Drug Administration and was employed to detect SCLC CTCs. In particular, CTCs were defined as nucleated cells coexpressing EpCAM and cytokeratins and negative for the white blood cell surface marker CD45 [2]. In SCLC patients, the detection rates of the CellSearch system ranged from 14.3 to 68.6 % [1]. However, during cancer invasion and metastasis, some cells undergo epithelial– mesenchymal transition (EMT), thus increasing the probability of false-negative CTC results. Among primary lung cancer patients, SCLC patients showed a higher CTC count than non-small cell lung cancer (NSCLC) cases [1]. A higher stage of lung cancer usually correlates with a higher number of CTCs. Yet, only a minority of these disseminated cells can develop into metastases [2]. Lung cancer cells shed into the circulation during surgery only persist for a short time [1]. Numerous investigations have evaluated the prognostic value of CTCs in lung cancer; however, the conclusions of these studies are controversial. For example, Naito et al. indicated that SCLC patients with[8 CTCs/7.5 ml blood had a significantly shorter survival time than the remaining patients with\8 CTCs after therapy [3]. Hou et al. drew the same conclusion using 50 CTCs/7.5 ml blood as the cut-off value, citing an overall survival of 5.4 versus 11.5 months for the high CTC cohort [4]. More studies using standardized methods are needed to validitate a correlation between CTC count and prognosis/disease response. In the latter study, CTCs were present in 85 % of patients and were abundant (range 0–44,896, mean ± SD: 1589 ± 5565), although a significant subpopulation of the CTCs was apoptotic [4]. Molecular characterization of CTCs may provide novel insights into progression of SCLC but investigations are hampered by insufficient tissue for research, because surgical resection and/or serial biopsies are rarely performed. In order to identify the functional properties of different CTC subsets, expansion of CTCs in cell culture systems is required. First ex vivo culture of CTCs has been described in breast cancer by Zhang et al. and Yu et al. [5]. However, these cultures required very high CTC concentrations in suitable blood samples ([1000 cells/7.5 ml in breast cancer), which were so far only found in a few patients. In colon cancer, CTCs isolated from the blood of one patient with a rapidly progressing tumor (out of 71 patients) displaying a CTC count[2300 cells/7.5 ml blood gave rise to a stable colon CTC line [6]. Another approach to expand the number of CTCs is xenotransplantation of patientderived CTCs into immunodeficient mice. Only blood & Gerhard Hamilton gerhard.hamilton@toc.lbg.ac.at

For a subpopulation of NSCLC patients genetic rearrangement of the anaplastic lymphoma kinase (ALK) was found as driver mutation, which can be targeted by the selective inhibitor crizotinib.This article presents an overview of the clinical studies that provided the characterization of the pharmacokinetic parameters for the administration of crizotinib to cancer patients and the factors influencing the clinical profiles of this drug.Crizotinib is administered orally as a capsule and clinical studies indicated 250 mg crizotinib BID continuously as the maximal tolerated dose in cancer patients. Bioavailability is ∼ 40% and pharmacokinetic parameters are influenced by food only to a minor degree. This dose of the drug corresponds to a significant inhibition of the mutated ALK, retards tumor growth and achieves clinical responses in the majority of patients. Crizotinib lactam is the single metabolite with minor inhibitory activity for the ALK fusion protein. Metabolization is executed mainly by CYP3A4/5 and is modulated by other drugs interacting with this cytochrome oxidase. Despite the one-fits-all approach in administration of crizotinib at a fixed dose the pharmacokinetic parameters indicate a stable steady state upon continuous administration, which achieves sufficient inhibition of the ALK drug target.

AIMS Nuclear imaging may have an increasing role in the diagnosis of hepatocellular carcinoma (HCC). The aim of this study was to compare prospectively the Gallium-67 citrate ((67)Ga) scintigraphy results with those obtained by positron emission tomography (PET) using (11)C-acetate in patients with HCC. METHODS We prospectively analysed 21 patients (mean age, 64+/-11 years) with histopathologically verified HCC undergoing (11)C-acetate PET and (67)Ga scintigraphy. (67)Ga scans were not performed in three of these 21 patients due to the exacerbation of the disease. Whole-body (11)C-acetate PET were performed following intravenous injection of 850 MBq of (11)C-acetate. For (67)Ga scintigraphy, whole-body, planar and single photon emission computed tomography imaging acquisitions were performed after intravenous application of a mean dose of 189 MBq (67)Ga. RESULTS (67)Ga scintigraphy found abnormalities only in 10 of 18 patients (56%) and detected 22 of 46 clinically involved sites (48%); it was false-positive in two patients. (11)C-acetate PET found abnormalities in 14 of 18 patients (78%) and detected 36 of 46 clinical lesions (78%); it was false-positive in one patients. In one patient with left supraclavicular lymph node metastases, neither the (67)Ga scintigraphy nor the conventional computed tomography have shown the lesions, which were clearly demonstrated by the (11)C-acetate PET. CONCLUSION Our results indicate significantly higher sensitivity and specificity of (11)C-acetate PET than (67)Ga scan in detection of HCC lesions. This study suggests that imaging with (11)C-acetate PET might play a potential role in the diagnostic workup of patients with HCC.

Small cell lung cancer (SCLC) is distinguished by excessive numbers of circulating tumor cells (CTCs) in extended and recurring disease. This malignancy has a poor prognosis due to rapid emergence of chemoradioresistant relapses after first-line chemotherapy. In vitro expansion of several CTC lines allowed for a detailed study of the contribution of these cells to metastasis. Generation of CTCs involves the establishment of co-cultures and recruitment of macrophages and specific cytokines. All cell lines show E-cadherin-positive epithelial-like cells and spontaneous assembling into very large tumorospheres. Such multicellular aggregates seem to be responsible for the observed broad resistance due to limited access to drugs, quiescent cell layers, and hypoxic cores.

Primary pulmonary hypertension is a fatal disease causing progressive right heart failure within 3 years after diagnosis. We describe a new concept for treatment of the disease using vasoactive intestinal peptide, a neuropeptide primarily functioning as a neurotransmitter that acts as a potent systemic and pulmonary vasodilator. Our rationale is based on the finding of a deficiency of the peptide in serum and lung tissue of patients with primary pulmonary hypertension, as evidenced by radioimmunoassay and immunohistochemistry. The relevance of this finding is underlined by an upregulation of corresponding receptor sites as shown by Northern blot analysis, Western blot analysis, and immunological techniques. Consequently, the substitution with the hormone results in substantial improvement of hemodynamic and prognostic parameters of the disease without side effects. It decreased the mean pulmonary artery pressure in our eight study patients, increased cardiac output, and mixed venous oxygen saturation. Our data provide enough proof for further investigation of vasoactive intestinal peptide and its role in primary pulmonary hypertension.

Chemotherapeutic interventions in cancer patients are limited by the appearance of chemoresistance. For instance, advanced lung and ovarian cancer patients relapse invariably after few cycles of platinum-based chemotherapy. Disseminated tumors are characterized by genetic instability/heterogeneity, thus containing or generating a repertoire of resistant subpopulations. At the cellular level, altered drug uptake, efflux, and metabolization, as well as modifications of drug targets, increased repair, and decreased cell death complement the limited perfusion and adverse hypoxic/acidic extracellular conditions at the tumor level in retaining cancer cell viability. Similarly, targeted therapy is rendered ineffective by mutations of the specific target protein within a few months or years of administration. Assessment of the expression profiles of resistant tumor cells revealed extensive changes in numerous pathways affecting hundreds of genes. Therefore, reversal of drug resistance will require individual profiles of drug resistance mediators and the combination of several specific drugs, targeting critical components to provide new therapeutic options.

Lymphoma of the mucosa-associated lymphoid tissue (MALT) type usually arises in MALT acquired through chronic antigenic stimulation triggered by persistent infection and/or autoimmune processes. Due to specific ligand–receptor interactions between lymphoid cells and high-endothelial venules of MALT, both normal and neoplastic lymphoid cells display a pronounced homing tendency to MALT throughout the body. In the case of neoplastic disease these homing properties may be responsible for lymphoma dissemination among various MALT-sites. According to this concept, we have standardized staging procedures in all patients diagnosed with MALT-type lymphoma. All patients with MALT-type lymphoma underwent standardized staging procedures before treatment. Staging included ophthalmologic examination, otolaryngologic investigation, gastroscopy with multiple biopsies, endosonography of the upper gastrointestinal tract, enteroclysis, colonoscopy, computed tomography of thorax and abdomen and bone marrow biopsy. Biopsy was performed in all lesions suggestive for lymphomatous involvement, and evaluation of all biopsy specimens was performed by a reference pathologist. 35 consecutive patients with histologically verified MALT-type lymphoma were admitted to our department. Twenty-four patients (68%) had primary involvement of the stomach, five (15%) had lymphoma of the ocular adnexa, three (8.5%) had lymphoma of the parotid, and three (8,5%) of the lung. Lymph-node involvement corresponding to stage EII disease was found in 13 patients (37%), only one patient with primary gastric lymphoma had local and supradiaphragmatic lymph-node involvement (stage EIII). Bone marrow biopsies were negative in all patients. Overall, eight of 35 patients (23%) had simultaneous biopsy-proven involvement of two MALT-sites: one patient each had lymphoma of parotid and lacrimal gland, conjunctiva and hypopharynx, conjunctiva and skin, lacrimal gland and lung, stomach and colon, and stomach and lung. The remaining two patients had bilateral parotideal lymphoma. Staging work-up was negative for lymph-node involvement in all of these eight patients. The importance of extensive staging in MALT-type lymphoma is emphasized by the demonstration of multiorgan involvement in almost a quarter of patients. In addition, our data suggest that extra-gastrointestinal MALT-type lymphoma more frequently occurs simultaneously at different anatomic sites than MALT-type lymphoma involving the GI-tract. © 2000 Cancer Research Campaign

BACKGROUND In an intermittent androgen suppression therapy (IAS) trial we observed regular cycles of tumor suppression and regrowth in the majority of patients (17 +/- 2.6 month), with a subpopulation of patients (14 of 72) exhibiting a prolonged response of 28 +/- 7 month (range 18-64+ month) to the first eight-month androgen suppression cycle. PURPOSE To compare clinical data and laboratory tests (testosterone, prostate-specific antigen, and tissue polypeptide-specific antigen) of matched IAS patients showing either regular treatment cycles (n = 16) or prolonged response (n = 14). RESULTS Periods of androgen suppression resulted in reversible reduction of serum testosterone (< 1 nmol/l), PSA (< 1 ng/ml) indicating partial growth arrest and apoptotic regression of the tumors. The long-term response subgroup showed significantly lower mean values of tumor gradings, of pretreatment PSA values (11.36 +/- 4.54 vs. 47.5 +/- 12.4 ng/ml PSA), of PSA nadirs during androgen suppression (0.5 vs. 1 ng/ml PSA), and of overall testosterone values (3.9 +/- 1.14 vs. 6.6 +/- 1.18 mmol/l pretreatment) associated with low TPS values. CONCLUSIONS Patients exhibiting prolonged response to a single cycle of androgen suppression during IAS are characterized by a lower tumor burden, slow growing and less aggressive tumors, lower testosterone serum concentrations and lower absolute PSA nadirs during androgen suppression compared to a matched control group of short-term responders.

Purpose: Camptothecin (CPT) is a potent topoisomerase I inhibitor that has recently been undergoing phase I clinical trials. Though CPT shows high activity against various tumor cells, its biotransformation is still unknown. To investigate the metabolism and biliary excretion of CPT, an isolated perfused rat liver system was used. Methods: CPT was added to the perfusion medium at a concentration of 20 μM, and bile and perfusate samples were collected for 90 min. CPT (lacton and carboxylate) and three novel metabolites were identified by mass spectroscopy and quantified by reversed-phase high-performance liquid chromatography (HPLC). Kinetic parameters of CPT and its biotransformation products were then estimated in bile and effluent perfusate. Results: Biliary secretion of CPT and its three metabolites reached a peak secretion of 37.6 ± 16.3, 0.94 ± 0.29, 0.19 ± 0.023 and 0.302 ± 0.076 nmol/g liver/min, respectively, after 20 min. The total amount of CPT and M1–M3 excreted into bile during 90 min of perfusion was 63.5 ± 15.4%, 1.8 ± 0.37%, 0.43 ± 0.06%, and 0.72 ± 0.15% of CPT cleared from the perfusate during 90 min, respectively. In the perfusate, only one metabolite (M3) could be detected (cumulative release into the perfusion medium: 0.37 ± 0.026 μmol/liver). Analysis of the biliary metabolites by mass spectroscopy supported the existence of dihydroxy-CPT derivatives (M1 and M2), whereas M3 appears to be a monohydroxy-analog. Conclusion: CPT is biotransformed to three novel metabolites, mainly excreted into bile. The possible pharmacological effects of these new metabolites need to be considered.

Long acting somatostatin-14 (SST) analogs are used clinically to inhibit tumor growth and proliferation of various tumor types via binding to specific receptors (R). We have developed a 111In-/90Y-labeled SST analog, DOTA-(d)βNal1-lanreotide (DOTALAN), for tumor diagnosis and therapy. 111In-/90Y-DOTALAN bound with high affinity (dissociation constant, Kd, 1–12 nm) to a number of primary human tumors (n = 31) such as intestinal adenocarcinoma (n = 17; 150-4000 fmol/mg protein) or breast cancer (n = 4; 250-9000 fmol/mg protein). 111In-/90Y-DOTALAN exhibited a similar high binding affinity (Kd, 1–15 nm) for the human breast cancer cell lines T47D and ZR75–1, the prostate cancer cell lines PC3 and DU145, the colonic adenocarcinoma cell line HT29, the pancreatic adenocarcinoma cell line PANC1, and the melanoma cell line 518A2. When expressed in COS7 cells, 111In-DOTALAN bound with high affinity to hsst2 (Kd, 4.3 nm), hsst3 (Kd, 5.1 nm), hsst4 (Kd, 3.8 nm), and hsst5 (Kd, 10 nm) and with lower affinity to hsst1...

BACKGROUND The present study evaluated monthly measurements of free and total prostate-specific antigen (PSA), and the tumor proliferation markers tissue polypeptide-specific antigen (TPS) and cytokeratin fragment 21-1 (CYFRA 21-1) in patients with advanced prostate cancer receiving intermittent androgen suppression therapy (IAS). METHODS Thirty-four men received alternating cycles of 8 month androgen suppression and treatment cessation (mean duration, 10.3 months) until PSA increased to >20 μg/l. Measurements of testosterone, percentage of free PSA, TPS, and CYFRA 21-1 were performed using ELISA and RIA assays. RESULTS Periods of androgen suppression resulted in reversible reductions of testosterone (from 6 ± 0.8 to

Multicellular spheroids (MCS) have been used as an in vitro model system of micrometastases and avascular tumor regions for studying cell adhesion-dependent resistance to cytotoxic drugs and possible reversal by chemosensitizers and adhesion-reversing agents. Multicellular drug resistance has been linked to limited accessibility of cell subpopulations, active drug efflux, quiescence of cells in deeper layers due to cell contact inhibition and adverse microenvironmental conditions like acidic extracellular pH, hypoxia and nutritional depletion. The shortcomings of MCS as a tumor model include limited knowledge of the mechanisms leading to necrosis/apoptosis of core cells, the production of an extracellular matrix (ECM) by tumor cells instead of intratumoral normal cell populations and the complex relationship of MCS parameters like size, growth regulation, synthesis of ECM components and others on the origin and pretreatment of the tumor cells and specific culture conditions.

The excessive uncontrolled activation of inflammatory cells and mediators after trauma or major surgery plays a key role in the development of adult respiratory distress syndrome and multiple organ system failure (MOSF). In the past elevated cytokine levels were shown to influence the outcome of these patients adversely. There are diverging results regarding the removal of circulating cytokines by various methods of hemopurification for clinical improvement of MOSF. Seven patients after trauma or major surgery underwent continuous venovenous hemofiltration (CVVH) for the treatment of severe organ failure of the heart and lungs (Murray score 2.74) but not for renal or liver failure. The cytokine levels were measured at the beginning and 15, 60, 120, and 240 minutes after initiation of CVVH (measure points MP1–5). Clinical improvement during the treatment was monitored, and correlation with cytokine levels was evaluated. Arterially measured tumor necrosis factor α rose from 11.14 ng/ml to 17.86 ng/ml (p < 0.05). Arterial interleukin-6 (IL-6) levels significantly decreased during CVVH from 1284.7 ng/ml to 557.9 ng/ml; IL-8 levels simultaneously decreased from an initial peak of up to 154.4 ng/ml at MP3 to 97.3 ng/ml at MP5. The drop in serum IL-6 and IL-8 levels closely correlated with clinical improvement. After 2 hours of CVVH the hemodynamic situation improved significantly, as revealed by a decrease in catecholamine expenditure, an increase in arterial pressure, and a decrease in pulmonary artery pressure. Moreover, 2 hours after the initiation of CVVH the oxygenation index rose significantly and correlated well with the drop in shunt fraction. The Murray score significantly fell to 1.86. The removal of IL-6 and IL-8 by CVVH after initial stimulation correlates with clinical improvement, which was demonstrated by significantly improved oxygenation and hemodynamics from 2 hours after the initiation of CVVH onward. The elimination of cytokines and several mediators by CVVH may contribute to the cardiopulmonary improvement of critically ill patients. In comparison with the clinical control group ( n = 7), which was comparable in terms of MOSF, no intervention led to a similar improvement in cardiorespiratory failure, and overall two of these patients died. Moreover, patients of the control group experienced a significant longer stay at in the intensive care unit.

Epidermal growth factor (EGF) exhibits a cytoprotective effect on gastrointestinal epithelia via a receptor-mediated mechanism. We investigated the effect of EGF on Clostridium difficile toxin A (TxA)- and toxin B (TxB)-induced damage of human colon. Ussing-chambered colonic mucosa was exposed serosally to EGF before and during luminal exposure to TxA and TxB. Resistance was calculated from potential difference and short-circuit current. Epithelial damage was assessed by light microscopy and alteration of F-actin by fluoresceinated phalloidin. Luminal exposure of colonic strips to TxA and TxB caused a time- and dose-dependent decrease in electrical resistance, necrosis and dehiscence of colonocytes, and disruption and condensation of enterocyte F-actin. These effects were inhibited by prior, but not simultaneous, serosal application of EGF (20 nM). Administration of the tyrosine kinase inhibitor genistein (10−6M) inhibited the protective effects of EGF. We conclude that EGF protects against TxA and TxB probably by stabilizing the cytoskeleton, the main target of these toxins.

For the development of lectin-mediated drug delivery systems, the proteolytic stability of the non-toxic lectins from Arachis hypogea, Lens culinaris, Dolichus biflorus, Solanum tuberosum (STL) and Triticum vulgare was investigated by in vitro exposure to gastrointestine-located enzymes. No degradation products were observed within 24 h of incubation on sodium dodecyl sulfate polyacrylamide gels. Binding to human colon carcinoma cell lines was investigated by flow cytometry. The fluorescein-labelled derivatives of N-acetylglucosamine-specific wheat germ agglutinin (WGA) and STL exhibited the highest cell-associated fluorescence intensity. As determined by dilution experiments, the number of WGA-binding sites on Caco-2, HT-29 and human colonocytes exceeded those for STL by 5-, 1.7- and 1.4-fold, respectively. By a competitive flow cytometric assay using N,N′, N″-triacetylchitotriose for inhibition, WGA-affinity exceeded STL-affinity by ten-fold. The affinity of each lectin to Caco-2, HT-29 and human colonocytes was about the same, indicating that similar lectin receptors were involved. Preventing N-glycosylation of the carcinoma cells by pretreatment with 0.001% tunicamycin for 40 h resulted in 30% inhibition of WGA- and STL-binding. When WGA was covalently attached to Sepharose beads (250–350 μm), the interaction with HT-29 and Caco-2 cells showed stable and tight binding. Therefore, especially considering the comparable affinity of human colonocytes and monolayer-forming Caco-2 and HT-29 cells, this system is proposed as a model for the development of lectin-mediated particulate pharmaceutical devices.

For use in development of receptor-specific bioadhesive drug delivery systems, the interaction between K99-fimbriae and porcine enterocytes is proposed as an in vitro model system for targeting drugs to specific regions of the intestine. K99-fimbriae resisted proteolytic attack of pepsin, trypsin, chymotrypsin, pancreatin and elastase as a prerequisite for peroral administration of bioadhesive formulations. At pH 2.5 native K99-fimbriae dissociated into the corresponding subunits still exhibiting high affinity to the K99-receptor. K99-fimbriae were found to bind specifically to viable duodenal and jejunal enterocytes of porcine small intestine using a competitive flow cytometric assay. Receptor-specificity of adhesion was confirmed by lacking affinity to gastric mucin as well as human colon carcinoma derived cell lines Caco-2, SW620, SW480 and human colonocytes. Upon coupling of methylprednisolone to the bioadhesive protein, increased binding by apparently non-specific mechanisms was observed due to lipophilicity of the substituent. Thus hydrophilicity of the coupled drug as well as the degree of substitution influence tissue targeting of receptor-specific bioadhesive formulations.

Grundlagen: Die schnelle Restitution ermoglicht den raschen Schlus von Epitheldefekten durch Enterozytenmigration. Wir untersuchten den Einflus extrazellularer Matrixproteine auf die Restitution der sauregeschadigten Mukosa des Kaninchenduodenums in vitro. Methodik: Schleimhautpraparationen des Kaninchenduodenums wurden in Ussing-Kammern eingebracht und nach einer 30minutigen Aquilibrierungsphase durch 10 min luminale Exposition mit 10 mM HCl geschadigt. Danach wurden die Gewebe 3 Stunden mit Puffer (Kontrollen) oder Puffer, der auf der luminalen Seite die Testsubstanzen enthielt, inkubiert (n=6 pro Gruppe). Potentialdifferenz und Kurzschlusstrom wurden gemessen, der Widerstand (R) nach dem Ohmschen Gesetz berechnet. Nach Versuchsende wurden HE-gefarbte histologische Schnitte hergestellt, das Ausmas der Schadigung mit einer speziellen Morphometrieanlage gemessen. Ergebnisse: Nach Schadigungsende waren 58±4%, nach 1 und 3 h 40±4% bzw. 36±2% geschadigt (Kontrollen, n=6). Nach HCl-Exposition losten sich die geschadigten Enterozyten der oberen 2 Zottendrittel ab, so das luminal denudierte Stummelzotten resultierten, welche 3 h nach Schadigung teilweise von flachen Enterozyten bedeckt waren. Luminales Rinderserumalbumin (38±5%), Antikorper gegen Kollagen IV (35±7%) und Fibronektin (38±4%) sowie jeweils 25, 50 und 100 µg/ml Kollagen IV (34±3, 35±2 bzw. 33±4%) und Fibronektin (39±2, 36±4 bzw. 36±2%) hatten keinen Einflus auf die Restitution (n=6). Antilaminin fuhrte zu einer signifikanten Zunahme der Schadigung auf 64±5% (p

Grundlagen: Clostridium difficile ist der Erreger der Antibiotika-assoziierten Enterokolitis. In Tierstudien schadigt Clostridium difficile Toxin A, aber nicht Toxin B die Schleimhaut des Gastrointestinaltraktes. Die Wirkung der Toxine auf die humane Kolonschleimhaut ist unbekannt. Deshalb untersuchten wir in dieser Studie die Wirkung von Clostridium difficile Toxin A (TxA) und Toxin B (TxB) auf die humane Kolonschleimhaut in vitro.

Platinum-based chemotherapeutics are the mainstay of treatment of a range of tumors achieving high response rates but limited in the course of disease by appearance of drug resistance. Tumor cells respond with reduced uptake and increased intracellular inactivation of the drugs, as well as increased DNA repair and general resistance to chemotherapyinduced cell death. Cisplatin is known to induce expression of cyclophilins, a group of proteins that have peptidyl-prolyl cis-trans isomerase (PPIase) and molecular chaperone activities, as stress response. Cyclophilin A (CypA) and other members of this family are inhibited by cyclosporin A (CsA) which sensitized diverse drug-resistant tumor cell lines in vitro to cisplatin. This effect of CsA was attributed to metabolic changes, inhibition of DNA repair, enhancement of apoptosis, altered intracellular signal transduction or increased production of reactive oxygen species (ROS), although no definitive explanation was provided so far. Several clinical trials employing cisplatin/carboplatin in combination with CsA yielded unsatisfactory results. Since viral replication was found to be dependent on cyclophilins of the host cells, effective new inhibitors, different from CsA or with low or absent immunosuppressive activity, are in development or clinical trials. Sanglifehrins are more potent than CsA and proved to increase toxicity of cisplatin against hepatocellular cancer cells in vitro. These novel cyclophilin inhibitors may offer new opportunities to achieve reversal of resistance to platinumbased drugs in refractory patients. Responsive cancer patients may be enriched in clinical trials by an identification of the downstream targets of Cyps responsible for chemoresistance.

Morpholino-disiloxane (ALIS-409) and piperazino-disiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate-early antigen (EBV-EA) expression induced by tetradecanoyl-phorbol-acetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study. The application of dimethyl-benzanthracene (DMBA; 390 nmol) as a tumor initiator was followed by exposure to TPA (1.7 nmol/l) as a tumor promoter. The experiments showed that ALIS-409 at a concentration of 85 nmol/l had a weak EBV-EA inhibitory effect in vitro and a moderate antitumor activity, compared to the positive control of DMBA plus TPA-treated mice. Flow cytometry by differential staining demonstrated interactions in co-cultures of MCF7 breast cancer and MRC5 human lung fibroblasts. The growth rate of tumor cells in mixed populations of MCF7 breast cancer and MRC5 normal fibroblast cells was reduced in the presence of ALIS-409, as compared to the control non-treated cell populations. The two disiloxanes were moderately-effective in chemoprevention in DMBA-induced and TPA-promoted in vivo tumor formation. Authors suggest that the inhibition of tumor cell and fibroblast interaction by ALIS409 might have some perspective in the development of anti-stromal therapy.

The physiological role of the multidrug resistance P-glycoprotein (P- gp), which is expressed by normal human T lymphocytes, is still largely unknown. To investigate whether or not P-gp is involved in the transport of cytokines, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA) in the absence or presence of P-gp inhibitors, and concentrations of cytokines (interleukin-2 [IL-2], IL- 4, IL-6, interferon-gamma [IFN-gamma]) in the supernatants of these cultures were quantitated by enzyme-linked immunosorbent assay. P-gp inhibitors included verapamil (Ver), tamoxifen (Tmx), and the P-gp specific monoclonal antibody UIC2. Release of IL-2 was significantly suppressed by these inhibitors at concentrations that were also effective in blocking efflux of Rhodamine-123 from normal T lymphocytes. IL-2 mRNA expression in lymphocytes was not different between PHA control and the cultures with P-gp inhibitors. Ver and Tmx did not interfere with T-cell activation as determined by CD25 and CD69 expression. In a nonhematological model, the P-gp expressing HCT-8 adenocarcinoma cell line, exogenously added IL-2 was shown to exert an inhibitory effect on P-gp mediated Rhodamine-123 efflux. In addition, transepithelial transport of IL-2 by electrophysiologically tight and polarized HCT-8 monolayers was examined. A time-dependent flux of IL-2 across dense monolayers, which was partially inhibited by Ver, was observed. We also investigated whether or not P-gp inhibitors suppressed release of other cytokines produced by activated T cells (IL- 4, IL-6, IFN-gamma). Release of IL-4 and IFN-gamma was significantly inhibited by Ver, Tmx, and UIC2; however, release of IL-6 remained unaffected. These data show P-gp mediated transmembrane flux of IL-2 in T lymphocytes and HCT-8 cells. We conclude that P-gp participates in the transport of cytokines (IL-2, IL-4, and IFN-gamma) in normal peripheral T lymphocytes.

Growth factors are mainly involved in the regulation of intestinal epithelial barrier function. This study investigated the effect of epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) on epithelial restitution of rabbit duodenum in vitro.Rabbit duodenal mucosal strips mounted in an Ussing chamber were luminally exposed to 10 mmol/L HCl for 10 minutes and then incubated with buffer alone or luminal buffer containing various concentrations of EGF and IGF-1 for 3 hours. Resistance was calculated from potential difference and short-circuit current. Damage was assessed by morphometry on HE-stained sections.HCl caused resistance to decrease from 112 +/- 2 to 51 +/- 4 ohms x cm2 10 minutes after injury (n = 6; P0.05). Postinjury treatment with 25 or 50 ng/mL luminal EGF for 3 hours stimulated resistance to recover to 94 +/- 3 and 104 +/- 3 ohms x cm2, respectively, vs. 81 +/- 3 omega x cm2 in controls (P0.05). Ten minutes after injury, 62% of the mucosa was damaged; 3 hours after injury, damage was reduced to 24% +/- 1.09% and 10% +/- 1.42% in the 25 and 50 ng/mL EGF group, respectively, vs. 38% +/- 0.93% in controls (n = 6 per group). EGF stimulated enterocyte migration. IGF-1 did not impair epithelial restitution.EGF, but not IGF-1, promoted epithelial restitution of rabbit duodenum in vitro.

Mechanisms of intracellular pH (pHi) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pHi regulation in colonic tumors. pHi changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [3H]thymidine uptake. Resting pHi in Hepes-buffered solution was 7.53 +/- 0.01 (n = 36). Both 1 mM amiloride and Na(+)-free solution inhibited pHi recovery from acidification and decreased pHi in resting cells. In HCO3-/CO2-buffered media resting pH1 was 7.42 +/- 0.01 (n = 36). Recovery from acidification was Na(+)-dependent, CI(-)-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 microM H2DIDS pHi recovery was completely inhibited. In Na(+)-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 (n = 6) and no alkalinization was observed in CI(-)-free medium. Addition of 5 microM tributyltin bromide (an anion/OH-exchange ionophore) caused pHi to decrease from 7.43 +/- 0.05 to 7.17 +/- 0.08 (n = 5). The effects of pH0 on steady-state pHi, pHi recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [CO2] and [HCO3-]. In general, increases in pH0 between 6.7 and 7.4 increased pHi recovery, steady-state pHi and growth rates. In summary, SW-620 cells have a resting pHi > 7.4 at 25 degrees C, which is higher than other intestinal cells. Acid extrusion in physiological bicarbonate media is accomplished by a pHi-sensitive Na+/H+ exchanger and a pHi-insensitive Na(+)-HCO3-cotransporter, both of which are operational in control cells at the resting pHi. No evidence for activity of a CI-/HCO3- exchanger was found in these cells, which could account for the high pHi observed and may explain why the cells continue to grow in acidic tumor environments.

The extragonadal synthesis of biological active steroid hormones from their inactive precursors in target tissues is named “intracrinology.” Of particular importance for the progression of estrogen-dependent cancers is the in situ formation of the biological most active estrogen, 17beta-estradiol (E2). In cancer cells, conversion of inactive steroid hormone precursors to E2 is accomplished from inactive, sulfated estrogens in the “sulfatase pathway” and from androgens in the “aromatase pathway.” Here, we provide an overview about expression and function of enzymes of the “sulfatase pathway,” particularly steroid sulfatase (STS) that activates estrogens and estrogen sulfotransferase (SULT1E1) that converts active estrone (E1) and other estrogens to their inactive sulfates. High expression of STS and low expression of SULT1E1 will increase levels of active estrogens in malignant tumor cells leading to the stimulation of cell proliferation and cancer progression. Therefore, blocking the “sulfatase pathway” by STS inhibitors may offer an attractive strategy to reduce levels of active estrogens. STS inhibitors either applied in combination with aromatase inhibitors or as novel, dual aromatase-steroid sulfatase inhibiting drugs are currently under investigation. Furthermore, STS inhibitors are also suitable as enzyme–based cancer imaging agents applied in the biomedical imaging technique positron emission tomography (PET) for cancer diagnosis.

Toxin A but not toxin B, appears to mediate intestinal damage in animal models of Clostridium difficile enteritis. The purpose of this study was to investigate the electrophysiologic and morphologic effects of purified C. difficile toxins A and B on human colonic mucosa in Ussing chambers. Luminal exposure of tissues to 16-65 nM of toxin A and 0.2-29 nM of toxin B for 5 h caused dose-dependent epithelial damage. Potential difference, short-circuit current and resistance decreased by 76, 58, and 46%, respectively, with 32 nM of toxin A and by 76, 55, and 47%, respectively, with 3 nM of toxin B, when compared with baseline (P < 0.05). 3 nM of toxin A did not cause electrophysiologic changes. Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficile colitis in man.