Biomolecular characterization of human glioblastoma cells in primary cultures: differentiating and antiangiogenic effects of natural and synthetic PPARgamma agonists.

Gliomas are the most commonly diagnosed malignant brain primary tumors. Prognosis of patients with high-grade gliomas is poor and scarcely affected by radiotherapy and chemotherapy. Several studies have reported antiproliferative and/or differentiating activities of some lipophylic molecules on glioblastoma cells. Some of these activities in cell signaling are mediated by a class of transcriptional factors referred to as peroxisome proliferator-activated receptors (PPARs). PPARgamma has been identified in transformed neural cells of human origin and it has been demonstrated that PPARgamma agonists decrease cell proliferation, stimulate apoptosis and induce morphological changes and expression of markers typical of a more differentiated phenotype in glioblastoma and astrocytoma cell lines. These findings arise from studies mainly performed on long-term cultured transformed cell lines. Such experimental models do not exactly reproduce the in vivo environment since long-term culture often results in the accumulation of further molecular alterations in the cells. To be as close as possible to the in vivo condition, in the present work we investigated the effects of PPARgamma natural and synthetic ligands on the biomolecular features of primary cultures of human glioblastoma cells derived from surgical specimens. We provide evidence that PPARgamma agonists may interfere with glioblastoma growth and malignancy and might be taken in account as novel antitumoral drugs.

Differential expression of the components of the plasminogen activating system in human thyroid tumour derived cell lines and papillary carcinomas.

We characterised the expression of the plasminogen activators (uPA and tPA), the uPA receptor (uPAR) and the PAs inhibitors (PAI-1 and PAI-2) in human thyroid cell lines derived from normal thyroid, follicular adenoma, follicular, papillary and anaplastic carcinomas. Urokinase PA activity was detected in the supernatant of normal thyrocytes and augmented in those of all tumour cells. Quantitative RT-PCR analysis showed that uPA, uPAR and PAI-1 mRNAs increased in all carcinoma cells. Similar results were found in 13 papillary thyroid carcinoma (PTC) tissues which were mirrored in Western blot experiments. A correlation was found between tumour size and uPA mRNA increase, and higher levels of uPA and uPAR mRNAs were found in metastatic PTC. In conclusion, thyroid carcinoma cell lines and PTC overexpress uPA, uPAR and PAI-1 and the correlation of uPA and its cognate receptor with tumour size and metastasis may suggest their potential prognostic relevance in thyroid cancer.

Molecular aspects of gefitinib antiproliferative and pro-apoptotic effects in PTEN-positive and PTEN-negative prostate cancer cell lines.

To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer (PCa) to more invasive forms. One of the major targets for the therapy in PCa can be epidermal growth factor receptor (EGFR), which signals via the phosphoinositide 3'-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, among others. Despite multiple reports of overexpression in PCa, the reliance on activated EGFR and its downstream signalling to the PI3K and/or MAPK/extracellular signal-regulated kinase (ERK) pathways has not been fully elucidated. We reported that the EGFR-selective tyrosine kinase inhibitor gefitinib (ZD1839; Iressa) is able to induce growth inhibition, G(1) arrest and apoptosis in PCa cells and that its effectiveness is associated primarily with phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression (and thus Akt activity). In fact PTEN-negative PCa cells are slowly sensitive to gefitinib treatment, because this molecule is unable to downregulate PI3K/Akt activity. PI3K inhibition, by LY294002 or after PTEN transfection, restores EGFR-stimulated Akt signalling and sensitizes the cells to pro-apoptotic action of gefitinib. The MAPK pathway seems to be involved primarily on cell-growth modulation because dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition (both not cell apoptosis) in PTEN-positive PCa cells and reduced EGF-mediated growth in PTEN-negative cells. Thus the effectiveness of gefitinib requires growth factor receptor-stimulated PI3K/Akt and MAPK signalling to be intact and functional. The loss of the PTEN activity leads to uncoupling of this signalling pathway, determining a partial gefitinib resistance. Moreover, gefitinib sensitivity may be maintained in these cells through its inhibitory potential in MAPK/ERK pathway activity, modulating proliferative EGFR-triggered events. Therefore, our data suggest that the inhibition of EGFR signalling can result in a significant growth reduction and in increased apoptosis in EGFR-overexpressing PCa cells with different modalities, which are regulated by PTEN status, and this may have relevance in the clinical setting of PCa.

Expression of matrix metalloproteinases and their specific inhibitors in normal and different human thyroid tumor cell lines.

In the present study we investigated, by means of zymography and reverse transcription-polymerase chain reaction (RT-PCR), the expression of different matrix metalloproteinases (MMPs) and of the specific tissue inhibitor of metalloproteinases [TIMPs] in human cell lines derived from normal thyrocytes (HTU5), follicular adenoma (HTU42), and follicular (FTC-133), papillary (B-CPAP), and anaplastic (CAL-62, 8305C) thyroid carcinomas. We demonstrated that normal thyrocytes constitutively express MMP-1, MMP-2, MMP-10, MMP-14, and TIMP-1, TIMP-2, TIMP-3, and TIMP-4, and this pattern of expression is profoundly modified in all thyroid tumor-derived cell lines. Analysis of the gelatinolytic activity in the different cell supernatants showed that the expressions of MMP-2 and MMP-9 are, respectively, increased or induced in all the neoplastic cell lines, except in CAL-62. Caseinolytic activity was found only in the supernatants of the 8305C and B-CPAP cells. Using RTPCR analysis we detected an increased expression of MMP-1 in cell lines derived from papillary and from one (8305C) of the two anaplastic carcinomas. MMP-13 mRNA was expressed only in the 8305C, FTC-133, and BCPAP cells. Among stromelysins, MMP-3 mRNA could not be detected in any cell line, while MMP-10 mRNA was expressed in all of them, although at variable levels. MMP-11 mRNA was absent in normal and follicular adenoma derived thyrocytes and induced in all carcinoma cell types. The expression of MMP-14 (MT1-MMP) mRNA was found significantly increased in all thyroid tumor cell lines with respect to HTU5 and HTU42 cells. The expression of TIMP-1 and TIMP-2 mRNAs was maintained in all cell lines tested, while that of TIMP-3 was lost in both anaplastic carcinoma cell lines and that of TIMP-4 was absent in the CAL-62. In conclusion, our data demonstrated a differential expression of MMPs and TIMPs in different thyroid tumor cell types with respect to normal thyrocytes. In particular, the induction of MMP-11 in all thyroid-derived carcinoma cell lines studied and of MMP-13 in all but one may represent, if confirmed in other thyroid tumor-derived cell lines and in thyroid tumor tissues, a new marker of thyrocyte transformation.

Apoptotic effects of selected strains of lactic acid bacteria on a human T leukemia cell line are associated with bacterial arginine deiminase and/or sphingomyelinase activities.

The aim of the present work was, first, to analyze the apoptotic effect in vitro of sonicated preparations of selected strains of lactic acid bacteria on normal and tumor human lymphocytes. Incubation with bacterial samples led to a relevant time-dependent apoptotic cell death of Jurkat cells but not normal human peripheral blood lymphocytes. Lactobacillus brevis (CD2) samples were more efficient in inducing apoptosis of Jurkat cells than were samples of Streptococcus thermophilus (S244). In an attempt to characterize the mechanisms underlying these effects, we found that the apoptotic death-inducing ability of S244 preparations could be attributed to the ability of high levels of neutral sphingomyelinase activity to generate relevant amounts of ceramide, a known apoptotic death messenger, in Jurkat cells. On the other hand, our results indicate that apoptosis induced by CD2 samples could also be associated with high levels of arginine deiminase activity, which in turn was able to downregulate polyamine synthesis in Jurkat cells.

R- and S-isomers of nonsteroidal anti-inflammatory drugs differentially regulate cytokine production.

2-arylpropionic acids, a well known class of non-steroidal anti-inflammatory drugs (NSAIDs), exist as a racemic mixture of their enantiomeric forms, with S-isomers primarily responsible for inhibition of prostaglandin (PG) production and of inflammatory events. In this study we show that S-isomers are also responsible for the paradoxical up-regulation of tumor necrosis factor (TNF) induced by ketoprofen, flurbiprofen and ibuprofen in murine peritoneal macrophages stimulated by bacterial endotoxin (LPS). This effect is in close correlation with cyclooxygenase inhibitory capacity of S-isomers and, from Northern blot analysis, seems to be mediated by the up-regulation of TNF mRNA. In addition, up-regulation of TNF production by S-isomers is associated with inhibition of interleukin-10 (IL-10) production. Conversely, we have observed that S-enantiomers reduce IL-6 production at a concentration 100 times higher than that able to inhibit cyclooxygenase activity. The unwanted pro-inflammatory effects of S-isomers through TNF and IL-10 production could therefore hinder their analgesic effect, that is, at least in part, related to IL-6 inhibition. In addition, TNF amplification by S-isomers could be correlated to the clinical evidence of their gastric toxicity. On the other hand, R-isomers did not affect TNF and IL-10 production even at cyclooxygenase-blocking concentration, while they reduced IL-6 production to the same levels as S-isomers. It is concluded that the regulation of cytokine production by S-isomers of 2-arylpropionic acids could partially mask their therapeutic effects and could be correlated to the clinical evidence of their higher gastric toxicity. On the other hand, IL-6 inhibition without the unwanted effects on TNF and IL-10 production shown by R-isomers could be correlated to the analgesic effect reported for R-2-arylpropionic acids.

Erk-dependent cytosolic phospholipase A2 activity is induced by CD95 ligand cross-linking in the mouse derived Sertoli cell line TM4 and is required to trigger apoptosis in CD95 bearing cells.

In the present study we demonstrated that CD95L cross-linking generated reverse signalling in the mouse derived Sertoli cell line TM4. Treatment of TM4 cells with mAb anti-CD95L induced activation of the cytosolic phospholipase A2 (cPLA2). Cytosolic PLA2 activation was controlled by the MAPK pathway as indicated by the ability of the specific MEK inhibitor, PD098059, to abolish cPLA2 activation. In addition, Western blot experiments showed a rapid increase in phosphorylated Erk1/2 following CD95L cross-linking, while no effect on the phosphorylation of other MAPK, p38 or JNK, was observed. CD95L cross-linking by mAb increased the levels of soluble CD95L and apoptotic activity of TM4 cell supernatants, which was blocked by co-incubation with the PLA2 inhibitor, AACOCF3 or PD098059. Finally, pre-treatment of TM4 cells with AACOCF3 or PD098059 completely abolished TM4-induced apoptosis of Jurkat T cells, thus indicating that the Erk/cPLA2 pathway is required for CD95L-induced apoptosis.

Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-gamma production.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells. Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.

Finasteride dose-dependently reduces the proliferation rate of the LnCap human prostatic cancer cell line in vitro.

OBJECTIVES: To assess the effects of finasteride, a 5-alpha-reductase inhibitor, and of classic antiandrogens on the growth rate of the LnCap human prostate carcinoma cell line, derived from a primary and well-differentiated neoplasm. METHODS: Cell proliferation experiments in vitro with and without the antiandrogens cyproterone acetate, hydroxyflutamide, and finasteride in the 0.0001 to 10.0 microM range. RESULTS: The growth rate of the LnCap cell line can be dose-dependently inhibited by 5-alpha-reductase inhibition (finasteride) and by antiandrogens (cyproterone acetate and hydroxyflutamide) in vitro, in defined conditions. CONCLUSIONS: Besides other human prostate cell lines derived from metastatic sites (PC3, DU145), also in the LnCap cell line an autonomous androgen-dependent mechanism of growth stimulation can be hypothesized, since testosterone and dihydrotestosterone are unable to stimulate the cell proliferation rate at the same molar concentrations. The clinical implications of these results in prostate cancer therapy and the possible future use of these molecules in the prevention of cancer incidence are discussed.

Early diagnosis of prostatic carcinoma may be achieved through in vitro culture of tumor cells harvested by prostatic massage.

As a new method for early diagnosis of prostatic carcinoma we succeeded in growing in vitro the epithelial cells which can be collected from prostatic fluid after rectal prostatic massage. We report here the updated and statistically analyzed series of data (174 patients) on this new approach, which allows all the harvested cells to express their biological features. The method reaches a sensitivity of 72-86% and a specificity of 88-100%. This noninvasive test, which is also suitable for mass screening, may be very useful for an early diagnosis of the neoplasm.

Imipenem reaches therapeutic concentrations in aqueous humor, as determined by HPLC.

The majority of ocular infections in the industrialized countries arise after surgical interventions. An appropriate antibacterial prophylaxis is therefore highly desirable in eye surgery. We used high performance liquid chromatography (HPLC) to measure the concentrations reached by imipenem, a modern wide-spectrum beta-lactam antibiotic, in plasma and aqueous humor of 26 patients scheduled for cataract surgery. Even a single 500 mg dose of imipenem achieved therapeutic levels of the molecule for the most common ophthalmic pathogens (1 microgram/ml or above) in the aqueous humor within one to two hours after administration. This drug may therefore be suitable for antibacterial prophylaxis in eye surgery.

Simultaneous determination of simvastatin and its hydroxy acid form in human plasma by high-performance liquid chromatography with UV detection.

Simvastatin (SV), an analogue of lovastatin, is the lactone form of 1',2',6',7',8',8a'-hexahydro-3,5-dihydroxy-2',6'-dimethyl-8'(2'',2''-di met hyl-1''-oxobutoxy)-1'-naphthalene-heptanoic acid (SVA) which lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. A fast, simple and accurate method for determining SV and SVA concentrations in human plasma has been developed and validated for use in the analysis of plasma samples from patients and healthy volunteers. This method involves an extraction procedure using a mixture of acetonitrile-water and reversed-phase high-performance liquid chromatography with UV detection. The procedure was linear from 20 to 1000 ng ml-1 for SV and from 25 to 1000 ng ml-1 for SVA, respectively. The method was accurate with relative errors of 5.0, 2.1 and 3.2% for human plasma controls containing 50, 250 and 500 ng ml-1 of SV, respectively. The corresponding precision was 2.3, 1.8 and 1.0% (RSD%). Similarly, relative standard deviations less than 2.3% and relative errors of less than 5.2% were obtained from human plasma controls containing SVA at identical concentrations. The method is suitable for pharmacology and pharmacokinetic studies of simvastatin.

Establishment and characterization of a human prostatic carcinoma cell strain obtained from exfoliated tumor cells after prostatic massage.

During a control campaign connected to our main program on early diagnosis of prostatic carcinoma through tissue culture of prostatic fluid samples obtained after prostatic massage (M. Bologna et al., Eur. Urol., 14, 474-476, 1988), we isolated and characterized a human prostatic carcinoma cell strain from a 58-year-old patient with a grade III prostatic carcinoma. The epithelial cell strain, named PMU-23, has been passaged in vitro for 31 subculture cycles during a period of approximately 8 months, after which cell proliferation slowed down irreversibly. The isolation of this cell strain constitutes a renewed confirmation of the validity of our method for the early diagnosis of prostatic carcinoma and demonstrates some intermediate features in the progression of prostatic tumors. In addition, the study of limited-lifespan tumor cell strains in culture may extend the knowledge on prostatic cell biology, particularly toward the identification of intermediate steps of tumor progression, for a better approach of tumor therapy and prevention of metastatic spread.

Determination of imipenem in human plasma, urine and tissue by high-performance liquid chromatography.

A simple and reliable HPLC method is described for the new beta-lactam antibiotic imipenem; suitable extraction procedures for the drug in human plasma, urine and prostatic tissue are described. The figures of merit for the assays are reported and examples given of their application.

Bombesin stimulates growth of human prostatic cancer cells in vitro.

Cell proliferation of the human prostatic carcinoma cell line PC3 and of the epithelial cell strain PMU 23 derived from a primary culture of a stage III prostatic carcinoma was enhanced dose dependently by adding 0.1 nM to 10.0 nM bombesin (BMBS) to the culture medium. The growth stimulation was specifically inhibited by antibodies versus Gastrin Releasing Peptide (GRP) crossreacting with BMBS. Presence of BMBS-positive neuroendocrine cells in human prostate and measurable amounts of BMBS-like peptides in prostatic fluid were reported previously. In a binding assay using 125I-GRP, it was possible to demonstrate the presence of saturable specific receptors on PC3 cells, numerically comparable with those measured on small cell lung cancer cell lines. By immunofluorescence, however, no BMBS immunoreactivity on PC3 cells could be demonstrated. These observations suggest that BMBS plays a role in prostatic epithelium growth and that prostatic carcinoma may have an autocrine or paracrine proliferation stimulus within the gland microenvironment.

Early diagnosis of prostatic carcinoma based on in vitro culture of viable tumor cells harvested by prostatic massage.

Prostatic cancer is diagnosed too late in most cases, so that therapy is frequently ineffective or even not undertaken at all because of the already advanced stage of the disease. An early diagnosis technique for prostatic cancer would therefore be highly desirable, also because all other available markers give very unsatisfactory results. Because of our experience in tissue culture of human prostatic specimens, by which we have shown good correlations with patient prognosis, we attempted to grow epithelial cells collected from prostatic fluid after rectal prostatic massage. Samples from prostatic cancer patients, diagnosed by needle biopsy, were grown in culture and were able to survive in vitro for at least 2 weeks, thus providing morphological and biochemical data concerning their neoplastic and differentiation features. The early data on this new approach, which we believe might represent a very useful test for the early diagnosis of the neoplasm, are reported here. The method is noninvasive and suitable for mass screening of the disease. Accuracy and reliability of the technique are currently being tested.

Study of plasma, urine and gastric juice concentrations of famotidine using high performance liquid chromatography.

An improved method for the high performance liquid chromatography (HPLC) determination of famotidine, a recently introduced inhibitor of histamine H2-receptors, has been devised. Plasma, urine and gastric juice concentrations of famotidine have been measured in a series of 46 patients hospitalized for gastrointestinal disorders and for other unrelated pathologies, after a single oral dose of 40 mg. Pharmacokinetic data confirmed previously described trends of distribution and metabolism of famotidine, attaining peak plasma levels 1.5-2.0 h after oral administration and high urine levels, in unmodified form, between two and twelve hours. This simple HPLC method may be adopted for monitoring plasma concentrations of famotidine in patients, for assessing patient compliance in taking the drug by measuring urine levels and for examining the relationship between plasma famotidine concentration and the antisecretory effect.

HPLC reveals famotidine in the urine up to five days after a single 20 mg oral dose.

Using a simple method for the HPLC determination of famotidine (FMTD), a new inhibitor of histamine H2-receptors, it is possible to evaluate the urine levels of the drug in patients undergoing treatment. FMTD is excreted mostly in the urine, in the unmetabolized form. The authors evaluated the endpoint of FMTD levels in the urine in five patients given a single oral dose of 20 mg and found measurable levels of the drug up to 106 h (5 days) after the patients began the medication. This method may be useful for assessing patient compliance in taking the drug: this will allow the gastroenterologist to distinguish patients with true relapses of peptic ulcer disease from false relapses, in clinical trials using this histamine H2-inhibitor.

Short-term tissue culture of prostatic carcinoma samples provides useful biological parameters related to patient prognosis.

In order to identify new data to be able to better evaluate patient prognosis in prostatic carcinoma (PRCA), we started a study 6 years ago correlating in vitro parameters from human PRCA samples grown in tissue culture with histological diagnosis of the same tumors [Eur. Urol. 11: 330-333, 1985]. The original study has been extended with more cases and updated with follow-up of the patients. To date, we evaluated 51 specimens of PRCA (18 grade I, 19 grade II, 13 grade III and 1 grade IV) and 8 of benign prostatic hypertrophy. Tissue samples were cultured in medium DME plus 10% fetal calf serum, 10% horse serum and 50 ng/ml each of hydrocortisone and insulin. Epithelial cells grown from the explants showed an average life in culture and morphological and biochemical features in good correlation with tumor grade. Short cultural life span, regular growth and positive secretion activity are typical of low-grade tumors, meanwhile the opposite is true for high-grade tumors. Of these patients, 15 were evaluable for prognosis, because they died or because they were followed-up for at least 3 years. In this group we compared tissue culture data with survival and found a fairly good correlation between growth parameters and clinical outcome of each case. Although more cases are needed to provide statistical significance to the results, the data we collected seem to indicate that low-grade tumors susceptible of poorer prognosis can be identified by a short-term tissue culture showing morphological atypias and long average life span. This method may be easily reproduced in any hospital equipped with a tissue culture unit.