Paola Muzi

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.

Increased Enterocyte Apoptosis and Fas-Fas Ligand System in Celiac Disease

Our aim was to evaluate whether increased enterocyte apoptosis was responsible for mucosal flattening in celiac disease (CD), and, since the mechanisms responsible for tissue injury in this condition are unknown, we studied the possibility that the Fas-Fas ligand (FasL) system may be involved. Endoscopic duodenal biopsy specimens from 12 patients with untreated and 12 with treated CD and 12 control subjects were evaluated for enterocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling assay and for Fas and FasL expression by immunohistochemistry. A coculture of isolated enterocytes (targets) and purified lamina propria mononuclear cells (LPMCs) (effectors) was performed in the absence or presence of an antagonistic ZB4 anti-Fas antibody. We found a significant correlation between the degree of villous atrophy, morphometrically evaluated, and the level of enterocyte apoptosis, suggesting that mucosal flattening is a consequence of exaggerated epithelial cell death. Most celiac enterocytes express Fas, and LPMCs express FasL. The abolishment of enterocyte apoptosis observed in the presence of ZB4 antibody suggests that enterocytes are potential targets of lymphocyte infiltrate. These results directly demonstrate that FasL-mediated apoptosis is a major mechanism responsible for enterocyte death in CD.

Epidermal growth factor modulates prostate cancer cell invasiveness regulating urokinase-type plasminogen activator activity. EGF-receptor inhibition may prevent tumor cell dissemination.

Urokinase-type plasminogen activator receptor (uPAR) and Epidermal Growth Factor Receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. We compared the uPA production, the presence of uPAR, AR, EGFR and Her2 with the chemotaxis and the Matrigel invasion in ten human PCa cell lines and observed that: (1) the levels of Her2, but not of EGFR, as well as the uPA secretion, cell motility and Matrigel invasion were statistically higher in AR negative than in AR positive PCa cells; (2) the uPA secretion and uPA Rexpression were positively related to Matrigel invasion; (3) the EGF was able to stimulate chemotaxis and Matrigel invasion in a dose-dependent manner; (4) the EGF-induced cell migration was statistically higher inAR negative than in AR positive cells with a similar increase with respect to basal value (about 2.6 fold); (5) the Matrigel invasion was statistically higher in AR negative than in AR positive PCa cells also if the increment of Matrigel invasion after EGF treatment was statistically higher in AR positive respect to AR negative cells; (6) the EGF induced uPA secretion and its membrane uptake through the increment of uPAR; and (7) these effects were blocked by EGFR/Her2 tyrosine kinase inhibitors with IC(50) lower than those needed to inhibit cell proliferation and required PI3K/Akt, MAPK and PI-PLC activities as verified by inhibition experiments. These enzymatic activities were regulated in different manners in PTEN positive and negative cells. In fact, the inhibition of PI3K blocked the EGF-induced invasiveness in PTEN positive cells but not in PTEN negative cells, in which PI3K activity was not influenced by EGFR/Her2 activation, whereas the inhibition of MAPK was able to block the invasive phenomena in both cell types. Taken together, our data suggest that the blockade of EGFR could attenuate the invasive potential of PCa cells. In addition, considering that the EGFR expression is related to higher Gleason grade of PCa and that EGFR levels are increased after anti androgenic therapy, this therapeutic approach could slow down the metastasis formation which represents the most dramatic event of PCa progression.

Plasminogen activator system modulates invasivecapacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and itscell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPAin matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulat-ingthe amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differ-entialproduction of uPA corresponds with the capacity to bind and activate plasminogen. In addition, weprovide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize theinvasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquireplasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin throughexogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observedin several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to thepericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmingenerated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the pro-teolyticcascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chainof uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which mayregulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies againstuPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growthin uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF)induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic can-cerpatients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growthof the primary tumor and dissemination of metastatic cells. ©Kluwer Academic Publishers

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.

Azacitidine improves antitumor effects of docetaxel and cisplatin in aggressive prostate cancer models

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells. In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2 antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were also tolerable in mice and superior to either agent alone. As DTX is the standard first-line chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest in this disease stage. Our results provide a rationale for clinical trials on combination treatments with azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.

Transglutaminase and coeliac disease: endomysial reactivity and small bowel expression

This study was aimed at verifying whether tissue transglutaminase (tTG) is the sole autoantigen eliciting anti‐endomysial antibodies in coeliac disease (CoD) and investigating tTG expression in normal and coeliac mucosa. Twelve anti‐endomysial‐positive coeliac sera and 12 anti‐endomysial‐negative control sera (10 μl, diluted 1:5–1:400 in PBS pH 7.3) were preincubated with 10, 20 or 50 μg guinea pig liver tTG at 4°C overnight. Monkey oesophagus tissue slides were then tested with tTG‐preincubated and non‐preincubated sera to search for IgA anti‐endomysial reactivity by indirect immunofluorescence. Moreover, six sections of monkey oesophagus were incubated with an anti‐tTG mouse MoAb, six sections with an anti‐cytokeratin mouse MoAb and six sections with only 3% bovine serum albumin. Finally, endoscopic duodenal biopsy sections obtained from 12 patients affected by untreated CoD, six patients affected by treated CoD and 10 biopsied controls were immunohistochemically stained with a peroxidase‐conjugated anti‐tTG MoAb. Our results show that (i) preincubation with tTG abolished endomysial immunofluorescence in most, but not in all, coeliac sera; (ii) the incubation of anti‐tTG MoAb with sections of monkey oesophagus resulted in an immunofluorescence staining pattern similar but not identical to that of anti‐endomysial‐positive coeliac sera; (iii) although tTG expression was present at muscularis mucosae and pericryptal fibroblast in both normal and coeliac mucosa, it was slightly more marked and evident in the latter. Although our absorption experiment was performed with guinea pig liver tTG, we confirm that tTG is the predominant antigen of endomysial antibodies, but we speculate that, at least in some patients, it is not the only one.

Additive antitumor effects of the epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), and the nonsteroidal antiandrogen, bicalutamide (Casodex), in prostate cancer cells in vitro.

Progression from an androgen‐dependent to an androgen‐independent state often occurs in patients with prostate cancer (PCa) who undergo hormonal therapy. We have investigated whether inhibition of the epidermal growth factor receptor (EGFR) signaling pathway affects the antitumor effect of a nonsteroidal antiandrogen. Gefitinib (Iressa), an EGFR tyrosine kinase inhibitor, and bicalutamide (Casodex), a nonsteroidal antiandrogen [androgen receptor (AR) antagonist], were administered alone and in combination to AR‐positive human PCa cell lines. FACS analysis showed lower EGFR expression levels on AR‐positive cells (LNCaP, CWR22, CWR22R 2152 and AR‐transfected DU145 cell lines) compared with AR‐negative cells (DU145, PC3 and TSU‐Pr1). Moreover, in AR‐transfected DU145 cells, chronic treatment with bicalutamide increased EGFR expression to levels similar to androgen‐independent DU145 cells. All AR‐positive PCa cell lines were sensitive to gefitinib (IC50 = 0.1–0.6 μM), whereas higher concentrations of bicalutamide were needed to reduce AR‐positive PCa cell line proliferation (IC50 = 0.8–2.0 μM). Low doses of gefitinib increased the antitumor effects of bicalutamide by strongly reducing the IC50 of bicalutamide (approximately 10‐fold). Similarly, bicalutamide increased the antiproliferative effects of gefitinib by reducing the IC50 of gefitinib (approximately 5‐fold). Taken together, our data suggest that in androgen‐dependent cell lines, addition of gefitinib in combination with bicalutamide results in concurrent dual inhibition of AR and EGFR/HER2 pathways. This causes a significant delay in the onset of EGFR‐driven androgen independence.

Akt down-modulation induces apoptosis of human prostate cancer cells and synergizes with EGFR tyrosine kinase inhibitors

PTEN is a well‐characterized tumor suppressor that negatively regulates cell growth and survival through the modulation of PI3K/Akt pathway.

Arachidonic acid modulates the crosstalk between prostate carcinoma and bone stromal cells

Diets high in n-6 fatty acids are associated with an increased risk of bone metastasis from prostate carces (PCa). The molecular mechanism underlying this phenomenon is largely unknown. Arachidonic acid (AA) and its precursor linoleic acid can be metabolized to produce pro-inflammatory cytokines that act as autocrine and paracrine regulators of cancer behavior. We and other authors have previously reported that factors released by PCa cells excite an aberrant response in bone marrow stromal cells (BMSCs). We planned to study how AA may modulate in vitro the interaction between PCa cells and human BMSCs. First, we observed that AA is a potent mitogenic factor for PCa cells through the production of both 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) metabolites. While 5-LOX controls cell survival through the regulation of the Bcl-2/Bax ratio, COX-2 activity stimulates the release of transforming growth factor-alpha (TGF-alpha) and pro-inflammatory cytokines. The blockade of COX-2 activity through a specific inhibitor is sufficient to repress AA-induced gene transcription. The over-expression of transforming growth factor -alpha (TGF-alpha), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) by AA-primed PCa cells resulted particularly effective in modifying cell behavior of cultured human BMSCs. In fact, we observed an increment in the cell number of BMSCs, due prevalently to the action of TGF-alpha, the number of osteoblasts, and the production of receptor activator for nuclear factor kappa B ligand (RANKL), events mainly controlled by inflammatory cytokines. These findings provide a possible molecular mechanism by which dietary n-6 fatty acids accumulating in bone marrow may influence the formation of PCa-derived metastatic lesions and indicate new molecular targets for the therapy of metastatic PCa.