Leda Biordi

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.

Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice.

Polymorphonuclear leukocyte infiltration and activation into colonic mucosa are believed to play a pivotal role in mediating tissue damage in human ulcerative colitis (UC). Ligands of human CXC chemokine receptor 1 and 2 (CXCR1/R2) are chemoattractants of PMN, and high levels were found in the mucosa of UC patients. To investigate the pathophysiological role played by CXCR2 in experimental UC, we induced chronic experimental colitis in WT and CXCR2−/− mice by two consecutive cycles of 4% dextran sulfate sodium administration in drinking water. In wild‐type (WT) mice, the chronic relapsing of DSS‐induced colitis was characterized by clinical signs and histopathological findings that closely resemble human disease. CXCR2−/− mice failed to show PMN infiltration into the mucosa and, consistently with a key role of PMN in mediating tissue damage in UC, showed limited signs of mucosal damage and reduced clinical symptoms. Our data demonstrate that CXCR2 plays a key pathophysiological role in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.

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.

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.

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.

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. Cell Death and Differentiation (2000) 7, 916–924

The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemo-resistant tumors. The aim of this study is to evaluate the role of Palomid 529 (P529), a novel TORC1/TORC2 inhibitor, in association with docetaxel (DTX) and cisplatin (CP). This work utilizes a wide panel of prostatic cancer cell lines with or without basal activation of Akt as well as two in vivo models of aggressive HRPC. The blockade of Akt/mTOR activity was associated to reduced cell proliferation and induction of apoptosis. Comparison of IC50 values calculated for PTEN-positive and PTEN-negative cell lines as well as the PTEN transfection in PC3 cells or PTEN silencing in DU145 cells revealed that absence of PTEN was indicative for a better activity of the drug. In addition, P529 synergized with DTX and CP. The strongest synergism was achieved when prostate cancer (PCa) cells were sequentially exposed to CP or DTX followed by treatment with P529. Treatment with P529 before the exposure to chemotherapeutic drugs resulted in a moderate synergism, whereas intermediated values of combination index were found when drugs were administered simultaneously. In vivo treatment of a combination of P529 with DTX or CP increased the percentage of complete responses and reduced the number of mice with tumor progression. Our results provide a rationale for combinatorial treatment using conventional chemotherapy and a Akt/mTOR inhibitor as promising therapeutic approach for the treatment of HRPC, a disease largely resistant to conventional therapies.

Chemokine MIP-2/CXCL2, Acting on CXCR2, Induces Motor Neuron Death in Primary Cultures

Objectives: Chemokines are implicated in many diseases of the central nervous system (CNS). Although their primary role is to induce inflammation through the recruitment of leukocytes by their chemotactic activity, they may also have direct effects on neuronal cells. We evaluated the expression of CXCR1 and CXCR2 and investigated the effect of CXCR2 activation by the agonist MIP-2 (CXCL2) on primary cultured motor neurons. To specifically assess the role of CXCR2 in the neurotoxicity induced by MIP-2, we used the CXCR1/2 inhibitor reparixin and studied the effect of the chemokine on motor neuron cultures from CXCR2-deficient mice. Methods: Primary motor neurons prepared from rat or mouse embryos were treated with MIP-2 and reparixin. Motor neuron viability and receptor expression were assessed by immunocytochemical techniques. Results: Rat primary motor neurons expressed CXCR2 receptors and recombinant rat MIP-2 induced dose-dependent neurotoxicity. This neurotoxicity was counteracted by reparixin, a specific CXCR1/2 inhibitor, and was not observed in motor neurons from CXCR2-deficient mice. Conclusions: CXCR2 activation might directly contribute to motor neuron degeneration. Thus, chemokines acting on CXCR2, including IL-8, may have direct pathogenic effects in CNS diseases, independent of the induction of leukocyte migration.

Effects of EGFR tyrosine kinase inhibitor erlotinib in prostate cancer cells in vitro

Erlotinib is a small‐molecule tyrosine kinase inhibitor targeted EGFR, known to be overexpressed in a variety of cancers, including prostate cancer. Clinical trials showed insignificant clinical benefit in patients with castration resistant prostate cancer both when EGFR inhibitors were administered as monotherapy or in association with antiandrogens or chemotherapeutics. Why, differently to other tumors, have EGFR inhibitors been so ineffective in human prostate cancer? This is the question that we have set in this report.

CXCR4 pharmacogical inhibition reduces bone and soft tissue metastatic burden by affecting tumor growth and tumorigenic potential in prostate cancer preclinical models.

The majority of prostate cancer (Pca) patient morbidity can be attributed to bone metastatic events, which poses a significant clinical obstacle. Therefore, a better understanding of this phenomenon is imperative and might help to develop novel therapeutic strategies. Stromal cell‐derived factor 1α (SDF‐1α) and its receptor CXCR4 have been implicated as regulators of bone resorption and bone metastatic development, suggesting that agents able to suppress this signaling pathway may be used as pharmacological treatments. In this study we studied if two CXCR4 receptor antagonists, Plerixafor and CTE9908, may affect bone metastatic disease induced by Pca in preclinical experimental models