Immunohistochemical Analysis of DNA Repair- and Drug-Efflux-Associated Molecules in Tumor and Peritumor Areas of Glioblastoma.

Glioblastoma (GBM), the most commonly occurring primary tumor arising within the central nervous system, is characterized by high invasiveness and poor prognosis. In spite of the improvement in surgical techniques, along with the administration of chemo- and radiation therapy and the incessant investigation in search of prospective therapeutic targets, the local recurrence that frequently occurs within the peritumoral brain tissue makes GBM the most malignant and terminal type of astrocytoma. In the current study, we investigated both GBM and peritumoral tissues obtained from 55 hospitalized patients and the expression of three molecules involved in the onset of resistance/unresponsiveness to chemotherapy: O6-methylguanine methyltransferase (MGMT), breast cancer resistance protein (BCRP1), and A2B5. We propose that the expression of these molecules in the peritumoral tissue might be crucial to promoting the development of early tumorigenic events in the tissue surrounding GBM as well as responsible for the recurrence originating in this apparently normal area and, accordingly, for the resistance to treatment with the standard chemotherapeutic regimen. Notably, the inverse correlation found between MGMT expression in peritumoral tissue and patients' survival suggests a prognostic role for this protein.

Multifaceted Functional Role of Semaphorins in Glioblastoma.

Glioblastoma (GBM) is the most malignant tumor type affecting the adult central nervous system. Despite advances in therapy, the prognosis for patients with GBM remains poor, with a median survival of about 15 months. To date, few treatment options are available and recent trials based on the molecular targeting of some of the GBM hallmark pathways (e.g., angiogenesis) have not produced any significant improvement in overall survival. The urgent need to develop more efficacious targeted therapies has led to a better molecular characterization of GBM, revealing an emerging role of semaphorins in GBM progression. Semphorins are a wide group of membrane-bound and secreted proteins, originally identified as axon guidance cues, signaling through their receptors, neuropilins, and plexins. A number of semaphorin signals involved in the control of axonal growth and navigation during development have been found to furthermore participate in crosstalk with different dysfunctional GBM pathways, controlling tumor cell proliferation, migration, and invasion, as well as tumor angiogenesis or immune response. In this review, we summarize the regulatory activities mediated by semaphorins and their receptors on the oncogenic pathways implicated in GBM growth and invasive/metastatic progression.

Cancer stem cells from peritumoral tissue of glioblastoma multiforme: the possible missing link between tumor development and progression.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.

Pivotal role of human stearoyl-CoA desaturases (SCD1 and 5) in breast cancer progression: oleic acid-based effect of SCD1 on cell migration and a novel pro-cell survival role for SCD5.

The influence of cell membrane fluidity on cancer progression has been established in different solid tumors. We previously reported that "cancer-associated fibroblasts" (CAFs) induced epithelial-mesenchymal transition and increased cell membrane fluidity and migration in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. We also found that the membrane fluidity regulating enzyme stearoyl-CoA desaturase 1 (SCD1) was upregulated in tumor cells co-cultured with CAFs and established its essential role for both intrinsic and CAF-driven tumor cell motility. Here, we further explored the mechanisms involved in the SCD1-based modulation of breast cancer cell migration and investigated the role of the other human SCD isoform, SCD5. We showed that the addition of oleic acid, the main SCD1 product, nullified the inhibitory effects produced on MCF-7 and MDA-MB-231 cell migration by SCD1 depletion (pharmacological or siRNA-based). Conversely, SCD5 seemed not involved in the regulation of cancer cell motility. Interestingly, a clear induction of necrosis was observed as a result of the depletion of SCD5 in MCF-7 cells, where the expression of SCD5 was found to be upregulated by CAFs. The necrotic effect was rescued by a 48-h treatment of cells with oleic acid. These results provide further insights in understanding the role of SCD1 in both intrinsic and CAF-stimulated mammary tumor cell migration, unveiling the metabolic basis of this desaturase-triggered effect. Moreover, our data suggest the ability of CAFs to promote the maintenance of tumor cell survival by the induction of SCD5 levels.

Characterization of inflammatory cell infiltrate of scleroderma skin: B cells and skin score progression.

BACKGROUND: The purpose of this study was to investigate the frequency and the distribution of inflammatory cell infiltrate in two sets of cutaneous biopsies derived from clinically affected and unaffected skin in patients with systemic sclerosis (SSc) and to test correlation between the cell infiltrate and the progression of skin involvement. METHODS: Skin was immunohistochemically assessed to identify CD68, CD3, CD20 and CD138-positive (+) cells in clinically affected and unaffected skin in 28 patients with SSc. Patients were followed for 6 months and the characteristics of the infiltrate were analyzed according to disease duration, clinical features and skin involvement progression. RESULTS: In all SSc cutaneous specimens, cellular infiltrates were found in a perivascular location predominantly in the mid and deeper portions of the dermis. All the analyzed biopsies showed a CD3+ and CD68+ cell infiltrate and the mean number of CD3+ and of CD68+ cells was higher in clinically involved skin (CD3+, 71.7 ± 34.6 and CD68+, 26.3 ± 8.4, respectively) than in clinically uninvolved skin (CD3+, 45.7 ± 36.0 and CD68+, 13.6 ± 6.1, respectively) (p < 0.001 for both comparisons). CD20+ cells were found in 17 (60.7%) patients and in these patients the mean number of CD20+ cells was higher in clinically involved (4.7 ± 5.9) than in uninvolved skin (1.9 ± 2.9), (p = 0.04). There was a greater number of CD20+ cells in patients with early SSc compared with patients with long-standing disease. CD138+ cells were found in 100% of biopsies of clinically involved skin and in 89.3% of biopsies of uninvolved skin. The mean number of CD138+ cells was higher in clinically involved skin (3.6 ± 2.3) than in clinically uninvolved skin (1.9 ± 1.7), (p < 0.001). Seven patients experienced more than 20% worsening in the skin score after 6 months of follow up; all of them had a CD20+ skin infiltrate on biopsy of clinically involved skin. CONCLUSIONS: Our results confirm that mononuclear cells are present in the skin of all patients with SSc, underlining the role of inflammatory cell infiltrates in skin involvement in SSc. B cells in the skin seem to characterize patients with early diffuse skin disease and to correlate with skin progression.

Analysis of angiogenesis related factors in glioblastoma, peritumoral tissue and their derived cancer stem cells.

The formation of new blood vessels represents a crucial event under both physiological and pathological circumstances. In this study, we evaluated by immunohistochemistry, and/or Western blotting and/or quantitative real time-PCR the expression of HIF1α, HIF2α, VEGF, VEGFR1 and VEGFR2 in surgical glioblastoma multiforme (GBM) and peritumoral tissue samples obtained from 50 patients as well as in cancer stem cells (CSCs) isolated from GBM (GCSCs) and peritumoral tissue (PCSCs) of 5 patients. We also investigated the contribution of both GCSCs and PCSCs on the behavior of endothelial cells (ECs) in vitro. Immunohistochemistry demonstrated the expression of angiogenesis markers in both GBM and peritumoral tissue. In addition, in vitro tube formation assay indicated that both GCSCs and PCSCs stimulate EC proliferation as well as tube-like vessel formation. An increased migration aptitude was mainly observed when ECs were cultured in the presence of GCSCs rather than in the presence of PCSCs. These findings suggest that relevant neoangiogenetic events may occur in GBM. In particular, VEGF/VEGFR co-expression in PCSCs leads to hypothesize the involvement of an autocrine signaling. Moreover, our results suggest that both GCSCs and PCSCs own the skill of activating the "angiogenic switch" and the capability of modulating EC behavior, indicating that both cell types are either responsive to angiogenic stimuli or able to trigger angiogenic response. Together with our previous findings, this study adds a further piece to the challenging puzzle of the characterization of peritumoral tissue and of the definition of its real role in GBM pathophysiology.

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, ßIII-Tubulin and GFAP); Ca(2+) signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (ßIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and ßIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca(2+)-channels but they exhibited increased intracellular Ca(2+) levels in response to ATP. These Ca(2+) signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca(2+)-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

Leuprorelin acetate long-lasting effects on GnRH receptors of prostate cancer cells: an atomic force microscopy study of agonist/receptor interaction.

High cell-surface GnRH receptor (GnRH-R) levels have been shown to have a major influence on the extent of GnRH agonist-mediated tumor growth inhibition. The ability of the GnRH agonist leuprorelin acetate (LA) to induce a post-transcriptional upregulation of GnRH-R at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells has been previously demonstrated by Western blotting. Here we performed single molecule force spectroscopy by using Atomic Force Microscopy (AFM), which has proven to be a powerful tool allowing for investigation of living cell surface biological features, such as the so far unclear GnRH agonist/receptor interaction. Thus, in the hormone-insensitive PC-3 cells, we characterized the strength of the LA-receptor binding, and the amount and distribution of the functional receptor molecules on the cell surface. The effect of a long and continuous treatment (up to 30 days) with the agonist (10(-11) and 10(-6) M) on the same parameters was also investigated. A GnRH-R increase was observed, reaching the maximum (∼80%) after 30 days of treatment with the highest dose of LA (10(-6) M). The analogue-induced increase in GnRH-R was also demonstrated by Western blotting. In addition, two different receptor bound strengths were detected by AFM, which suggests the existence of two GnRH-R classes. A homogeneous distribution of the unbinding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the membrane of these living cells may warrant the maintenance of the response to LA also in androgen-unresponsive PCa. Moreover, the determination of ligand/receptor bond strength could shed light on the poorly understood event of LA/GnRH-R interaction and/or address structural/chemical agonist optimizations.

Epithelial-stromal interactions in human breast cancer: effects on adhesion, plasma membrane fluidity and migration speed and directness.

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of "cadherin switching", another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.

Interactions between normal human fibroblasts and human prostate cancer cells in a co-culture system.

BACKGROUND: Stroma affects the development and the structure of many organs and plays an important role in regulating epithelial malignancies, including those derived from the prostate. Fibroblasts represent the major cell type of the stromal compartment. Aiming at clarifying the relationships between normal fibroblasts and epithelial cancer cells, we utilized a co-culture system, which included both androgen-sensitive (LNCaP) and -insensitive (PC-3, DU-145) prostate cancer cell lines and a human gingival fibroblast cell line (FG). MATERIALS AND METHODS: The morphological aspects of the cultures were analyzed under an inverted phase-contrast microscope; the proliferation in conditioned media (CM) was assessed by cell counts, and the E-cadherin expression was evaluated by immunocytochemistry. RESULTS: In co-culture, androgen-sensitive LNCaP cells grew in a network on the top of the monolayer formed by FG, while colonies of androgen-insensitive PC-3 and DU-145 cells were surrounded by FG cells. After six days, the LNCaP cell number was apparently lower in the co-cultures than in the plates where they grew alone. Both LNCaP and FG cells underwent morphological changes. After the same period of time, the growth of PC-3 and DU-145 cells overcame the growth of FG cells, which were almost abolished. The CM of FG inhibited the proliferation of LNCaP cells, after three days by 33% (p<0.01) and after six days by up to 82% (p<0.01), but had no effect on the PC-3 and DU-145 cell growth. The CM of all three prostate cancer cell lines reduced the growth of FG. Growth reduction in DU-145 cells was the most effective (50% inhibition after three days, p<0.01, and 55% after six days, p<0.01). FG did not express E-cadherin, while strong E-cadherin staining was detected in LNCaP cells. PC-3 cells exhibited E-cadherin nuclear staining, while sporadic membrane expression of the specific protein was observed in DU-145 cells. In co-culture, there seemed to be a reduction in the nuclear E-cadherin reactivity of PC-3 cells. CONCLUSION: Our data confirm the existence of a dialogue between normal fibroblasts and prostate cancer cells, which results in both a peculiar modality of growth and a regulation of proliferation, probably due to factors secreted in the culture medium. The variation in E-cadherin expression found in PC-3 cells co-cultured with FG merits further investigation.

B cells in systemic sclerosis: a possible target for therapy.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition in the skin and other visceral organs and it is associated with immune activation characterized by autoantibody production, release of various cytokines and T-lymphocyte activation. Several recent lines of evidence in animal models and in SSc patients indicate a potential role for B cells in the SSc. B cells have arisen as a possible player in tissue fibrosis in some experimental models and, since IL-6 produced by B cells, along with TGF-ß, may induce matrix synthesis and less collagen degradation, targeting B cells could be one way to reduce ECM deposition and reduce the inflammatory background. Both SSc patients and tight-skin mice, a genetic model of SSc, have intrinsic B-cell abnormalities characterized by chronic B-cell activation. SSc patients present an increased number of naïve B cells and an activation of memory B cells, despite a reduction in their number. B cells from SSc patients exhibit increased expression of CD19. Remarkably, CD19 loss or B-cell depletion using antimouse CD20 antibody suppresses the development of skin hyperplasia and autoimmunity in tight-skin mice. Additionally, recent studies revealed a possible beneficial effect of anti-human CD20 antibody (Rituximab) therapy on skin fibrosis and lung involvement in SSc patients. These studies reported also the safety of Rituximab in SSc patients. All these findings suggest a possible role of antiCD20 treatment in SSc patients.

Leuprorelin acetate affects adhesion molecule expression in human prostate cancer cells.

Reduced cell-cell adhesion allows malignant epithelial cells to invade the basal membrane and penetrate the stroma. This implies the potential of the cells to escape from the primary tumor as well as spreading ability. Herein, we investigated the effects of leuprorelin acetate (LA), a GnRH agonistic analogue, alone or in combination with dihydrotestosterone (DHT), on the expression of molecules involved in cell adhesion (E-cadherin, N-cadherin, α-, ß- and γ-catenin) or in migration/invasion (c-met, CD44v6 and caveolin-1) in androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (CaP) cells. We demonstrated by immunoblotting that, in LNCaP cells, molecules present in the adherens junctions (E-cadherin, α-, ß- and γ-catenin) were expressed, while α-catenin was absent in PC-3 cells which expressed N-cadherin and c-met. In LNCaP cells, no changes in E-cadherin levels were produced by 10(-9) M DHT while LA (10(-11) or 10(-6) M) up-regulated the protein level (up to 26-30% after 48 h). In the same cells, ß- and γ-catenin expression was enhanced either by DHT (24 and 20%, respectively) or LA (up to 18 and 40%, respectively), while α-catenin was not affected. Antagonistic effects were consistently observed between DHT and LA when the two hormones were jointly administered to the cells. Consistent results were obtained by immunocytochemistry. Co-immunoprecipitation analysis, used to verify the integrity of the LNCaP cell adhesion complex, demonstrated the association of E-cadherin with catenins. In PC-3 cells, adhesion molecule expression, analyzed by immunoblotting, was unaffected by LA, while a down-regulation of c-met (up to 28%) was observed after 24 h of treatment but which did not hold up over time (48-144 h). Our findings demonstrate the efficacy of LA in upregulating E-cadherin, ß- and γ-catenin in LNCaP cells. This effect, that may be considered as another aspect of the direct antitumor activity of the GnRH analogue in hormone-dependent CaP cells, may contribute to maintenance/restoration of the normal tissue architecture counteracting the tumor cell spreading tendency.

B cell depletion in diffuse progressive systemic sclerosis: safety, skin score modification and IL-6 modulation in an up to thirty-six months follow-up open-label trial.

INTRODUCTION: An over-expression of CD19 has been shown in B cells of systemic sclerosis (SSc) and B cells are thought to contribute to the induction of skin fibrosis in the tight skin mouse model. The aim was to define the outcome on safety and the change in skin score after rituximab therapy in SSc patients and to correlate the clinical characteristics with the levels of interleukin (IL)-6 and with the immune cell infiltrate detected by immunohistochemistry. METHODS: Nine patients with SSc with mean age 40.9 +/- 11.1 years were treated with anti-CD20, 1 g at time 0 and after 14 days. Skin biopsy was performed at baseline and during the follow-up. B-cell activating factor (BAFF) and IL-6 levels were also determined at the follow-up times. RESULTS: After 6 months patients presented a median decrease of the skin score of 43.3% (range 21.1-64.0%), and a decrease in disease activity index and disease severity index. IL-6 levels decreased permanently during the follow up. After treatment, a complete depletion of peripheral blood B cells was observed in all but 2 patients. Only 3 patients presented CD20 positive cells in the biopsy of the involved skin at baseline. CONCLUSIONS: Anti-CD20 treatment has been well tolerated and SSc patients experienced an improvement of the skin score and of clinical symptoms. The clear fall in IL-6 levels could contribute to the skin fibrosis improvement, while the presence of B cells in the skin seems to be irrelevant with respect to the outcome after B cell depletion. TRIAL REGISTRATION: ISRCTN77554566.

GnRH receptor expression in human prostate cancer cells is affected by hormones and growth factors.

GnRH receptors (GnRH-R) have been found in various malignancies, including prostate cancer (PCa). They mediate the direct antitumor effects of GnRH analogs. Nevertheless, few reports concern drug-induced modulation of GnRH-R levels. In this study, we investigated GnRH-R expression in androgen-sensitive (LNCaP) and -insensitive (PC-3) PCa cells treated for 4 and 6 days with a GnRH agonist (Leuprorelin acetate, LA, 10(-11) or 10(-6) M), Dihydrotestosterone (DHT, 10(-9) M), Cyproterone acetate (CA, 10(-7) M), and Epidermal growth factor (EGF, 10 ng/ml), either alone or combined. The RT-PCR analysis showed no variation in GnRH-R mRNA levels of both treated LNCaP and PC-3 cells. On the contrary, immunoblotting indicated that in LNCaP and PC-3 cells, LA upregulated membrane GnRH-R expression (up to 92%). In androgen-sensitive cells, DHT induced a GnRH-R increase (up to 119%) always comparable to that occurring in the presence of CA. GnRH-R upregulation by LA/DHT or CA/DHT association was similar to that promoted by the single agents. In PC-3 cells, EGF upregulated GnRH-R (up to 110%). A prolonged treatment (for 12 days) determined a greater EGF-induced increase in GnRH-R levels (142%). Lower (or no) receptor enhancement occurred when LA and EGF were associated. Our findings indicate that LA post-transcriptionally upregulates its own membrane receptor in androgen-sensitive and -insensitive PCa cells, counteracting the receptor enhancement produced by DHT and EGF. The effects, obtained with a relatively long and continuous treatment, may have implications in the choice of therapy modality with GnRH analogs and may render the receptor a novel therapeutic target, particularly in hormone-refractory PCa.

Leuprorelin acetate affects ERK1/2 activity in prostate cancer cells.

The mechanisms by which a GnRH analogue, leuprorelin acetate (LA), antagonizes the mitogenic effect of dihydrotestosterone (DHT) or epidermal growth factor (EGF) in prostate cancer cells is poorly understood. The mitogen-activated protein kinase system has a central role in growth regulation and, for this reason, we investigated the involvement of the extracellular signal-regulated kinase (ERK1/2) pathway in the response of both androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer cells to LA alone or combined with EGF or DHT. The evaluation of ERK activation was performed by using Western blot analysis and immunocytochemistry. EGF specifically induced ERK1/2 activity in both models and this effect was counteracted by an inhibitor of EGF-receptor phosphorylation. The addition of LA produced an appreciable reduction of ERK phosphorylation promoted by EGF in LNCaP cells, while it generally determined an increase in ERK activity in androgen-unresponsive PC-3 cells. The slight ERK activation induced by DHT in LNCaP cells was counteracted by LA and this effect was evident only by immunocytochemistry. Our findings suggest that the antiproliferative effect of LA in prostate cancer cells stimulated by hormones and growth factors may be, at least in part, mediated by the reduction of ERK1/2 activation in LNCaP cells and linked to the unexpected increase of this activity produced by the analogue in PC-3 cells.

Reverse transcriptase-PCR analysis of apoptosis-regulating gene expression in human benign prostatic hyperplasia.

BACKGROUND: To determine whether an imbalanced interaction between proapototic and antiapoptotic signals may account for the loss of the normal cell growth control in benign prostatic hyperplasia (BPH), the expression of some apoptosis-regulating genes (bcl-2, bax, c-myc, fas) was investigated. PATIENTS AND METHODS: BPH specimens were obtained from 20 patients who underwent trans-urethral resection of the prostate (TURP) or adenomectomy. Gene expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and its correlation with age and serum PSA level was also investigated. RESULTS: Genes were found to be differentially expressed in BPH tissues. In particular, the antiapoptotic gene bcl-2, which was found in 18/20 samples, gave the weakest signal (p < 0.05 - p < 0.001, Wilcoxon's signed rank test), whereas the cell cycle regulator c-myc was detected in all the specimens and was the most highly expressed (p < 0.001). A positive relationship between the expression of bcl-2 and that of the two proapoptotic genes bax and fas was observed (p < 0.05, Spearman's rank correlation test), as well as between c-myc and fas levels (p < 0.005). Moreover, bax expression positively correlated with age and PSA (p < 0.02), which have also been shown to directly correlate (p < 0.01). CONCLUSION: The higher expression of the oncogene c-myc suggests the activation of mitogenic signals within hyperplastic prostate tissue which a relatively high expression of the proapoptotic genes bax and fas fails to counterbalance.

Apoptosis-related gene expression affected by a GnRH analogue without induction of programmed cell death in LNCaP cells.

BACKGROUND: In this study we confirmed the ability of a Gonadotropin Releasing Hormone (GnRH) agonist, leuprorelin acetate (LA), to counteract or even suppress the 5alpha-dihydrotestosterone (DHT)-stimulated growth of androgen-sensitive prostate cancer cells (LNCaP). Since the cellular mechanisms mediating this effect are not well defined, we investigated the activity of LA, also in combination with DHT or with cyproterone acetate (CA), on the expression of genes (bcl-2, bax, c-myc) which may contribute to the proliferative behaviour of LNCaP cells. In addition, experiments aimed to evaluate the action of the analogue on apoptosis were performed. MATERIALS AND METHODS: Gene expression was evaluated by RT-PCR and Western blotting on cells treated with LA (10(-11) or 10(-6) M), alone or combined with 10(-9) M DHT or 10(-7) M CA. The occurrence of apoptosis following treatment with LA (10(-11), 10(-6) or 10(-5) M), alone or combined with 10(-9) M DHT, was assessed by DNA fragmentation analysis. RESULTS: Both the mRNA and protein of the anti-apoptotic gene bcl-2 were induced (30-125%) by DHT after 24-144 h. LA decreased bcl-2 mRNA (10-40%), while it did not unequivocally affect protein expression. The analogue always reduced (13-74%) both mRNA and protein levels obtained under DHT treatment. The mRNA and protein of the pro-apoptotic gene bax were down-regulated by DHT (15-40%), while LA generally induced bax protein but not its mRNA. LA counteracted DHT activity, even increasing bax protein levels over the controls. c-myc mRNA and protein were enhanced by DHT (15-45%) but down-regulated by LA (10-40%). Once more, the androgen effect was antagonized by LA, sometimes reducing c-myc content below the controls. CA produced the most similar effects to those triggered by DHT. The hormonal treatment did not induce any DNA fragmentation. CONCLUSION: In spite of gene modulation, apoptosis was not observed under LA treatment, in agreement with the lack of a cell growth effect when the analogue was used alone. Nevertheless, the observed changes in gene expression may be directly or indirectly involved in the antiproliferative effect of LA on androgen-stimulated cells.