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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.

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.

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 HIF1a, HIF2a, 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.

Despite the acknowledged impact of the tumor stroma on breast cancer development and progression, the molecular basis of such effects remain partially unexplained. We previously reported that breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and an increase in cell membrane fluidity and migration speed in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. More recently, in order to better define the mechanisms responsible for the CAF-promoted tumor cell migration, we investigated the role of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, and demonstrated its CAF-triggered up-regulation as well as its crucial role in the migratory ability of the above tumor cells. Besides SCD1 induction, a CAF-promoted enhancement in the protein level and/or activity of the SCD1 transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), was observed. Moreover, the influence of stroma-derived signals in cancer cell migration speed was proved by cell tracking analysis in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor, where a marked reduction or abolishment of the fibroblast-triggered increase in cancer cell migration speed was observed. In the last part of the study, in order to verify if soluble CAF-derived factors stimulate breast cancer cell migration in a SCD1-dependent manner, tumor cells were exposed to CAF-conditioned medium (CM) and their migration evaluated by scratch assay in the presence of a small molecule inhibitor of SCD1. Moreover, to asses if the induction of SCD1 expression by CAFs might occur via SREBP1, the desaturase levels were also determined in SREBP1-inhibited tumor cells. These latest investigations indicate that SCD1 contributes to the promotion of breast cancer cell migration by CAF-derived soluble factors, since the desaturase inhibition completely suppressed the stim

Background: Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. Methods: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1- inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-b or basic fibroblast growth factor. Results: A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in c

It is widely accepted that glioblastoma (GBM) develops from cancer stem cells (CSCs), a subset of stem-like cells displaying high resistance to treatment. In fact, despite aggressive therapy, 90% of patients relapse within 2 cm from tumor edge. Our recent findings showed the existence of a CSC type, residing in GBM peritumor tissue (PCSCs), that bears distinct characteristics from CSCs of the tumor mass (GCSCs). It should be considered the possibility that, after surgical resection, PCSCs might represent a reservoir of cells able to recapitulate the tumor. In this setting, characterization of PCSCs appears to be crucial in order to identify novel effective therapeutic targets. Thus, our aim was to investigate GCSCs and PCSCs role in angiogenesis, a key event in both GBM and peritumor tissue, whose vasculature shows features similar to those found in the tumor mass. In particular, we analyzed, by immunocytochemistry (ICC), Western blotting or real-time PCR, the expression of molecules involved in hypoxia and angiogenesis, such as HIF1α, HIF2α, and VEGF along with its receptors (VEGFR1, VEGFR2). ICC has highlighted the presence and the specific localization of these molecules in both GCSCs and PCSCs. The two cell populations showed comparable levels of VEGF. The transcript of VEGFR1 was in general expressed at higher levels in GCSCs than in PCSCs, while VEGFR2 mRNA and protein did not show a unique trend of expression. The expression of VEGF and its receptors in both GCSCs and PCSCs suggests that, besides well-known paracrine loops, autocrine signalings are also involved in tumor angiogenesis. Moreover, the expression of angiogenesis markers in PCSCs suggests these cells to have a direct role in peritumor tissue new vessel formation. In this regard, PCSCs should be considered a promising therapeutic target to counteract the angiogenesis-supported tumor progression. Supported by FIRB Accordi di Programma 2010

The key role played by the stroma in breast cancer development and progression has been widely recognized. Recently, we reported that the cross-talk between epithelial and stromal cells affects structural and functional features correlated with the invasive phenotype of breast cancer cells by co-culturing mammary cancer cells with different metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs) [1]. This study was designed to deepen the knowledge of the role played notably by CAFs in promoting breast tumor cell migratory skill through the analysis of the expression/activity of downstream potential target molecules and of the contribution of paracrine signalings. Thus, we investigated the influence of fibroblasts on the expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the level and activity of its transcription factor, SREBP1, in breast cancer cells. The ability of CAFs to promote a 2-3 fold increase in SCD1 mRNA and protein expression as well as an induction of SREBP1 DNA binding activity has been shown in the two cancer cell lines. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration. To clarify the possible role of tumor-stroma paracrine interaction in the previously reported improvement of cancer cell migratory ability, cocultures were set up in presence of neutralizing antibodies against hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor. Cell tracking analysis demonstrated that the CAF-mediated increase in tumor cell migration speed was reduced or abolished by neutralizing the above soluble factors. These results provide new insights in understanding the role of CAFs in promoting tumor cell invasiveness and may help to devise new targeted therapeutic approaches. References [1] Angelucci et al. (2012), PLoS

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Despite increasing knowledge of the GBM biomolecular characterization and breakthrough in treatment, the prognosis in the great majority of patients is poor as almost all tumors recur in peritumor tissue. This site of alterations seems to harbor CSCs that show some differences when compared to CSCs derived from GBM. By means of immunocytochemistry, Western blotting and RT-PCR, we have compared CSCs derived from GBM (GCSCs) to those isolated from peritumor tissue (PCSCs) as to the expression of molecules linked to stemness (Nestin, Musashi-1, SOX2), proliferation and apoptosis (pERK1/2 and pJNKs), migration/invasion, angiogenesis, and hypoxia (reelin, GD3, NG2, VEGF, VEGFR1/2, HIF1α, and HIF2α). Furthermore, comparison of growth rate, frequency of tumor initiating cells (TICs), tumorigenicity in Scid/bg mice, and ultrastructure between the two CSC populations was performed. Finally, we analyzed the effect of different chemotherapeutic agents (temozolomide, etoposide, irinotecan and carboplatin) on proliferation of both GCSCs and PCSCs. All the molecules analyzed were expressed in the two populations of CSCs. Among the investigated markers, nestin, pJNKs, Musashi-1, NG2 and VEGFR1 were found to be significantly increased in GCSCs if compared with PCSCs. Moreover, GCSCs grew at a higher rate and showed a greater frequency of TICs. Animal survival time revealed that mice receiving GCSCs died significantly earlier than mice inoculated with PCSCs. Only PCSCs ultrastructure showed fully organized desmosomes and gap junctions. All the chemotherapeutic agents used inhibited proliferation of both GCSCs and PCSCs, although with different efficiency. Moreover, each drug had similar effect on the GCSCs and PCSCs derived from the same patient. These findings support the need for a complete characterization of GCSCs and PCSCs to optimi

Background: Despite the recognized contribution of the stroma to breast cancer development and progression, the effective therapeutic targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and increase in cell membrane fluidity and migration in both well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. Materials and Methods: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. Migration was evaluated by wound healing assay in SCD1-inhibited (by small interfering RNA, siRNA, or pharmacologically) tumor cells. To assess the role of stromal-derived signals in cancer cell migration speed, cell tracking analysis was performed in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor. Results: A 2-3 fold increase in SCD1 mRNA and protein expression as well as an induction of SREBP1 DNA binding activity has been induced, particularly by CAFs, in the two cancer cell lines. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralizing the above growth factors. Conclusions: These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic stra

It is widely accepted that glioblastoma (GBM) develops from cancer stem cells (CSCs), a subset of stem-like cells displaying high resistance to treatment. Our recent findings showed the existence of a CSC type, residing in GBM peritumor tissue (PCSCs), that bears distinct characteristics from CSCs of the tumor mass (GCSCs) and that, after surgical resection, might represent a reservoir of cells able to recapitulate the tumor. In this setting, characterization of PCSCs appears to be crucial in order to identify novel effective therapeutic targets. Thus, our aim was to investigate GCSCs and PCSCs role in angiogenesis, a key event in both GBM and peritumor tissue, whose vasculature shows features similar to those found in the tumor mass. In particular, we analyzed, by immunocytochemistry (ICC), Western blotting or real-time PCR, the expression of molecules involved in hypoxia and angiogenesis, such as HIF1α, HIF2α, and VEGF along with its receptors (VEGFR1, VEGFR2). ICC has highlighted the presence and the specific localization of these molecules in both GCSCs and PCSCs. The two cell populations showed comparable levels of VEGF. The transcript of VEGFR1 was in general expressed at higher levels in GCSCs than in PCSCs, while VEGFR2 mRNA and protein did not show a unique trend of expression. The presence of VEGF and its receptors in both GCSCs and PCSCs suggests that, besides well-known paracrine loops, autocrine signalings are also involved in tumor angiogenesis. Moreover, the expression of angiogenesis markers in PCSCs suggests these cells to have a direct role in peritumor tissue new vessel formation. In this regard, PCSCs should be considered a promising therapeutic target to counteract the angiogenesis-supported tumor progression. Supported by FIRB Accordi di Programma 2010

In breast tumor, interactions occurring between malignant cells and stromal mi-croenvironment heavily influence various aspects of cancer biology. We recently reported that this cross-talk affects structural and functional features correlated with the invasive phenotype of breast cancer cells by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts, particularly those isolated from breast tumor stroma (cancer-associated fibroblasts, CAFs). In the present study, we aimed to elucidate some aspects of the epithelium-stroma interaction, related to two linked CAF-triggered effects we previously described: a) increase in cancer cell plasma membrane fluidity; b) increase in speed and directness of cancer cell migration. Thus, we tested the effect of fibroblast/breast cancer cell co-culturing on the expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating the fatty acid membrane composition whose up-regulation has been reported in different cancer cells, generally leading to loosely packed membranes. Western blot analysis revealed a dramatic CAF-promoted increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells. Quantitative real-time PCR demonstrated similar variations in the SCD1 transcript levels. With the aim of clarifying the possible role of soluble factors in the CAF-induced increase in speed and directness of cancer cell migration, we performed co-culture assays in presence of a neutralizing antibody against hepatocyte growth factor (HGF) which is released by CAFs and notoriously affects cancer cell motili-ty. We demonstrated that speed and directness increases were strongly reduced or completely abolished by the addition of the anti-HGF neutralizing antibody to the culture medium. These results provide new insights in understanding the role of CAFs, the main host stromal component of breast cancer, in promoting the tumor cell invasive attitude and may help in defining furthe

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. © 2013 Lama et al.

Glioblastoma (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with VEGF antibody reduces GBM tumor growth. However, the clinical benefits are transient and invariably followed by tumor recurrence. Recently, it has been demonstrated that in glioblastoma (GBM), cancer stem cells (CSCs) are responsible for either tumorigenesis or neoangiogenesis. In particular, a fraction of CSCs is represented by a population of endothelial progenitors. In our previous studies, we showed the presence of nestin+ cell and nestin+ and endoglin (CD105) + vasculature, not only in the GBM but also in the tissue surrounding the tumor, suggesting the involvement of CSC in endothelial differentiation also in peritumor tissue. In this study, with the aim to understand CSCs involvement in angiogenesis, we investigated the expression of angiogenesis markers (VEGF, VEGFR1, VEGFR2, HIF1α and HIF2α) by immunocytochemistry, western blot or RT- PCR analyisis, in CSCs derived from GBM (GCSC) and peritumor tissue (PCSC) of 4 patients. Both GCSCs and PCSCs were immunopositive for HIF1α and HIF2α, VEGF and its receptors VEGFR1 and VEGFR2, which underline the presence of autocrine and paracrine growth factor loops. Immunoblotting showed that VEGF is expressed at higher levels in GCSCs than PCSCs, with the exception of a patient who showed an up-regulation in PCSCs. Moreover, real time PCR showed that both VEGFR1 and VEGFR2 were expressed at low level in the two types of CSCs with a down-regulation of VEGFR1 in PCSCs. These results indicate the presence of all angiogenic markers in PCSCs suggesting their involvement in the GBM local recurrence. The inhibition of multiple angiogenic pathways may be a new line of attack on this deadly cancer.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured 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 breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured 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 breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

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 veloci

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

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce an increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6-30 days, the effect of LA (10-11 and 10-6M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with 10-6M LA. Two different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength. A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to LA in androgen-unresponsive PCa also, which might be useful in clinical practice. Moreover, the definition of parameters as ligand/receptor bond st

The migratory/invasive behaviour of breast cancer cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of tumor cells (i.e. adhesion, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive and poorly invasive (MCF-7) or ER-negative and highly invasive (MDA-MB-231) breast cancer cells with normal fibroblasts (NFs) isolated from breast healthy skin or breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to affect E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells. Two-photon microscopy imaging of monolayer cells labeled with Laurdan, a membrane fluorescent probe, showed that tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while neoplastic cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the epithelial cell/fibroblast cross-talk determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-invasive effect of CAFs on both well- and poorly differentiated cancer cells was exteriorized by reduction of cell adhesion, induct

The links between biomechanics and human diseases have been the subject of considerable scientific research effort for a number of decades. Recently, the application of soft glass rheology and polymers dynamics concepts from physics and engineering to the study of biological and physiological problems has provided valuable insights into the mechanics of cell function, their mechanical response to external stimuli and their physical and mechanical processes.Here we report the characteristic self-similar cell stress-stiffening response of the two subsequent cytoskeleton dynamics: the early entropic and the later soft glass-like dynamics. We propose that the master relation obtained by a single parameter normalization reflects, in context with the hierarchical nature of cytoskeleton architecture, the intrinsic tight dependence of the two mechanical behaviours

The links between biomechanics and human diseases have been the subject of considerable scientific research effort for a number of decades. Recently, the application of soft glass rheology and polymers dynamics concepts from physics and engineering to the study of biological and physiological problems has provided valuable insights into the mechanics of cell function, their mechanical response to external stimuli and their physical and mechanical processes.Here we report the characteristic self-similar cell stress-stiffening response of the two subsequent cytoskeleton dynamics: the early entropic and the later soft glass-like dynamics. We propose that the master relation obtained by a single parameter normalization reflects, in context with the hierarchical nature of cytoskeleton architecture, the intrinsic tight dependence of the two mechanical behaviours

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce a post-transcriptional increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6, 12, 18, 24 and 30 days, the effect of LA (10-11 and 10-6 M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto conical AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the number of GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with the highest dose of LA (10-6M). At least 2 different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength (~37 piconewton). A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and LA-treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to the analogue in androgen-unresponsive PCa a

The links between biomechanics and human diseases have been the subject of considerable scientific research effort for a number of decades. Recently, the application of soft glass rheology and polymers dynamics concepts from physics and engineering to the study of biological and physiological problems has provided valuable insights into the mechanics of cell function, their mechanical response to external stimuli and their physical and mechanical processes.Here we report the characteristic self-similar cell stress-stiffening response of the two subsequent cytoskeleton dynamics: the early entropic and the later soft glass-like dynamics. We propose that the master relation obtained by a single parameter normalization reflects, in context with the hierarchical nature of cytoskeleton architecture, the intrinsic tight dependence of the two mechanical behaviours

The migratory/invasive behaviour of breast cancer cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of tumor cells (i.e. adhesion, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive and poorly invasive (MCF-7) or ER-negative and highly invasive (MDA-MB-231) breast cancer cells with normal fibroblasts (NFs) isolated from breast healthy skin or breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to affect E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells. Two-photon microscopy imaging of monolayer cells labeled with Laurdan, a membrane fluorescent probe, showed that tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while neoplastic cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the epithelial cell/fibroblast cross-talk determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-invasive effect of CAFs on both well- and poorly differentiated cancer cells was exteriorized by reduction of cell adhesion, induct

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce an increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6-30 days, the effect of LA (10-11 and 10-6M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with 10-6M LA. Two different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength. A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to LA in androgen-unresponsive PCa also, which might be useful in clinical practice. Moreover, the definition of parameters as ligand/receptor bond st

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce a post-transcriptional increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6, 12, 18, 24 and 30 days, the effect of LA (10-11 and 10-6 M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto conical AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the number of GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with the highest dose of LA (10-6M). At least 2 different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength (~37 piconewton). A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and LA-treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to the analogue in androgen-unresponsive PCa a

Stromal fibroblasts have been reported to interfere with proliferation and invasion of cancer cells. Our objective was to elucidate, using a three-dimensional coculture system (nodules), the interaction between a human, estrogen receptor-positive, well differentiated breast cancer cell line (MCF-7) and fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in terms of cell growth and adhesion. The cell proliferating fraction of each cell type was evaluated by immunohistochemical detection of two cell cycle-associated nuclear proteins: Ki67 and proliferating cell nuclear antigen (PCNA). The expression of epithelial adhesion molecule E-cadherin was investigated in MCF-7 cells by confocal immunofluorescence microscopy. The MCF-7 cells/CAFs mixed nodules showed a significantly larger proportion (50%, p<0.001, Tukey’s multiple comparison test) of Ki67-positive tumor cells with respect to that observed in MCF-7cells/NFs (30.6%, p<0.001) or MCF-7 (34.8%, p<0.001) nodules. MCF-7 cells seemed to have no influence on the growth of both kinds of fibroblasts. Similar results were obtained with PCNA, even though a more heterogeneous and less intense nuclear staining was observed. Immunofluorescence analysis showed that in MCF-7 cells/NFs nodules E-cadherin expression level in tumor cells was about 2.5 fold higher than that observed in MCF-7 cultured alone and about 16 fold higher than that showed by MCF-7 cells cocultured with CAFs. These data support the hypothesis that the activated fibroblasts residing in the breast tumor stroma are able to transmit mitogenic signals to the epithelial cancer cells. Concerning cell adhesion, our results clearly demonstrate the ability of CAFs to negatively affect cell-cell contacts of epithelial cells by strongly reducing E-cadherin expression. The crosstalk of tumor cells with NFs seems to play a crucial role in preventing disruption of cell junctions, cell sha

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.

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.

We provide evidence that recombinant human interferon-beta (rHuIFN-beta) is able to increase androgen receptor (AR) expression, interfere with the acquisition of a neuroendocrine (NE) phenotype, and improve adhesion potential of androgen-insensitive prostate cancer cells (PC-3). The effect of rHuIFN-beta (10-1000 IU/mL) on AR, chromogranin A (CgA), E-cadherin (E-cad), N-cadherin (N-cad), and c-met levels was investigated by Western blotting after 48, 96, and 144 h. In agreement with our previous results, rHuIFN-beta (10-1000 IU/mL) induced a dramatic increase in AR (up to 5.3-fold, p < 0.001) that was already evident with the lowest cytokine concentration (10 IU/mL). A reduction in CgA levels (up to 45%, p < 0.002) was produced by 100 and 1000 IU/mL after 48-144 h. E-cad upregulation (up to 90%, p < 0.05) was observed starting from 96 h of treatment with 100 and 1000 IU/mL rHuIFN-beta and persisted until 144 h. An rHuIFN-beta-dependent reduction occurred in N-cad and c-met signal after a 48-96 h of treatment. This effect was particularly strong after 144 h of exposure to 1000 IU/mL rHuIFN-beta (81.5%, N-cad; 58%, c-met) (p < 0.002). Reverse transcription-PCR (RT-PCR) analysis of c-met expression demonstrated that the IFN-induced c-met downregulation mostly occurs at the transcriptional level (reduction up to nearly 50%, p < 0.000). Together, these results indicate that rHuIFN-beta may reduce the motility and invasiveness of poorly differentiated prostate cancer cells and interfere with the acquisition of an NE phenotype, often characterized by a low AR level.

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 mitogenactivated 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.

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 apoptosisregulating 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.