Phenotypic characterization of human prostatic stromal cells in primary cultures derived from human tissue samples

Emerging evidence has shown that the tumor microenvironment plays a crucial role in prostate cancer (PCa) development and progression. However, the mechanism(s) through which stromal cells regulate epithelial cells and the differences among prostatic stromal cells of different histological/pathological origin in PCa progression remain unclear. Therefore, it is necessary to characterize the stromal cell populations present in benign prostatic hyperplasia (BPH) and PCa. To this end, we used cultures from stromal cells obtained from BPH-derived (15 cases) and PCa-derived (30 cases) primary cultures. In culture, stromal cells are a mixture of fibroblasts, myofibroblasts (MFs) and muscle cells. Fibroblasts are characterized for the expression of vimentin, MFs for the co-expression of α-smooth muscle actin (α-SMA) and vimentin, whereas muscle cells for the expression of α-SMA and desmin. Fibroblasts were present in large amounts in the BPH-compared to the PCa-derived cultures, whereas MFs were more representative of PCa-as opposed to BPH-derived cultures. Some α-SMA-positive cells retained the expression of basal cytokeratin K14. This population was defined as myoepithelial cells and was associated with senescent cultures. The percentage of MFs was higher in high-grade compared to moderate-and low-grade PCa-derived cultures, whereas the number of myoepithelial cells was lower in high-grade compared to moderate-and low-grade PCa-derived cultures. In addition, we analyzed the expression of p75NTR, as well as the expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of MMPs (TIMPs). p75NTR expression was elevated in the stromal cultures derived from PCa compared to those derived from BPH and in cultures derived from cases with Gleason scores.7 compared to those derived from cases with Gleason scores <7, as well as in cultures with a high concentration of MFs compared to those with a high concentration of fibroblasts. MMP-2 was secreted by all primary cultures, whereas MMP-9 secretion was observed only in some PCa-derived stromal cells, when the percentage of MFs was significantly higher compared to BPH-derived cultures. TIMP1, TIMP2 and TIMP3 were secreted in elevated amounts in the BPH-compared to the PCa-derived stromal cultures, suggesting the differential regulation of extracellular matrix (ECM) degradation. When we used 22rv1 and PC3 PCa xenograft models for the isolation and characterization of murine cancer-associated fibroblasts (CAFs) we noted that the angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, vimentin, tenascin, calponin, desmin and Masson's trichrome. In conclusion, MF stromal cells from PCa participate in the progression and metastasis of PCa, modualting inflammation, angiogenesis and epithelial cancer cell proliferation

Targeted Molecular Therapy in Glioblastoma.

This special issue was aimed at updating researchers on current topics and progress made in basic, preclinical, and clinical glioblastoma research. It also provided a platform for pharmaceutical and translational scientists to submit original research articles, review articles, and clinical studies, focusing on the evaluation of new molecular pathways as pharmacological targets for treatment strategies which may improve the management of aggressive, drug-resistant GBM, in the hope that a deeper knowledge of GBM biology may eventually lead to effective targeted therapeutic approaches based on the inhibition of tumor-specific proteins or molecular pathways

Increased expression and activity of p75NTR are crucial events in azacitidine-induced cell death in prostate cancer

The high affinity nerve growth factor (NGF) NGF receptor, p75NTR, is a member of the tumor necrosis factor (TNF) receptor superfamily that shares a conserved intracellular death domain capable of inducing apoptosis and suppressing growth in prostate epithelial cells. Expression of this receptor is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines. We aimed to verify the role of p75NTR in the azacitidine-mediated antitumor effects on 22Rv1 and PC3 androgen-independent prostate cancer cells. In the present study, we reported that the antiproliferative and pro-apoptotic effects of 5-azacytidine (azacitidine) were more marked in the presence of physiological concentrations of NGF and were reduced when a blocking p75NTR antibody or the selective p75NTR inhibitor, Ro 08-2750, were used. Azacitidine increased the expression of p75NTR without interfering with the expression of the low affinity NGF receptor TrkA and induced caspase 9-dependent caspase 3 activity. Taken together, our results suggest that the NGF network could be a candidate for future pharmacological manipulation in aggressive prostate cancer

The possible prognostic role of histone deacetylase and transforming growth factor β/Smad signaling in high grade gliomas treated by radio-chemotherapy: a preliminary immunohistochemical study

Glioblastoma (GBM) is the most common and aggressive tumor of the central nervous system. Unfortunately, patients affected by this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies. Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Histone Deacetylases (HDAC) dependent epigenetic modifications and transforming growth factor (TGF)-β/Smad pathway seem to play an important role in GBM tumorigenesis, resistance to common therapies and poor clinical outcome. The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis and prognosis of GBM. Immunohistochemistry (IHC) was performed on microdissected GBM samples, collected from 14 patients (6 men and 8 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into two groups: short and long OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the TGF-β/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the long survivors. The short OS group exhibits a decrease in Smad 7 expression and also low levels of p21 immunostaining, which represents a common target of the two pathways. The IHC data was confirmed by quantitative analysis and Immunoblotting. Our preliminary results suggest that both HDAC4 and HDAC6 together with the TGF-β/Smad pathway may be involved in progression of GBM and this cross talking could be a useful prognostic marker in this deadly disease

Increased levels of DNA methyltransferases are associated with the tumorigenic capacity of prostate cancer cells

DNA methylation might be the earliest somatic genome changes in prostate cancer that also play an important role in the process of tumor invasion, growth and metastasis. In recent years, several inhibitors of DNA methyltransferases (DNMTis) have been developed and evaluated in pre-clinical models and in clinical trials. While these compounds are effective in the treatment of hematological conditions, clinical trials in solid tumors and in prostate cancer have shown limited or no efficacy. This may be attributed to inappropriate dose regimens leading to toxicity-related adverse events. As with other anti-target compounds, one of the obstacles encountered with DNMTis in prostate cancer could be the inability to select patients for the clinical studies as well as the inability to monitor the efficacy of the drug if not the conclusion of the study. Primary cultures derived from human prostatic tissues harvested from patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) as well as neoplastic and non-neoplastic prostate cell lines were tested for DNMT expression/activity and to monitor azacitidine molecular efficacy. We observed that in primary cultures the levels of DNMT activity as well as the protein levels of DNMT1, DNMT3a and DNMT3b were higher in cultures derived from PCa compared to BPH tissue samples and significantly higher in cultures derived from PCa with Gleason scores ≥7 compared to those observed in cultures derived from Gleason scores <7. In addition, DNMT activity as well as DNMT1, DNMT3a and DNMT3b levels were higher in PCa cell lines compared to their non-neoplastic counterparts. Although DNMT activity was higher in high tumorigenic/aggressive PCa cell lines compared to low tumorigenic/aggressive cell lines, only the levels of DNMT3a and DNMT3b were significantly higher in the first group of cells, suggesting that DNMT1 activity is related to the transition to non-neoplastic versus neoplastic phenotype whereas the de novo methylation enzymes were mainly related to progression. Nevertheless, the comparison in the more aggressive PC3 cell derivatives (PC3-LN4 cells) also possessed higher levels of DNMT1 compared to PC3 and PC3M from which these cells were derived. Collectively, our results confirm previous data on the increased methylation in more aggressive tumors supporting the use of DNMTis in advanced prostate cancer. In addition, since glutathione S-transferase-π (GSTP1) was re-expressed or its protein levels were increased after treatment with non-toxic azacitidine doses and since GSTP1 can easily be measured in patient sera, the monitoring of this protein may aide in the evaluation of therapy in future clinical trials

Enhancement of radiosensitivity by the novel anticancer quinolone derivative vosaroxin in preclinical glioblastoma models

Purpose: Glioblastoma multiforme (GBM) is the most aggressive brain tumor. The activity of vosaroxin, a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, was investigated in GBM preclinical models as a single agent and combined with radiotherapy (RT). Results: Vosaroxin showed antitumor activity in clonogenic survival assays, with IC50 of 10-100 nM, and demonstrated radiosensitization. Combined treatments exhibited significantly higher γH2Ax levels compared with controls. In xenograft models, vosaroxin reduced tumor growth and showed enhanced activity with RT; vosaroxin/RT combined was more effective than temozolomide/RT. Vosaroxin/ RT triggered rapid and massive cell death with characteristics of necrosis. A minor proportion of treated cells underwent caspase-dependent apoptosis, in agreement with in vitro results. Vosaroxin/RT inhibited RT-induced autophagy, increasing necrosis. This was associated with increased recruitment of granulocytes, monocytes, and undifferentiated bone marrow-derived lymphoid cells. Pharmacokinetic analyses revealed adequate blood-brain penetration of vosaroxin. Vosaroxin/RT increased disease-free survival (DFS) and overall survival (OS) significantly compared with RT, vosaroxin alone, temozolomide, and temozolomide/RT in the U251-luciferase orthotopic model. Materials and Methods: Cellular, molecular, and antiproliferative effects of vosaroxin alone or combined with RT were evaluated in 13 GBM cell lines. Tumor growth delay was determined in U87MG, U251, and T98G xenograft mouse models. (DFS) and (OS) were assessed in orthotopic intrabrain models using luciferasetransfected U251 cells by bioluminescence and magnetic resonance imaging. Conclusions: Vosaroxin demonstrated significant activity in vitro and in vivo in GBM models, and showed additive/synergistic activity when combined with RT in O6- methylguanine methyltransferase-negative and -positive cell lines

The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

Background: The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6- methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity. Methods: Tinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments. Results: We demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity. Conclusions: Our data suggest that tinostamustine deserves further investigation in patients with glioblastoma

Hypoxia sustains glioblastoma radioresistance through ERKs/DNA-PKcs/HIF-1α functional interplay

The molecular mechanisms by which glioblastoma multiforme (GBM) refracts and becomes resistant to radiotherapy treatment remains largely unknown. This radioresistance is partly due to the presence of hypoxic regions, which are frequently found in GBM tumors. We investigated the radiosensitizing effects of MEK/ERK inhibition on GBM cell lines under hypoxic conditions. Four human GBM cell lines, T98G, U87MG, U138MG and U251MG were treated with the MEK/ERK inhibitor U0126, the HIF-1α inhibitor FM19G11 or γ-irradiation either alone or in combination under hypoxic conditions. Immunoblot analysis of specific proteins was performed in order to define their anti‑oncogenic or radiosensitizing roles in the different experimental conditions. MEK/ERK inhibition by U0126 reverted the transformed phenotype and significantly enhanced the radiosensitivity of T98G, U87MG, U138MG cells but not of the U251MG cell line under hypoxic conditions. U0126 and ERK silencing by siRNA reduced the levels of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70 and K80 proteins and clearly reduced HIF-1α activity and protein expression. Furthermore, DNA-PKcs siRNA-mediated silencing counteracted HIF-1α activity and downregulated protein expression suggesting that ERKs, DNA-PKcs and HIF-1α cooperate in radioprotection of GBM cells. Of note, HIF-1α inhibition under hypoxic conditions drastically radiosensitized all cell lines used. MEK/ERK signal transduction pathway, through the sustained expression of DNA-PKcs, positively regulates HIF-1α protein expression and activity, preserving GBM radioresistance in hypoxic condition

Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer

Background and aims: Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon. Material and methods: Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives. Results and conclusions: We show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients

Biological rationale for the use of DNA methyltransferase inhibitors as new strategy for modulation of tumor response to chemotherapy and radiation

Epigenetic modifications play a key role in the patho-physiology of many tumors and the current use of agents targeting epigenetic changes has become a topic of intense interest in cancer research. DNA methyltransferase (DNMT) inhibitors represent a promising class of epigenetic modulators. Research performed yielded promising anti-tumorigenic activity for these agents in vitro and in vivo against a variety of hematologic and solid tumors. These epigenetic modulators cause cell cycle and growth arrest, differentiation and apoptosis. Rationale for combining these agents with cytotoxic therapy or radiation is straightforward since the use of DNMT inhibitor offers greatly improved access for cytotoxic agents or radiation for targeting DNA-protein complex. The positive results obtained with these combined approaches in preclinical cancer models demonstrate the potential impact DNMT inhibitors may have in treatments of different cancer types. Therefore, as the emerging interest in use of DNMT inhibitors as a potential chemo- or radiation sensitizers is constantly increasing, further clinical investigations are inevitable in order to finalize and confirm the consistency of current observations