Publications by authors named "Lucio Tentori"

54 Publications

Targeting the vascular endothelial growth factor receptor-1 by the monoclonal antibody D16F7 to increase the activity of immune checkpoint inhibitors against cutaneous melanoma.

Pharmacol Res 2020 09 30;159:104957. Epub 2020 May 30.

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. Electronic address:

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a membrane receptor for VEGF-A, placenta growth factor (PlGF) and VEGF-B that plays a crucial role in melanoma invasiveness, vasculogenic mimicry and tumor-associated angiogenesis. Furthermore, activation of VEGFR-1 is involved in the mobilization of myeloid progenitors from the bone marrow that infiltrate the tumor. Myeloid-derived suppressor cells and tumor-associated macrophages have been involved in tumor progression and resistance to cancer treatment with immune checkpoint inhibitors (ICIs). We have recently demonstrated that the anti-VEGFR-1 monoclonal antibody (mAb) D16F7 developed in our laboratories is able to inhibit melanoma growth in preclinical in vivo models and to reduce monocyte/macrophage progenitor mobilization and tumor infiltration by myeloid cells. Aim of the study was to investigate whether the anti-VEGFR-1 mAb D16F7 affects the activity of protumoral M2 macrophages in vitro in response to PlGF and inhibits the recruitment of these cells to the melanoma site in vivo. Finally, we tested whether, through its multi-targeted action, D16F7 mAb might increase the efficacy of ICIs against melanoma. The results indicated that VEGFR-1 expression is up-regulated in human activated M2 macrophages compared to activated M1 cells and exposure to the D16F7 mAb decreases in vitro chemotaxis of activated M2 macrophages. In vivo treatment with the anti-VEGFR-1 mAb D16F7 of B6D2F1 mice injected with syngeneic B16F10 melanoma cells resulted in tumor growth inhibition associated with the modification of tumor microenvironment that involves a decrease of melanoma infiltration by M2 macrophages and PD-1+ and FoxP3+ cells. These alterations result in increased M1/M2 and CD8+/FoxP3+ ratios, which favor an antitumor and immunostimulating milieu. Accordingly, D16F7 mAb increased the antitumor activity of the ICIs anti-CTLA-4 and anti-PD-1 mAbs. Overall, these data reinforce the role of VEGFR-1-mediated-signalling as a valid target for reducing tumor infiltration by protumoral macrophages and for improving the efficacy of immunotherapy with ICIs.
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http://dx.doi.org/10.1016/j.phrs.2020.104957DOI Listing
September 2020

Role of VEGFR-1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib.

J Cell Mol Med 2020 01 23;24(1):465-475. Epub 2019 Nov 23.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF-A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR-1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour-associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro-angiogenic pathways, we have investigated VEGFR-1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF-A and express higher VEGFR-1 levels compared with their BRAFi-sensitive counterparts. Transient VEGFR-1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR-1 expression, by stable gene transfection in receptor-negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR-1 are more invasive than VEGFR-1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF-A and PlGF. These data suggest that VEGFR-1 up-regulation might contribute to melanoma progression and spreading after acquisition of a drug-resistant phenotype. Thus, VEGFR-1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR-1 positive tumours with acquired resistance to vemurafenib.
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http://dx.doi.org/10.1111/jcmm.14755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933379PMC
January 2020

Effects of Glutathione Transferase-Targeting Nitrobenzoxadiazole Compounds in Relation to PD-L1 Status in Human Melanoma Cells.

Chemotherapy 2019 22;64(3):138-145. Epub 2019 Oct 22.

Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy,

Background: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine.

Methods: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis.

Results: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC50 values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells.

Conclusions: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.
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http://dx.doi.org/10.1159/000503339DOI Listing
December 2019

Cytotoxicity and Differentiating Effect of the Poly(ADP-Ribose) Polymerase Inhibitor Olaparib in Myelodysplastic Syndromes.

Cancers (Basel) 2019 Sep 16;11(9). Epub 2019 Sep 16.

Hematology Section, Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy.

Myelodysplastic syndromes (MDS) are highly heterogeneous myeloid diseases, characterized by frequent genetic/chromosomal aberrations. Olaparib is a potent, orally bioavailable poly(ADP-ribose) polymerase 1 (PARP1) inhibitor with acceptable toxicity profile, designed as targeted therapy for DNA repair defective tumors. Here, we investigated olaparib activity in primary cultures of bone marrow mononuclear cells collected from patients with MDS ( = 28). A single treatment with olaparib induced cytotoxic effects in most samples, with median IC of 5.4 µM (2.0-24.8 µM), lower than plasma peak concentration reached in vivo. In addition, olaparib induced DNA damage as shown by a high proportion of γH2AX positive cells in samples with low ICs. Olaparib preferentially killed myeloid cells causing a significant reduction of blasts and promyelocytes, paralleled by an increase in metamyelocytes and mature granulocytes while sparing lymphocytes that are not part of the MDS clone. Consistently, flow cytometry analysis revealed a decrease of CD117+/CD123+ immature progenitors ( < 0.001) and induction of CD11b+/CD16+ ( < 0.001) and CD10+/CD15+ ( < 0.01) neutrophils. Morphological and immunophenotypic changes were associated with a dose-dependent increase of PU.1 and CEBPA transcription factors, which are drivers of granulocytic and monocytic differentiation. Moreover, the combination of olaparib with decitabine resulted in augmented cytotoxic and differentiating effects. Our data suggest that olaparib may have therapeutic potential in MDS patients.
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http://dx.doi.org/10.3390/cancers11091373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769925PMC
September 2019

Experimental Evidence of the Antitumor, Antimetastatic and Antiangiogenic Activity of Ellagic Acid.

Nutrients 2018 Nov 14;10(11). Epub 2018 Nov 14.

Department of Systems Medicine, University of Rome Tor Vergata, 00173 Rome, Italy.

Ellagic acid (EA) is a naturally occurring polyphenolic compound endowed with strong antioxidant and anticancer properties that is present in high quantity in a variety of berries, pomegranates, and dried fruits. The antitumor activity of EA has been mostly attributed to direct antiproliferative and apoptotic effects. Moreover, EA can inhibit tumour cell migration, extra-cellular matrix invasion and angiogenesis, all processes that are crucial for tumour infiltrative behaviour and the metastatic process. In addition, EA may increase tumour sensitivity to chemotherapy and radiotherapy. The aim of this review is to summarize the in vitro and in vivo experimental evidence supporting the anticancer activity of pure EA, its metabolites, and EA-containing fruit juice or extracts in a variety of solid tumour models. The EA oral administration as supportive therapy to standard chemotherapy has been recently evaluated in small clinical studies with colorectal or prostate cancer patients. Novel formulations with improved solubility and bioavailability are expected to fully develop the therapeutic potential of EA derivatives in the near future.
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http://dx.doi.org/10.3390/nu10111756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266224PMC
November 2018

Drug-induced xenogenization of tumors: A possible role in the immune control of malignant cell growth in the brain?

Pharmacol Res 2018 05 9;131:1-6. Epub 2018 Mar 9.

Department of Systems Medicine, Medical Oncology, University of Rome Tor Vergata, Rome, Italy.

In recent years, immune checkpoint inhibitors (ICpI) have provided the ground to bring tumor immunity back to life thanks to their capacity to afford a real clinical benefit in terms of patient's survival. Essential to ICpI success is the presence of tumor-associated neoantigens generated by non-synonymous mutations, since a direct relationship between mutation load of malignant cells and susceptibility to ICpI has been confidently established. However, it has been also suggested that high intratumor heterogeneity (ITH) associated with subclonal neoantigens could not elicit adequate immune responses. Several years ago we discovered that in vivo treatment of leukemic mice with triazene compounds (TZC) produces a marked increase of leukemia cell immunogenicity [a phenomenon termed Drug-Induced Xenogenization (DIX)] through point mutations able to generate strong tumor neoantigens (Drug-Induced Neoantigens, DIN). Immunogenic mutations are produced by TZC-dependent methylation of O6-guanine of DNA, that is suppressed by the DNA repair protein methyl-guaninemethyltransferase (MGMT). This minireview illustrates preclinical investigations conducted in animal models where DIN-positive murine leukemia cells were inoculated intracerebrally into histocompatible mice. The analysis of the literature indicates that the growth of xenogenized malignant cells is controlled by anti-DIN graft responses and by intra-cerebral or intravenous adoptive transfer of anti-DIN cytotoxic T lymphocytes. This survey reminds also that PARP inhibitors increase substantially the antitumor activity of TZC and can be administered with the intent of suppressing more efficiently tumor load and possibly reducing ITH through downsizing the polyclonality of xenogenized tumor cell population. Finally, the present report illustrates a hypothetical clinical protocol that could be considered as an example of future development of DIXbased tumor immuno-chemotherapy in brain malignancies. The protocol involves oral or intravenous administration of TZC along with loco-regional (i.e. intracerebral "wafer") treatment with agents able to increase tumor cell sensitivity to the cytotoxic and xenogenizing effects of TZC (i.e. MGMT and PARP inhibitors) without enhancing the systemic toxicity of these DNA methylating compounds.
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http://dx.doi.org/10.1016/j.phrs.2018.03.005DOI Listing
May 2018

Poly(ADP-ribose) polymerase inhibitor olaparib hampers placental growth factor-driven activation of myelomonocytic cells.

Oncol Rep 2018 May 5;39(5):2261-2269. Epub 2018 Mar 5.

Department of Systems Medicine, University of Rome 'Tor Vergata', I‑00133 Rome, Italy.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor activated by the angiogenic factors VEGF‑A and placental growth factor (PlGF). While VEGF‑A binds to both VEGFR‑1 and VEGFR‑2, PlGF interacts exclusively with VEGFR‑1 triggering a signaling pathway involved in: i) tumor‑associated angiogenesis; ii) chemotaxis and invasion of the extracellular matrix (ECM) by cancer cells; and iii) mobilization of bone marrow‑derived myeloid cells that generate tumor‑associated macrophages. By using a novel anti‑VEGFR‑1 monoclonal antibody (D16F7 mAb), which hampers receptor activation without avoiding ligand binding, we recently demonstrated that VEGFR‑1 blockade reduced myeloid progenitor mobilization and monocyte/macrophage cell infiltration of tumor grafts in vivo. Since poly(ADP‑ribose) polymerase (PARP)‑1 exerts a pro‑inflammatory role favoring monocyte activation, in the present study we investigated whether the PARP inhibitor (PARPi) olaparib hampers PlGF‑induced activation of human myelomonocytic cells. HL‑60 cells induced to differentiate towards the monocytic/macrophage lineage were tested and the results were confirmed in freshly isolated monocytes obtained from healthy donors. Cells were treated with olaparib, at clinically achievable concentrations, before exposure to PlGF and were analyzed for migration and ECM invasion in response to PlGF. Olaparib effects were compared to those obtained with D16F7 mAb used as single agent or in combination with the PARPi. The results indicate that differentiated HL‑60 cells and monocytes expressed VEGFR‑1 and migrated in response to PlGF. Moreover, olaparib and D16F7 inhibited PlGF‑induced chemotaxis and ECM invasion in a dose‑dependent manner and with similar efficacy. However, in combination studies the PARPi and D16F7 did not exert synergistic effects. Olaparib also hampered PlGF‑induced monocyte adhesion to fibronectin, while it did not affect NF‑κB activation in response to the angiogenic factor. These data suggest that olaparib likely interferes with the same pathway affected by the anti‑VEGFR‑1 mAb and that inhibition of PlGF-induced monocyte activation may contribute to PARPi antitumor activity.
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http://dx.doi.org/10.3892/or.2018.6291DOI Listing
May 2018

The Anti-Vascular Endothelial Growth Factor Receptor-1 Monoclonal Antibody D16F7 Inhibits Glioma Growth and Angiogenesis In Vivo.

J Pharmacol Exp Ther 2018 01 12;364(1):77-86. Epub 2017 Oct 12.

Departments of Systems Medicine (M.G.A., L.T., C.C., G.G.) and Experimental Medicine and Surgery (E.B., M.S.), University of Rome Tor Vergata, Rome, Italy; and Laboratory of Molecular Oncology, "Istituto Dermopatico dell'Immacolata"-Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy (F.R., P.M.L.)

The vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1) is a tyrosine kinase receptor that does not play a relevant role in physiologic angiogenesis in adults, whereas it is important in tumor angiogenesis. In high-grade glioma VEGFR-1 expression by tumor endothelium and neoplastic cells contributes to the aggressive phenotype. We recently generated an anti-VEGFR-1 monoclonal antibody (D16F7 mAb) characterized by a novel mechanism of action, since it hampers receptor activation without interfering with ligand binding. The mAb is able to inhibit chemotaxis and extracellular matrix invasion of glioma cells in vitro stimulated by VEGF-A and by the VEGFR-1-selective ligand placental growth factor (PlGF). In this study, we have investigated the influence of D16F7 on glioma growth and angiogenesis in vivo using C6 glioma cells transfected with the human VEGFR-1. D16F7 was able to inhibit receptor activation and migration and extracellular matrix invasion of C6 cells overexpressing the receptor in response to PlGF and VEGF-A. In nude mice, treatment with 10 and 20 mg/kg D16F7 as a single agent was well tolerated and significantly inhibited glioma growth ( < 0.001). Strikingly, in an intracranial orthotopic model, mice dosed with 20 mg/kg D16F7 demonstrated a 65% increase in median survival time compared with vehicle-treated controls ( < 0.001) with a high percentage of long-term survivors (46%). These effects were associated with glioma cell apoptosis and decreased tumor-associated vessel formation. Overall, these results highlight the therapeutic potential of D16F7 in glioma treatment, deserving further investigation after a humanization process as single agent or in combination therapies.
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http://dx.doi.org/10.1124/jpet.117.244434DOI Listing
January 2018

Modulation of GDF11 expression and synaptic plasticity by age and training.

Oncotarget 2017 Aug 3;8(35):57991-58002. Epub 2017 Aug 3.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

The Growth Differentiation Factor 11 (GDF11) has been controversially involved in the aging/rejuvenation process. To clarify whether GDF11 is differently expressed during aging, we have evaluated GDF11 levels in skeletal muscles and hippocampi of young and old mice, sedentary or subjected to a 12-weeks triweekly training protocol. The results of real-time PCR and Western blot analyses indicate that skeletal muscles of sedentary old mice express higher levels of GDF11 compared to young animals ( < 0.05). Conversely, in hippocampi no significant differences of GDF11 expression are detected. Analysis of long-term potentiation, a synaptic plasticity phenomenon, reveals that population spikes in response to a tetanic stimulus are significantly higher in sedentary young mice than in old animals ( < 0.01). Training induces a significant improvement of long-term potentiation in both young and old animals ( < 0.05), an increase ( < 0.05) of skeletal muscle GDF11 levels in young mice and a reduction of GDF11 expression in hippocampi of old mice ( < 0.05). Overall, data suggest that GDF11 can be considered an aging biomarker for skeletal muscles. Moreover, physical exercise has a positive impact on long-term potentiation in both young and old mice, while it has variable effects on GDF11 expression depending on age and on the tissue analyzed.
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http://dx.doi.org/10.18632/oncotarget.19854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601628PMC
August 2017

The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells.

J Exp Clin Cancer Res 2017 08 10;36(1):106. Epub 2017 Aug 10.

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.

Background: Glioblastoma (GBM) is a highly migratory, invasive, and angiogenic brain tumor. Like vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF) promotes GBM angiogenesis. VEGF-A is a ligand for both VEGF receptor-1 (VEGFR-1) and VEGFR-2, while PlGF interacts exclusively with VEGFR-1. We recently generated the novel anti-VEGFR-1 monoclonal antibody (mAb) D16F7 that diminishes VEGFR-1 homodimerization/activation without affecting VEGF-A and PlGF binding.

Methods: In the present study, we evaluated the expression of VEGFR-1 in human GBM tissue samples (n = 42) by immunohistochemistry, in cell lines (n = 6) and GBM stem cells (GSCs) (n = 18) by qRT-PCR and/or western blot analysis. In VEGFR-1 positive GBM or GSCs we also analyzed the ability of D16F7 to inhibit GBM invasiveness in response to VEGF-A and PlGF.

Results: Most of GBM specimens stained positively for VEGFR-1 and all but one GBM cell lines expressed VEGFR-1. On the other hand, in GSCs the expression of the receptor was heterogeneous. D16F7 reduced migration and invasion of VEGFR-1 positive GBM cell lines and patient-derived GSCs in response to VEGF-A and PlGF. Interestingly, this effect was also observed in VEGFR-1 positive GSCs transfected to over-express wild-type EGFR (EGFRwt) or mutant EGFR (ligand binding domain-deficient EGFRvIII). Furthermore, D16F7 suppressed intracellular signal transduction in VEGFR-1 over-expressing GBM cells by reducing receptor auto-phosphorylation at tyrosine 1213 and downstream Erk1/2 activation induced by receptor ligands.

Conclusion: The results from this study suggest that VEGFR-1 is a relevant target for GBM therapy and that D16F7-derived humanized mAbs warrant further investigation.
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http://dx.doi.org/10.1186/s13046-017-0577-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553938PMC
August 2017

Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth.

Nutrients 2016 Nov 22;8(11). Epub 2016 Nov 22.

Department of Systems Medicine, University of Rome Tor Vergata, Rome 00173, Italy.

Ellagic acid (EA) is a polyphenolic compound that can be found as a naturally occurring hydrolysis product of ellagitannins in pomegranates, berries, grapes, green tea and nuts. Previous studies have reported the antitumor properties of EA mainly using in vitro models. No data are available about EA influence on bladder cancer cell invasion of the extracellular matrix triggered by vascular endothelial growth factor-A (VEGF-A), an angiogenic factor associated with disease progression and recurrence, and tumor growth in vivo. In this study, we have investigated EA activity against four different human bladder cancer cell lines (i.e., T24, UM-UC-3, 5637 and HT-1376) by in vitro proliferation tests (measuring metabolic and foci forming activity), invasion and chemotactic assays in response to VEGF-A and in vivo preclinical models in nude mice. Results indicate that EA exerts anti-proliferative effects as a single agent and enhances the antitumor activity of mitomycin C, which is commonly used for the treatment of bladder cancer. EA also inhibits tumor invasion and chemotaxis, specifically induced by VEGF-A, and reduces VEGFR-2 expression. Moreover, EA down-regulates the expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint involved in immune escape. EA in vitro activity was confirmed by the results of in vivo studies showing a significant reduction of the growth rate, infiltrative behavior and tumor-associated angiogenesis of human bladder cancer xenografts. In conclusion, these results suggest that EA may have a potential role as an adjunct therapy for bladder cancer.
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http://dx.doi.org/10.3390/nu8110744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133127PMC
November 2016

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding.

Oncotarget 2016 Nov;7(45):72868-72885

Laboratory of Molecular Oncology, "Istituto Dermopatico dell'Immacolata"-IRCCS, Rome, Italy.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation.
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http://dx.doi.org/10.18632/oncotarget.12108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341950PMC
November 2016

Exploiting Microglial Functions for the Treatment of Glioblastoma.

Curr Cancer Drug Targets 2017 ;17(3):267-281

Institute of Pharmacology, Catholic University Medical School, Rome, Italy.

Background: Glioblastoma multiforme (GBM) is the most common brain tumor in adults and is associated with a very low survival rate. The heterogeneity of the tumor microenvironment, its resistance to drug and radiation therapy, and its robust invasiveness all contribute to the poor outcome. Large numbers of glioma associated microglia and macrophages (GAMs) can accumulate within the tumor where they appear to have an important role in prognosis.

Methods: An extensive revision of current available literature on this topic has been carried out, using the PubMed database. Articles exploring the contribution of GAMs to GBM biology as well as evidence that GAMs can be pharmacologically modulated to inhibit tumor growth are critically discussed in this review article.

Results: GAMs constitute the largest portion of tumor infiltrating cells contributing up to 30% of the entire glioma mass. Upon interaction with neoplastic cells, GAMs acquire a unique phenotype of activation including both M1 and M2 specific markers. Different profiles of activation usually co-exist in the same tumor that is dependent upon GAM location or stage of disease. In addition to regulating immune responses which may control or favor astrocyte malignant transformation, GAMs are directly involved in the degradation of the extracellular matrix (ECM), a crucial mechanism that allows the expansion of tumors and parenchyma invasion. Several pharmacological strategies have been developed which interfere with GAM recruitment at the tumor site, cell polarization and immune function, and ECM remodeling by GAM-secreted factors. The most promising therapeutic approaches appear to target both GBM cells and GAM biological properties.

Conclusion: GAMs significantly contribute to GBM biology (favoring tumor growth and invasiveness). Data reviewed in the present article suggest that these cells represent a valuable alternative/ additional target for the development of more effective treatments for GBM.
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http://dx.doi.org/10.2174/1568009616666160813191240DOI Listing
October 2017

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Biochem Pharmacol 2015 May 17;95(1):16-27. Epub 2015 Mar 17.

The NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of "Tor Vergata", Rome, Italy; Department of Experimental Medicine and Surgery, University of "Tor Vergata", Rome, Italy. Electronic address:

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.
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http://dx.doi.org/10.1016/j.bcp.2015.03.004DOI Listing
May 2015

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvβ5 integrin.

Int J Cancer 2015 Mar 28;136(6):E545-58. Epub 2014 Nov 28.

Laboratory of Molecular Oncology, "Istituto Dermopatico dell'Immacolata"-IRCCS, Rome, Italy.

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP-1), a coreceptor of vascular endothelial growth factor A (VEGF-A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP-1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule-like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvβ5 integrin was found to be responsible for about 80% of the capability of NRP-1 expressing cells to adhere on vitronectin. In these cells αvβ5 expression level was twice higher than in low-invasive control cells and contributed to the ability of melanoma cells to form tubule-like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF-A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανβ5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP-1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.
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http://dx.doi.org/10.1002/ijc.29252DOI Listing
March 2015

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

J Exp Clin Cancer Res 2014 Sep 17;33:71. Epub 2014 Sep 17.

Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.

Methods: Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.

Results: Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.

Conclusions: It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.
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http://dx.doi.org/10.1186/s13046-014-0071-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172901PMC
September 2014

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide.

BMC Cancer 2014 Mar 5;14:151. Epub 2014 Mar 5.

Department of System Medicine, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

Background: Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy.

Methods: Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni's post-test or the non-parametric Kruskal-Wallis analysis and Dunn's post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman's rank test.

Results: All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman's test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01).

Conclusions: The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.
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http://dx.doi.org/10.1186/1471-2407-14-151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975727PMC
March 2014

PARP Inhibitors in Cancer Therapy: Magic Bullets but Moving Targets.

Front Oncol 2013 14;3:279. Epub 2013 Nov 14.

Laboratory for Skin Cancer Research, CHU-Q (CHUL) Research Centre, Laval University , Quebec City, QC , Canada.

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http://dx.doi.org/10.3389/fonc.2013.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827545PMC
December 2013

Challenging resistance mechanisms to therapies for metastatic melanoma.

Trends Pharmacol Sci 2013 Dec 7;34(12):656-66. Epub 2013 Nov 7.

Department of System Medicine, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

Melanoma is the most aggressive form of skin cancer and, if spread outside the epidermis, has a dismal prognosis. Before the approval of the anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody ipilimumab and the BRAF inhibitors vemurafenib and dabrafenib, no other agents had demonstrated better results in terms of overall survival than the DNA-methylating compound dacarbazine (or its oral analog temozolomide). However, most patients with metastatic melanoma do not obtain long-lasting clinical benefit from ipilimumab and responses to BRAF inhibitors are short lived. Thus, combination therapies with inhibitors of DNA repair (e.g., poly(ADP-ribose) polymerase [PARP] inhibitors), novel immunomodulators (monoclonal antibodies against programmed death-1 [PD-1] or its ligand PD-L1), targeted therapies (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase [MEK] or phosphatidylinositol 3-kinase [PI3K]/AKT/mammalian target of rapamycin [mTOR] inhibitors) or antiangiogenic agents are currently being investigated to improve the efficacy of antimelanoma therapies. This review discusses the implications of simultaneously targeting key regulators of melanoma cell proliferation/survival and immune responses to counteract resistance.
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http://dx.doi.org/10.1016/j.tips.2013.10.003DOI Listing
December 2013

Platelet-derived growth factor C and calpain-3 are modulators of human melanoma cell invasiveness.

Oncol Rep 2013 Dec 10;30(6):2887-96. Epub 2013 Oct 10.

Laboratory of Molecular Oncology, 'Istituto Dermopatico dell'Immacolata'- IRCCS, Rome, Italy.

The molecular mechanisms responsible for the elevated metastatic potential of malignant melanoma are still not fully understood. In order to shed light on the molecules involved in the acquisition by melanoma of a highly aggressive phenotype, we compared the gene expression profiles of two cell clones derived from the human cutaneous metastatic melanoma cell line M14: a highly invasive clone (M14C2/MK18) and a clone (M14C2/C4) with low ability to invade the extracellular matrix (ECM). The highly invasive phenotype of M14C2/MK18 cells was correlated with overexpression of neuropilin-1, activation of a vascular endothelial growth factor (VEGF)-A/VEGFR-2 autocrine loop and secretion of matrix metalloprotease-2. Moreover, in an in vivo murine model, M14C2/MK18 cells displayed a higher growth rate as compared with M14C2/C4 cells, even though in vitro both clones possessed comparable proliferative potential. Microarray analysis in M14C2/MK18 cells showed a strong upregulation of platelet-derived growth factor (PDGF)-C, a cytokine that contributes to angiogenesis, and downregulation of calpain-3, a calcium-dependent thiol-protease that regulates specific signalling cascade components. Inhibition of PDGF-C with a specific antibody resulted in a significant decrease in ECM invasion by M14C2/MK18 cells, confirming the involvement of PDGF-C in melanoma cell invasiveness. Moreover, the PDGF-C transcript was found to be upregulated in a high percentage of human melanoma cell lines (17/20), whereas only low PDGF-C levels were detected in a few melanocytic cultures (2/6). By contrast, inhibition of calpain-3 activity in M14C2/C4 control cells, using a specific chemical inhibitor, markedly increased ECM invasion, strongly suggesting that downregulation of calpain-3 plays a role in the acquisition of a highly invasive phenotype. The results indicate that PDGF-C upregulation and calpain-3 downregulation are involved in the aggressiveness of malignant melanoma and suggest that modulators of these proteins or their downstream effectors may synergise with VEGF‑A therapies in combating tumour-associated angiogenesis and melanoma spread.
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http://dx.doi.org/10.3892/or.2013.2791DOI Listing
December 2013

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

Int J Oncol 2013 Jul 8;43(1):210-8. Epub 2013 May 8.

Department of System Medicine, University of Rome 'Tor Vergata', I-00133 Rome, Italy.

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.
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http://dx.doi.org/10.3892/ijo.2013.1932DOI Listing
July 2013

MSH3 expression does not influence the sensitivity of colon cancer HCT116 cell line to oxaliplatin and poly(ADP-ribose) polymerase (PARP) inhibitor as monotherapy or in combination.

Cancer Chemother Pharmacol 2013 Jul 1;72(1):117-25. Epub 2013 May 1.

Department of System Medicine, University of Rome, Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.

Purpose: Defective expression of the mismatch repair protein MSH3 is frequently detected in colon cancer, and down-regulation of its expression was found to decrease sensitivity to platinum compounds or poly(ADP-ribose) polymerase inhibitors (PARPi) monotherapy. We have investigated whether MSH3 transfection in MSH3-deficient colon cancer cells confers resistance to oxaliplatin or PARPi and whether their combination restores chemosensitivity.

Methods: MSH3-deficient/MLH1-proficient colon cancer HCT116(MLH1) cells were transfected with the MSH3 cDNA cloned into the pcDNA3.1(-) vector. MSH3/MLH1-deficient HCT116, carrying MLH1 and MSH3 mutations on chromosome 3 and 5, respectively, and HCT116 in which wild-type MLH1 (HCT116+3), MSH3 (HCT116+5) or both genes (HCT116+3+5) were introduced by chromosome transfer were also tested. Sensitivity to oxaliplatin and to PARPi was evaluated by analysis of clonogenic survival, cell proliferation, apoptosis and cell cycle.

Results: MSH3 transfection in HCT116 cells did not confer resistance to oxaliplatin or PARPi monotherapy. MSH3-proficient HCT116+5 or HCT116+3+5 cells, which were more resistant to oxaliplatin and PARPi in comparison with their MSH3-deficient counterparts, expressed higher levels of the nucleotide excision repair ERCC1 and XPF proteins, involved in the resistance to platinum compounds, and lower PARP-1 levels. In all cases, PARPi increased sensitivity to oxaliplatin.

Conclusions: Restoring of MSH3 expression by cDNA transfection, rather than by chromosome transfer, did not affect colon cancer sensitivity to oxaliplatin or PARPi monotherapy; PARP-1 levels seemed to be more crucial for the outcome of PARPi monotherapy.
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http://dx.doi.org/10.1007/s00280-013-2175-0DOI Listing
July 2013

Ipilimumab: a novel immunostimulatory monoclonal antibody for the treatment of cancer.

Pharmacol Res 2012 Jan 10;65(1):9-22. Epub 2011 Sep 10.

Pharmacology and Medical Oncology Section, Department of Neuroscience, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

Ipilimumab (Yervoy, developed by Medarex and Bristol-Myers Squibb) is a fully human monoclonal IgG1κ antibody against the cytotoxic T-lymphocyte antigen-4 (CTLA-4), an immune-inhibitory molecule expressed in activated T cells and in suppressor T regulatory cells. Interaction of the monoclonal antibody with CTLA-4 blocks inhibitory signals generated through this receptor and enhances T cell activation, leading to increased antitumor responses. Ipilimumab has been approved by FDA in March 2011 as monotherapy (3mg/kg every 3 weeks for 4 doses) for the treatment of advanced (unresectable or metastatic) melanoma both in pre-treated or chemotherapy naïve patients. Four months later, ipilimumab has received a rapid approval by the European Commission, after a positive opinion from the Committee for Medicinal Products for Human Use. However, the indication in the EU is limited to previously-treated patients with advanced melanoma. Ipilimumab is the first agent that has demonstrated to improve overall survival in patients with metastatic melanoma, which has a very poor prognosis, in randomized phase III clinical trials. The patterns of tumour response to ipilimumab differ from those observed with cytotoxic chemotherapeutic agents, since patients may have a delayed yet durable response and obtain long-term survival benefit despite an initial tumour growth. The major draw-back of ipilimumab is the induction of immune-related adverse effects; the latter can be life-threatening, unless promptly managed with immunosuppressive agents (most frequently corticosteroids) according to specific guidelines. Further development of ipilimumab includes its use in the neoadjuvant or adjuvant high-risk melanoma setting and for the treatment of other refractory and advanced solid tumours, either as single agent or in combination with additional immunostimulating agents or molecularly targeted therapies.
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http://dx.doi.org/10.1016/j.phrs.2011.09.002DOI Listing
January 2012

Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1.

Mutat Res 2011 Jul 5;712(1-2):40-8. Epub 2011 May 5.

Department of Public Health and Cell Biology, University of Rome "Tor Vergata", Via Montpellier 1, 00133, Rome, Italy.

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.04.006DOI Listing
July 2011

The glutathione transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) increases temozolomide efficacy against malignant melanoma.

Eur J Cancer 2011 May 25;47(8):1219-30. Epub 2011 Jan 25.

Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy.

First line treatment of metastatic melanoma includes the methylating agent dacarbazine or its analogue temozolomide (TMZ) with improved pharmacokinetics and tolerability. However, the prognosis of the metastatic disease is poor and several trials are evaluating TMZ in polychemotherapy protocols. The novel glutathione transferase P1-1 (GSTP1-1) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has recently shown activity against melanoma through c-Jun N-terminal kinase activation. In this study we have investigated the in vitro and in vivo efficacy of NBDHEX and TMZ combination against melanoma. The results indicated that NBDHEX and TMZ exerted in vitro synergistic anti-proliferative effects in murine B16 and human A375 melanoma cells. In B16 cells TMZ as single agent caused cell accumulation at the G(2)/M phase of cell cycle, whereas NBDHEX induced mainly apoptotic effects. NBDHEX provoked a higher level of p53 phosphorylation with respect to TMZ and the drug combination caused a more than additive increase of p53 activation. The in vivo efficacy of NBDHEX and TMZ has been investigated in an orthotopic B16 model. Treatment with NBDHEX provoked a reduction of tumour growth comparable to that obtained with TMZ, whereas the drug combination significantly increased tumour growth inhibition with respect to the single agents, without worsening TMZ myelotoxicity. Immunohistochemical analysis of tumour grafts revealed a profound reduction of Cyclin D1 and CD31 in all treatment groups; VEGF expression was, instead, markedly decreased only in NBDHEX or NBDHEX and TMZ treated samples. These findings indicate that NBDHEX represents a good candidate for combination therapies including TMZ, offering new perspectives for the treatment of melanoma.
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http://dx.doi.org/10.1016/j.ejca.2010.12.008DOI Listing
May 2011

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant.

Nucleic Acids Res 2009 Nov 18;37(20):6849-58. Epub 2009 Sep 18.

Department of Biology, University of Rome Tor Vergata, CNR National Research Council, INFM National Institute for the Physics of Matter, Rome 00133, Italy.

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5' OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.
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http://dx.doi.org/10.1093/nar/gkp669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777420PMC
November 2009

Pharmacological inhibition of poly(ADP-ribose) polymerase activity down-regulates the expression of syndecan-4 and Id-1 in endothelial cells.

Int J Oncol 2009 Mar;34(3):861-72

Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata, 00167 Rome, Italy.

Poly(ADP-ribose) polymerase (PARP) is a family of nuclear proteins which regulate a number of cell functions, such as DNA repair, transcription, remodelling of chromatin structure, cell division and cell death. We and others have recently demonstrated that down-regulation of cellular PARP activity, using pharmacological inhibitors, impairs a number of endothelial functions and angiogenesis. In the present study, we investigated the potential mechanisms underlying the anti-angiogenic effect exerted by the potent PARP inhibitor GPI 15427, analyzing gene expression in human endothelial cells shortly after treatment with this compound. Analysis of gene and protein expression indicated that a 2-h exposure of human endothelial cells to GPI 15427 induced a rapid decrease of syndecan-4 (SDC-4), a transmembrane protein involved in modulation of cell signalling during angiogenesis that plays a role in endothelial cell migration and adhesion. Moreover, treatment with the PARP inhibitor induced a reduction of a helix-loop-helix transcription factor, the inhibitor of DNA binding-1 (Id-1), also implicated in the control of endothelial functions. We suggest that the inhibitory effect exerted by GPI 15427 on the angiogenic process is likely due to the reduced activity of specific transcription factors, such as Oct-1 and CREB that contribute to the regulation of SDC-4 and Id-1 expression, respectively. In conclusion, these results strongly suggest that PARP activity is capable of modulating molecules required for endothelial cell migration, adhesion, proliferation or differentiation during the angiogenic process.
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http://dx.doi.org/10.3892/ijo_00000213DOI Listing
March 2009

Recent approaches to improve the antitumor efficacy of temozolomide.

Curr Med Chem 2009 ;16(2):245-57

Department of Neuroscience, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

Temozolomide (TMZ) is an oral anticancer agent approved for the treatment of newly diagnosed glioblastoma in combination with radiotherapy. Moreover, TMZ has shown comparable efficacy with respect to dacarbazine, the reference drug for metastatic melanoma. Due to its favorable toxicity and pharmacokinetic profile, TMZ is under clinical investigation for brain metastasis from solid tumors and refractory leukemias. TMZ interacts with DNA generating a wide spectrum of methyl adducts mainly represented by N-methylpurines. However, its antitumor activity has been mainly attributed to O(6)-methylguanine, since tumor cell sensitivity inversely correlates with the levels of O(6)-alkylguanine DNA alkyltransferase and requires an intact mismatch repair system. Therefore, an increasing number of studies have been performed in order to identify patients who will benefit from TMZ treatment on the basis of their molecular/genetic profile. Unfortunately, resistance to the methylating agent occurs relatively often and strongly affects the rate and durability of the clinical response in cancer patients. Thus, different approaches have been developed to abrogate resistance or to increase the efficacy of TMZ and for many of them investigation is still underway. Herein, we provide an overview on the recent findings of preclinical and clinical studies on TMZ in combination with inhibitors of DNA repair, chemotherapeutic drugs with different mechanisms of action or radiotherapy, anti-angiogenic agents and other biological modulators.
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http://dx.doi.org/10.2174/092986709787002718DOI Listing
March 2009

Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide.

Eur J Cancer 2008 Sep 3;44(13):1914-21. Epub 2008 Aug 3.

Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata-IRCCS, Via dei Monti di Creta 104, 00167 Rome, Italy.

Vascular endothelial growth factor receptor-1 (VEGFR-1) exists in two isoforms: a membrane-bound isoform (mVEGFR-1) and a soluble one (sVEGFR-1). mVEGFR-1 is involved in endothelial cell migration and survival supported by VEGF-A and placenta growth factor (PlGF), whereas the biologic function of sVEGFR-1 has not been fully elucidated. We previously reported that sVEGFR-1 induces endothelial cell motility and promotes endothelial cell adhesion. In this study, we tested a set of VEGFR-1-derived peptides for their ability to interfere with endothelial cell migration. Peptide B3 was found to specifically inhibit cell migration induced by sVEGFR-1 and by mVEGFR-1-specific ligands. Moreover, peptide B3 markedly hampered angiogenesis in vitro and in vivo and was found to interfere with VEGFR-1 homodimerisation. Altogether, these data demonstrate that peptide B3 might be a useful tool for the specific inhibition of VEGFR-1 function and might represent a basis for the development of new anti-angiogenic compounds.
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http://dx.doi.org/10.1016/j.ejca.2008.06.032DOI Listing
September 2008

Stable depletion of poly (ADP-ribose) polymerase-1 reduces in vivo melanoma growth and increases chemosensitivity.

Eur J Cancer 2008 Jun 24;44(9):1302-14. Epub 2008 Apr 24.

Department of Neuroscience, University of Rome Tor Vergata, Via Montpellier 1, Rome, Italy.

Poly(ADP-ribose) polymerase (PARP)-1, which plays a key role in DNA repair, inflammation and transcription, has recently been shown to be involved in angiogenesis. The aim of this study was to investigate PARP-1 role in melanoma aggressiveness and chemoresistance in vivo using clones stably silenced for PARP-1 expression. Whilst the growth characteristics of PARP-1-deficient melanoma cells were comparable to those of PARP-1-proficient cells in vitro, their tumourigenic potential in vivo was significantly compromised. In fact, mice challenged intra-muscle with PARP-1-deficient cells showed a delayed development of measurable tumour nodules, which were also significantly reduced in size with respect to those of mice inoculated with PARP-1-proficient cells. Moreover, animals challenged intra-cranially with PARP-1-deficient cells, a model that mimics CNS localisation of melanoma, showed an increased survival. Immunohistochemical analyses of PARP-1-depleted melanoma grafts indicated a reduced expression of the angiogenesis marker PECAM-1/CD31 and of the pro-inflammatory mediators TNF-alpha and GITR. Notably, PARP-1-silenced melanoma was extremely sensitive to temozolomide, an anticancer agent used for the treatment of metastatic melanoma. These results provide novel evidence for a direct role of PARP-1 in tumour aggressiveness and chemoresistance.
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http://dx.doi.org/10.1016/j.ejca.2008.03.019DOI Listing
June 2008