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

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

Poly(ADP-ribose) polymerase (PARP) inhibitors as modulators of tumor drug resi stance to chemotherapy

Poly(ADP-ribose) polymerase (PARP) inhibitors are a promising class of anticancer agents currently in clinical trials either in combination with chemotherapy [e.g., the methylating compound temozolomide (TMZ), the topoisomerase I (TOPO I) poisons irinotecan (CPT-11) or topotecan] and radiotherapy or as monotherapy. In fact, PARP inhibitors exert cytotoxic effects as single agents in BRCA mutated tumors, which are defective in the homologous recombination repair (HR) of DNA double strand breaks (DSBs). In preclinical models we have demonstrated that PARP inhibitors enhance the antitumor activity of TMZ or of TOPO I inhibitors especially in mismatch repair (MMR) deficient tumors, including colorectal cancer that is frequently characterized by MMR dysfunction. Moreover, the PARP inhibitor GPI 15427 increases the efficacy also of CPT-11 and TMZ combination against MMR deficient colon cancer in vitro and in tumor xenografts. hMLH1 is mainly involved in the processing of O6-methylguanine:C/T mispairs responsible for the cytotoxic effects of TMZ. Recently, it has been suggested that MSH3 might be involved in the repair of DSBs induced by intra-strand cross-links provoked by platinum derivatives through the intervention of HR. The role of the different MMR components in the susceptibility of colon cancer cells to TOPO I poisons have not been clarified, yet. The human colon cancer cell lines HCT116 (known to have a homozygous mutation in the MMR hMLH1 gene on chromosome 3 and homozygous frameshift mutations in the MMR hMSH3 gene on chromosome 5), the HCT116 derived cell lines in which only the wild-type hMLH1 (HCT116+3) or both the wild-type hMLH1 and hMSH3 genes (HCT116+3+5) have been replaced, via chromosome transfer, were used to test their susceptibility to anticancer drugs with different mechanisms of action. The hMLH1 and hMSH3 deficient HCT116 cells and the HCT116+3+5 cells were more sensitive to SN38 (the active metabolite of CPT-11) than HCT116+3 cells that, instead, were highly sensitive to TMZ. Interestingly, the hMLH1 and hMSH3 proficient HCT116+3+5 cells were more resistant to oxaliplatin than the other cell lines. HCT116, characterized by a higher PARP-1 expression with respect to the other cell lines, were the most sensitive to the PARP inhibitor GPI 15427 as single agent. Stable silencing of PARP-1 expression resulted in increased chemosensitivity. The results suggest that that hMLH1, hMSH3 or PARP-1 status may predict differential sensitivity to chemotherapeutic agents

Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells

Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. We previously demonstrated that MMR-deficient tumour cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G(2) phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analogue BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient turnout cells to DNA damage induced by BLM in G(2). The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51,a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no additional effect. Similarly, treatment with BVDU did not affect BLM clastogenicity, in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was observed also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient versus MMR-deficient cells to BLM-induced chromatid breaks in the G(2) phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR. (c) 2009 Elsevier B.V. All rights reserved

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

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

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.

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

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

We recently demonstrated that poly(ADP-ribose) polymerase (PARP)-1 is involved in angiogenesis and melanoma aggressiveness. In fact, a reduction of blood vessel neo-formation in response to angiogenic stimuli is observed in PARP-1 KO mice and down-regulation of cellular PARP activity, using pharmacological inhibitors, impairs a number of endothelial functions. Notably, abrogation of PARP-1 expression by stable gene silencing reduces the in vivo aggressiveness of melanoma and this effect is associated with a decreased vasculature formation within the tumour. In this study we have investigated the potential mechanisms underlying the anti-angiogenic effect exerted by the PARP inhibitor GPI 15427, analyzing gene expression in human endothelial cells shortly after treatment with this compound. The results indicate that 2 h exposure of endothelial cells to GPI 15427 provokes a rapid decrease of syndecan-4 (SDC-4), a transmembrane protein involved in modulation of cell signalling during angiogenesis, and a reduction of the inhibitor of DNA binding-1 (Id-1), a helix-loop-helix transcription factor also implicated in the control of endothelial functions. Moreover, we evaluated chemosensitivity and cell cycle modifications induced by treatment with the methylating agent temozolomide, as single agent or in combination with GPI 15427, in melanoma cells stably silenced for PARP-1 expression. Silencing of PARP-1 in melanoma results in enhanced sensitivity to the methylating agent temozolomide and in an early cell accumulation at the G2/M phase after drug treatment. The addition of GPI 15427, which inhibits both PARP-1 and PARP-2, increases sensitivity to temozolomide both in PARP-1-proficient and -deficient melanoma cells, but it induces a different cell cycle modulation depending on PARP-1 expression. In conclusion, these findings provide a novel implication for PARP-1 in cancer development and underscore the importance of targeting both PARP-1 and PARP-2 for cancer therapy. Supported by: Italian Ministry of Education and Research (PRIN project to GG) and Italian Ministry of Health (RC07-3.16 project to PML)

Novel Glutathione S-Transferase Inhibitor Enhances Temozolomide Efficacy Against Malignant Melanoma 52nd Annual Meeting of the Italian Cancer Society. Lost in translation: bridging the gap between cancer research and effective therapies

First line treatment of metastatic melanoma includes the methylating agent dacarbazine, which has been replaced in several countries by 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 and apoptosis induction. Methods: In vitro chemosensitivity to the drug combination of B16 melanoma cells was studied by short or long term proliferation assays; apoptosis or cell cycle perturbations were analysed by flow-cytometry, whereas JNK and p53 activation was assessed by immunoblotting. The in vivo toxicity and antitumor efficacy of TMZ and NBDHEX was investigated in an orthotopic B16 model. Immunohistochemical analysis was performed in tumour specimens from treated or untreated animals. Results: Data indicated that NBDHEX and TMZ exerted in vitro synergistic effects. While cell accumulation at the G2/M phase of cell cycle was observed with TMZ, apoptosis prevailed over G2/M arrest when NBDHEX was associated with TMZ. Moreover, NBDHEX provoked a higher level of p53 phosphorylation with respect to TMZ and the drug combination caused a further increase of p53 activation. In vivo treatment with NBDHEX provoked a reduction of tumour growth comparable to that obtained with TMZ, whereas the drug combination significantly increased growth inhibition with respect to the single agents, without worsening TMZ myelotoxicity. Immunohistochemical analysis of tumour grafts revealed a profound reduction of Cyclin D and CD31 in all treatment groups, whereas VEGF expression was markedly decreased in NBDHEX or NBDHEX+TMZ treated samples. Conclusions These findings indicate that NBDHEX represents a good candidate for combination therapies including TMZ, offering new perspectives for the treatment of melanoma

POLY(ADP-RIBOSE)POLYMERASE SIGNALING OF TOPOISOMERASE 1-DEPENDENT DNA DAMAGE IN CARCINOMA CELLS

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2+/+ and p53+/+. HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT + PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT + PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72 h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors. © 2010 Elsevier Inc. All rights reserved