Carlo Leonetti

In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines

BackgroundNitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity.MethodsIn vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks.ResultsIn the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all.ConclusionsNCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

Neuroprotective Effect of Vitamin E Supplementation in Patients Treated With Cisplatin Chemotherapy

Purpose: The aim of this study is to evaluate the neuroprotective effect of antioxidant supplementation with vitamin E in patients treated with cisplatin chemotherapy. Methods: Between April 1999 and October 2000, forty-seven patients were randomly assigned to either group one, which received vitamin E supplementation during cisplatin chemotherapy, or to group two, which received cisplatin chemotherapy alone. Alpha-tocopherol (vitamin E; 300 mg/d) was administered orally before cisplatin chemotherapy and continued for 3 months after the suspension of treatment. For preclinical studies, nude mice carrying the human melanoma tumor were treated with cisplatin alone or in combination with vitamin E. Results: Twenty-seven patients completed six cycles of cisplatin chemotherapy: 13 patients in group one and 14 patients in group two. The incidence of neurotoxicity was significantly lower in group one (30.7%) than it was in group two (85.7%; P < .01). The severity of neurotoxicity, measured with a comprehensive n...

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors gamma-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

Bcl-2 overexpression enhances the metastatic potential of a human breast cancer line.

Bcl‐2 protein has been shown to contribute to oncogenesis because it can transform and immortalize cells in cooperation with c‐myc, ras, or viral genes. However, in vivo studies have not yet established whether bcl‐2 can play a role in metastasis. Here we investigate the potential metastatic role of bcl‐2. We introduced the human bcl‐2 gene into a low bcl‐2 expressing human breast cancer cell line MCF7 ADR. We demonstrate that two bcl‐2 overexpressing clones injected intravenously or intramuscularly into nude mice induce a significantly higher number of experimental and spontaneous lung metastases compared to the control transfectant clone. We demonstrate that bcl‐2 overexpressing clones are more invasive and migratory in response to chemotactic stimuli than the control transfectant clone. Furthermore, zymographic analysis shows that secretion of 72 and 92 kDa gelatinases increases in the two bcl‐2 overexpressing transfectants. Tumors originating from bcl‐2 overexpressing clones also show a decrease in the latency period of tumor appearance. In conclusion, our data show that bcl‐2 overexpression enhances both tumorigenicity and metastatic potential of MCF7 ADR cells by inducing metastasis‐associated properties.—Del Bufalo, D., Biroccio, A., Leonetti, C., Zupi, G. Bcl‐2 overexpression enhances the metastatic potential of a human breast cancer line. FASEB J. 11, 947–953 (1997)

Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway.

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma

Poly(ADP‐ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisom‐erase I inhibitor irinotecan (CPT‐11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT‐11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10‐(4‐methyl‐piperazin‐1‐ylm‐ethyl)‐2H‐7‐oxa‐1,2‐diaza‐benzo[de]anthracen‐3‐one) increases the efficacy of CPT‐11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP‐1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose‐limiting intestinal toxicity of CPT‐11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN‐38 (the active metabolite of CPT‐11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/day×5 days per os) in combination with TMZ (10 mg/kg/day×5 days) + CPT‐11 (4 mg/kg/day×5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2 × 3 days) prevented intestinal injury and diarrhea induced by CPT‐11 (30 mg/kg/day × 3 days) reducing inflammation and PARP‐1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP‐ribose) antibody(Ab). Inconclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.—Tentori, L., Leonetti, C., Scarsella, M., Muzi, A., Mazzon, E., Vergati, M., Forini, O., Lapidus, R., Xu, W., Dorio, A. S., Zhang, J., Cuzzo‐crea, S., Graziani, G. Inhibition of poly(ADP‐ribose) polymerase prevents irinotecan‐induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma. FASEB J. 20, E1024–E1036 (2006)

The future of antisense therapy: combination with anticancer treatments

The current direction in cancer research is rational drug design, which is based on the evidence that transformed cells are characterized by alterations of genes devoted to the regulation of both cell proliferation and apoptosis. A variety of approaches have been carried out to develop new agents selective for cancer cells. Among these, antisense oligonucleotides (ASOs) are one of such class of new agents able to inhibit specifically the synthesis of a particular cancer-associated protein by binding to protein-encoding RNA, thereby preventing RNA function. In the past decade, several ASOs have been developed and tested in preclinical and clinical studies. Many have shown convincing in vitro reduction in target gene expression and promising activity against a wide variety of tumors. However, because of the multigenic alterations of tumors, the use of ASOs as single agents does not seem to be effective in the treatment of malignancies. Antisense therapy that interferes with signaling pathways involved in cell proliferation and apoptosis are particularly promising in combination with conventional anticancer treatment. An overview of the progress of ASOs used in combination therapy is provided.

G-Quadruplex Ligand RHPS4 Potentiates the Antitumor Activity of Camptothecins in Preclinical Models of Solid Tumors

Purpose: The formation of G-quadruplex structures at telomeric DNA sequences blocks telomerase activity, offering an original strategy to design and develop new antitumor agents. The pentacyclic acridinium salt RHPS4 is one of the most effective and selective G4 ligands able to rapidly disrupt telomere architecture, resulting in apoptosis of cancer cells. Here, we studied the therapeutic index of RHPS4 and its integration with chemotherapeutics in preclinical model of solid tumors. Experimental Design: The antitumoral activity of RHPS4 was evaluated on human xenografts of different histotypes and compared with that of standard antineoplastic agents. Moreover, the effect of RHPS4/chemotherapeutics combinations on cell survival was studied and the most favorable combination was evaluated on tumor-bearing mice. Results: RHPS4 was active in vivo as single agent and showed a high therapeutic efficacy when compared with conventional drugs. Moreover, RHPS4 had antitumoral activity in human melanoma xenografts inherently resistant to chemotherapy and exhibited antimetastatic activity. RHPS4 also showed a strong synergistic interaction with camptothecins and this effect was strictly dependent on the drug sequence employed. Treatment of mice with irinotecan followed by RHPS4 was able to inhibit and delay tumor growth and to increase mice survival. Conclusions: Our data show that RHPS4 has a good pharmacodynamic profile and in combination therapy produces a strong antitumoral activity, identifying this drug as promising agent for clinical development.

Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes.

UNLABELLED Zoledronic acid (ZOL) is a potent amino-bisphosphonate used for the treatment of bone metastases with recently reported antitumor activity. However, the short plasma half-life and rapid accumulation in bone limits the use of ZOL as an antitumor agent in extraskeletal tissues. Therefore, we developed stealth liposomes encapsulating ZOL (LipoZOL) to increase extraskeletal drug availability. Compared to free ZOL, LipoZOL induced a stronger inhibition of growth of a range of different cancer cell lines in vitro. LipoZOL also caused significantly larger inhibition of tumor growth and increased the overall survival in murine models of human prostate cancer and multiple myeloma, in comparison with ZOL. Moreover, a strong inhibition of vasculogenetic events without evidence of necrosis in the tumor xenografts from prostate cancer was recorded after treatment with LipoZOL. We demonstrated both antitumor activity and tolerability of LipoZOL in preclinical animal models of both solid and hematopoietic malignancies, providing a rationale for early exploration of use of LipoZOL as a potential anticancer agent in cancer patients. FROM THE CLINICAL EDITOR The short plasma half-life and rapid accumulation in bone limits the use of zoledronic acid as an antitumor agent in extraskeletal tissues. Therefore, stealth liposomes encapsulating ZOL (LipoZOL) have been developed to increase extraskeletal drug availability.

PARP1 is activated at telomeres upon G4 stabilization: Possible target for telomere-based therapy

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.