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Annamaria Biroccio,
Manuela Porru,
Angela Rizzo,
Erica Salvati,
Carmen D'Angelo,
Augusto Orlandi,
Daniela Passeri,
Marco Franceschin,
Malcolm F G Stevens,
Eric Gilson,
Giovanni Beretta, Gabriella Zupi,
Claudio Pisano,
Franco Zunino,
Carlo Leonetti
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ABSTRACT: We previously reported that the G-quadruplex (G4) ligand RHPS4 potentiates the antitumor activity of camptothecins both in vitro and in tumor xenografts. The present study aims at investigating the mechanisms involved in this specific drug interaction.
Combination index test was used to evaluate the interaction between G4 ligands and standard or novel Topo I inhibitors. Chromatin immunoprecipitation was performed to study the presence at telomeres of various types of topisomerase, while immunolabeling experiments were performed to measure the activation of DNA damage both in vitro and in tumor xenografts.
We report that integration of the Topo I inhibitor SN-38, but not the Topo II poison doxorubicin with telomere-based therapy is strongly effective and the sequence of drug administration is critical in determining the synergistic interaction, impairing the cell ability to recover from drug-induced cytotoxicity. The synergistic effect of this combination was also observed by using novel camptothecins and, more interestingly, mice treated with ST1481/RHPS4 combination showed an inhibition and delay of tumor growth as well as an increased survival. The study of the mechanism(s) revealed that treatment with G4 ligands increased Topo I at the telomeres and the functional relevance of this observation was directly assessed by showing that standard and novel camptothecins stabilized DNA damage both in vitro and in xenografts.
Our results demonstrate an outstanding efficacy of Topo I inhibitors/G4 ligands combination, which likely reflects an enhanced and persistent activation of DNA damage response as a critical determinant of the therapeutic improvement.
Clinical Cancer Research 02/2011; 17(8):2227-36. · 7.74 Impact Factor
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ABSTRACT: Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.
By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.
We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90.
PLoS ONE 01/2010; 5(7):e11772. · 4.09 Impact Factor
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Anna Tesei,
Carlo Leonetti, Gabriella Zupi,
Marco Scarsella,
Giovanni Brigliadori,
Paola Ulivi,
Francesco Fabbri,
Chiara Arienti,
Dino Amadori,
Alessandro Passardi,
Rosella Silvestrini,
Wainer Zoli
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ABSTRACT: Despite the low efficacy of conventional antitumour drugs, chemotherapy remains an essential tool in controlling advanced gastric and oesophageal cancers. We aimed to provide a biological rationale based on the sorafenib-taxotere interaction for the clinical treatment of gastric cancer. In vitro experiments were performed on four human gastric cancer cell lines (GK2, AKG, KKP and NCI-N87). Cytotoxicity was evaluated by sulforhodamine B (SRB) assay, cell cycle perturbations, apoptosis and mitotic catastrophe were assessed by flow cytometric and microscopic analyses, and protein expression was studied by Western blot. In the in vivo experiments, nude mice xenografted with the most resistant line were treated with sorafenib and docetaxel singly or in association. Sorafenib inhibited cell growth (IG(50) values ranged from 3.4 to 8.1 μM) and caused down-regulation of MAP-K/ERK phosphorylation and of mcl-1 and p-bad expression after a 48-hr exposure. Apoptosis induction was associated with caspase-3 and -9 activation and mitochondrial membrane depolarization. The drug combination enhanced apoptosis (up to 80%) and produced a synergistic interaction when low doses of the taxane preceded administration of the antityrosine kinase. This synergism was probably due to the induction of an anomalous multidiploid G0-G1 peak and to consequent mitotic catastrophe, which increased sensitivity to sorafenib. Consistent with in vitro results, the docetaxel-sorafenib sequence exhibited high therapeutic efficacy in NCI-N87 mouse xenografts producing tumour weight inhibition (> 65%), tumour growth delay (up to 25 days) and increased mouse survival (30%). Our findings suggest the potential clinical usefulness of treatment with sorafenib and docetaxel for advanced gastric cancer.
Journal of Cellular and Molecular Medicine 12/2009; 15(2):316-26. · 4.13 Impact Factor
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Marta Chevanne,
Michele Zampieri,
Riccardo Caldini,
Angela Rizzo,
Fabio Ciccarone,
Angela Catizone,
Carmen D'Angelo,
Tiziana Guastafierro,
Anna Biroccio,
Anna Reale, Gabriella Zupi,
Paola Caiafa
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ABSTRACT: The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 µM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken toghether, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 µM PJ-34 for 24 h to obtain the same citotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment. J. Cell. Physiol. 222: 401–410, 2010. © 2009 Wiley-Liss, Inc.
Journal of Cellular Physiology 11/2009; 222(2):401 - 410. · 3.87 Impact Factor
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Marta Chevanne,
Michele Zampieri,
Riccardo Caldini,
Angela Rizzo,
Fabio Ciccarone,
Angela Catizone,
Carmen D'Angelo,
Tiziana Guastafierro,
Anna Biroccio,
Anna Reale, Gabriella Zupi,
Paola Caiafa
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ABSTRACT: The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment.
Journal of Cellular Physiology 11/2009; 222(2):401-10. · 3.87 Impact Factor
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Ludovica Ciuffreda,
Donatella Del Bufalo,
Marianna Desideri,
Cristina Di Sanza,
Antonella Stoppacciaro,
Maria Rosaria Ricciardi,
Sabina Chiaretti,
Simona Tavolaro,
Barbara Benassi,
Alfonso Bellacosa,
Robin Foà,
Agostino Tafuri,
Francesco Cognetti,
Andrea Anichini, Gabriella Zupi,
Michele Milella
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ABSTRACT: The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC(50) in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G(1)-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27(KIP1)) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.
Neoplasia (New York, N.Y.) 09/2009; 11(8):720-31. · 5.48 Impact Factor
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ABSTRACT: We examined the autocrine/paracrine role of interleukin-8 (CXCL8) and the functional significance of CXCL8 receptors, CXCR1 and CXCR2, in human malignant melanoma proliferation, migration, invasion and angiogenesis. We found that a panel of seven cell lines, even though at different extent, secreted CXCL8 protein, and expressed CXCR1 and CXCR2 independently from the CXCL8 expression, but depending on the oxygen level. In fact, hypoxic exposure increases the expression of CXCR1 and CXCR2. The cell proliferation of both M20 and A375SM lines, expressing similar levels of both CXCR1 and CXCR2 but secreting low and high amounts of CXCL8, respectively, was significantly enhanced by CXCL8 exposure and reduced by CXCL8, CXCR1 and CXCR2 neutralising antibodies, indicating the autocrine/paracrine role of CXCL8 in melanoma cell proliferation. Moreover, an increased invasion and migration in response to CXCL8 was observed in several cell lines, and a further enhancement evidenced under hypoxic conditions. A CXCL8-dependent in vivo vessel formation, evaluated through a matrigel assay, was also demonstrated. Furthermore, when neutralising antibodies against CXCR1 or CXCR2 were used, only the involvement of CXCR2, but not CXCR1 was observed on cell migration and invasion, while both receptors played a role in angiogenesis. In summary, our data demonstrate that CXCL8 induces cell proliferation and angiogenesis through both receptors and that CXCR2 plays an important role in regulating the CXCL8-mediated invasive and migratory behaviour of human melanoma cells. Thus, blocking the CXCL8 signalling axis promises an improvement for the therapy of cancer and, in particular, of metastatic melanoma.
European journal of cancer (Oxford, England: 1990) 09/2009; 45(14):2618-27. · 4.12 Impact Factor
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ABSTRACT: 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.
Nucleic Acids Research 08/2009; 37(16):5353-64. · 8.03 Impact Factor
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Carlo Leonetti,
Marco Scarsella,
Giuseppe Riggio,
Angela Rizzo,
Erica Salvati,
Maurizio D'Incalci,
Lidia Staszewsky,
Roberta Frapolli,
Malcolm F Stevens,
Antonella Stoppacciaro,
Marcella Mottolese,
Barbara Antoniani,
Eric Gilson, Gabriella Zupi,
Annamaria Biroccio
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ABSTRACT: 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.
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.
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.
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.
Clinical Cancer Research 12/2008; 14(22):7284-91. · 7.74 Impact Factor
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ABSTRACT: We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.
Journal of Neurochemistry 10/2008; 107(3):871-82. · 4.06 Impact Factor
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ABSTRACT: Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.
Journal of Biological Chemistry 07/2008; 283(32):21873-80. · 4.77 Impact Factor
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Erica Salvati,
Carlo Leonetti,
Angela Rizzo,
Marco Scarsella,
Marcella Mottolese,
Rossella Galati,
Isabella Sperduti,
Malcolm F G Stevens,
Maurizio D'Incalci,
Maria Blasco,
Giovanna Chiorino,
Serge Bauwens,
Béatrice Horard,
Eric Gilson,
Antonella Stoppacciaro, Gabriella Zupi,
Annamaria Biroccio
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ABSTRACT: 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.
Journal of Clinical Investigation 12/2007; 117(11):3236-47. · 15.39 Impact Factor
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ABSTRACT: This study aims to investigate the role of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for glutathione (GSH) synthesis, in the c-Myc-dependent response to antineoplastic agents. We found that specific c-Myc inhibition depleted cells of GSH by directly reducing the gene expression of both heavy and light subunits of the gamma-GCS enzyme and increased their susceptibility to antineoplastic drugs with different mechanisms of action, such as cisplatin (CDDP), staurosporine (STR), and 5-fluorouracil (5-FU). The effect caused by c-Myc inhibition on CDDP and STR response, but not to 5-FU treatment, is directly linked to the impairment of the gamma-GCS expression, because up-regulation of gamma-GCS reverted drug sensitivity, whereas the interference of GSH synthesis increased drug susceptibility as much as after c-Myc down-regulation. The role of gamma-GCS in the c-Myc-directed drug response depends on the capacity of drugs to trigger reactive oxygen species (ROS) production. Indeed, although 5-FU exposure did not induce any ROS, CDDP- and STR-induced oxidative stress enhanced the recruitment of c-Myc on both gamma-GCS promoters, thus stimulating GSH neosynthesis and allowing cells to recover from ROS-induced drug damage. In conclusion, our data demonstrate that the gamma-GCS gene is the downstream target of c-Myc oncoprotein, driving the response to ROS-inducing drugs. Thus, gamma-GCS impairment might specifically sensitize high c-Myc tumor cells to chemotherapy.
Molecular Pharmacology 11/2007; 72(4):1015-23. · 4.88 Impact Factor
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ABSTRACT: The response of hormone-refractory prostate cancer (HRPC) to chemotherapy remains modest, necessitating the search for new forms of treatment to improve the prognosis. Since an increased expression of oncogenes, including c-myc and bcl-2, accompanies the transition to HRPC, we evaluated whether the concomitant downregulation of these oncogenes by antisense strategy sensitized HRPC to chemotherapy.
PC-3 prostate cancer cells were exposed in vitro to c-myc (INX-6295) and bcl-2 (G3139) antisense oligodeoxynucleotides (ODNs) and docetaxel given alone or in combination. Therapeutic efficacy of the different treatments was also evaluated in xenografts.
We show that the triple combination of drugs given in the sequence G3139/docetaxel/INX-6295 was the most active in reducing the survival of PC-3. Likewise, the combination triggered apoptosis in more than 80% of cells. A marked tumor weight inhibition was observed in PC-3 xenografts after G3139/docetaxel/INX-6295 treatment, with a complete tumor regression being noted in half the mice. A 111% overall increase in life survival and a complete cure in two out of eight mice was also reported. This treatment remained effective even when started at a very late stage of tumor growth producing about 80% tumor weight inhibition (TWI), with tumor regression being maintained for 1 month. Finally, the antitumor effect resulted in a significant increase (70%) in mice survival.
These data indicate that the combined targeting of genes involved in uncontrolled proliferation and evasion of apoptosis renders HRPC responsive to chemotherapy making this treatment a promising antineoplastic strategy.
The Prostate 10/2007; 67(13):1475-85. · 3.48 Impact Factor
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ABSTRACT: In this paper, we investigated whether bcl-xL can be involved in the modulation of the angiogenic phenotype of human tumor cells. Using the ADF human glioblastoma and the M14 melanoma lines, and their derivative bcl-xL-overexpressing clones, we showed that the conditioned medium of bcl-xL transfectants increased in vitro endothelial cell functions, such as proliferation and morphogenesis, and in vivo vessel formation in Matrigel plugs, compared with the conditioned medium of control cells. Moreover, the overexpression of bcl-xL induced an increased expression of the proangiogenic interleukin-8 (CXCL8), both at the protein and mRNA levels, and an enhanced CXCL8 promoter activity. The role of CXCL8 on bcl-xL-induced angiogenesis was validated using CXCL8-neutralizing antibodies, whereas down-regulation of bcl-xL through antisense oligonucleotide or RNA interference strategies confirmed the involvement of bcl-xL on CXCL8 expression. Transient overexpression of bcl-xL led to extend this observation to other tumor cell lines with different origin, such as colon and prostate carcinoma. In conclusion, our results showed that CXCL8 modulation by bcl-xL regulates tumor angiogenesis, and they point to elucidate an additional function of bcl-xL protein.
Molecular Cancer Research 09/2007; 5(8):761-71. · 4.29 Impact Factor
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Michele Caraglia,
Monica Marra,
Carlo Leonetti,
Giuseppina Meo,
Anna Maria D'Alessandro,
Alfonso Baldi,
Daniele Santini,
Giuseppe Tonini,
Raffaello Bertieri, Gabriella Zupi,
Alfredo Budillon,
Alberto Abbruzzese
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ABSTRACT: Zoledronic acid (ZOL) has proved activity in bone metastases from prostate cancer through inhibition of mevalonate pathway and of prenylation of intracellular proteins. We have reported that ZOL synergizes with R115777 farnesyltransferase inhibitor (FTI, Zarnestra) in inducing apoptosis and growth inhibition on epidermoid cancer cells. Here, we have studied the effects of the combination of these agents in prostate adenocarcinoma models and, specifically, on androgen-independent (PC3 and DU145) and -dependent (LNCaP) prostate cancer cell lines. We have found that ZOL and R115777 were synergistic in inducing both growth inhibition and apoptosis in prostate adenocarcinoma cells. These effects were paralleled by disruption of Ras-->Erk and Akt survival pathways, consequent decreased phosphorylation of both mitochondrial bcl-2 and bad proteins, and caspase activation. Finally, ZOL/R115777 combination induced cooperative effects also in vivo on tumor growth inhibition of prostate cancer xenografts in nude mice with a significant survival increase. These effects were paralleled by enhanced apoptosis and inactivation of both Erk and Akt. In conclusions, the combination between ZOL and FTI leads to enhanced anti-tumor activity in human prostate adenocarcinoma cells likely through a more efficacious inhibition of ras-dependent survival pathways and consequent bcl-related proteins-dependent apoptosis.
Journal of Cellular Physiology 05/2007; 211(2):533-43. · 3.87 Impact Factor
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Honor Hugo,
Alina Cures,
Nirosha Suraweera,
Yvette Drabsch,
Damian Purcell,
Theo Mantamadiotis,
Wayne Phillips,
Alex Dobrovic, Gabriella Zupi,
Thomas J Gonda,
Barry Iacopetta,
Robert G Ramsay
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ABSTRACT: Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron 1. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron 1 is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes.
Genes Chromosomes and Cancer 01/2007; 45(12):1143-54. · 3.31 Impact Factor
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Annamaria Biroccio,
Angela Rizzo,
Raffaella Elli,
Catherine-Elaine Koering,
Aurélie Belleville,
Barbara Benassi,
Carlo Leonetti,
Malcolm F G Stevens,
Maurizio D'Incalci, Gabriella Zupi,
Eric Gilson
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ABSTRACT: The inhibition of the telomere-binding protein TRF2, by expressing the dominant negative form TRF2(DeltaBDeltaC), has been used as a model of anti-telomere strategy to induce a reversion of the malignant phenotype of M14 and JR5 human melanoma lines. Over-expression of TRF2(DeltaBDeltaC) induced apoptosis and reduced tumourigenicity exclusively in JR5 cells. p53 and Rb status and apoptotic response to DNA damage did not seem to account for the different response of the two lines to TRF2 inhibition. Interestingly, JR5 cells possess shorter and more dysfunctional telomeres compared to M14 line. Moreover, the treatment with the G-quadruplex-interacting agent (G4-ligand) RHPS4 sensitises M14 cells to TRF2 inhibition. These results demonstrate that TRF2 can impair tumuorigenicity of human cancer cells. They further suggest that a basal level of telomere instability favours an efficient response to TRF2 inhibition and that a combined anti-TRF2 and G4-ligand therapy would have synergistic inhibitory effects on tumour cell growth.
European Journal of Cancer 09/2006; 42(12):1881-8. · 5.54 Impact Factor
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ABSTRACT: Mammalian target of rapamycin (mTOR) is increasingly recognized as a master regulator of fundamental cellular functions, whose deregulation may underlie neoplastic transformation and progression. Hence, mTOR has recently emerged as a promising target for therapeutic anticancer interventions in several human tumors, including breast cancer. Here, we investigated the antiangiogenic potential of temsirolimus (also known as CCI-779), a novel mTOR inhibitor currently in clinical development for the treatment of breast cancer and other solid tumors. Consistent with previous reports, sensitivity to temsirolimus-mediated growth inhibition varied widely among different breast cancer cell lines and was primarily due to inhibition of proliferation with little, if any, effect on apoptosis induction. In the HER-2 gene-amplified breast cancer cell line BT474, temsirolimus inhibited vascular endothelial growth factor (VEGF) production in vitro under both normoxic and hypoxic conditions through inhibition of hypoxia-stimulated hypoxia-inducible factor (HIF)-1alpha expression and transcriptional activation. Interestingly, these effects were also observed in the MDA-MB-231 cell line, independent of its inherent sensitivity to the growth-inhibitory effects of temsirolimus. A central role for mTOR (and the critical regulator of cap-dependent protein translation, eIF4E) in the regulation of VEGF production by BT474 cells was further confirmed using a small interfering RNA approach to silence mTOR and eIF4E protein expression. In addition to its effect on HIF-1alpha-mediated VEGF production, temsirolimus also directly inhibited serum- and/or VEGF-driven endothelial cell proliferation and morphogenesis in vitro and vessel formation in a Matrigel assay in vivo. Overall, these results suggest that antiangiogenic effects may substantially contribute to the antitumor activity observed with temsirolimus in breast cancer.
Cancer Research 07/2006; 66(11):5549-54. · 7.86 Impact Factor
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Carlo Leonetti,
Marco Scarsella, Gabriella Zupi,
Wainer Zoli,
Dino Amadori,
Laura Medri,
Francesco Fabbri,
Marco Rosetti,
Paola Ulivi,
Lorenzo Cecconetto,
Manlio Bolla,
Anna Tesei
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ABSTRACT: We previously showed that NCX 4040 inhibits in vitro and in vivo tumor growth and induces apoptosis in human colon cancer cell lines. On the basis of these results, NCX 4040 antitumor activity in combination with 5-fluorouracil (5-FU) or oxaliplatin was evaluated in vitro and in vivo in human colon cancer models. The cytotoxicity of different NCX 4040 and 5-FU or oxaliplatin combination schemes was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr, and LRWZ) by the sulforhodamine B assay, and apoptosis was assessed by flow cytometry. NCX 4040 and 5-FU combination was always additive in vitro regardless of the scheme used. Sequential NCX 4040-->oxaliplatin treatment produced a strong synergism in three cell lines, with a ratio index ranging from 3.7 to 4. The synergistic effect was accompanied by apoptosis induction (up to 40%). In the in vivo experiments, xenografted mice were treated with the sequential combination of NCX 4040 and oxaliplatin, and apoptosis was evaluated immunohistochemically in excised tumors. Furthermore, in WiDr xenografts, this sequence caused a significantly higher reduction ( approximately 60%) in tumor growth compared with single-drug treatments and produced extensive apoptotic cell death (15.3%), significantly higher (P < 0.01) than that observed in untreated tumors (2.7%) or in tumors treated with NCX 4040 (5.1%) or oxaliplatin (5.7%) alone. These data show that NCX 4040 sensitizes colon cancer cell lines to the effect of antitumor drugs and suggests that their combination could be useful for the clinical management of colon cancer.
Molecular Cancer Therapeutics 05/2006; 5(4):919-26. · 5.23 Impact Factor
