Gabriella Zupi

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.

Antiangiogenic Potential of the Mammalian Target of Rapamycin Inhibitor Temsirolimus

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.

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)

Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line.

We have previously demonstrated that bcl‐2 overexpression enhances the metastatic potential of the MCF7 ADR human breast cancer cell line resistant to adriamycin by inducing metastasis‐associated properties. To further elucidate the relationship between bcl‐2 expression and the metastatic potential of the MCF7 ADR line, we evaluated whether bcl‐2 could be also involved in the modulation of the angiogenic phenotype. Four bcl‐2‐overex‐pressing clones, a control transfectant clone, and the MCF7 ADR parental line were used for in vitro and in vivo experiments. Bcl‐2 overexpression enhanced the synthesis of the hypoxia‐stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl‐2‐overex‐pressing clones, and reverse transcription‐polymer‐ase chain reaction showed higher levels of the VEGF121 and VEGF165 mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl‐2‐trans‐fected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl‐2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl‐2 transfectants. The effect of bcl‐2 on angiogenesis was not mediated by p53 protein. These results demonstrate that bcl‐2 and hypoxia can act synergis‐tically to modulate VEGF expression and the in vivo angiogenic response in the MCF7 ADR line.—Biroccio, A., Candiloro, A., Mottolese, M., Sapora, O., Albini, A., Zupi, G., Del Bufalo, D. Bcl‐2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line. FASEB J. 14, 652–660 (2000)

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.

Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity.

The aim of this paper was to study the molecular mechanisms by which bcl‐2 increases hypoxiainduced vascular endothelial growth factor (VEGF) expression. We demonstrated that bcl‐2 overexpression in M14 human melanoma cell line enhances hypoxia‐induced VEGF mRNA stability and promoter activation. In particular, the half‐life of the message was longer in bcl‐2 transfectants (approximately 330 min) than in control cells (approximately 180 min). In addition, bcl‐2 overexpression increased VEGF promoter activity through the hypoxia‐inducible factor‐1 (HIF‐1) transcription factor. Increased HIF‐1α protein expression and DNA binding activity were detected in bcl‐2 overexpressing cells compared with control cells. An enhanced functional activity of secreted VEGF was found both in in vitro and in vivo angiogenic assays, and the use of VEGF specific antibodies validated the role of VEGF on bcl‐2‐induced angiogenesis. Taken together our results indicate that bcl‐2 plays an important role in melanoma angiogenesis, and that VEGF mRNA stabilization and HIF‐1‐mediated transcriptional activity are two important control points in bcl‐2/hypoxia‐induced VEGF expression.

CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery

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.

ZD1839 (IRESSA), an EGFR-selective tyrosine kinase inhibitor, enhances taxane activity in bcl-2 overexpressing, multidrug-resistant MCF-7 ADR human breast cancer cells.

Constitutive bcl‐2 overexpression increases the tumorigenic and metastatic potential of doxorubicin‐resistant, estrogen‐independent, MCF‐7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl‐2‐overexpressing MCF‐7 ADR clones and control neomycin‐transfected MCF‐7 ADR neo cells. The 2 bcl‐2‐overexpressing MCF‐7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF‐7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF‐7 ADR neo and bcl‐2‐overexpressing MCF‐7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor‐α (TGF‐α). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa™, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR‐TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose‐dependent manner (IC50 of approximately 0.1 μM). This effect was accompanied by a dose‐dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF‐α, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl‐2‐overexpressing MCF‐7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose‐dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl‐2 expression in bcl‐2‐overexpressing MCF‐7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone‐independent, multidrug‐resistant, human breast cancer.

Functional activity of CXCL8 receptors, CXCR1 and CXCR2, on human malignant melanoma progression.

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.