Annamaria Biroccio

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)

β-Arrestin links endothelin A receptor to β-catenin signaling to induce ovarian cancer cell invasion and metastasis

The activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) has a critical role in ovarian tumorigenesis and progression. To define the molecular mechanism in ET-1-induced tumor invasion and metastasis, we focused on β-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, β-arrestin is recruited to ETAR to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3β and β-catenin stabilization. The engagement of β-arrestin in these two signaling complexes concurs to activate β-catenin signaling pathways. We then demonstrate that silencing of both β-arrestin-1 and β-arrestin-2 inhibits ETAR-driven signaling, causing suppression of Src, mitogen-activated protein kinase (MAPK), AKT activation, as well as EGFR transactivation and a complete inhibition of ET-1-induced β-catenin/TCF transcriptional activity and cell invasion. ETAR blockade with the specific ETAR antagonist ZD4054 abrogates the engagement of β-arrestin in the interplay between ETAR and the β-catenin pathway in the invasive program. Finally, ETAR is expressed in 85% of human ovarian cancers and is preferentially co-expressed with β-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing β-arrestin-1 mutant metastasize at a reduced rate, highlighting the importance of this molecule in promoting metastases. ZD4054 treatment significantly inhibits metastases, suggesting that specific ETAR antagonists, by disabling multiple signaling activated by ETAR/β-arrestin, may represent new therapeutic opportunities for ovarian cancer.

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.

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.

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.

bcl‐2 over‐expression enhances NF‐κB activity and induces mmp‐9 transcription in human MCF7ADR breast‐cancer cells

bcl‐2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over‐expression of bcl‐2 in a human breast‐cancer cell line (MCF7ADR) enhances its tumorigenicity and metastatic potential by inducing metastasis‐associated properties such as increased secretion of the matrix metalloproteinase‐9 (mmp‐9). In the present study, we investigated the effect of bcl‐2 over‐expression on the activity of the transcription factor NF‐κB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over‐expression of bcl‐2 in the MCF7ADR cell line, enhances NF‐κB‐dependent transcriptional activity. Mobility‐shift analysis revealed an increase of NF‐κB DNA‐binding in bcl‐2‐over‐expressing clones that correlated with lower levels of the NF‐κB cytoplasmic inhibitor IκBα. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl‐2 with bax, or deletion of the N‐terminal BH4 domain, completely eliminate the ability of this molecule to up‐regulate NF‐κB‐dependent transactivation. Since mmp‐9 is a NF‐κB‐regulated gene, we also investigated whether bcl‐2 over‐expression up‐regulated mmp‐9 transcription. We found that induction of mmp‐9 mRNA correlates with the activation of an mmp‐9‐promoter‐reporter‐gene construct in transient transfection assay, and a mutation of the (−600)mmp‐9‐NF‐κB binding element abolishes this effect. The overall data indicate that bcl‐2‐mediated regulation of NF‐κB‐transcription‐factor activity may represent an important mechanism for the promotion of malignant behavior in MCF‐7ADR cells. Int. J. Cancer 86:188–196, 2000.