RESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Bencenosulfonatos/administración & dosificación , Piridinas/administración & dosificación , Neoplasias Gástricas/tratamiento farmacológico , Taxoides/administración & dosificación , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Bencenosulfonatos/farmacología , Bencenosulfonatos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Sinergismo Farmacológico , Humanos , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Piridinas/farmacología , Piridinas/uso terapéutico , Rodaminas , Sorafenib , Taxoides/farmacología , Taxoides/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Acridinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , G-Cuádruplex/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Telómero/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Daño del ADN , Replicación del ADN/efectos de los fármacos , Humanos , Ligandos , Transducción de Señal , Telómero/química , Telómero/metabolismoRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Nucléolo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Melanoma/enzimología , Mitosis/efectos de los fármacos , Fenantrenos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Muerte Celular , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Humanos , Melanoma/patología , Microscopía por Video , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Acridinas/metabolismo , Antineoplásicos/metabolismo , Daño del ADN , G-Cuádruplex , Telómero/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , ADN/química , ADN/genética , ADN/metabolismo , Reparación del ADN , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Complejo Shelterina , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Trasplante Heterólogo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
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.
Asunto(s)
Acridinas/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/administración & dosificación , Sinergismo Farmacológico , Femenino , G-Cuádruplex/efectos de los fármacos , Humanos , Inmunohistoquímica , Ligandos , Masculino , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glioblastoma/metabolismo , Interleucina-8/biosíntesis , FN-kappa B/metabolismo , Proteína bcl-X/metabolismo , Western Blotting , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-8/genética , Neovascularización Patológica/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/fisiología , Transfección , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Glioblastoma/irrigación sanguínea , Interleucina-8/metabolismo , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteína bcl-X/metabolismo , Biomarcadores de Tumor/metabolismo , Northern Blotting , Western Blotting , Células Cultivadas , Colágeno , Combinación de Medicamentos , Endotelio Vascular , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Interleucina-8/genética , Laminina , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , Proteoglicanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Venas Umbilicales , Proteína bcl-X/genéticaRESUMEN
We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Melanoma/enzimología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Apoptosis/fisiología , Línea Celular Tumoral , ADN/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
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.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Sirolimus/análogos & derivados , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Proteínas Quinasas/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
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.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Aspirina/análogos & derivados , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Óxido Nítrico/metabolismo , Nitrocompuestos/farmacología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Aspirina/farmacología , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Humanos , Masculino , Ratones , Ratones Desnudos , Compuestos Organoplatinos/farmacología , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Melanoma/patología , Proteína 2 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Southern Blotting , Western Blotting , Vectores Genéticos , Humanos , Melanoma/terapia , Trasplante de Neoplasias , Telómero/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PURPOSE: bcl-2 and c-myc oncogenes are frequently overexpressed in different human tumors, including melanoma. Here, we evaluate the combined efficacy of two antisense oligonucleotides targeting bcl-2 mRNA (ODN bcl-2) and c-myc mRNA (ODN c-myc) in combination with cis-diammine dichloroplatinum (cisplatin, DDP) on three human melanoma lines (LM, NG, and M20). EXPERIMENTAL DESIGN: Two different sequences were designed to treat tumor-bearing mice: in the first one, ODN bcl-2 at a dose of 0.2 mg/day x4, followed by DDP given i.p. at a dose of 3.3 mg/kg/day x3 and ODN c-myc i.v. at 0.5 mg/day x7, whereas the other sequence consisted of ODN c-myc given as first agent followed by DDP and ODN bcl-2 at the same doses. Mice received three complete cycles of treatment in 1-week intervals. RESULTS: The treatment sequence with ODN bcl-2/DDP/ODN c-myc combination completely inhibited growth in NG tumor and induced a 35-day delay in LM tumor growth. In contrast, the M20 tumor growth was unaffected by the combination. A discrete amount of c-Myc and bcl-2 protein expression in both LM and NG tumors was detected, whereas no detectable levels of the two proteins were observed in M20 tumors. Compared with the other combination, the sequence (ODN bcl-2/DDP/ODN c-myc) produced the most effective results, producing a significant decrease in bcl-2 and c-Myc protein expression, which in turn significantly increased the survival of NG- and LM-bearing mice, with 4 mice out of 11 and 1 out of 7 mice being cured, respectively. Finally, this combination increased the apoptotic rate and produced an antiangiogenetic effect. CONCLUSIONS: These results show that an antisense approach to the treatment of melanoma xenografts overexpressing either bcl-2 or c-myc oncogenes represents a successful strategy to improve the response to chemotherapy in melanoma, with particular attention to the treatment sequence.
Asunto(s)
Melanoma/patología , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias Cutáneas/patología , Animales , Esquema de Medicación , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/genética , Ratones , Ratones Desnudos , ARN Mensajero , Neoplasias Cutáneas/genética , Análisis de Supervivencia , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Neoplasias/genética , Neoplasias/terapia , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , División Celular/genética , Clusterina , Terapia Combinada , Genes bcl-2 , Genes myc , Glicoproteínas/genética , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Transducción de Señal/genéticaRESUMEN
We have recently reported that bcl-2 overexpression and hypoxia synergistically interact to modulate vascular endothelial growth factor (VEGF) and in vivo angiogenesis in tumour cells through VEGF mRNA stabilization and hypoxia-inducible factor 1-mediated transcriptional activity. Bcl-2 antisense treatment has shown promising clinical results in patients with malignant melanoma. In the present study, we demonstrated that the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 inhibits bcl-2 expression and angiogenesis in two bcl-2 overexpressing clones derived from the M14 human melanoma cell line. The antiangiogenic effect was determined in in vitro and in vivo angiogenesis assays. In particular, a reduction of hypoxia-induced VEGF secretion was observed after 4625 treatment, and the conditioned medium (CM) of bcl-2 overexpressing clones treated with 4625 and exposed to hypoxic conditions resulted in decreased endothelial cell proliferation when compared to CM of untreated control cells. In addition, we found that CM of 4625 antisense-treated bcl-2 transfectants inhibited in vivo vessel formation in matrigel plugs implanted subcutaneously in C57/B16 mice. Our findings confirm that bcl-2 plays a crucial role in melanoma angiogenesis and demonstrate for the first time that downregulation of bcl-2 by antisense treatment has potential to inhibit angiogenesis independent of its effect on cell survival. The use of 4625 in cancer therapy is suggested as an approach to facilitate simultaneously tumour cell apoptosis and inhibit tumour angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Melanoma/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteína bcl-XRESUMEN
c-Myc is involved in the control of telomerase activity through its ability to induce the expression of the catalytic subunit of the enzyme, the human telomerase reverse transcriptase (hTERT). Our aim was to study whether telomerase plays a critical role in c-Myc-dependent tumorigenicity of melanoma cells. By using M14-derived clones, expressing low levels of c-Myc, we demonstrated that the down-regulation of c-Myc reduced cell proliferation rate, cloning efficiency and tumorigenicity and increased the apoptotic rate. Decreased tumorigenic potential correlated with reduced hTERT gene expression, telomerase activity and telomere shortening. Introduction of wild-type hTERT into these cells increased their proliferation rate and partially re-established their tumorigenic potential, at early passages, even though the apoptotic rate of the population remained unaltered. After several in vitro passages, hTERT-mediated cell proliferation made the tumorigenic potential of the c-Myc low-expressing clones comparable to that of the M14 parental line. Over-expression of the mutant biologically inactive hTERT did not drive cells to proliferate. In conclusion, our results demonstrate that the reconstitution of high levels of telomerase activity reverses the low tumorigenicity due to low c-Myc expression.
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Melanoma/patología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Telomerasa/fisiología , Animales , Apoptosis , División Celular , Células Clonales/metabolismo , Células Clonales/patología , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Genes myc , Humanos , Melanoma/metabolismo , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-myc/deficiencia , Proteínas Recombinantes de Fusión/fisiología , Telomerasa/genética , Telómero/ultraestructura , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Ensayo de Tumor de Célula MadreRESUMEN
The purpose of this study was to evaluate the antitumoral activity of different gemcitabine-based combination on an experimental model of human breast cancer, in order to identify the most effective treatment and to provide a rationale for clinical investigations. To this end, CG5 breast cancer cells were treated in vitro with gemcitabine followed by epirubicin, doxorubicin, docetaxel or paclitaxel. The reversed sequence was also investigated. Results, analyzed by multiple drug effect/combination index (CI) isobologram, demonstrated that the combination gemcitabine/paclitaxel was the most active showing synergism with a CI of about 0.5 in the two sequences employed. Moreover, the synergistic interaction of gemcitabine and paclitaxel was correlated to a block of the cells in the G0/G1 compartment of cell cycle and to an increase of apoptotic cells compared to each drug. Based on these evidences, the antitumoral efficacy of gemcitabine/paclitaxel combination has been studied in vivo. Mice bearing CG5 human breast xenografts treated with paclitaxel and gemcitabine in combination showed a significant higher inhibition of tumor growth (approximately 70%) compared to that with either agent alone (25%). In conclusion, this study suggests that paclitaxel is the most promising agent for combination protocols with gemcitabine and supports the use of gemcitabine/paclitaxel combination in the clinical management of advanced breast cancer.
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Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Paclitaxel/uso terapéutico , Animales , Antraciclinas/uso terapéutico , Desoxicitidina/uso terapéutico , Femenino , Humanos , Ratones , Taxoides/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The anti-apoptotic protein Bcl-2 has been implicated in the intrinsic resistance of melanoma to chemotherapy. The aim of this study was to investigate the effects of anti-Bcl-2 oligonucleotide oblimersen on the antitumour activity of gimatecan, a novel lipophilic camptothecin currently undergoing clinical phase II studies. Results showed a reduced sensitivity of melanoma cells to gimatecan following Bcl-2 transfection and inversely, increased cell sensitivity to gimatecan in combination with oblimersen. In in vivo studies performed in two melanoma xenografts expressing different Bcl-2 levels, the antitumour activity of oblimersen itself was modest, but the combination with gimatecan produced a significant therapeutic advantage. The combination therapy inhibited tumour growth and delayed regrowth of the two tumours tested. The enhancement of antitumour activity was observed at doses that were tolerated well. The effects of oblimersen on antitumour activity and toxicity of gimatecan were dose-dependent. The capability of oblimersen to improve the efficacy of gimatecan supports the therapeutic potential of the drug combination in the treatment of human melanoma.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/análogos & derivados , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Administración Oral , Animales , Camptotecina/administración & dosificación , División Celular , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos , Femenino , Humanos , Melanoma/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Tionucleótidos/administración & dosificación , Trasplante HeterólogoRESUMEN
BACKGROUND: Nitric 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. METHODS: In 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. RESULTS: In 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. CONCLUSIONS: NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.
RESUMEN
The aim of this paper was to study the molecular mechanisms by which bcl-2 increases hypoxia-induced 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-1a 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.
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Proteínas de Unión al ADN/fisiología , Factores de Crecimiento Endotelial/genética , Regulación Neoplásica de la Expresión Génica , Linfocinas/genética , Melanoma/genética , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Estabilidad del ARN , Factores de Transcripción , Animales , Hipoxia de la Célula , Embrión de Pollo , Factores de Crecimiento Endotelial/metabolismo , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Linfocinas/metabolismo , Melanoma/sangre , Melanoma/metabolismo , Modelos Biológicos , Neovascularización Patológica , Neovascularización Fisiológica , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PURPOSE: Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity in recent clinical trials against high grade gliomas, metastatic melanoma, and brain lymphoma. In this study, we tested whether systemic administration of GPI 15427, a novel poly(ADP-ribose) polymerase (PARP-1) inhibitor capable of crossing the blood-brain barrier, could enhance the efficacy of TMZ against metastatic melanoma, glioblastoma multiforme, and lymphoma growing in the brain. EXPERIMENTAL DESIGN: Murine B16 melanoma or L5178Y lymphoma cells were injected intracranially in syngeneic mice. An orthotopic xenograft of the human SJGBM2 glioblastoma multiforme was implanted in nude mice. Animals were treated with TMZ + GPI 15427 using a schedule of 40 mg/kg/i.v. GPI 15427 + 100 mg/kg/i.p. TMZ for 3 days. The efficacy of drug treatment was assessed by: (a) the increase of mouse survival and life span; and (b) the suppression of melanoma metastases to lung after i.v. injection of B16 cells. RESULTS: In all models, systemic administration of GPI 15427 shortly before TMZ significantly increased life span of tumor-bearing mice with respect to untreated controls or to groups treated with either GPI 15427 or TMZ only. Moreover, GPI 15427 increased the antimetastatic effect of TMZ. CONCLUSIONS: These data indicate that systemic administration of the poly(ADP-ribose) polymerase-1 inhibitor GPI 15427 significantly enhances TMZ antitumor efficacy against solid or hematological neoplasias even when located at the central nervous system site.