RESUMO
For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
Assuntos
Inativação Gênica , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Humanos , Indicadores e Reagentes , Camundongos , Oligonucleotídeos/análise , Oligonucleotídeos Antissenso/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , TransfecçãoRESUMO
Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.
Assuntos
Células Endoteliais/metabolismo , Fibrinolíticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hepatopatia Veno-Oclusiva/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Animais , Transplante de Medula Óssea/efeitos adversos , Bovinos , Linhagem Celular Transformada , Córnea/metabolismo , Córnea/patologia , Células Endoteliais/patologia , Fibrinolíticos/uso terapêutico , Hepatopatia Veno-Oclusiva/tratamento farmacológico , Hepatopatia Veno-Oclusiva/etiologia , Hepatopatia Veno-Oclusiva/patologia , Humanos , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Polidesoxirribonucleotídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells. G3139 is an 18-mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global phase III trial in advanced melanoma (GM301) has evaluated G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood because it is unlikely that Bcl-2 silencing is the sole mechanism for chemosensitization in melanoma cells. EXPERIMENTAL DESIGN: The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high-affinity cell surface receptors and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and human umbilical vascular endothelial cells were determined. RESULTS: G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in three-dimensional collagen gels, increases the mitogenesis of human umbilical vascular endothelial cells similarly, and induces vessel sprouts in the rat aortic ring model. CONCLUSIONS: G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with lactate dehydrogenase observed clinically.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Tionucleotídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Becaplermina , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-sis , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Tionucleotídeos/genética , Tionucleotídeos/metabolismoRESUMO
PURPOSE: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. EXPERIMENTAL DESIGN: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. RESULTS: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors. CONCLUSIONS: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.
Assuntos
Melanoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Sequência de Bases , Citocromos c/metabolismo , DNA Antissenso/genética , Regulação para Baixo , Inativação Gênica , Humanos , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Tionucleotídeos/genética , Tionucleotídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Veno-occlusive disease of the liver (VOD) remains a troubling and potentially fatal complication of high-dose chemotherapy and hematopoietic stem cell transplantation conditioning regimens. No effective therapy has been available for these patients to date, and the best supportive care measures remain woefully inadequate. Defibrotide (DF) (Gentium, S.p.A., Como, Italy), a polydisperse mixture of all the single-stranded phosphodiester oligodeoxyribonucleotides that can be obtained from the controlled depolymerization of porcine intestinal mucosal genomic DNA, seems to offer a safe and effective treatment for some patients suffering from severe VOD, a condition for which no accepted standard therapy currently exists. Early clinical studies evaluating the efficacy of DF for the treatment of severe VOD in patients undergoing hematopoietic stem cell transplantation have been very encouraging. Approximately 45% of the patients treated in multiple initial phase II clinical trials achieved a complete response at day +100, demonstrating normalization of serum bilirubin and resolution of the clinical syndrome. However, although multi-institutional, these represented single arm studies. A large, FDA-approved, pivotal, prospective, multi-institutional, global phase III trial of DF vs. historical controls (best available therapy) commenced in the first quarter of 2006 and should provide further validation of DF's efficacy. The drug seems to have few significant side effects, and almost all test subjects who have received this treatment have tolerated it well. Although the mechanism of action remains unclear, the drug exerts minimal systemic anticoagulant effects yet appears to induce numerous antithrombotic and profibrinolytic effects both in vitro and in vivo. It may function as an adenosine receptor agonist and causes increased concentrations of endogenous prostaglandins, which modulate thrombomodulin, platelets, and fibrinolysis. It also appears to block lipopolysaccharide (LPS)-induced tissue factor (TF) expression. However, despite the fact the DF is composed of oligonucleotides, its mechanism of action, which at the present time is unclear, is not related to Watson-Crick base pair-dependent downregulation of gene expression but is rather likely a result of its polyanionic nature.
Assuntos
Hepatopatia Veno-Oclusiva/tratamento farmacológico , Polidesoxirribonucleotídeos/uso terapêutico , Hepatopatia Veno-Oclusiva/diagnóstico , Hepatopatia Veno-Oclusiva/fisiopatologia , Humanos , Polidesoxirribonucleotídeos/farmacologia , Prognóstico , Resultado do TratamentoRESUMO
Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Tionucleotídeos/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linfócitos B/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/imunologia , Linhagem Celular Tumoral , Citosina/química , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Metilação , Camundongos , Camundongos Nus , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tionucleotídeos/administração & dosagem , Tionucleotídeos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
bcl-xL is a M(r) 26,000 bcl-2 homologue that is highly expressed in prostate cancer cells. In previous studies, the down-regulation of its expression by antisense oligonucleotides led to resistance. In this work, the 445-bp 5' terminus of the bcl-xL cDNA was cloned in the antisense orientation and stably transfected into DU145 and LNCaP prostate cancer cells. In the DU145 (and to a lesser extent the LNCaP) transfectants, phenotypic changes (versus mock-transfected cells) included an increase in doubling time (from 36 to 175 h) in the clone in which bcl-xL protein expression was 25% of control. The transfectants did not demonstrate characteristic apoptotic changes, as demonstrated by 4',6-diamidino-2-phenylindole staining, lack of either DNA laddering, caspase-3 activation, or poly(ADP)ribose and lamin cleavage, and the absence of a significant sub-G(0) population. Cell cycle analysis demonstrated an increase in a tetraploid population (from 28% to 66%), as well as the appearance of a hypertetraploid population. Levels of cIAP-1 protein were almost undetectable in the mock cells but increased at least 25-fold in the DU145 transfectants. The down-regulation of bcl-xL in both DU145 (and to a much lesser extent in LNCaP) cells led to their resistance to cytotoxic agents, including docetaxel, mitoxantrone, etoposide, vinblastine, and carboplatin. Reversion of bcl-xL expression in stable DU145 transfectants to nearly the levels found in the mock-transfected cells was accomplished by retroviral infection of the cells with a bcl-xL sense cDNA under control of a prolific promoter. This led to a dramatic increase in the growth rate and in BrdUrd incorporation, as well as a sharp decrease in the expression of cIAP-1 protein. Overall, these findings highlight the adaptability of prostate cancer cells to loss of bcl-xL and suggest that in addition to its prosurvival role, bcl-xL protein may also be involved in the regulation of the rate of cellular proliferation.
Assuntos
Resistência a Múltiplos Medicamentos , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Antissenso/genética , Antineoplásicos/farmacologia , Apoptose/genética , Divisão Celular/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Ploidias , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Proteína bcl-XRESUMO
In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4',6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only approximately 4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Melanoma/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tionucleotídeos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Citocromos c/análise , Citocromos c/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Humanos , Lipídeos/farmacologia , Melanoma/genética , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , TransfecçãoRESUMO
Antisense oligonucleotides have been evaluated as antineoplastic agents in a series of clinical trials, with mixed results. However, phase III trials incorporating G3139, a phosphorothioate oligomer targeted to the initiation codon region of the bcl-2 mRNA, have recently been completed in advanced melanoma, myeloma, and chronic lymphocytic leukemia (CLL). This article discusses the mechanism of the antisense effect and its dependence on the cellular internalization of oligonucleotides and the activity of RNase H. It also describes the properties, specific and nonspecific, of phosphorothioate oligonucleotides, the predominant species in current clinical trials, and discusses pharmacokinetic data obtained from earlier phase I and II trials employing these molecules. While the application of antisense technology to the treatment of human cancer is conceptually straightforward, in practice there are many complicated, mechanistically based questions that must be considered.
Assuntos
Inativação Gênica , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Oncogenes/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacologia , Tionucleotídeos/uso terapêuticoRESUMO
G3139 is an antisense Bcl-2 phosphorothioate oligonucleotide that has been combined with DTIC in a phase III clinical trial in melanoma. However, its actual mechanism of action in melanoma is controversial. Treatment of 518A2 melanoma cells with either G3139 or G4126 (a two-base mismatch) and then with light-activated DTIC caused these cells (but not SK-Mel-30 or 346.1 cells) to be protected against the cytotoxic effects of DTIC. This cytoprotection was not recapitulated with a phosphodiester congener of G3139 nor with a small interfering RNA (siRNA) also targeted to the Bcl-2 mRNA. Administering the drugs in reverse order also did not produce cytoprotection, and an 18- mer phosphorothioate homopolymer of thymidine was also inactive. Subsequently, it was discovered that gemcitibine and cis-platinum also induced cytoprotection to DTIC in this cell line, suggesting that the cytoprotection is a stress response to chemical proapoptotic stress. Cytoprotection was completely inhibited by O(6)-benzylguanine, an inhibitor of O(6)-guanosine alkyltransferase (OGAT) activity. However, a direct assay of OGAT activity demonstrated that 518A2 melanoma cells are essentially completely devoid of it, either basally or induced. The cytoprotection may thus be caused by a chemical stress-induced increase in mismatch repair activity.
Assuntos
Antineoplásicos/farmacologia , Dacarbazina/toxicidade , Resistencia a Medicamentos Antineoplásicos , Melanoma/patologia , Tionucleotídeos/farmacologia , Antineoplásicos/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Guanina/análogos & derivados , Guanina/metabolismo , Guanina/farmacologia , Humanos , Melanoma/genética , Melanoma/metabolismo , Oligonucleotídeos Antissenso/farmacologia , GencitabinaRESUMO
G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. Because of the ability of this antisense construct to downregulate the expression of Bcl-2 mRNA and protein, it has entered phase III clinical trials in a number of human cancers, including advanced melanoma. However, the actual mechanism of this agent is far from certain. In this work, we demonstrate that G3139 induces the relatively rapid release of cytochrome c into the cytoplasm of treated 518A2 melanoma cells. This release activates the intrinsic pathway of apoptosis, eventually leading to a mitochondrial permeability transition and cell death. By employing an siRNA strategy, we also show that this entire process appears to be Bcl-2 independent, as downregulation of Bcl-2 protein expression does not alter the induction of apoptosis by G3139. Furthermore, forced overexpression of Bcl-2 protein contributes relatively little to chemoresistance in this cell line. While these results may or may not be reflective of the in vivo situation, the value of Bcl-2 as a target in advanced melanoma must at least be questioned.
Assuntos
Melanoma/genética , Melanoma/terapia , Oligonucleotídeos Antissenso/farmacologia , Tionucleotídeos/farmacologia , Animais , Apoptose , Caspases/metabolismo , Sobrevivência Celular , Ensaios Clínicos como Assunto , Citoplasma/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells. EXPERIMENTAL DESIGN: In this work, we down-regulated the expression of bcl-2 protein by 80-90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells. RESULTS: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock-infected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously. CONCLUSIONS: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Antissenso/metabolismo , Tionucleotídeos/farmacologia , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Oligonucleotídeos/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
PURPOSE: G3139 is an antisense bcl-2 phosphorothioate oligodeoxyribonucleotide that is currently being evaluated in Phase III clinical trials in several human cancers. The aim of the present work was to further identify the apparent non-bcl-2-dependent mechanism of this action of this compound in PC3 prostate cancer cells. EXPERIMENTAL DESIGN: We performed Affymetrix U95A oligonucleotide microarray studies on mRNA isolated from cells treated with G3139 and related oligonucleotides. RESULTS: Hierarchical clustering revealed the presence of a set of genes of which the expression was elevated on both 1 and 3 days after oligonucleotide treatment. Significantly, the persistence of expression of the up-regulation of these genes, many of which are members of the IFN cascade, was greater for G3139 than for any other oligomer evaluated. Furthermore, many of the genes with the greatest up-regulation of expression are also those of which the expression is up-regulated after treatment of cells with IFNs. Treatment of PC3 cells with either IFN-beta or -gamma recapitulated some of the aspects of the molecular and phenotypic changes observed after treatment with a G3139/Lipofectin complex. These include down-regulation of bcl-2 protein expression itself, down-regulation of protein kinase C alpha protein expression (but not that of other protein kinase C isoforms), alteration in p21/Waf1/Cip1 protein expression, up-regulation of MHC-I cell surface expression, and profound suppression of cell growth in the absence of a notable increase in cellular apoptosis. However, G3139 (when complexed with Lipofectin) did not induce the up-regulation of expression of either type I or type II IFNs, nor could IFNs be found in conditioned media from treated cells. CONCLUSIONS: Oligonucleotide microarray experiments demonstrated that G3139 could induce elements of the IFN cascade in PC3 cells in vitro. In addition, the cellular phenotype obtained after treatment with exogenous IFN could, at least in part, recapitulate that obtained after G3139 treatment. Nevertheless, the oligonucleotide microarray experiments we performed also demonstrated that there are extremely large qualitative and quantitative differences between the two treatments.
Assuntos
Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Interferon beta/uso terapêutico , Interferon gama/uso terapêutico , Oligonucleotídeos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/terapia , Algoritmos , Anexina A5/química , Anticorpos Monoclonais/química , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Ilhas de CpG , Relação Dose-Resposta a Droga , Regulação para Baixo , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Isoformas de Proteínas , Proteína Quinase C/biossíntese , Proteína Quinase C-alfa , Proteínas Proto-Oncogênicas c-bcl-2/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
PURPOSE: Inhibition of the function of Bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy. G3139 (Genasense) is a phosphorothioate anti-Bcl-2 antisense oligonucleotide, but its mechanism of action is uncertain. The aim of the present work is to investigate inhibition of Bcl-2 expression in 518A2 melanoma cells, the cell line on which recent phase II and phase III clinical trials employing this agent were based. EXPERIMENTAL DESIGN: We down-regulated the expression of Bcl-2 protein by two different strategies in these cells: one employing G3139 and controls, and the other using a small interfering RNA approach. Cell viability after treatment with oligonucleotides or small interfering RNA and cytotoxic agents including gemcitibine, DDP, docetaxel, and thapsigargin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A 518A2 melanoma cell line stably overexpressing Bcl-2 protein was constructed and treated with either these cytotoxic agents or G3139. RESULTS: The cytotoxic effects of either G3139 or small interfering RNA treatment of 518A2 melanoma cells are Bcl-2 independent. In addition, in the Bcl-2-overexpressing cells, only a modest increment in chemoresistance was observed, and treatment with G3139 not only did not down-regulate Bcl-2 expression but produced essentially identical toxicity as was observed in the wild-type or mock-transfected cells. CONCLUSIONS: Our results suggest that the mechanism whereby G3139 produces drug-induced cytotoxicity in the 518A2 melanoma line is not dependent on levels of Bcl-2. These findings emphasize the nonsequence specific effects of this phosphorothioate oligonucleotide and call into question the validity of Bcl-2 as a target in this cell line.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas/patologia , Tionucleotídeos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Docetaxel , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Humanos , Melanoma/genética , Melanoma/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Taxoides/farmacologia , Tapsigargina/farmacologia , Células Tumorais Cultivadas , GencitabinaRESUMO
G3139 is an 18-mer phosphorothioate oligodeoxyribonucleotide, which is targeted to the initiation codon region of the bcl-2mRNA. Although treatment of PC3 prostate cancer cells with G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2 protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of pro-caspase 3 cleavage products and of Annexin V cell surface expression. In addition, ATP production and the mitochondrial membrane potential DeltaPsim were preserved. Despite this, G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft agar. G4232, a variant of G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2 protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms. G3139 caused production of reactive oxygen species in growth-arrested cells and oxidation of nuclear guanosine to 8-hydroxy-2'-deoxyguanosine, as determined by 1F7 monoclonal antibody staining. Bromodeoxyuridine incorporation studies demonstrated that G3139 induced a G1-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several proteins encoded by S-phase genes, including c-myb and poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of G3139 in PC3 cells.
Assuntos
Ilhas de CpG , Oligonucleotídeos Antissenso/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Tionucleotídeos/farmacologia , Ágar/farmacologia , Motivos de Aminoácidos , Anexina A5/farmacologia , Northern Blotting , Western Blotting , Bromodesoxiuridina/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Corantes/farmacologia , DNA/química , Metilação de DNA , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Fase G1 , Guanosina/química , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mutação , Oligonucleotídeos/química , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio , Fase S , Fatores de Tempo , TransfecçãoRESUMO
Currently employed treatment options for patients with advanced and metastatic cancer such as surgery, radiation, hormone therapy, and chemotherapy are limited. In particular, the well known limitations of chemotherapy are at least in part due to a lack of specificity. The activation of dominant oncogenes and inactivation of tumor suppressor genes may represent novel targets for cancer therapy. Antisense therapy has been widely used to specifically and selectively inhibit the expression of selected genes at the messenger RNA level. Combinations of antisense oligonucleotides with chemotherapeutic agents may offer important advantages in cancer treatment. Several antisense drugs, especially oblimersen (G3139), have shown interesting results in experiments in animals, and have entered clinical trials. However, control oligonucleotides must be carefully chosen to separate antisense effects from the many potential nonspecific effects of oligonucleotides. This review summarizes the advantages and limitations of antisense therapy and its use in the treatment of androgen-independent prostate cancer.
Assuntos
Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Masculino , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Relação Estrutura-AtividadeRESUMO
In order to overcome the problems of enzymatic degradation and short plasma half life, which can limit the delivery of antisense oligonucleotides, and the potential immuno-stimulatory effects of CpG motifs, we utilized a polyethylene glycol (PEG) technology that employed various releasable linkers (rPEG). 5'-20 kDa-PEGylation of an anti-Bcl-2 5'-aminoalkyl-oligonucleotide with the same sequence as G3139 (Compound 1) did not alter its binding to the heparin-binding protein bFGF, nor the release of cytochrome c from isolated mitochondria treated with the conjugates. However, in 518A2 melanoma cells in vitro, PEGylation resulted in greatly diminished cellular uptake. In striking contrast, PEGylation of 1 resulted in dramatically improved pharmacokinetic profiles in vivo, with a prolonged half-life (t1/2), increased plasma concentration, and increased area under the plasma concentration-time curve (AUC). In an in vivo melanoma 518A2 xenograft mouse model, treatment with either 5'-20 kDa-PEG-1 or 1 demonstrated similar tumor growth inhibition. Furthermore, in an in vitro mouse splenocyte culture system, attachment of a PEG moiety to 1 through releasable linkers abolished the immunostimulatory response that was observed for G3139. Our results demonstrate the potential of the in vivo use of PEGylated oligonucleotides, and point out the profound differences between in vitro and in vivo models of oligonucleotide activity.
Assuntos
Antineoplásicos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Polietilenoglicóis/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Polietilenoglicóis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
G3139 (Genasense), an 18mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 messenger RNA (mRNA), downregulates Bcl-2 protein and mRNA expression in many cell lines. However, both the in vitro and in vivo mechanisms of action of G3139 are still uncertain. The isosequential L-deoxyribose enantiomer L-G3139, which does not downregulate Bcl-2 expression, was synthesized to study the role of the Bcl-2 protein in melanoma cells. Both D-G3139 and L-G3139 bind nonspecifically to basic fibroblast growth factor with approximately the same K(c), and cause highly effective inhibition of net formation in 518A2 melanoma cells on Matrigel. The uptakes of D-G3139 and L-G3139 in melanoma cells were also similar. However, unlike D-G3139, L-G3139 does not produce poly ADP-ribose polymerase-1 and procaspase-3 cleavage at 9.5 h after the initiation of the transfection, but can activate the intrinsic pathway of apoptosis at approximately 48 h. Furthermore, treatment of A375 melanoma human xenografts in severe combined immunodeficiency (SCID) mice demonstrates that tumor growth is not inhibited by L-G3139, whereas D-G3139 significantly inhibits the rate of tumor growth. Furthermore, the immunostimulatory properties of L-G3139 appear to be nil, which differs dramatically from those of D-G3139. In conclusion, profound differences exist between D-G3139 and L-G3139 in vivo despite their similarities in vitro.
Assuntos
Melanoma Experimental/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Interleucina-12/metabolismo , Interleucina-16/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microscopia de Contraste de Fase , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , Ligação Proteica , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Estereoisomerismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochrome c precedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochrome c in the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochrome c from isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochrome c can transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.
Assuntos
Melanoma/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tionucleotídeos/farmacologia , Canais de Ânion Dependentes de Voltagem/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Fosfatos/químicaRESUMO
The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.