RESUMO
INTRODUCTION: Temozolomide (TMZ) is a life prolonging DNA alkylating agent active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately acquired TMZ resistance severely undermines its clinical efficacy. Using an in vitro model, we tested whether poly (ADP-ribose) polymerase-1 and -2 (PARP) inhibition could suppress the emergence of resistance to enhance the effectiveness of TMZ. METHODS: Using the MGMT-methylated GBM line U251N, in which TMZ resistance can be induced, we developed a method to rapidly recreate mechanisms of TMZ resistance seen in GBMs, including MMR mutations and MGMT re-expression. We then assessed whether TMZ resistant U251N sub-clones could be re-sensitized to TMZ by co-treatment with the PARP inhibitor ABT-888, and also whether the emergence of resistance could be suppressed by PARP inhibition. RESULTS: U251N cultures chronically exposed to TMZ developed discrete colonies that expanded during TMZ treatment. These colonies were isolated, expanded further as sub-clones, and assessed for mechanisms of TMZ resistance. Most resistant sub-clones had detectable mutations in one or more mismatch repair (MMR) genes, frequently MSH6, and displayed infrequent re-expression of MGMT. TMZ resistance was associated with isolated poly(ADP-ribose) (pADPr) up-regulation in one sub-clone and was unexplained in several others. TMZ resistant sub-clones regressed during co-treatment with TMZ and ABT-888, and early co-treatment of U251N parental cultures suppressed the emergence of TMZ resistant colonies. CONCLUSION: In a model of acquired resistance, co-treatment with TMZ and a PARP inhibitor had two important benefits: re-sensitization of TMZ resistant cells and suppression of TMZ resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Células Tumorais CultivadasRESUMO
In vitro studies indicate that the inflammatory response to zymosan, a fungal wall preparation, is dependent on Toll-like receptor (TLR) 2, and that this response is enhanced by the dectin-1 receptor. Complement may also play an important role in this inflammatory response. However, the relevance of these molecules within the in vivo pulmonary environment remains unknown. To examine pulmonary in vivo inflammatory responses of the lung to zymosan, zymosan was administered by intratracheal aerosolization to C57BL/6, TLR2- TLR4-, MyD88-, and complement-deficient mice. Outcomes included bronchoalveolar fluid cell counts. We next examined effects of dectin-1 inhibition on response to zymosan in alveolar macrophages in vitro and in lungs of C57BL/6, TLR2-, and complement-deficient mice. Finally, the effect of alveolar macrophage depletion on in vivo pulmonary responses was assessed. Marked zymosan-induced neutrophil responses were unaltered in TLR2-deficient mice despite a TLR2-dependent response seen with synthetic TLR2 agonists. TLR4, MyD88, and complement activation were not required for the inflammatory response to zymosan. Although dectin-1 receptor inhibition blocked the inflammatory response of alveolar macrophages to zymosan in vitro, in vivo pulmonary leukocyte recruitment was not altered even in the absence of TLR2 or complement. Depletion of alveolar macrophages did not affect the response to zymosan. Neither complement, macrophages, nor TLR2, TLR4, MyD88, and/or dectin-1 receptors were involved in the pulmonary in vivo inflammatory response to zymosan.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Pulmão/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Toll-Like/fisiologia , Zimosan/farmacologia , Animais , Líquido da Lavagem Broncoalveolar , Lectinas Tipo C , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
PURPOSE: Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. EXPERIMENTAL DESIGN: Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. RESULTS: Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. CONCLUSION: Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Experimentais/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Neoplasias da Mama/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , GMP Cíclico/metabolismo , Feminino , Humanos , Dinitrato de Isossorbida/metabolismo , Dinitrato de Isossorbida/farmacologia , Masculino , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Doadores de Óxido Nítrico/sangue , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroglicerina/sangue , Nitroglicerina/metabolismo , Nitroglicerina/farmacologia , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.
Assuntos
Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Gradação de Tumores , RNA Interferente Pequeno/genéticaRESUMO
Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 (+/-) Trp53 (+/-) mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/ß) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/ß in response to MYXV infection. To confirm IFNα/ß did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/ß via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/ß is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.
Assuntos
Neoplasias Encefálicas/terapia , Resistência à Doença/imunologia , Glioma/terapia , Myxoma virus/crescimento & desenvolvimento , Proteínas de Neurofilamentos/imunologia , Terapia Viral Oncolítica , Proteína Supressora de Tumor p53/imunologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistência à Doença/genética , Feminino , Glioma/genética , Glioma/imunologia , Glioma/patologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/deficiência , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Interferon-alfa/imunologia , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Myxoma virus/imunologia , Proteínas de Neurofilamentos/deficiência , Proteínas de Neurofilamentos/genética , Transplante Isogênico , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs). METHODS: We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts. RESULTS: The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo. CONCLUSIONS: Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.
Assuntos
Neoplasias Encefálicas/prevenção & controle , Glioblastoma/prevenção & controle , Células-Tronco Neoplásicas/patologia , Terapia Viral Oncolítica , Infecções por Poxviridae/prevenção & controle , Sirolimo/uso terapêutico , Infecções Tumorais por Vírus/prevenção & controle , Animais , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/virologia , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Feminino , Citometria de Fluxo , Imunofluorescência , Glioblastoma/patologia , Glioblastoma/virologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Camundongos , Camundongos SCID , Myxoma virus/fisiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/virologia , Infecções por Poxviridae/patologia , Infecções por Poxviridae/virologia , Células Tumorais Cultivadas , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Tumor hypoxia has been correlated with metastasis and resistance to chemotherapy. Hypoxia is also associated with human prostate cancers, which are highly resistant to chemotherapy. We hypothesized that hypoxia contributes to chemoresistance in prostate cancer cells and this hypoxia induced chemoresistance can be inhibited by low concentrations of nitric oxide (NO) mimetics. MATERIALS AND METHODS: Human PC-3 and mouse TRAMP-C2 prostatic adenocarcinoma cells were incubated in 20% or 0.5% O(2) for 12 hours with or without glyceryl trinitrate (GTN) (0.1 nM). This treatment was followed by a 1-hour incubation with doxorubicin and survival was assessed by clonogenic assays. Western blot analysis was used to measure NO synthase levels. The effect of hypoxia and GTN on cell cycle distribution was determined by flow cytometry. RESULTS: Hypoxic pre-incubation of the 2 cell lines resulted in increased survival following exposure to doxorubicin. Co-incubation of PC-3 and TRAMP-C2 cells with GTN (0.1 nM) inhibited the hypoxia induced resistance to doxorubicin. Each cell line expressed all 3 NO synthase isoforms at levels that were not significantly affected by O(2) concentrations. Cell cycle analysis revealed that there was no significant difference in the distribution of PC-3 cells at each stage of the cycle. However, incubation under hypoxia resulted in a small decrease in the number of TRAMP-C2 cells in S-phase. CONCLUSIONS: These findings indicate that NO may have an important role in the regulation of chemosensitivity in prostate cancer cells. Furthermore, the results suggest that GTN administration may represent a means of chemosensitizing prostatic carcinomas.