RESUMO
The first oncolytic virotherapy employing HSV-1 (oHSV-1) was approved recently by the FDA to treat cancer, but further improvements in efficacy are needed to eradicate challenging refractory tumors, such as glioblastomas (GBM). Microglia/macrophages comprising approximately 40% of a GBM tumor may limit virotherapeutic efficacy. Here, we show these cells suppress oHSV-1 growth in gliomas by internalizing the virus through phagocytosis. Internalized virus remained capable of expressing reporter genes while viral replication was blocked. Macrophage/microglia formed a nonpermissive OV barrier, preventing dissemination of oHSV-1 in the glioma mass. The deficiency in viral replication in microglial cells was associated with silencing of particular viral genes. Phosphorylation of STAT1/3 was determined to be responsible for suppressing oHSV-1 replication in macrophages/microglia. Treatment with the oxindole/imidazole derivative C16 rescued oHSV-1 replication in microglia/macrophages by inhibiting STAT1/3 activity. In the U87 xenograft model of GBM, C16 treatment overcame the microglia/macrophage barrier, thereby facilitating tumor regression without causing a spread of the virus to normal organs. Collectively, our results suggest a strategy to relieve a STAT1/3-dependent therapeutic barrier and enhance oHSV-1 oncolytic activity in GBM.Significance: These findings suggest a strategy to enhance the therapeutic efficacy of oncolytic virotherapy in glioblastoma. Cancer Res; 78(3); 718-30. ©2017 AACR.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Macrófagos/patologia , Microglia/patologia , Terapia Viral Oncolítica , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virologia , Proliferação de Células , Feminino , Glioblastoma/metabolismo , Glioblastoma/virologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Nus , Microglia/metabolismo , Microglia/virologia , Vírus Oncolíticos/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncolytic herpes simplex virus type 1 (oHSV-1) therapy is an emerging treatment modality that selectively destroys cancer. Here we report use of a glioma specific HSV-1 amplicon virus (SU4-124 HSV-1) to selectively target tumour cells. To achieve transcriptional regulation of the SU4-124 HSV-1 virus, the promoter for the essential HSV-1 gene ICP4 was replaced with a tumour specific survivin promoter. Translational regulation was achieved by incorporating 5 copies of microRNA 124 target sequences into the 3'UTR of the ICP4 gene. Additionally, a 5'UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. Our results confirmed enhanced expression of survivin and eIF4E in different glioma cells and increased micro-RNA124 expression in normal human and mouse brain tissue. SU4-124 HSV-1 had an increased ICP4 expression and virus replication in different glioma cells compared to normal neuronal cells. SU4-124 HSV-1 exerted a strong antitumour effect against a panel of glioma cell lines. Intracranial injection of SU4-124 HSV-1 did not reveal any sign of toxicity on day 15 after the injection. Moreover, a significantly enhanced antitumour effect with the intratumourally injected SU4-124 HSV-1 virus was demonstrated in mice bearing human glioma U87 tumours, whereas viral DNA was almost undetectable in normal organs. Our study indicates that incorporation of multiple cancer-specific regulators in an HSV-1 system significantly enhances both cancer specificity and oncolytic activity.
Assuntos
Glioma/terapia , Herpesvirus Humano 1/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Fator 2 de Crescimento de Fibroblastos/genética , Glioma/genética , Glioma/virologia , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Inibidoras de Apoptose/genética , Camundongos , Vírus Oncolíticos/fisiologia , Regiões Promotoras Genéticas/genética , Ratos , Survivina , Carga Tumoral/genética , Células VeroRESUMO
Exposure of cancer cells to anticancer agents in cultures induces detachment of cells that are usually considered dead. These drug-induced detached cells (D-IDCs) may represent a clinical problem for chemotherapy since they may survive anoikis, enter the circulation, invade other tissues and resume proliferation, creating a metastasis, especially in tissues where the bioavailability of anticancer agents is not enough to eliminate all cancer cells. In this study we evaluated the antiproliferative effect of menadione : sodium orthovanadate (M : SO) combination on A549 lung cancer cells as well as the ability of M : SO to induce cell detachment. In addition, we followed the fate and chemosensitivity of M : SO-induced detached cells. Using transwell chambers, we found that a fraction of the M : SO-induced detached cells were viable and, furthermore, were able to migrate, re-attach, and resume proliferation when re-incubated in drug-free media. The total elimination of A549 detachment-resistant cells and M : SO-induced detached cells were successfully eliminated by equivalent M : SO concentration (17.5 µM : 17.5 µM). Thus, M : SO prevented cell migration. Similar results were obtained on DBTRG.05MG human glioma cells. Our data guarantee further studies to evaluate the in vivo occurrence of D-IDCs, their implications for invasiveness and metastasis and their sensitivity to anticancer drugs.
RESUMO
Gliomas are the most common primary brain tumor, and their treatment is still a challenge. Here, we evaluated the antiproliferative effect of a novel combination of two potent oxidative stress enhancers: menadione (M) and sodium orthovanadate (SO). We observed both short-term and prolonged growth inhibitory effects of M or SO alone as well as in combination (M:SO) on DBTRG.05MG human glioma cells. A stronger antiproliferative effect was observed in the short-term proliferation assay with the M:SO combination compared to either investigated agent alone. In the long-term proliferation assay, a 10-day exposure to M:SO at concentrations of 10 µM:17.5 µM or 17.5 µM:10 µM was enough to kill 100% of the cells; no cell regrowth was observed after re-incubation in drug-free media. When used in combination, the single concentration of M and SO could be decreased by 2.5- to 5-fold of those used for each experimental drug alone and still obtain a similar antiproliferative effect. The underlying molecular mechanism was investigated by co-incubating M:SO with dithiothreitol (DTT) and genistein. Both substances partially neutralized the effects of the M:SO combination, showing additive effects. This observation suggests a role of oxidative stress and tyrosine kinase stimulation in the M:SO cytotoxic effect. Our results indicate that M:SO combination is an attractive alternative for glioma treatment that encourages further study. The neutralizing effects of genistein and DTT reveal a possibility for their use in the minimization of potential M:SO systemic toxicity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Glioma/tratamento farmacológico , Glioma/patologia , Vanadatos/uso terapêutico , Vitamina K 3/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ditiotreitol/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Genisteína/farmacologia , Humanos , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Fatores de Tempo , Vanadatos/farmacologia , Vitamina K 3/farmacologiaRESUMO
In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 µM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Glutationa/química , Inibidores da Topoisomerase II/química , Antígenos de Neoplasias , DNA Topoisomerases Tipo II , Ensaios Enzimáticos , Humanos , OxirreduçãoRESUMO
Menadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC (50) of approximately 10 µM but 50 µM had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1-2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC(50) values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment.