RESUMEN
Transforming growth factor-ß1 (TGF-ß1) induces stromal fibroblast-to-myofibroblast transdifferentiation in the tumor-stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro-RNAs (miRNAs) and their roles in TGF-ß1-induced myofibroblast differentiation in tumor-stroma interaction are unclear. Using quantitative real-time RT-PCR, we demonstrated that the expression of micro-RNA-21 (miR-21) was upregulated in activated fibroblasts after treatment with TGF-ß1 or conditioned medium from cancer cells. To determine the potential roles of miR-21 in TGF-ß1-mediated gene regulation during myofibroblast conversion, we showed that miR-21 expression was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic. Interestingly, downregulation of miR-21 with the inhibitor effectively inhibited TGF-ß1-induced myofibroblast differentiation while upregulation of miR-21 with a mimic significantly promoted myofibroblast differentiation. We further demonstrated that MiR-21 directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myofibroblasts. Taken together, these results suggested that miR-21 participated in TGF-ß1-induced myofibroblast transdifferentiation in cancer stroma by targeting PDCD4.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Diferenciación Celular , Fibroblastos/citología , Regulación de la Expresión Génica , MicroARNs/fisiología , Miofibroblastos/citología , Proteínas de Unión al ARN/metabolismo , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Células Cultivadas , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Medios de Cultivo Condicionados/farmacología , Femenino , Feto , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Pulmón/metabolismo , Pulmón/patología , Miofibroblastos/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/patología , Factor de Crecimiento Transformador beta/genética , Microambiente TumoralRESUMEN
Molecular targeted therapy (MTT) represents the new generation of anti-cancer arsenals. In this study, we report an alternative approach using a hybrid toxin that utilises the high-affinity of receptor-binding fragment of Clostridium perfringens enterotoxin (CPE). CPE naturally binds to CLDN-4 through the C-terminal 30 amino acid. However, recent studies have shown that CLDN-4 is also overexpressed on a range of cancer cells. We thus constructed a cDNA comprising C-CPE and a well characterised toxic domain of Pseudomonas aeruginosa exotoxin A (C-CPE-ETA'). The recombinant C-CPE-ETA' fusion protein was shown to retain the specificity of binding to CLDN-4 and initiating rapid penetration into cytosol in five different CLDN-4 positive cancer cells (Breast-MCF7, Skin-A431, Colon-SW480, Prostate-PC3 and DU145) but not to CLDN-4 negative cells (Hela, HUVEC). C-CPE-ETA' was strongly cytotoxic towards CLDN-4 positive cancer cell, as opposed to cells lacking CLDN-4 expression. Furthermore, we demonstrated that the recombinant fusion protein had significant anti-cancer ability in CLDN-4 positive cancer models in vivo. Subcutaneously implanted MCF7 and SW480 xenograft tumours were significantly decreased or abolished after three repeated injection of the hybrid toxin. Taken together, our results convincingly show that the hybrid toxin targets CLDN-4 positive cancer through receptor-binding, and causes significant tumour cell apoptosis, suggesting its potential as an alternative molecular targeted therapy against a plethora of CLDN-4 positive cancers.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Enterotoxinas/uso terapéutico , Exotoxinas/uso terapéutico , Proteínas de la Membrana/biosíntesis , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Factores de Virulencia/uso terapéutico , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Animales , Apoptosis , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Claudina-4 , Enterotoxinas/genética , Enterotoxinas/metabolismo , Exotoxinas/genética , Exotoxinas/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
In the quest to developing novel cancer therapies, oncolytic Clostridia are re-emerging as promising candidates due to their ability to specifically target and lyse tumours with great efficacy. Clostridial spores have the innate abilities that exploit the unique tumour microenvironment by directly penetrating, colonising and killing tumour cells. These unique features have prompted many studies to investigate their oncolytic potency. In addition, Clostridia possess a number of characteristics that enable them to be developed as oncolytic gene delivery vectors, such as their unlimited capacity, antibiotic sensitivity, and extracellular existence. Similarly, numerous strategies are being devised and tested to take advantage of these features with modern molecular technologies. In this review, we detail the traits that distinguish Clostridia from other agents, and describe the potential therapeutic effects that Clostridial spores demonstrate, but are not achievable with other treatment modalities. Furthermore, we will also summarize the recent advances in the use of Clostridial spores as viable therapeutic candidates, incorporating the latest progresses in genetic engineering tools, such as ClosTron. Finally, we will highlight some avenues deserving further studies in order to realise the ultimate goal of utilizing oncolytic Clostridia clinically in patients.
Asunto(s)
Antineoplásicos , Clostridium/genética , Terapia Genética , Vectores Genéticos/genética , Neoplasias/terapia , Esporas Bacterianas , Clostridium/inmunología , Ingeniería Genética , HumanosRESUMEN
Original observation of patients' spontaneous recovery from advanced tumours after an infection or a "fever" inspired extensive research. As a result, Coley's toxin for the therapy of sarcomas and live Bacillus Calmette-Guerin (BCG) for bladder cancer were born. In addition, three genera of anaerobic bacteria have been shown to specifically and preferentially target solid tumours and cause significant tumour lyses. Initial research had focused on determining the best tumour colonizing bacteria, and assessing the therapeutic efficacy of different strategies either as a single or combination treatment modalities. However, although clinical trials were carried out as early as the 1960s, lack of complete tumour lyses with injection of Clostridial spores had limited their further use. Recent progress in the field has highlighted the rapid development of new tools for genetic manipulation of Clostridia which have otherwise been a hurdle for a long time, such as plasmid transformation using electroporation that bore the problems of inefficiency, instability and plasmid loss. A new Clostridium strain, C. novyi-NT made apathogenic by genetic modification, is under clinical trials. New genetic engineering tools, such as the group II intron has shown promise for genetic manipulation of bacteria and forecast the dawn of a new era for a tumour-targeted bacterial vector system for gene therapy of solid tumours. In this review we will discuss the potential of genetically manipulated bacteria that will usher in the new era of bacterial therapy for solid tumours, and highlight strategies and tools used to improve the bacterial oncolytic capability.
Asunto(s)
Bacterias Anaerobias/genética , Clostridium/genética , ADN Bacteriano/uso terapéutico , Terapia Genética/métodos , Neoplasias/terapia , Animales , Bacterias Anaerobias/patogenicidad , Hipoxia de la Célula , Clostridium/patogenicidad , Ingeniería Genética , Vectores Genéticos , Humanos , Intrones , Necrosis , Neoplasias/microbiología , Neoplasias/patología , Esporas BacterianasRESUMEN
Hypoxia is a hallmark of solid cancer and characterized by regions of low oxygen and necrosis due to insufficient blood perfusion. Intratumoral hypoxia triggers the transcription of genes responsible for cell survival. The transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) is a key regulator of this response. HIF activation is associated with resistance to radio- and chemotherapy and poor clinical outcome, and may therefore provide an attractive therapeutic target. Clostridium-based oncolysis is a promising therapeutic strategy for the treatment of hypoxic tumors where these microorganisms naturally home. Here, we report for the first time the isolation of transconjugants of two excellent tumor colonizing Clostridium strains, C. novyi-NT and C. sporogenes, expressing single chain antibodies specific for human HIF-1alpha. This is a first step towards Clostridium-directed antibody therapy (CDAT) that holds promise as a carrier of cancer therapeutics targeting the most resistant regions in human solid cancer.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Clostridium/clasificación , Clostridium/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Neoplasias/inmunología , Neoplasias/microbiología , Humanos , Especificidad de la EspecieRESUMEN
Bacterial-based tumor-targeted therapy is an area of growing interest and holds promise for the treatment of solid tumors. Upon systemic administration, various types of non-pathogenic obligate anaerobes and facultative anaerobes have been shown to infiltrate and selectively replicate within solid tumors. The tumor specificity is based upon the unique physiology of solid tumors, which is often characterized by regions of hypoxia and necrosis. Prokaryotic vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models. Although the amount of clinical experience with bacterial vectors is limited to date, the available data clearly demonstrated the feasibility of bacterial-mediated therapy in humans. There are several issues however that are still unknown and remain major challenges. In this review, using Clostridium and modified Salmonella as prototypical agents, we will discuss the major advantages, challenges and shortcomings of bacterial systems for tumor-specific therapy. In addition, we will highlight the requirements needed to advance the approach into clinical trials.
Asunto(s)
Clostridium/fisiología , Neoplasias/terapia , Salmonella/fisiología , HumanosRESUMEN
To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells that are most resistant to conventional therapies. We demonstrate that the exponential growth of the attenuated bacteria is identical under normoxia and hypoxia. A hypoxia-inducible promoter (HIP-1) was created from a portion of the endogenous Salmonella pepT promoter and was shown to drive reporter gene expression under both acute and chronic hypoxia, but not under normoxia. Genetic engineering of the TATA- and FNR-box within HIP-1 allowed fine-tuning of gene induction, resulting in hypoxic induction factors of up to 200-fold. Finally, we demonstrate that HIP-1 can drive hypoxia-mediated gene expression in bacteria which have colonized human tumor xenografts in mouse models. Expression of both GFP and RFP under control of HIP-1 demonstrated an approximately 15-fold increase relative to a constitutive promoter when tumors were made hypoxic. Moreover, the use of a constitutive promoter resulted in reporter gene expression in both tumors and normal tissues, whereas reporter gene expressing using HIP-1 was confined to the tumor.