RESUMEN
Epigenetic modification is an important process during hematopoietic cell differentiation. Histone deacetylase (HDAC) inhibitors have previously been shown to enhance expansion of umbilical cord blood-derived hematopoietic stem cells (HSCs). However, the effect of HDAC inhibitors on pluripotent stem cells (PSCs) in this context is less understood. For years, investigators have considered PSC-derived natural killer (NK) and T-cell therapies. These "off-the-shelf" cellular therapies are now entering the clinic. However, the in vitro commitment of PSCs to the hematopoietic lineage is inefficient and represents a major bottleneck. We investigated whether HDAC inhibitors (HDACi) influence human PSC differentiation into CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), focusing on hemogenic endothelium (HE). Pluripotent stem cells cultured in the presence of HDACi showed a 2-5 times increase in HSPCs. Concurrent with this, HDACi-treated PSCs increased expression of 7 transcription factors (HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1, and LCOR) recently shown to convert HE to HSPCs. ChIP-qPCR showed that SAHA upregulated acetylated-H3 at the promoter region of the above key genes. SAHA-treated human PSC-derived CD34+CD45+ cells showed primary engraftment in immunodeficient mice, but not serial transplantation. We further demonstrate that SAHA-derived HSPCs could differentiate into functional NK cells in vitro. The addition of SAHA is an easy and effective approach to overcoming the bottleneck in the transition from PSC to HSPCs for "off-the-shelf" cellular immunotherapy.
Asunto(s)
Hemangioblastos , Trasplante de Células Madre Hematopoyéticas , Células Madre Pluripotentes , Animales , Antígenos CD34/metabolismo , Diferenciación Celular , Células Cultivadas , Hemangioblastos/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , RatonesRESUMEN
Suberoylanilide hydroxamic acid (SAHA) is one of the most widely used histone deacetylase inhibitors. However, the potential advantage of SAHA has not been sufficiently validated as an adjunct to gene therapy of head and neck squamous cell carcinoma (HNSCC). SAHA has been shown to boost the efficiency of gene transfer by upregulating the expression of coxsackie adenoviral receptor on treated cells. The p53 family genes, p63 and p73, have been shown to have characteristics similar to p53, and although they are not confirmed as tumor suppressors, DNA-damaging signals induce their overexpression. We previously reported that the adenovirus-mediated transfer of p63 or p73 showed an effective cancer-killing effect similar to that of p53. In this study, we combined SAHA with adenoviral delivery of p63 or p73 to enhance the efficiency of gene therapy. This combination resulted in a significantly enhanced cancer-killing effect in HNSCC cell lines but had no effect on normal human fibroblasts. SAHA treatment added to ad-p63/p73 gene delivery caused an increase in p21 expression and cleaved poly-ADP ribose polymerase. Our results indicate that adjuvant SAHA treatment could be developed as a therapeutic strategy to enhance the efficiency of adenoviral gene transfer in the treatment of cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Proteínas de Unión al ADN/genética , Terapia Genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Ácidos Hidroxámicos/uso terapéutico , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Adenoviridae/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/uso terapéutico , Quimioterapia Combinada , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Luciferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/uso terapéutico , Proteínas Nucleares/metabolismo , Proteínas Nucleares/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptores Virales/metabolismo , Resultado del Tratamiento , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/uso terapéutico , VorinostatRESUMEN
Glucocorticoids (GCs) modulate the synthesis of many pro-inflammatory cytokines and influence multiple transduction pathways. GCs negatively or positively influence the transcription factors of their target genes. All of these transcription signals are closely connected to cancer survival or death. We investigated the action of dexamethasone (DEX) on head and neck cancer cell lines. When SNU-1041 and SNU-1076 were treated with DEX, the cell lines showed different patterns of responses. DEX inhibition of cell growth depended on concentration in SNU-1041, but not in SNU-1076. Furthermore, DEX suppressed vascular endothelial growth factor (VEGF) secretion from SNU-1041, but not from SNU-1076. We explored the mechanism that explains these distinct differences. After DEX treatment, the differences of NF-kappaB (p65), glucocorticoid receptor and p-AKT were not observed between the cell lines. However, phospho-signal transducer and activator of transcription 3 (STAT3) decreased in SNU-1041 only. Moreover, STAT3 inhibition using si-RNA suppressed VEGF secretion. When STAT3 was overexpressed after DEX treatment, the level of VEGF in the culture media was restored. Taken together, we suggest that p-STAT3 can be a mediating factor which regulates VEGF secretion in the DEX treatment. Because the relationship between the three molecules DEX, STAT3 and VEGF is scarcely known, our findings clarified one of the signaling pathways of DEX, which is often used in clinical conditions.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Neoplasias de Cabeza y Cuello/metabolismo , Factor de Transcripción STAT3/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de Cabeza y Cuello/genética , Humanos , ARN Interferente Pequeño , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
A major obstacle to the success of gene therapy strategies that directly target cancer cells is the low gene transfer rate. To address this problem, we had previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 (Delta24RGD), and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Suicide/pro-drug gene therapy has an important additional benefit to the therapy of cancer. This relates to the transfer and expression of non-mammalian genes encoding enzymes that convert non-toxic pro-drugs into cellular toxins. We investigated the interaction between CRAD (Delta24RGD) and a replication-defective E1-deleted adenovirus (ad-HSTK) containing a suicide gene (HSTK: herpes simplex virus thymidine kinase gene) with respect to therapeutic gene production and tumor cell killing efficacy. Combined transduction of CRAD and ad-HSTK increased the transduction efficiency of HSTK and increased its sensitivity to ganciclovir (GCV) more efficiently than ad-HSTK alone. Transfer of medium of CRAD and ad-HSTK co-transduced cells induced the transfer of HSTK (media transferable bystander effect), and enhanced its sensitivity to GCV. In an animal tumor model, combined intratumoral injection of CRAD and ad-HSTK followed by GCV administration induced prolonged expression of HSTK and stronger growth suppression of established lung cancer xenografts than single injections. These data demonstrate that the selective replication of ad-HSTK due to the presence of mutant E1, produced by a Delta24RGD and HSTK/GCV suicide gene system, resulted in a striking improvement in anti-tumor effects in vitro and in vivo.
Asunto(s)
Adenoviridae , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Viroterapia Oncolítica/métodos , Simplexvirus , Timidina Quinasa , Replicación Viral/fisiología , Adenoviridae/enzimología , Adenoviridae/fisiología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Simplexvirus/enzimología , Simplexvirus/fisiología , Timidina Quinasa/genética , Timidina Quinasa/metabolismoRESUMEN
To overcome the low efficiency of gene therapy, we combined a conditionally replicating adenovirus (CRAd) and an adenoviral vector with a therapeutic gene. CRAd has an oncolytic activity in cancer cells with abnormal Rb activity and helps the replication of therapeutic genes incorporated in the E1-deleted adenovirus. We investigated the anticancer effect of a combination of CRAd and adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand (ad-TRAIL). We expected to see increased gene expression in cancer cells as well as an antitumor effect. With the combined application of CRAd and ad-luciferase in head and neck cancer cell lines, we observed considerably increased luciferase activity that was 10- to 50-fold greater than with ad-luciferase alone. The combination of CRAd and ad-TRAIL showed significant suppression of growth in cell lines and increased the sub-G(1) portion of cells 30-fold compared to any single treatment. The expression of TRAIL was highly amplified by the combined treatment and was accompanied by expression of molecules related to apoptosis. In a xenograft animal model, mice treated with CRAd and ad-TRAIL showed complete regression of established tumors, whereas mice treated with CRAd or ad-TRAIL alone did not. In conclusion, this combined strategy using CRAd and adenovirus carrying a therapeutic gene increased the gene transfer rate and enhanced antitumor effects. We expect that this combination strategy could be extended to a multitarget cancer gene therapy by combining multiple adenoviruses and CRAd.
Asunto(s)
Adenoviridae/fisiología , Proteínas E1 de Adenovirus/fisiología , Carcinoma de Células Escamosas/terapia , Terapia Genética , Neoplasias de Cabeza y Cuello/terapia , Viroterapia Oncolítica , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Replicación Viral , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The family of signal transducers and activators of transcription (STAT) are transcription factors. Among them, STAT1 is associated with an apoptosis pathway, while STAT3 is associated with tumorigenicity in various cancer cells. In order to investigate the primary roles of STAT1 and STAT3 in head and neck squamous cell carcinoma (HNSCC), we blocked STAT3 with two JAK inhibitors: AG490 (JAK2-STAT3 pathway inhibitor) and JAK total inhibitor. When we inhibited STAT3 with AG490, significant cell death was observed. However, in the case of JAK kinase total inhibitor, no cell growth retardation was observed. We focused on the role of STAT1 in this phenomenon. Suppression of STAT1 by si-RNA resulted in increased cell survival. Furthermore, the growth inhibitory effect of AG490 was reduced by treatment with si-RNA of STAT1. These results reveal that STAT1 is required to promote the tumor killing effect of STAT3 inhibition in HNSCC.
Asunto(s)
Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Factor de Transcripción STAT1/fisiología , Factor de Transcripción STAT3/antagonistas & inhibidores , Tirfostinos/farmacología , Western Blotting , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , ARN Interferente Pequeño , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
We previously showed that nitric oxide (NO) induces overexpression of cyclooxygenase-2 (COX-2) and production of prostaglandin E(2) in cancer cells. Here, we investigated the mechanisms by which NO induces COX-2 expression in cancer cells. We found that the cAMP-response element (CRE) is a critical factor in NO-induced COX-2 expression in all cells tested. We found that in cancer cells, three transcription factors (TFs) - cAMP response element-binding protein (CREB), activating transcription factor-2 (ATF-2) and c-jun, bound the CRE in the COX-2 promoter, and their activities were increased by addition of the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP). NO-induced activation of soluble guanylate cyclase (sGC), p38 and c-Jun NH(2)-terminal kinase (JNK) upregulated the three TFs, leading to COX-2 overexpression. Addition of dibutyryl-cGMP (db-cGMP) induced COX-2 expression in a manner similar to SNAP; this induction was blocked by a p38 inhibitor (SB202190), but not by a JNK inhibitor (SP600125). NO-induced cGMP was found to activate CREB and ATF-2 in a p38, but not c-jun-dependent manner, while NO induced JNK in a cGMP-independent manner, leading to subsequent activation of c-jun and ATF-2. These results suggest that the low concentrations of endogenous NO present in cancer cell may induce the expression of many genes, including COX-2, which promotes the growth and survival of tumor cells.