RESUMEN
We investigated the novel recombinant oncolytic adenovirus Ad-delo-sr39TK-RGD, armed with a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39TK) as a suicide gene, and explored its antitumor efficacy in combination with HSV1-sr39TK/ganciclovir (GCV) gene therapy and temozolomide (TMZ). Ad-delo-sr39TK-RGD is an E1-mutated conditionally replicating adenovirus dependent on the human Y-box binding protein 1 (YB-1). Thus, we utilized the YB-1 dependency of the vector to target human glioma cells in vitro, using two-dimensional cell culture and three-dimensional multicellular spheroids, and demonstrated the strong replication competence and oncolytic potential of the virus. The cytotoxicity mediated by HSV1-sr39TK and its prodrug GCV enhanced the oncolytic effect even at <0.1 µg ml(-1) GCV and induced cell killing of > 95% after adding GCV 0-1 days following infection. An increased bystander effect of viral replication and GCV in co-cultured infected and uninfected cells was observed. Co-administrating Ad-delo-sr39TK-RGD with TMZ and GCV, spheroid growth was reduced drastically. Gamma counting of infected spheroids demonstrated successful accumulation of the radiotracer (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine mediated by HSV1-sr39TK. Hence, our results show that the combination of YB-1-dependent virotherapy with suicide genes and TMZ effectively induces glioma cell killing and may allow for in vivo non-invasive imaging within a limited time frame.
Asunto(s)
Adenoviridae/genética , Antineoplásicos/farmacología , Genes Transgénicos Suicidas , Glioma/genética , Glioma/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Proteína 1 de Unión a la Caja Y/genética , Antineoplásicos/administración & dosificación , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular/genética , Efecto Citopatogénico Viral , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Glioma/mortalidad , Glioma/patología , Humanos , Esferoides Celulares , Temozolomida , Transducción Genética , Células Tumorales Cultivadas , Replicación ViralRESUMEN
BACKGROUND: Histology-based classifications and clinical parameters of head and neck squamous cell carcinoma (HNSCC) are limited in their clinical capacity to provide information on prognosis and treatment choice of HNSCC. The primary aim of this study was to analyse Y-box-binding protein-1 (YB-1) protein expression in different grading groups of HNSCC patients, and to correlate these findings with the disease-specific survival (DSS). METHODS: We investigated the expression and cellular localisation of the oncogenic transcription/translation factor YB-1 by immunohistochemistry on tissue micro arrays in a total of 365 HNSCC specimens and correlated expression data with clinico-pathological parameters including DSS. RESULTS: Compared with control tissue from healthy individuals, a significantly (P<0.01) increased YB-1 protein expression was observed in high-grade HNSCC patients. By univariate survival data analysis, HNSCC patients with elevated YB-1 protein expression had a significantly (P<0.01) decreased DSS. By multivariate Cox regression analysis, high YB-1 expression and nuclear localisation retained its significance as a statistically independent (P<0.002) prognostic marker for DSS. Within grade 2 group of HNSCC patients, a subgroup defined by high nuclear and cytoplasmic YB-1 levels (co-expression pattern) in the cells of the tumour invasion front had a significantly poorer 5-year DSS rate of only 38% compared with overall 55% for grade 2 patients. Vice versa, the DSS rate was markedly increased to 74% for grade 2 cancer patients with low YB-1 protein expression at the same localisation. CONCLUSION: Our findings point to the fact that YB-1 expression in combination with histological classification in a double stratification strategy is superior to classical grading in the prediction of tumour progression in HNSCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Sobrevivientes , Proteína 1 de Unión a la Caja Y/metabolismo , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Modelos de Riesgos ProporcionalesRESUMEN
We have earlier described the oncolytic adenovirus vector dl520 that was rendered cancer-specific by deletion of the transactivation domain CR3 of the adenoviral E1A13S protein; this deletion causes antitumor activity in drug-resistant cells displaying nuclear YB-1 expression. We hypothesized that the anticancer activity of dl520 could be further improved by introducing the RGD motif in the fiber knob and by deletion of the adenoviral E1B19K protein (Ad-Delo3-RGD). In this study, the in vitro and in vivo antitumor activity of Ad-Delo3-RGD was investigated focussing on two pancreatic cancer cell lines MiaPaCa-2 and BxPC3 alone and in combination with cytotoxic drugs. Furthermore, luciferin-based bioluminescence imaging was established to study the therapeutic response in vivo. In addition, to confirm the specificity of Ad-Delo3-RGD for YB-1 a tetracycline-inducible anti-YB-1 shRNA-expressing cell variant EPG85-257RDB/tetR/YB-1 was used. This TetON regulatable expression system allows us to measure adenoviral replication by real-time PCR in the absence of YB-1 expression. The results confirmed the YB-1 dependency of Ad-Delo3-RGD and showed that Ad-Delo3-RGD has potent activity against human pancreatic cancer cells in vitro and in vivo, which was augmented by the addition of paclitaxel. However, although high replication capacity was measured in vitro and in vivo, complete tumor regression was not achieved, indicating the need for further improvements to treat pancreatic cancer effectively.
Asunto(s)
Adenoviridae/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas Nucleares/biosíntesis , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Línea Celular Tumoral , Terapia Combinada , Proteínas de Unión al ADN/genética , Vectores Genéticos/genética , Células HeLa , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Nucleares/genética , Paclitaxel/farmacología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína 1 de Unión a la Caja YRESUMEN
Malignant melanoma displays strong resistance against various antineoplastic drugs. The mechanisms conferring this intrinsic resistance are unclear. To better understand the molecular events associated with drug resistance in melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.e., vindesine, etoposide, fotemustine and cisplatin, was characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Of 269 mRNA fragments found to be altered in expression level by DDRT-PCR, a total of 11 cDNA clones was characterized after confirmation of a differential expression pattern by Northern blot analyses. These clones include 3 genes (DSM-1, DSM-3 and DSM-5) of known function, 4 previously sequenced genes (DSM-2, DSM-4, DSM-6 and DSM-7) of uncharacterized function and 4 novel genes (DSM-8-DSM-11) without match in GenBank. All of these genes exhibited altered mRNA expression in high level etoposide-resistant cells, whereby 7 genes (DSM-1-DSM-6 and DSM-8) were found to be decreased in the transcription rate in these etoposide-resistant cells. The mRNA synthesis of the remaining genes (DSM-7 and DSM-9-DSM11) was enhanced in high level etoposide-resistant melanoma cells. The expression of 5 (DSM-5 and DSM-7-DSM-10) of the cloned cDNA encoding mRNAs was modulated in various independently established drug-resistant melanoma cells, indicating to be associated with drug resistance. Further characterization of these genes may yield inside into the biology and development of drug resistance in malignant melanoma.
Asunto(s)
Antineoplásicos/toxicidad , Resistencia a Múltiples Medicamentos , Etopósido/toxicidad , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Transcripción Genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Resistencia a Antineoplásicos , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
We report the distribution pattern of the target antigen of an IgG1 monoclonal antibody, Ki-OC III raised in BALB/C mice against solubilised ovarian adenocarcinoma, in normal tissue and in a collection of human tumour types. Special reference was made to benign and malignant ovarian tumours. The reactive antigen protein purified to homogeneity for a planned amino acid analysis showed three bands between 37-40 kDa. Immunohistochemical analysis revealed a specificity of 98% and a sensitivity of 77%. The tracer kinetics of the radiolabelled antibody were tested on a human ovarian carcinoma cell line in athymic mice. The results were compared to cell lines derived from breast and stomach carcinomas as well as to a human glioblastoma cell line. The results show the preferential uptake by ovarian cancer cells followed by breast cancer cell lines. In animal models, scintigraphic monitoring and direct measurement of the radio-labelled monoclonal antibody showed a preferential accumulation in tumours, in addition to high level signals in the liver, kidney, spleen and heart which were related to degradation, excretion and high circulation. The Ki-OC III reactive antigen could be a potential candidate for immunomonitoring of ovarian and possibly also of breast cancer, for in vivo tumour imaging as well as for histopathologic examinations.