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1.
Int J Oncol ; 36(1): 233-44, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19956852

RESUMO

Pancreatic cancer is an aggressive malignancy with a dismal prognosis. To improve treatment options new treatments, such as adenoviral (Ad) gene therapy are necessary. However, low expression of the coxsackie and adenovirus receptor (CAR) in pancreatic cancer cells (PC) limits the therapeutic efficacy of these vectors. The aim of this study was to improve transduction of PC by recombinant adenoviruses by inserting peptides into the HI loop that binds to receptors highly expressed on pancreatic cancer and were shown to target these carcinomas in vivo. We report the successful incorporation into the HI loop of peptide Tyr-Ser-Ala (YSA), a peptide ligand targeting the EphrinA2 (EphA2) receptor, and K237, a peptide targeting to the vascular endothelial growth factor receptor-II (VEGFRII). Subsequently, we showed that both peptides enhanced the transduction of a number of human PC lines that abundantly express the targeted receptor. Additional competition studies confirmed that the YSA peptide redirects Ad-YSA from CAR and specifically targets the EphA2 receptor. Due to this transduction efficiency of Ad-YSA is increased not only in human pancreatic cancer cell lines but more importantly also in pancreatic cancer resection specimens. Since the YSA peptide has been shown to specifically target pancreatic cancer in patients, it may be expected that Ad-YSA will also display increased tropism for this tumour.


Assuntos
Adenoviridae/metabolismo , Técnicas de Transferência de Genes , Neoplasias Pancreáticas/genética , Receptor EphA2/genética , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Peptídeos/química , Receptor EphA2/metabolismo , Proteínas Recombinantes/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
World J Gastroenterol ; 15(22): 2754-62, 2009 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-19522026

RESUMO

AIM: To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo. METHODS: YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor (CAR) and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS: Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude (nu/nu) mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION: Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.


Assuntos
Adenoviridae/genética , Vetores Genéticos , Neoplasias Pancreáticas/metabolismo , Receptor EphA2/metabolismo , Transdução Genética , Adenoviridae/metabolismo , Animais , Linhagem Celular Tumoral , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Receptor EphA2/genética
3.
Dig Surg ; 25(4): 278-92, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18635930

RESUMO

BACKGROUND: The prognosis of patients with pancreatic cancer is poor. This is mainly caused by the late diagnosis, the aggressive biology and the lack of effective treatment modalities. Adenoviral gene therapy has the potential to selectively treat both primary tumor and (micro)metastatic tissue. METHODS: This review provides an overview of what has been achieved so far in the field of adenoviral gene therapy for pancreatic cancer. RESULTS: Transductional targeting allows decreased toxicity due to vector dissemination to non-target cells and permits delivery with a lower viral dose. It can evade or diminish the immune response, which remains a major problem. Transcriptional targeting evolves quickly but essential drawbacks such as the lack of an efficient animal model delay clinical application. Few clinical trials utilizing adenoviruses have been performed in patients with pancreatic cancer today. Worldwide, only seven phase III trials are being performed investigating adenoviral vectors in cancer patients. CONCLUSION: A clear therapeutic effect of adenoviral gene therapy in pancreatic cancer has not yet been achieved, because the step from bench to bedside has encountered drawbacks. Combinations of the different targeting strategies and techniques to evade the immune system harbor the future for adenoviral gene therapy in patients with pancreatic cancer.


Assuntos
Adenocarcinoma/terapia , Adenoviridae/genética , Terapia Genética , Neoplasias Pancreáticas/terapia , Ensaios Clínicos Fase III como Assunto , Marcação de Genes , Genes Supressores de Tumor , Humanos , Transdução Genética , Resultado do Tratamento
4.
J Surg Res ; 140(1): 50-4, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17418868

RESUMO

PURPOSE: Adenoviral gene therapy could potentially play a role in the treatment of esophageal cancer and Barrett's esophagus. The adenoviruses can be categorized in different serotypes. The goal of the present study was to investigate the transduction efficacy of different adenoviral serotypes in different models of esophageal cancer and Barrett's esophagus. METHODS: Chimeras of the adenoviral serotype 5 backbone and fibers of serotypes 5, 16, 35, 40, and 50 were constructed with PCR technology. For esophageal cancer, cell lines were used originating from with adenocarcinoma and squamous cell carcinoma, respectively. Differentiating Caco-2 cells were used as an in vitro model for Barrett's esophagus. GFP was used as a reporter gene and transduction efficacy was assessed by flow cytometry. RESULTS: Overall transduction was rather efficient in the cancer cell lines. Especially serotype 16 and 50 exhibited an improved transduction compared with the other serotypes. In the Caco-2 cell lines, we observed a decreased transduction upon differentiation of the cells. All serotypes had a very limited transduction and no serotype had an additional value in this setting. CONCLUSIONS: Some serotypes could have an additional value in the development of gene therapy for esophageal cancer. Especially serotype 16 and 50 exhibited an improved transduction in esophageal cancer cells compared with the native serotype 5. In the setting of Barrett's esophagus, none of the serotypes had an improved potency as in differentiated intestinal cells all serotypes had a very limited transduction.


Assuntos
Adenoviridae/classificação , Adenoviridae/genética , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/terapia , Terapia Genética/métodos , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Esôfago de Barrett/patologia , Esôfago de Barrett/terapia , Células CACO-2 , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias Esofágicas/patologia , Humanos , Sorotipagem , Transdução Genética/métodos
5.
J Hepatol ; 44(1): 126-33, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16168519

RESUMO

BACKGROUND/AIMS: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector. METHODS: mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes. RESULTS: mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines. CONCLUSIONS: The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.


Assuntos
Adenoviridae/genética , Neoplasias dos Ductos Biliares/terapia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/terapia , Terapia Genética , Vetores Genéticos , Regiões Promotoras Genéticas , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Genes Reporter , Humanos , Técnicas In Vitro , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Transcrição Gênica , Resultado do Tratamento , Células Tumorais Cultivadas
6.
Cancer Gene Ther ; 12(9): 778-86, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15877083

RESUMO

Adenoviral gene therapy could potentially be used for treatment of patients with a Barrett's esophagus. In order to study the feasibility of this approach it is important to study adenoviral intestinal transduction both in vitro and in vivo. In the present study, we used differentiating Caco-2 cells, closed intestinal loops and a Barrett's esophagus rat model to test transduction of adenoviruses expressing green fluorescent protein. We observed a decreased adenoviral transduction from 18.6 to 2.3% in undifferentiated and differentiated Caco-2 cells, respectively. This could be improved by the use of the mucolytic agent N-acetylcysteine (NAC) and the polycation diethylaminoethyl-dextran (DEAE-dextran), which improved transduction in differentiated cells five- and ten-fold, respectively. Also an RGD-retargeted adenovirus showed an improved transduction in differentiated cells. In closed intestinal loops adenoviral transduction was limited and the use of NAC and DEAE-dextran or RGD targeting had little effect. The Barrett's esophagus rat model consisted of an esophagojejunostomy, which results in a Barrett's esophagus and esophageal tumors within 6 months. Adenoviral transduction in this model was limited and mainly localized in the basal layer of normal esophagus and stromal tissue of a Barrett's segment. We conclude that although the adenovirus shows promising results in vitro, the current adenoviral vectors are probably not suitable for patients with Barrett's esophagus.


Assuntos
Adenoviridae/genética , Esôfago de Barrett/terapia , Terapia Genética , Transdução Genética/métodos , Acetilcisteína/farmacologia , Animais , Células CACO-2 , DEAE-Dextrano/farmacologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Mucosa Intestinal/metabolismo , Ratos
7.
Cancer Gene Ther ; 11(4): 289-96, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14765131

RESUMO

Adenoviral gene therapy might be a promising therapeutic strategy for esophageal carcinoma. However, adenoviral transduction efficacy in vivo is still limited. This efficacy can be improved by the insertion of an Arg-Gly-Asp (RGD) peptide in the HI-loop of the viral fiber knob. Indeed in established esophageal cell lines, we observed an up to six-fold improved transduction using the RGD-targeted adenovirus. Established cell lines, however, are easily transformed and do not represent the more complex in vivo histology and anatomy. Therefore, we set up an esophageal explant model using esophageal biopsies from patients. Viability is a limiting factor for this system. Cultured squamous epithelium, intestinal metaplasia and squamous cell carcinoma had a sufficient viability to study adenoviral transduction. Viability of the cultured adenocarcinoma biopsies was poor. Adenoviral transduction in the explant model was poor and was localized in particular cells. The transduction of the nontargeted and RGD-targeted adenovirus was similar in localization and efficacy. In conclusion, we established an esophageal explant system to test the transduction of adenoviral vectors ex vivo. The transduction was limited and localized in specific cells. RGD-targeted adenovirus did not show an improved transduction in this system.


Assuntos
Adenoviridae/genética , Carcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Carcinoma/patologia , Carcinoma/terapia , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Imunoquímica , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Receptores Virais/análise , Transdução Genética/métodos
8.
J Mol Med (Berl) ; 82(1): 56-63, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14647920

RESUMO

Biliary tract cancer, or cholangiocarcinoma, has a poor prognosis. Resection is the only curative treatment, but only a minority of patients are eligible. Chemotherapy and gamma-irradiation are merely palliative, as they are unable to remove the malignancy completely. The chicken anemia virus-derived protein apoptin induces apoptosis in a wide range of human tumor cells and is not hindered by mutations inactivating p53 or by overexpression of Bcl-2, changes known to frustrate chemotherapy and radiation therapy. We examined whether apoptin kills human biliary tract cancer cells. Expression of apoptin by means of plasmids caused extensive cell death in three independent cholangiocarcinoma cell lines, CC-LP, CC-SW, and Mz-ChA-1, regardless of their oncogenic mutations, which included inactivated p16 and p53 and the disruption of the transforming growth factor beta signaling pathway. In vitro delivery of apoptin by an adenoviral vector completely eradicated cholangiocarcinoma cells. Moreover, coexpression of the broad-spectrum caspase inhibitor p35 with apoptin only delayed the induced cell death. Changes in nuclear morphology still occurred early after transfection, and nuclei eventually disintegrated, suggesting that apoptin-induced cell death in these cells is not blocked by mutations in either the initiation or execution phase of apoptosis. The efficient induction of cell death by apoptin in cholangiocarcinoma cell lines makes apoptin an attractive candidate for molecular therapy of biliary tract cancer.


Assuntos
Neoplasias do Sistema Biliar/metabolismo , Proteínas do Capsídeo/metabolismo , Morte Celular/fisiologia , Colangiocarcinoma/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Neoplasias do Sistema Biliar/patologia , Neoplasias do Sistema Biliar/terapia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/uso terapêutico , Inibidores de Caspase , Linhagem Celular Tumoral , Vírus da Anemia da Galinha/metabolismo , Colangiocarcinoma/patologia , Colangiocarcinoma/terapia , Vetores Genéticos , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/metabolismo
9.
Ann Surg ; 238(6): 815-24; discussion 825-6, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14631218

RESUMO

OBJECTIVE: To evaluate if an integrin-retargeted adenoviral vector could establish a more efficient and tumor-specific gene transfer in esophageal carcinoma cells. SUMMARY BACKGROUND DATA: Although preclinical data indicated that adenoviral gene therapy could be a promising novel treatment modality for various malignancies, clinical results are often disappointing. An important problem is the decreased tumoral expression of the Coxsackie and adenovirus receptor (CAR), which mediates adenoviral entry. Retargeting the adenoviral vector to other cellular receptors, by inserting an arginine-glycine-aspartate (RGD) tripeptide in the fiber knob, might overcome this problem. METHODS: Four esophageal carcinoma cell lines and 10 fresh surgical resection specimens were cultured. All were infected with the native adenovirus (Ad) and the retargeted adenovirus (AdRGD), encoding for the reporter genes luciferase or Green Fluorescent Protein to analyze gene transfer efficiency. RESULTS: In all cell lines, an increase in viral expression per cell and an increase in the percentage of transduced cells were seen with the retargeted adenovirus. Also, in the primary cultures of carcinoma cells, a more efficient gene transfer was seen when the retargeted vector was used. This phenomenon was less pronounced in normal cells, indicating that the RGD virus transduces tumor cells more efficiently than normal cells. CONCLUSIONS: This study demonstrates that an RGD retargeted adenovirus infects human esophageal carcinoma cells with enhanced efficiency, while in normal esophageal cells this effect is less pronounced. Therefore, this retargeted vector is expected to have a better performance in vivo, when compared with nonretargeted vectors used for cancer gene therapy so far.


Assuntos
Adenocarcinoma/terapia , Adenoviridae , Neoplasias Esofágicas/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Linhagem Celular Tumoral , Técnicas de Cultura , Humanos
10.
J Mol Med (Berl) ; 81(9): 558-65, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12879152

RESUMO

The herpes simplex virus protein VP22 has the intriguing ability to deliver proteins from an expressing cell to neighboring cells. Fusion of VP22 to Apoptin, a protein that induces apoptosis in tumor cells but not in normal cells, might enhance the delivery of Apoptin. To analyze this hypothesis two fusion proteins of VP22 and full-length Apoptin were constructed, namely VP22-VP3 and VP3-VP22, and their apoptosis-inducing ability and intercellular spreading behavior were analyzed by transfection in tumor cells. While both of the Apoptin-VP22 fusion proteins retained the capacity to kill tumor cells, neither of them showed intercellular trafficking. To determine whether the presence of a nuclear localization signal in the C-terminus of Apoptin caused nuclear retention of the fusion protein and the subsequent lack of intercellular spreading, VP22 was fused to the biologically active N-terminal part (residues 1-69) of Apoptin (VP3n), which lacks the nuclear localization signal. However, analysis of the VP3n-VP22 fusion constructs gave no evidence of intercellular transport. A more careful inspection of different fractions of cell lysates expressing Apoptin with or without fusion to VP22 revealed that both the full-length Apoptin protein and its fusion with VP22 are insoluble. Despite the fact that VP3n was found to be soluble on its own, which could make it amenable to transport by VP22, the VP3n-VP22 fusion proteins were present exclusively in the insoluble fraction. We hypothesize that the N-terminal multimerization domain of Apoptin cooperates with VP22 to facilitate aggregation with cellular proteins, thereby inducing insolubility. From these results we conclude that, depending on the fusion partner, VP22 can have a negative effect on the solubility of fusion proteins, which consequently precludes intercellular trafficking. Such properties should be taken into account when establishing new VP22-mediated protein transduction systems.


Assuntos
Proteínas do Capsídeo/genética , Vetores Genéticos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Transporte Biológico , Células COS , Proteínas do Capsídeo/metabolismo , Técnicas de Transferência de Genes , Proteínas Recombinantes de Fusão/genética , Solubilidade , Proteínas Estruturais Virais/genética
11.
Biochem Biophys Res Commun ; 295(5): 1156-9, 2002 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-12135615

RESUMO

Mice lacking ApoA-V, a novel HDL-associated apolipoprotein identified by our group and independently by Pennacchio et al. [Science 294 (2001) 169], were recently shown to be hypertriglyceridemic. To study the role of ApoA-V in triglyceride homeostasis, we compared lipid profiles in mice expressing normal and highly elevated levels of ApoA-V. For this purpose, adenoviral vectors expressing sense or antisense ApoA-V cDNA were constructed. Treatment of mice with sense adenoviral constructs resulted in circa 20-fold higher serum ApoA-V levels compared with mice injected with either PBS or antisense adenoviral constructs. ApoA-V overexpressing mice had markedly decreased (-70%) serum triglyceride levels caused primarily by lowered triglyceride content of the VLDL fraction. Furthermore, in these mice cholesterol levels were found to be lowered in all lipoprotein fractions with the largest mass decrease in the HDL fraction. This resulted in a 40% drop of serum cholesterol content. These findings suggest a role of ApoA-V in regulating levels of circulating triglycerides and cholesterol.


Assuntos
Apolipoproteínas A/fisiologia , Apolipoproteínas , Colesterol/sangue , Homeostase/fisiologia , Triglicerídeos/sangue , Adenoviridae/genética , Animais , Apolipoproteína A-V , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Masculino , Camundongos , Modelos Animais
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