RESUMO
BACKGROUND: Lung cancer is the most common oncological cause of death in the Western world. Early diagnosis is critical for successful treatment. However, no effective screening methods exist. A promising approach could be the use of volatile organic compounds as diagnostic biomarkers. To date there are several studies, in which dogs were trained to discriminate cancer samples from controls. In this study we evaluated the abilities of specifically trained dogs to distinguish samples derived from lung cancer patients of various tumor stages from matched healthy controls. METHODS: This single center, double-blind clinical trial was approved by the local ethics committee, project no FF20/2016. The dog was conditioned with urine and breath samples of 36 cancer patients and 150 controls; afterwards, further 246 patients were included: 41 lung cancer patients comprising all stages and 205 healthy controls. From each patient two breath and urine samples were collected and shock frozen. Only samples from new subjects were presented to the dog during study phase randomized, double-blinded. This resulted in a specific conditioned reaction pointing to the cancer sample. RESULTS: Using a combination of urine and breath samples, the dog correctly predicted 40 out of 41 cancer samples, corresponding to an overall detection rate of cancer samples of 97.6% (95% CI [87.1, 99.9%]). Using urine samples only the dog achieved a detection rate of 87.8% (95% CI [73.8, 95.9%]). With breath samples, the dog correctly identified cancer in 32 of 41 samples, resulting in a detection rate of 78% (95% CI [62.4, 89.4%]). CONCLUSIONS: It is known from current literature that breath and urine samples carry VOCs pointing to cancer growth. We conclude that olfactory detection of lung cancer by specifically trained dogs is highly suggestive to be a simple and non-invasive tool to detect lung cancer. To translate this approach into practice further target compounds need to be identified.
Assuntos
Biomarcadores , Expiração , Neoplasias Pulmonares , Percepção Olfatória , Compostos Orgânicos Voláteis , Cães Trabalhadores , Animais , Cães , Humanos , Broncoscópios , Detecção Precoce de Câncer , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Testes de Função Respiratória , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/urina , Cães Trabalhadores/fisiologiaRESUMO
Background: Loss of LLGL1 has been associated with loss of cellular adhesion and dissemination of cells from colorectal cancer and malignant melanoma. Regulation and relevance of LLGL1 were analyzed in gastric cancer patients with lymphatic and distant dissemination. Furthermore, LLGL1 expression was analyzed in relation to the cellular adhesion protein E-cadherin. Methods: LLGL1 and E-cadherin transcription levels were evaluated in 56 gastric cancer patients and five gastric cancer cell lines. IHC staining for LLGL1 was performed on 39 gastric cancer specimens. LLGL1 was stably transfected into LLGL1 negative gastric cancer cell line SNU16 (del(17) (p11.2)) for functional in vitro assays and a xenograft bioassay. Results: Gastric cancer specimens and cell lines displayed LLGL1 and E-cadherin expression levels with variable intensity. In gastric mucosa, LLGL1 exhibited weak cytoplasmic and strong cortical staining. Loss of LLGL1 expression occurred in 65% of gastric cancers and significantly correlated with loss of E-cadherin expression (P=0.00009). Loss of LLGL1 expression was associated with the diffuse type of gastric cancer (P=0.029) with peritoneal carcinomatosis (M1; P=0.006) and with female gender (P=0.017). Stable reexpression of LLGL1 in SNU16 cells significantly increased both plastic surface adhesion and extracellular matrix proteins laminin and fibronectin, but had no impact on in vitro proliferation, apoptosis, or invasion or on in vivo proliferation or differentiation in our xenograft bioassay. Conclusion: LLGL1 is coexpressed with E-cadherin. Loss of expression of either protein is associated with diffuse gastric cancer and peritoneal metastases. LLGL1 does not impact on proliferation or epithelial-mesenchymal transition (EMT) rather increasing cellular adhesion.
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Antígenos CD/genética , Caderinas/genética , Proteínas do Citoesqueleto/genética , Neoplasias Peritoneais/patologia , Neoplasias Gástricas/patologia , Idoso , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Cetuximab-induced skin rash Gd3+ occurs in ≥16% patients (pts) (Heinemann et al., Lancet Oncol 15(10):1065-1075, 2014; Van Cutsem et al. J Clin Oncol 27(19):3117-25; 2009b). Survival, response, and toxicity parameters were re-evaluated under a pre-defined skin prophylaxis consistent of vitamin K1 ointment and oral doxycycline. METHODS: This is a national, multicenter, phase 4, first-line mCRC (K-RAS wt) trial. Pts received irinotecan 180 mg/m² (d1), FA 400 mg/m² (d1), 5-FU 400 mg/m² (d1), 5-FU 2400 mg/m² (d1-2), and cetuximab [400 mg/m² (d1), and then 250 mg/m² qw], prophylactic 0.1% vitamin K1 ointment qd, and oral doxycycline 100 mg bid. PRIMARY OBJECTIVE: 1-year PFS rate; secondary objectives: skin side-effects (grade, onset), objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) time, and overall survival (OS) time and safety. RESULTS: Twenty centers recruited 55 patients. Recruitment started Q1 2011 and ended Q3 2013 due to slow accrual. Characteristics were in line with CRYSTAL trial except for age and colonic location. 1-year PFS rate was 25.9%, mOS 21.8 months (m), and mPFS 8.5 m. ORR was 63.0%, DCR 77.8%. Rash Gd2+ occurred in 42.6% [median onset was 4.0 weeks (w)]; paronychia Gd2+ occurred in 22.2% (median onset 15.4w.); skin fissures Gd2+ occurred in 31.5% (median onset 19.9 weeks) 7% pts abandoned cetuximab treatment due to toxicity. CONCLUSION: Our data reveal encouraging improvements in skin reactions and their time to occurrence due to a pre-defined skin care.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/análogos & derivados , Cetuximab/administração & dosagem , Cetuximab/efeitos adversos , Neoplasias Colorretais/tratamento farmacológico , Toxidermias/prevenção & controle , Higiene da Pele/métodos , Adenocarcinoma/patologia , Administração Cutânea , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Quimioprevenção/métodos , Neoplasias Colorretais/patologia , Doxiciclina/administração & dosagem , Exantema/induzido quimicamente , Exantema/prevenção & controle , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Metástase Neoplásica , Pomadas , Resultado do Tratamento , Vitamina K 1/administração & dosagemRESUMO
MOTIVATION: Co-regulated genes are not identified in traditional microarray analyses, but may theoretically be closely functionally linked [guilt-by-association (GBA), guilt-by-profiling]. Thus, bioinformatics procedures for guilt-by-profiling/association analysis have yet to be applied to large-scale cancer biology. We analyzed 2158 full cancer transcriptomes from 163 diverse cancer entities in regard of their similarity of gene expression, using Pearson's correlation coefficient (CC). Subsequently, 428 highly co-regulated genes (|CC| ≥ 0.8) were clustered unsupervised to obtain small co-regulated networks. A major subnetwork containing 61 closely co-regulated genes showed highly significant enrichment of cancer bio-functions. All genes except kinesin family member 18B (KIF18B) and cell division cycle associated 3 (CDCA3) were of confirmed relevance for tumor biology. Therefore, we independently analyzed their differential regulation in multiple tumors and found severe deregulation in liver, breast, lung, ovarian and kidney cancers, thus proving our GBA hypothesis. Overexpression of KIF18B and CDCA3 in hepatoma cells and subsequent microarray analysis revealed significant deregulation of central cell cycle regulatory genes. Consistently, RT-PCR and proliferation assay confirmed the role of both genes in cell cycle progression. Finally, the prognostic significance of the identified KIF18B- and CDCA3-dependent predictors (P = 0.01, P = 0.04) was demonstrated in three independent HCC cohorts and several other tumors. In summary, we proved the efficacy of large-scale guilt-by-profiling/association strategies in oncology. We identified two novel oncogenes and functionally characterized them. The strong prognostic importance of downstream predictors for HCC and many other tumors indicates the clinical relevance of our findings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Cinesinas/genética , Neoplasias/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinogênese , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Progressão da Doença , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Cinesinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias/mortalidade , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoAssuntos
Anticorpos Monoclonais/efeitos adversos , Colite/induzido quimicamente , Toxidermias/etiologia , Melanoma/tratamento farmacológico , Infiltração de Neutrófilos , Pioderma Gangrenoso/induzido quimicamente , Neoplasias Cutâneas/tratamento farmacológico , Toxidermias/imunologia , Feminino , Humanos , Ipilimumab , Pessoa de Meia-IdadeRESUMO
BACKGROUND & AIMS: Therapeutic options for hepatocellular carcinoma (HCC) still remain limited. Development of gene targeted therapies is a promising option. A better understanding of the underlying molecular biology is gained in in vitro experiments. However, even with targeted manipulation of gene expression varying treatment responses were observed in diverse HCC cell lines. Therefore, information on gene expression profiles of various HCC cell lines may be crucial to experimental designs. To generate a publicly available database containing microarray expression profiles of diverse HCC cell lines. METHODS: Microarray data were analyzed using an individually scripted R program package. Data were stored in a PostgreSQL database with a PHP written web interface. Evaluation and comparison of individual cell line expression profiles are supported via public web interface. RESULTS: This database allows evaluation of gene expression profiles of 18 HCC cell lines and comparison of differential gene expression between multiple cell lines. Analysis of commonly regulated genes for signaling pathway enrichment and interactions demonstrates a liver tumor phenotype with enrichment of major cancer related KEGG signatures like 'cancer' and 'inflammatory response'. Further molecular associations of strong scientific interest, e.g. 'lipid metabolism', were also identified. CONCLUSIONS: We have generated CellMinerHCC (http://www.medicalgenomics.org/cellminerhcc), a publicly available database containing gene expression data of 18 HCC cell lines. This database will aid in the design of in vitro experiments in HCC research, because the genetic specificities of various HCC cell lines will be considered.
Assuntos
Carcinoma Hepatocelular/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Terapia Genética/métodos , Humanos , Internet , Neoplasias Hepáticas/metabolismo , Análise em Microsséries/métodos , Biologia de Sistemas/métodosRESUMO
Multiple activations of individual genes during embryonic liver and HCC development have repeatedly prompted speculations about conserved embryonic signatures driving cancer development. Recently, the emerging discussion on cancer stem cells and the appreciation that generally tumors may develop from progenitor cells of diverse stages of cellular differentiation has shed increasing light on the overlapping genetic signatures between embryonic liver development and HCC. However there is still a lack of systematic studies investigating this area. We therefore performed a comprehensive analysis of differentially regulated genetic signaling pathways in embryonic and liver cancer development and investigated their biological relevance.Genetic signaling pathways were investigated on several publically available genome wide microarray experiments on liver development and HCC. Differentially expressed genes were investigated for pathway enrichment or underrepresentation compared to KEGG annotated pathways by Fisher exact evaluation. The comparative analysis of enrichment and under representation of differentially regulated genes in liver development and HCC demonstrated a significant overlap between multiple pathways. Most strikingly we demonstrated a significant overlap not only in pathways expected to be relevant to both conditions such as cell cycle or apoptosis but also metabolic pathways associated with carbohydrate and lipid metabolism. Furthermore, we demonstrated the clinical significance of these findings as unsupervised clustering of HCC patients on the basis of these metabolic pathways displayed significant differences in survival.These results indicate that liver development and liver cancer share similar alterations in multiple genetic signaling pathways. Several pathways with markedly similar patterns of enrichment or underrepresentation of various regulated genes between liver development and HCC are of prognostic relevance in HCC. In particular, the metabolic pathways were identified as novel prognostically relevant players in HCC development.
Assuntos
Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fígado/embriologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Camundongos , Prognóstico , Transdução de SinaisRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood. OBJECTIVE: To characterise immune responses in the diethylnitrosamine (DEN)-liver cancer mouse model. DESIGN: Tumour development and immune cell functions upon DEN treatment were compared between C57BL/6 wild-type (WT), chemokine scavenging receptor D6-deficient, B cell- (Igh6), CD4 T cell- (MHC-II) and T-/B cell-deficient (Rag1) mice. Relevance for human HCC was tested by comparing gene array results from 139 HCC tissues. RESULTS: The induction of premalignant lesions after 24 weeks and of HCC-like tumours after 42 weeks by DEN in mice was accompanied by significant leucocyte infiltration in the liver and upregulation of distinct intrahepatic chemokines (CCL2, CCL5, CXCL9). Macrophages and CD8 (cytotoxic) T cells were most prominently enriched in tumour-bearing livers, similar to samples from human HCC. Myeloid-derived suppressor cells (MDSC) increased in extrahepatic compartments of DEN-treated mice (bone marrow, spleen). The contribution of immune cell subsets for DEN-induced hepatocarcinogenesis was functionally dissected. In D6(-/-) mice, which lack the chemokine scavenging receptor D6, hepatic macrophage infiltration was significantly increased, but tumour formation and progression did not differ from that of WT mice. In contrast, progression of hepatic tumours (numbers, diameters, tumour load) was strikingly enhanced in T-/B cell-deficient Rag1(-/-) mice upon DEN treatment. When mice deficient for B cells (Igh6(-/-), µMT) or major histocompatibility complex II were used, the data indicated that T cells prevent initial tumour formation, while B cells critically limit growth of established tumours. Accordingly, in tumour-bearing mice antibody production against liver-related model antigen was enhanced, indicating tumour-associated B cell activation. In agreement, T and B cell pathways were differentially regulated in gene array analyses from 139 human HCC tissues and significantly associated with patients' survival. CONCLUSIONS: Distinct axes of the adaptive immune system, which are also prognostic in human HCC, actively suppress DEN-induced hepatocarcinogenesis by controlling tumour formation and progression.
Assuntos
Imunidade Adaptativa , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Animais , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Carcinógenos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL9/metabolismo , Dietilnitrosamina , Progressão da Doença , Humanos , Leucócitos/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Análise de Sobrevida , Linfócitos T Citotóxicos/metabolismoRESUMO
BACKGROUND: Cancer cells are characterized by massive dysegulation of physiological cell functions with considerable disruption of transcriptional regulation. Genome-wide transcriptome profiling can be utilized for early detection and molecular classification of cancers. Accurate discrimination of functionally different tumor types may help to guide selection of targeted therapy in translational research. Concise grouping of tumor types in cancer maps according to their molecular profile may further be helpful for the development of new therapeutic modalities or open new avenues for already established therapies. METHODS: Complete available human tumor data of the Stanford Microarray Database was downloaded and filtered for relevance, adequacy and reliability. A total of 649 tumor samples from more than 1400 experiments and 58 different tissues were analyzed. Next, a method to score deregulation of KEGG pathway maps in different tumor entities was established, which was then used to convert hundreds of gene expression profiles into corresponding tumor-specific pathway activity profiles. Based on the latter, we defined a measure for functional similarity between tumor entities, which yielded to phylogeny of tumors. RESULTS: We provide a comprehensive, easy-to-interpret functional cancer map that characterizes tumor types with respect to their biological and functional behavior. Consistently, multiple pathways commonly associated with tumor progression were revealed as common features in the majority of the tumors. However, several pathways previously not linked to carcinogenesis were identified in multiple cancers suggesting an essential role of these pathways in cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'. CONCLUSION: The functional cancer map provides a systematic view on molecular similarities across different cancers by comparing tumors on the level of pathway activity. This work resulted in identification of novel superimposed functional pathways potentially linked to cancer biology. Therefore, our work may serve as a starting point for rationalizing combination of tumor therapeutics as well as for expanding the application of well-established targeted tumor therapies.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Linhagem Celular Tumoral , Genoma Humano , Humanos , FilogeniaRESUMO
Microarray studies have successfully shed light on various aspects of the molecular mechanisms behind the development of hepatocellular carcinoma (HCC), such as the identification of novel molecular subgroups and the genetic profiles associated with metastasis and venous invasion. These experiments, mainly comprising genome wide profiling, potentially represent the basis of novel targeted therapeutic strategies in HCC. In response, we summarize the multiple reported expression profiles in HCC associated with HCC development, novel subgroups, venous invasion and metastasis.
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BACKGROUND & AIMS: p73 belongs to the p53 family of transcription factors known to regulate cell cycle and apoptosis. The Trp73 gene has two promoters that drive the expression of two major p73 isoform subfamilies: TA and ΔN. In general, TAp73 isoforms show proapoptotic activities, whereas members of the N-terminally truncated (ΔN) p73 subfamily that lack the transactivation domain show antiapoptotic functions. We found that upregulation of ΔNp73 in hepatocellular carcinoma (HCC) correlated with reduced survival. Here, we investigated the molecular mechanisms accounting for the oncogenic role of ΔNp73 in HCC. RESULTS: ΔNp73ß can directly interfere with the transcriptional activation function of the TA (containing the transactivation domain) isoforms of the p53 family and consequently inhibit transactivation of proapoptotic target genes. Interference of ΔNp73ß with apoptosis-/chemosensitivity takes place at several levels of apoptosis signaling. ΔNp73ß negatively regulates the genes encoding for the death receptors CD95, TNF-R1, TRAIL-R2 and TNFRSF18. Furthermore, ΔNp73ß represses the genes encoding caspase-2, -3, -6, -8 and -9. Concomitantly, ΔNp73ß inhibits apoptosis emanating from mitochondria. CONCLUSIONS: Thus, ΔNp73 expression in HCC selects against both the death receptor and the mitochondrial apoptosis activity of the TA isoforms. Our data suggest that ΔNp73 isoforms repress apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways thereby contributing to chemoresistance. The clinical importance of these data is evidenced by our finding that the ΔNp73ß target gene signature can predict the prognosis of patients suffering from HCC.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/patologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Sequência de Bases , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Dominantes/genética , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Mitocôndrias/enzimologia , Modelos Biológicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Prognóstico , Isoformas de Proteínas/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Systems biology approaches offer novel insights into the development of chronic liver diseases. Current genomic databases supporting systems biology analyses are mostly based on microarray data. Although these data often cover genome wide expression, the validity of single microarray experiments remains questionable. However, for systems biology approaches addressing the interactions of molecular networks comprehensive but also highly validated data are necessary. RESULTS: We have therefore generated the first comprehensive database for published molecular associations in human liver diseases. It is based on PubMed published abstracts and aimed to close the gap between genome wide coverage of low validity from microarray data and individual highly validated data from PubMed. After an initial text mining process, the extracted abstracts were all manually validated to confirm content and potential genetic associations and may therefore be highly trusted. All data were stored in a publicly available database, Library of Molecular Associations http://www.medicalgenomics.org/databases/loma/news, currently holding approximately 1260 confirmed molecular associations for chronic liver diseases such as HCC, CCC, liver fibrosis, NASH/fatty liver disease, AIH, PBC, and PSC. We furthermore transformed these data into a powerful resource for molecular liver research by connecting them to multiple biomedical information resources. CONCLUSION: Together, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.
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Bases de Dados Genéticas , Hepatopatias/genética , Biologia Computacional , Mineração de Dados , Humanos , Interface Usuário-ComputadorRESUMO
We investigated the downstream mechanisms by which chemotherapeutic drugs elicit apoptosis in hepatocellular carcinoma (HCC). Genomic signatures of HCC cell lines treated with different chemotherapeutic drugs were obtained. Analyses of apoptosis pathways were performed and RNA interference was used to evaluate the role of the p53 family. Endogenous p53, p63 and p73 were upregulated in response to DNA damage by chemotherapeutic drugs. Blocking p53 family function led to chemoresistance in HCC. Stimulation and blocking experiments of the CD95-, the TNF- and the TRAIL-receptor systems revealed that cytotoxic drugs, via the p53 family members as transactivators, can trigger expression of each of these death receptors and consequently sensitize HCC cells toward apoptosis. Furthermore, our findings demonstrate a link between chemotherapy, the p53 family and the mitochondrial apoptosis pathway in HCC. Chemotherapeutic treatment induces expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of Apaf1, BNIP1, Pdcd8 and RAD. Thus, upon DNA damage, p53, p63 and p73 promote apoptosis via the extrinsic and the intrinsic signaling pathway. In addition, not only proapoptotic genes were upregulated, but also genes known to exert antiapoptotic functions. Bleomycin-induced upregulation of BCL-XL/BCLXL1 and MDM2 suggests that it is the ratio of proapoptotic and antiapoptotic proteins that regulates the apoptosis response of HCC cells toward chemotherapy, thereby playing a decisive role between treatment sensitivity vs. drug resistance. The clinical importance of these data is evidenced by our finding that the bleomycin target gene signature can predict the prognosis of patients suffering from HCC.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/fisiologia , Bleomicina/farmacologia , Carcinoma Hepatocelular/patologia , Caspases/fisiologia , Proteína de Domínio de Morte Associada a Fas/fisiologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
The completely assembled human genome has made it possible for modern medicine to step into an era rich in genetic information and high-throughput genomic analysis. These novel and readily available genetic resources and analytical tools may be the key to unravel the molecular basis of hepatocellular carcinoma (HCC). Moreover, since an efficient treatment for this disease is lacking, further understanding of the genetic background of HCC will be crucial in order to develop new therapies aimed at selected targets. We report on the current status and recent developments in HCC genetics. Special emphasis is given to the genetics and regulation of major signalling pathways involved in HCC such as p53, Wnt-signalling, TGFbeta, Ras, and Rb pathways. Furthermore, we describe the influence of chromosomal aberrations as well as of DNA methylation. Finally, we report on the rapidly developing field of genomic expression profiling in HCC, mainly by microarray analysis.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/fisiopatologia , Aberrações Cromossômicas , Metilação de DNA , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias Hepáticas/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Activation of the p53 network plays a central role in the inflammatory stress response associated with ulcerative colitis and may modulate cancer risk in patients afflicted with this chronic disease. Here, we describe the gene expression profiles associated with four microenvironmental components of the inflammatory response (NO*, H2O2, DNA replication arrest, and hypoxia) that result in p53 stabilization and activation. Isogenic HCT116 and HCT116 TP53-/- colon cancer cells were exposed to the NO* donor Sper/NO, H2O2, hypoxia, or hydroxyurea, and their mRNA was analyzed using oligonucleotide microarrays. Overall, 1,396 genes changed in a p53-dependent manner (P < 0.001), with the majority representing a "unique" profile for each condition. Only 14 genes were common to all four conditions. Included were eight known p53 target genes. Hierarchical sample clustering distinguished early (1 and 4 hours) from late responses (8, 12, and 24 hours), and each treatment was differentiated from the others. Overall, NO* and hypoxia stimulated similar transcriptional responses. Gene ontology analysis revealed cell cycle as a key feature of stress responses and confirmed the similarity between NO* and hypoxia. Cell cycle profiles analyzed by flow cytometry showed that NO* and hypoxia induced quiescent S-phase and G2-M arrest. Using a novel bioinformatic algorithm, we identified several putative p53-responsive elements among the genes induced in a p53-dependent manner, including four [KIAA0247, FLJ12484, p53CSV (HSPC132), and CNK (PLK3)] common to all exposures. In summary, the inflammatory stress response is a complex, integrated biological network in which p53 is a key molecular node regulating gene expression.
Assuntos
Inflamação/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular , Hipóxia Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Peróxido de Hidrogênio , Inflamação/etiologia , Inflamação/genética , Doadores de Óxido Nítrico , Óxidos de Nitrogênio , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermina/análogos & derivados , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Heat shock proteins (HSPs) have two unique roles as constituents of tumor vaccines: (i) to shuttle associated tumor antigens into professional antigen-presenting cells (APCs) and (ii) to activate professional APCs. Here we investigated the shuttle function of the HSP gp96 (glycoprotein 96) for a human melanoma peptide antigen MART-1 that was noncovalently bound to gp96 in vitro. This in vitro complexing reaction was optimized using the radioiodinated MART-1 peptide and human gp96. Up to 20% of gp96 molecules could bind the peptide, assuming a 1:1 molar ratio. The binding was temperature-dependent and thus reversible. At -20 degrees C, 95% of the peptide remained complexed after 24 h, but 25% and 60% of the peptide dissociated at 37 degrees C within 6 and 24 h, respectively. This observation suggests that under the physiological conditions in APCs, spontaneous peptide dissociation from gp96 complexes may facilitate the delivery of peptide antigen into antigen presentation pathways. The gp96/MART-1 complexes stimulated an HLA A2-restricted MART-1-specific CTL clone dependent on the amount of complexed peptide and the presence of HLA-A2-positive APCs. The reaction was peptide-specific and could be blocked by an excess of untreated native gp96. These results show for the first time that peptide antigens from in vitro reconstituted gp96/peptide antigen complexes can be cross-presented by human APCs. These findings extend the scientific basis for further evaluating the use of either endogenous or in vitro reconstituted gp96/tumor-antigen complexes as tumor vaccines.
Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Epitopos/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/isolamento & purificação , Linhagem Celular Transformada , Células Clonais , Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ativação Linfocitária , Antígeno MART-1 , Substâncias Macromoleculares , Proteínas de Neoplasias/metabolismo , TemperaturaRESUMO
Primary hepatocellular carcinoma (HCC) is one of the most common malignancies and has the fourth highest mortality rate worldwide. The major risk factors, including chronic infections with the hepatitis B or C virus, are exposure to dietary aflatoxin B1(AFB1), vinyl chloride, or alcohol consumption. Southern China and sub-Saharan Africa have the highest dietary AFB1 exposure, making it and hepatitis B virus (HBV) the major causes of cancer mortality in these geographic areas. Recent studies have discovered genetic and epigenetic changes involved in the molecular pathogenesis of HCC, including somatic mutations in the p53 tumor suppressor gene (TP53). AFB1 induces typical G:C to T:A transversions at the third base in codon 249 of p53. Chronic active hepatitis B and C (HCV) infection, and further inflammatory and oxyradical disorders including Wilson disease (WD) or hemochromatosis, generate reactive oxygen/nitrogen species that can damage DNA and mutate the p53 gene. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC. The integrated HBx is frequently mutated and has a diminished ability to function as a transcriptional cotransactivator and to activate the NF-kappa B pathway. However, the mutant HBx proteins still retain their ability to bind to and abrogate p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology and molecular pathogenesis of HCC. The resultant molecular changes in the ras and Wnt signal-transduction pathways, and the p53 and Rb tumor suppressor pathways significantly contribute to liver carcinogenesis
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína Supressora de Tumor p53/genética , Apoptose/fisiologia , Ciclo Celular/fisiologia , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteína Supressora de Tumor p53/fisiologiaRESUMO
One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.