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Distant metastasis and local recurrence are still the major causes for failure of treatment in patients with nasopharyngeal carcinoma (NPC), making it urgent to further elicit the molecular mechanisms of NPC metastasis. Using a gene microarray including transcription factors and known markers for cancer stem cells, prostate stem cell antigen (PSCA) was found to be significantly down-regulated in metastatic NPC in lymph node, compared to its primary tumour, and in NPC cell lines with high metastatic ability compared to those with low metastatic ability. NPC patients with low PSCA expression had a consistently poor metastasis-free survival (p = 0.003). Knockdown and overexpression of PSCA respectively enhanced and impaired the migration and invasion in vitro and the lung metastasis in vivo of NPC cells. Mechanistically, the enhancement of NPC metastasis by knocking down PSCA probably involved epithelial-mesenchymal transition (EMT), by up-regulating N-cadherin and ZEB1/2 and by activating RhoA. The down-regulation of PSCA in NPC cells resulted directly from the binding of Slug to the PSCA promoter. PSCA may be a potential diagnostic marker and therapeutic target for patients with NPC.
Assuntos
Antígenos de Neoplasias/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/metabolismo , Animais , Biomarcadores Tumorais/análise , Western Blotting , Carcinoma , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação para Baixo , Feminino , Proteínas Ligadas por GPI/biossíntese , Xenoenxertos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/mortalidade , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família SnailRESUMO
Background: Tamarix chinensis Lour. is a 3-6-meter-tall small tree with high salt- and alkali- tolerance and aggressive invasiveness, mainly distributed in the eastern part of China in warm-temperate and subtropical climate zones, yet there is little information available regarding genetic diversity and population structure. Methods: A total of 204 individuals of nine T. chinensis populations were investigated for genetic diversity and population structure using a set of 12 highly polymorphic microsatellite markers. Results: The total number of alleles detected was 162, the average number of effective allele was 4.607, the average polymorphism information content (PIC) value of the 12 loci was 0.685, and the mean observed heterozygosity (Ho) and the mean expected heterozygosity (He) was 0.653 and 0.711, respectively. Analysis of molecular variance (AMOVA) showed a 5.32% genetic variation among T. chinensis populations. Despite a low population differentiation, Bayesian clustering analysis, discriminant analysis of principal components (DAPC) and the unweighted pair group method with arithmetic mean (UPGMA) clearly identified three genetic clusters correlated to the populations' geographic origin: the northern populations including those from Yellow River Delta, the Fangshan (FS) population from Beijing, the Changyi (CY) population from Bohai Bay, the Huanjiabu (HHJ) population from Hangzhou Bay, and the remaining two populations from Hangzhou Bay. There was a significant relationship between the genetic distance and geographical distance of the paired populations. Gene flow (Nm) was 4.254 estimated from FST. Conclusion: T. chinensis possessed high genetic diversity comparable to tree species, and although the population differentiation is shallow, our results classified the sampled populations according to sampling localities, suggesting the different origins of the study populations.
Assuntos
Tamaricaceae , Humanos , Teorema de Bayes , Tamaricaceae/genética , Repetições de Microssatélites/genética , Agressão , Variação Genética/genéticaRESUMO
Background: To analyze the relationship between V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) status and radioresistance in non-small cell lung cancer (NSCLC), we identified potential genotypic differences and pathways involved. Methods: We retrospectively analyzed epidermal growth factor receptor (EGFR) and KRAS status in patients undergoing definitive radiotherapy for NSCLC between 2004 and 2018. Cox proportional hazard models were used to evaluate local progression-free survival (LPFS). Using clonogenic survival and measurement of γH2AX foci, we analyzed the difference in radiosensitivity between NSCLC cell lines with different KRAS status. The Cancer Genome Atlas (TCGA) analysis was used to explore the potential pathways involved. Results: The results showed that of the 286 patients identified, 68 (24%) had local tumor progression (mean ± standard deviation (SD), 27 ± 17.4 months); of these patients, KRAS mutations were found in 14 (23%), and KRAS status was associated with LPFS. After adjusting for concurrent chemotherapy, gross tumor volume, and mutation status in multivariate analysis, KRAS mutation was associated with shorter LPFS (hazard ratio: 1.961; 95% confidence interval: 1.03 - 2.17; P = 0.032). KRAS mutation showed higher radioresistance in vitro. TCGA data showed that the ERK1/2 pathway, phosphatidylinositol I3 kinase (PI3K)/mTOR, p38 MAPK pathway, cell cycle checkpoint signaling, DNA damage, repair pathways, and EGFR/PKC/AKT pathway were differentially expressed in patients with KRAS mutations or cell lines compared with their expression in the wild-type group. Conclusions: Diverse analyses identified that KRAS mutation was associated with radioresistance in NSCLC. KRAS mutation status may be helpful as a biomarker of radioresistance and a potential target to increase radiosensitivity.
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S-phase kinase-associated protein 2 (Skp2), which plays a role in cell cycle regulation, is commonly overexpressed in a variety of human cancers and associated with poor prognosis. However, its role in nasopharyngeal carcinoma (NPC) is not well understood. In this study, we examined the clinical significance of Skp2, with a particular emphasis on overall survival (OS) and disease-free survival (DFS), in NPC cases in South China, where NPC is an epidemic. Additionally, we explored the function of Skp2 in maintaining a cancer stem cell-like phenotype in NPC cell lines. Skp2 expression was assessed for 127 NPC patients using tissue microarrays and immunohistochemistry and analyzed together with clinicopathologic features, OS, and DFS. Skp2 expression was detectable, or positive, in 75.6% of patients. Although there was no correlation between Skp2 and any clinicopathologic factor, Skp2 expression significantly portended inferior OS (P = 0.013) and DFS (P = 0.012). In the multivariate model, Skp2 expression remained significantly predictive of poor OS [P = 0.009, risk ratio (RR) = 4.06] and DFS (P = 0.008, RR = 3.56), and this was also true for clinical stage (P = 0.012 and RR=3.201 for OS; P = 0.002 and RR=1.94 for DFS) and sex (P = 0.016 and RR=0.31 for OS; P = 0.006 and RR = 0.27 for DFS). After Skp2 knockdown, a colony formation assay was used to evaluate the self-renewal property of stem-like cells in the NPC cell lines CNE-1 and CNE-2. The colony formation efficiency in CNE-1 and CNE-2 cells was decreased. In Skp2-transfected CNE-1 and CNE-2 cells, side population (SP) proportion was increased as detected by flow cytometry. Skp2 is an independent prognostic marker for OS and DFS in NPC. Skp2 may play a role in maintaining the cancer stem cell-like phenotype of NPC cell lines.
Assuntos
Neoplasias Nasofaríngeas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno , Proteínas Quinases Associadas a Fase S/metabolismo , Adolescente , Adulto , Idoso , Carcinoma , Linhagem Celular Tumoral , China , Intervalo Livre de Doença , Feminino , Seguimentos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Estadiamento de Neoplasias , RNA Interferente Pequeno/genética , Proteínas Quinases Associadas a Fase S/genética , Fatores Sexuais , Taxa de Sobrevida , Análise Serial de Tecidos , Transfecção , Adulto JovemRESUMO
[This corrects the article on p. 2942 in vol. 7, PMID: 25031713.].
RESUMO
The endophytic bacterial diversity of rose was analyzed by high-throughput sequencing of 16S rDNA and functional prediction of the bacterial community. The number of bacterial sequence reads obtained from 18 rose samples ranged from 63,951 to 114,833, and reads were allocated to 1982 OTUs based on sequences of the V3-V4 region. The highest Shannon Index was found in Luogang rose (1.93), while the lowest was found in Grasse rose (0.35). The bacterial sequence reads were grouped into three different phyla: Firmicutes, Proteobacteria, and Actinobacteria. At the genus level, Bacillus and Staphylococcus had the highest abundance across all 18 samples; Bacillus was particularly abundant in Daguo rose (99.09%), Rosa damascena (99.65%), and Fenghua rose (99.58%). Unclassified OTUs were also found in all samples. PICRUSt gene prediction revealed that each endophyte sample contained multiple KEGG functional modules related to human metabolism and health. A high abundance of functional genes were involved in (1) Amino Acid Metabolism, (2) Carbohydrate Metabolism, (3) Cellular Processes and Signaling, (4) Energy Metabolism, and (5) Membrane Transport, indicating that the endophytic community comprised a wide variety of microorganisms and genes that could be used for further studies. The rose endophytic bacterial community is rich in diversity; community composition varies among roses and contains functional information related to human health.
Assuntos
Bactérias/genética , Endófitos/genética , Rosa/microbiologia , Biodiversidade , China , Análise por Conglomerados , DNA Bacteriano/genética , DNA Ribossômico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , RNA Ribossômico 16S/genética , Rosa/genética , Análise de Sequência de DNARESUMO
Nasopharyngeal carcinoma is a disease with a remarkable geographic and ethnic distribution, and has a high incidence in southern China. Infection with Epstein-Barr virus (EBV) is an important contributing factor. The profile of EBV strains in Cantonese patients from Guangdong, the nasopharyngeal carcinoma endemic region in southern China, is described on the sequence variations in latent membrane protein 1 carboxyl-terminus. The results show that China 1 was the dominant EBV strain detected in both the tumor biopsies and samples of throat washings, whereas multiple strains, including China 1, China 2, B95-8, and Med, were detected in blood samples. In addition, a new strain named China 4 was found in blood samples. These findings suggest that the host population is susceptible to the predominant China 1 strain in the nasopharyngeal carcinoma endemic region of China, but its relationship with the host remains to be characterized further.
Assuntos
Doenças Endêmicas , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/classificação , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virologia , Sangue/virologia , China/epidemiologia , DNA Viral/genética , Infecções por Vírus Epstein-Barr/epidemiologia , Genótipo , Herpesvirus Humano 4/isolamento & purificação , Humanos , Neoplasias Nasofaríngeas/epidemiologia , Faringe/virologia , Análise de Sequência de DNA , Proteínas da Matriz Viral/genéticaRESUMO
Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma-associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism -32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The -32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this -32A variant creates an activator protein (AP-1)-binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the -32G/A genotype compared with nasopharyngeal carcinoma cells with the -32G/G genotype. Collectively, these results provide evidence that the -32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied.
Assuntos
Carcinoma/genética , Carcinoma/patologia , Predisposição Genética para Doença , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Fatores de Transcrição/genética , Éxons , Humanos , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
BACKGROUND: Bcl-3 as an oncoprotein is overexpressed in nasopharyngeal carcinoma (NPC). Nedd4 binding protein 2 (N4BP2), which is located in the NPC susceptibility locus, is a Bcl-3 binding protein. This study is aimed to explore the association between N4BP2 genetic polymorphism and the risk of NPC. METHODS: We performed a hospital-based case-control study, including 531 sporadic NPC and 480 cancer-free control subjects from southern China. PCR-sequencing was carried out on Exons, promoter region and nearby introns of the N4BP2 gene. The expression pattern of N4BP2 and Bcl-3 was also analyzed. RESULTS: We observed a statistically significant difference in haplotype blocks ATTA and GTTG between cases and controls. In addition, three novel SNPs were identified, two of which were in exons (loc123-e3l-snp2, position 39868005, A/G, Met171Val; RS17511668-SNP2, position 39926432, G/A, Glu118Lys), and one was in the intron6 (RS794001-SNP1, position 39944127, T/G). Moreover, N4BP2 was at higher levels in a majority of tumor tissues examined, relative to paired normal tissues. CONCLUSION: These data suggest that haplotype blocks ATTA and GTTG of N4BP2 is correlation with the risk of sporadic nasopharyngeal carcinoma in the Southern Chinese population and N4BP2 has a potential role in the development of NPC.
Assuntos
Povo Asiático/genética , Proteínas de Transporte/genética , Predisposição Genética para Doença , Haplótipos , Neoplasias Nasofaríngeas/epidemiologia , Neoplasias Nasofaríngeas/genética , Adulto , Proteína 3 do Linfoma de Células B , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , China/epidemiologia , Enzimas Reparadoras do DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) and the viral nuclear antigen 1 (EBNA1) plays a crucial role in viral latency. Three EBNA1 subtypes, P-ala, V-thr and V-val have been detected from healthy carriers in Guangzhou area. A close relation of V-val EBNA1 with NPC was suggested by its preference to infect NPC cells. We therefore investigated the functional difference among these three EBNA1 subtypes in human epithelial cell line. The three coding sequences of the EBNA1 subtypes were cloned into the pGFP-C2 vector, and transfected into 293 cells, respectively. Effect of EBNA1 expression on cell proliferation was examined. The maintenance activity and expression level of EBNA1-plasmid in 293 cells were evaluated by using GFP as a reporter. The expression of P-ala, V-thr or V-val EBNA1 had no effect on 293 cell growth, while the relative average intensity of fluorescence after 14-day selection in V-val-EBNA1/293 cells was statistically higher than P-ala-EBNA1/293 (P<0.05, t test). We suggest that V-val EBNA1 with the functional advantage compared with prototype shown in this study might contribute to the tumorigenesis of NPC by increasing the expression of itself or other viral or cellular genes.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Neoplasias Nasofaríngeas/virologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Antígenos Nucleares do Vírus Epstein-Barr/genética , Variação Genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Plasmídeos/genética , Isoformas de Proteínas , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Signaling pathways activated by the Toll-like receptor 4 (TLR4) involve the induction of anti-cancer immunity. While screening for nasopharyngeal carcinoma (NPC) susceptibility genes, we isolated TLR4 and found that the 3'-untranslated region (3'-UTR) of exon 4 contained two polymorphisms that may alter its translation efficiency, potentially leading to NPC. To test this hypothesis, we conducted a hospital-based case-controlled study on NPC patients and cancer-free controls. We determined that the variant allele 11350C and the 11350GC genotype were associated with a significantly increased risk for NPC. We also determined significant differences between the male gender group and the remaining patient cases and controls, and between subjects equal to or younger than 47 years old and the cases and controls. Secondly, we cloned the entire 3'-UTR into a luciferase reporter system, and compared the luciferase activities between the wild-type 3'-UTR construct (WILD) and a construct containing the 11350C variant (MUT). Both constructs caused lower reporter gene activities, as compared to the positive control pGL3-promoter plasmid. Sixty hours after the transfections, the MUT construct reduced the reporter gene activity by 40% compared to that of the WILD construct (P<0.05). Functional analyses of the 11350C variant suggested that the TLR4 3'-UTR is a potent regulator of gene expression, as the mutated TLR4 3'-UTR was associated with decreased mRNA stability, and may down-regulate TLR4 expression resulting in EBV metainfective antiviral immunologic deficits and a high risk of NPC.
Assuntos
Regiões 3' não Traduzidas/genética , Variação Genética , Neoplasias Nasofaríngeas/genética , Receptor 4 Toll-Like/genética , Sequência de Bases , Primers do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Masculino , Mutação , Neoplasias Nasofaríngeas/epidemiologia , Fatores de RiscoRESUMO
OBJECTIVE: To investigate the genetic polymorphism of CYP2F1 gene, a member of CYP450 gene family in the healthy population and the patients with nasopharyngeal carcinoma (NPC) of Guangdong province, and furthermore analyze the relationship between CYP2F1 genetic polymorphism and the risk of developing NPC. METHODS: By direct gene sequencing, all of 10 exons of CYP2F1 gene were detected in 40 peripheral blood specimens of patients with primary NPC. For the genetic polymorphism with high allelic frequency, mismatch PCR-RFLP technique was developed to identify the different frequency between 368 NPC cases and 344 cancer-free controls. RESULTS: There were totally 35 SNPs identified in all of 10 exons and exon-intron junctions of CYP2F1 gene from 40 NPC patients, which included 10 missense mutations and 1 frame shift mutation. The most important mutation was C insertion located in 15-16 bp, which caused the frame shift. The allelic frequency of C insertion was 25%. However, there was no significant difference found between 368 NPC cases and 344 controls in allelic frequency of 15-16 bp C insertion mutation (P>0.05). CONCLUSION: A lot of genetic polymorphism of CYP2F1 gene is found in Guangdong population of China. However, no single genetic polymorphism associated with the individual susceptibility to NPC can be identified. The cooperated operations with multiple genetic polymorphisms of one or more genes may be critical factors contributing to the development and progression of NPC.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Polimorfismo de Nucleotídeo Único , Povo Asiático/genética , Sequência de Bases , China , Família 2 do Citocromo P450 , Frequência do Gene , Predisposição Genética para Doença/genética , Humanos , Neoplasias Nasofaríngeas/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a spheroid formation assay and a xenograft tumor model. Mechanistically, KCTD12 suppresses CRC cell stemness markers, such as CD44, CD133 and CD29, by inhibiting the ERK pathway, as the ERK1/2 inhibitor U0126 abolishes the increase in expression of CRC cell stemness markers induced by the down-regulation of KCTD12. Indeed, a decreased level of KCTD12 is detected in CRC tissues compared with their adjacent normal tissues and is an independent prognostic factor for poor overall and disease free survival in patients with CRC (p = 0.007). Taken together, this report reveals that KCTD12 is a novel regulator of CRC cell stemness and may serve as a novel prognostic marker and therapeutic target for patients with CRC.
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Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Regulação para Baixo , Sistema de Sinalização das MAP Quinases , Células-Tronco Neoplásicas/patologia , Proteínas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Mesilato de Imatinib/farmacologia , Masculino , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , PrognósticoRESUMO
The role of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) inhibiting migration in hepatocellular carcinoma (HCC) remains unknown. Expression of EFEMP1 in HCC cell lines were quantified by western blotting and real-time PCR. The role of EFEMP1 in HCC cell migration was explored in vitro via siRNA and adding purified EFEMP1 protein. The associated molecule expression was detected by western blotting after downregulation of EFEMP1 and also tested by immunohistochemistry. Eight pairs of HCC non-HCC liver samples and 215 HCC samples were subjected to immunohistochemistry. EFEMP1 was highly expressed in 7,721 and HepG2 HCC cell lines while HuH7 HCC cell line expressed the lowest level of EFEMP1 compared with the others. Downregulating EFEMP1 by siRNA markedly increased the migration ability of HCC cells while adding purified EFEMP1 protein inhibited HCC cell migration. Downregulation of EFEMP1 increased the expression of ERK1/2, MMP2 and MMP9. Furthermore, U0126 (a highly selective and potent inhibitor of pERK1/2) could abrogate the migration ability enhanced by siRNA. Accordingly, MMP2 and MMP9 were inversely expressed with EFEMP1 expression by immunohistochemistry. EFEMP1 downregulated in HCC tissues, and lower EFEMP1 expression was significantly associated with HCC patients with ascites (P=0.050), vascular invasion (P=0.044), poorer differentiation (P=0.002) and higher clinical stage (P=0.003).
Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proteínas da Matriz Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Butadienos/farmacologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Nitrilas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
High frequency loss of 3p21.3 region is a common event in various kinds of tumors including nasopharyngeal carcinoma (NPC). RASSF1A has been identified as a putative tumor suppressor gene residing in this region. Chromosome alterations and epigenetic changes are commonly observed as mechanisms for inactivation of RASSF1A function. In this study, we applied the PCR-cloning-sequencing strategy to examine somatic mutations in RASSF1A in NPC tissues as compared with the sequences detected in the matched peripheral blood lymphocytes. Our results revealed a high incidence of RASSF1A mutation in primary tumor tissues of NPC. There are totally 35 mutations identified in 74% (17/23) of these NPC cases, including 30 transitions, three transversions and two deletions. Most of these mutations result in amino acid changes: three nonsense (stop codon) mutations, two-1 bp deletion (frameshift), 26 missense and the remaining four are synonymous (silent). No obvious 'hot-spot' mutations were observed in this study. A similarly high rate (74%) of promoter methylation of RASSF1A was also detected in the same group of NPC tissues, but no significant correlation between mutation and methylation was detected. Our results suggest various mechanisms involved in inactivation of RASSF1A function and indicate a critical role of RASSF1A in NPC development.
Assuntos
Carcinoma/patologia , Genes Supressores de Tumor , Mutação , Neoplasias Nasofaríngeas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/patologiaRESUMO
The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from--45 to 1469, designated Ad-S(N)), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S(N) once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.
Assuntos
Adenoviridae/metabolismo , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Glicoproteínas de Membrana/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/administração & dosagem , Adenoviridae/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Vetores Genéticos , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Testes de Neutralização , Ratos , Ratos Wistar , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus , Vacinação , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Apoptose/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células PC12 , Ratos , RoscovitinaRESUMO
PURPOSE: Aspirin has recently been reported to reduce both the incidence and the risk of metastasis in colon cancer. However, there is no evidence at the cellular levels or in the animal models for such an effect of aspirin on cancer metastasis. EXPERIMENTAL DESIGN: MTT assay, colony formation assay, and apoptosis assay were employed to analyze the effects of aspirin on the osteosarcoma cell viability in vitro. The NF-κB activity was measured by the NF-κB p65 luciferase reporter. Western blotting was used to analyze the proteins in cells. The migration and invasion abilities of osteosarcoma cells in vitro were measured by the Transwell assay. Xenograft-bearing mice were used to assess the roles of aspirin in both tumor growth and metastasis of osteosarcoma in vivo (n = 5-8 mice/group). An unpaired Student t test or ANOVA with the Bonferroni post hoc test were used for the statistical comparisons. RESULTS: Aspirin reduced cell viability in a dose- and time-dependent manner in osteosarcoma cell lines, and aspirin synergistically sensitized osteosarcoma cells to cisplatin (DDP) in vitro and in vivo (P < 0.001). Moreover, aspirin markedly repressed the migration and invasion of osteosarcoma cells in vitro (P < 0.001), and dramatically diminished the occurrence of osteosarcoma xenograft metastases to the lungs in vivo (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma migration, invasion, and metastasis through the NF-κB pathway. CONCLUSIONS: Aspirin suppresses both the growth and metastasis of osteosarcoma through the NF-κB pathway at the cellular level and in the animal models.
Assuntos
Aspirina/farmacologia , Neoplasias Ósseas/metabolismo , NF-kappa B/metabolismo , Osteossarcoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Metástase Neoplásica , Osteossarcoma/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms of NPC metastasis are poorly understood. Here, using our customized gene microarray containing all of the known human transcription factors and the current markers for epithelial-mesenchymal transition, we report that TEL2 was down-regulated in highly metastatic NPC cells and the metastatic tissues in lymph node. Mechanistically, TEL2 inhibits the cell migration and invasion in vitro and metastasis in vivo by directly suppressing the SERPINE1 promoter in NPC. Consistently, an inverse correlation was observed between the protein levels of TEL2 and SERPINE1 using clinical NPC samples. Collectively, we have provided the first evidence that TEL2 plays a key role in NPC metastasis by directly down-regulating SERPINE1, and that this novel axis of TEL2 / SERPINE1 may be valuable to develop new strategies for treating NPC patients with metastasis.