RESUMO
BACKGROUND: DTL has been found to be related with multiple cancers. However, comprehensive analyses, which identify the prediction value of DTL in diagnosis, prognosis, immune infiltration and treatment, have rarely been reported so far. METHODS: Combined with the data online databases, the gene expression, gene mutation, function enrichment and the correlations with the immunity status and clinical indexes of DTL were analyzed. Expression of DTL and the degree of immune cell infiltration were examined by immunofluorescence (IF) and immunohistochemistry (IHC) and analyzed by statistical analysis. Furthermore, the influences of DTL on the cell cycle, cell proliferation and apoptosis were detected by live cell imaging, IF and flow cytometric (FC) analysis. Genomic stability assays were conducted by chromosome slide preparation. RESULTS: DTL was widely expressed in various cells and tissues, while it was overexpressed in tumor tissues except acute myeloid leukemia (LAML). Pan-cancer bioinformatics analysis showed that the expression of DTL was correlated with the prognosis, immunotherapy, and clinical indexes in various cancers. In addition, gene set enrichment analysis (GSEA) uncovered that DTL was enriched in oocyte meiosis, pyrimidine metabolism, the cell cycle, the G2M checkpoint, mTORC1 signaling and E2F targets. Furthermore, the overexpression of DTL, and its association with immune cell infiltration and clinical indexes in liver hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA) and stomach adenocarcinoma (STAD) were verified in our study. It was also verified that overexpression of DTL could regulate the cell cycle, promote cell proliferation and cause genomic instability in cultured cells, which may be the reason why DTL plays a role in the occurrence, progression and treatment of cancer. CONCLUSIONS: Collectively, this study suggested that DTL is of clinical value in the diagnosis, prognosis and treatment of various cancers, and may be a potential biomarker in certain cancers.
Assuntos
Carcinoma Hepatocelular , Carcinoma de Células de Transição , Neoplasias Hepáticas , Neoplasias da Bexiga Urinária , Humanos , Prognóstico , Biomarcadores , Imunoterapia , Proteínas NuclearesRESUMO
Chronic infection of hepatitis B virus (HBV) is associated with an increased incidence of hepatocellular carcinoma (HCC). HBV encodes an oncoprotein, hepatitis B x protein (HBx), that is crucial for viral replication and interferes with multiple cellular activities including gene expression, histone modifications, and genomic stability. To date, it remains unclear how disruption of these activities contributes to hepatocarcinogenesis. Here, we report that HBV exhibits antiresection activity by disrupting DNA end resection, thus impairing the initial steps of homologous recombination (HR). This antiresection activity occurs in primary human hepatocytes undergoing a natural viral infection-replication cycle as well as in cells with integrated HBV genomes. Among the seven HBV-encoded proteins, we identified HBx as the sole viral factor that inhibits resection. By disrupting an evolutionarily conserved Cullin4A-damage-specific DNA binding protein 1-RING type of E3 ligase, CRL4WDR70 , through its H-box, we show that HBx inhibits H2B monoubiquitylation at lysine 120 at double-strand breaks, thus reducing the efficiency of long-range resection. We further show that directly impairing H2B monoubiquitylation elicited tumorigenesis upon engraftment of deficient cells in athymic mice, confirming that the impairment of CRL4WDR70 function by HBx is sufficient to promote carcinogenesis. Finally, we demonstrate that lack of H2B monoubiquitylation is manifest in human HBV-associated HCC when compared with HBV-free HCC, implying corresponding defects of epigenetic regulation and end resection. Conclusion: The antiresection activity of HBx induces an HR defect and genomic instability and contributes to tumorigenesis of host hepatocytes.
Assuntos
Carcinoma Hepatocelular/virologia , Instabilidade Genômica/genética , Hepatite B/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Transativadores/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Epigênese Genética , Hepatite B/patologia , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Litostatina/genética , Neoplasias Hepáticas/patologia , Camundongos , Sensibilidade e Especificidade , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
BACKGROUND: Many diseases have been associated with intestinal microbial dysbiosis. Host-microbial interactions regulate immune function, which influences the development of gastric cancer. AIMS: The aims were to investigate the characteristics of intestinal microbiota composition in gastric cancer patients and correlations between the intestinal microbiota and cellular immunity. METHODS: Fecal samples were collected from 116 gastric cancer patients and 88 healthy controls from Shanxi Province, China. The intestinal microbiota was investigated by 16S rRNA gene sequencing. Peripheral blood samples were also collected from the 66 gastric cancer patients and 46 healthy controls. The populations of peripheral T lymphocyte subpopulations and NK cells were analyzed by flow cytometry. RESULTS: The intestinal microbiota in gastric cancer patients was characterized by increased species richness, decreased butyrate-producing bacteria, and the enrichment of other symbiotic bacteria, especially Lactobacillus, Escherichia, and Klebsiella. Lactobacillus and Lachnospira were key species in the network of gastric cancer-associated bacterial genera. The combination of the genera Lachnospira, Lactobacillus, Streptococcus, Veillonella, and Tyzzerella_3 showed good performance in distinguishing gastric cancer patients from healthy controls. There was no significant difference in enterotype distribution between healthy controls and gastric cancer patients. The percentage of CD3+ T cells was positively correlated with the abundance of Lactobacillus and Streptococcus, and CD3+ T cells, CD4+ T cells, and NK cells were associated with Lachnospiraceae taxa. CONCLUSIONS: Our study revealed a dysbiotic intestinal microbiota in gastric cancer patients. The abundance of some intestinal bacterial genera was correlated with the population of peripheral immune cells.
Assuntos
Microbioma Gastrointestinal/genética , Redes Reguladoras de Genes/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adulto , Idoso , China/epidemiologia , Fezes/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/microbiologiaRESUMO
As an effective antimalarial drug, Dihydroartemisinin (DHA) is readily isolated from the traditional Chinese medicine of Artemisia annua. DHA is not only an autophagy promoter but also a substance with strong antitumor efficiency. The relationship between autophagy and inflammasomes has been suggested in hepatocellular carcinoma (HCC). However, there are few reports describing relationships between inflammasomes and autophagy in HCC therapy. The present study demonstrated that DHA suppressed cell proliferation in HepG2215 cells in a dose- and time-dependent manner. The inhibitory activity is mediated by autophagy, in which reactive oxygen species (ROS) production induced nuclear and mitochondrial DNA damage. Then, DHA were first shown to promote AIM2/caspase-1 inflammasome. Compared with the DHA group, the autophagy inhibitor 3-MA significantly inhibited the expressions of activated Caspase-1, a pyroptotic marker proteins. Meanwhile, repression of mTOR by rapamycin promoted autophagy and AIM2/caspase-1 activation. The caspase-1 inhibitor Z-YVAD-FMK also notably blocked autophagy cell death characterized by the downexpression of Beclin-1 and LC3-II. Additionally, the study demonstrated that DHA suppressed pseudopodium formation and cell mobility. Therefore, we first reveal a novel mechanism that DHA promotes AIM2/caspase-1 inflammasome, which contributes to autophagy in HepG2215 cells. Moreover, nuclear and mitochondrial DNA damage was also involved in this process via ROS production.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Clorometilcetonas de Aminoácidos/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/metabolismo , Caspase 1/metabolismo , Caspases/metabolismo , Proteínas de Ligação a DNA , Células Hep G2 , Humanos , Inflamassomos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVES: To analyze the cellular function of the newly discovered DNA damage repair factor WDR70, and investigate the mutation in ovarian cancer to verify if function loss of the WDR70gene was associated with ovarian cancer. METHODS: The WDR70 gene was silenced by using siRNA technique or overexpressed its wild and mutation type by with lentivirus and plasmid in hunman cells. The subcellular localization and biochemical function of WDR70 was analyzes by indirect immunofluorescence and immunoblotting. The expression level of WDR70 and the mutations of its cDNA was checked with RT-PCR sequencing for 1 normal ovarian tissue and 16 ovarian cancer specimen. RESULTS: We found gene silencing of WDR70 or overexpression of WDR70 mutation type disrupts the phosphorylation level of homologous recombination functional protein RPA32 and the ability of recruitment at DNA damage site of recombinase RAD51, the loss of function of WDR70 also causes the elevation of the chromosome breakage in metaphase. Meanwhile, we also noticed that the existence of multiple mutations in genomic WDR70 in ovarian cancer specimen. CONCLUSIONS: Our results defined that in vitro system, WDR70 is a DNA damage repair gene, silencing of WDR70 or overexpression of WDR70 mutation type disrupts homologous recombination and chromosomal instability; the frequent mutations of WDR70 gene in genome of ovarian cancer specimens could also lead to DNA repair defeat and gene instability. Consequently WDR70 gene could represent an anti-cancer mechanism for ovarian cancer.
Assuntos
Dano ao DNA , Reparo do DNA , Neoplasias Ovarianas/genética , Feminino , Humanos , MutaçãoRESUMO
AIM: Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms. METHODS: Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy. RESULTS: Treatment of SMMC-7721 cells with Nutlin-3 (10 µmol/L) or etoposide (40 µmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells. CONCLUSION: Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.
Assuntos
Carcinoma Hepatocelular/metabolismo , Cromatina/metabolismo , Imidazóis/farmacologia , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , HumanosRESUMO
The purpose of this study was to investigate the effects of a long-term high-fructose diet on the insulin-signaling pathway of the hippocampus. Sprague-Dawley rats were fed either on a control (0% fructose solution) or high-fructose diet (10% fructose solution). Food intake and body mass were measured regularly. Eight months later, peripheral insulin sensitivity, the activity of the hippocampal insulin pathway, and memory tasks were assessed. Compared to the control group, the high fructose group exhibited more weight gain, peripheral insulin resistance, metabolic disorders, and memory impairments. In addition, insulin signaling in the hippocampus was attenuated in the high fructose group. These results suggested that a high-fructose diet induced peripheral insulin resistance and an abnormal insulin-signaling pathway in the hippocampus which exacerbated memory deficits in the rats.
Assuntos
Carboidratos da Dieta/efeitos adversos , Frutose/efeitos adversos , Hipocampo/efeitos dos fármacos , Resistência à Insulina , Transtornos da Memória/fisiopatologia , Animais , Peso Corporal , Carboidratos da Dieta/administração & dosagem , Frutose/administração & dosagem , Teste de Tolerância a Glucose , Hipocampo/metabolismo , Insulina/metabolismo , Masculino , Memória/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Aumento de PesoRESUMO
Hepatitis B virus (HBV) infection is a major public health problem by affecting 350 million people worldwide. The mechanisms that regulate HBV gene expression and viral replication remain poorly understood. HBx is known as the central regulator for HBV replication and is associated with the CUL4-DDB1 ubiquitin ligase through H-box motif. Here, we show that blocking the activity of DDB1 by RNA interfering inhibited viral production and gene expression of HBV, and direct association of HBx with DDB1 promoted viral activities, indicating that DDB1 function is required for viral production. On the other hand, HBx interfered with DDB1-dependent polyubiquitination of PRMT1, arginine methyltransferase 1, suggesting that HBx can also block the function of a subset of CUL4-DDB1 E3 ligases. Thus, we conclude that HBx regulates the function of DDB1 in both positive and negative manners in the context of distinct CUL4-DDB1 complexes and plays different roles in HBV replication cycle.
Assuntos
Proteínas Culina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Transativadores/fisiologia , Sequência de Bases , Linhagem Celular , Primers do DNA , Vírus da Hepatite B/fisiologia , Humanos , Proteína-Arginina N-Metiltransferases/fisiologia , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias , Replicação ViralRESUMO
Smu1 is an evolutionarily conserved gene that encodes a member of the WD40-repeat protein family. Disruption of Smu1 function leads to multiple cellular defects including chromosomal instability, aberrant DNA replication and alternative RNA splicing events. In this paper, we show that Smu1 is a chromatin-bound protein that functions as a negative regulator of DNA replication. Knockdown of Smu1 gene expression promotes excessive incorporation of dNTP analogue, implicating the acceleration of DNA synthesis. Smu1-silenced cells show an excessive activation of replication checkpoint in response to ultraviolate (UV) or hydroxyurea treatment, indicating that abnormal stimulation of DNA replication leads to instability of genomic structure. Hence, we propose that Smu1 participates in the protection of genomic integrity by negatively regulating the process of DNA synthesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células HEK293 , Humanos , Hidroxiureia/farmacologia , Microscopia de Fluorescência , Ligação Proteica , Interferência de RNA , Raios UltravioletaRESUMO
Secondary pneumonia due to Staphylococcus aureus (S. aureus) causes significant morbidity and mortality. The aim of the research was designed a novel DNA vaccine encoding the Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein to provide protection against both influenza and secondary infection with S. aureus. The DNA vaccine vector efficiently expressed the encoded antigen in mammalian cells, as determined by RT-PCR, Western blotting and immunofluorescence analysis. Mice were immunized with the vaccine by intramuscular injection before challenge with IAV and S. aureus. The pulmonary and the splenocyte culture IFN-γ levels were significant higher in immunized mice than their respective controls. Although the antibody titer in the HI test was low, the sera of mice immunized with the novel vaccine vector were effective in neutralisation assay in vitro. The vaccine could reduce the loss of body weight in mice during IAV challenge. Both Western blotting and RT-PCR showed that the vaccine markedly enhanced toll like receptor 2 (TLR2) expression in splenocytes after the secondary infection with S. aureus. The survival rate of mice with high TLR2 expression (pEGFP/Ag85A-HA2 or iPR) was significantly increased compared with mice immunized with pEGFP/HA2 after challenge with S. aureus. However, the pulmonary IL-10 concentration and S. aureus titer were significantly decreased in immunized mice, and expression of TLR2 was increased after challenge with S. aureus. These results demonstrated that Ag85A could strengthen the immune response to IAV and S. aureus, and TLR2 was involved in the host response to S. aureus.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Vacinas Antiestafilocócicas/imunologia , Vacinas de DNA/imunologia , Aciltransferases/genética , Animais , Anticorpos Antivirais/sangue , Antígenos de Bactérias/genética , Carga Bacteriana , Modelos Animais de Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Intramusculares , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/prevenção & controle , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Análise de Sobrevida , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Objective To identify the sets of lymphocytes that could systematically evaluate immune function of colorectal cancer patients, based on the expression of colorectal cancer T cells, natural killer (NK) cells, and NKT cell surface protein receptors. Methods Peripheral blood samples from 144 patients with colorectal cancer and 87 healthy controls were collected, and the differences in surface receptors of lymphocyte subsets in peripheral blood of patients and healthy controls were analyzed by means of flow cytometry and cell culture. Results Compared with healthy control group, the percentage of peripheral blood total lymphocytes, CD16brightCD56dimNK cells and NKT cells decreased in patients with colorectal cancer. The percentage of T cells, CD16brightCD56dimNK cells and NKT cell surface inhibitory receptors T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains (TIGIT) increased; T cells, NK cells, NKT cell surface chemokine receptor C-C motif chemokine receptor 7 (CCR7) slightly decreased. Conclusion There are differences in the proportion of NK cell subsets and the expression profile of surface receptors in peripheral blood of patients with colorectal cancer.
Assuntos
Neoplasias Colorretais , Subpopulações de Linfócitos , Humanos , Células Matadoras Naturais , Contagem de Linfócitos , Receptores de QuimiocinasRESUMO
Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus-bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8-dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Neoplasias Hepáticas/genética , Transativadores/genética , Transativadores/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/genética , Hepatite B/genética , Recombinação Homóloga , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory autoreactive T cells that mediate the selective destruction of insulin-producing ß cells via both direct and indirect mechanisms. Many immune cells and proinflammatory cytokines are involved in the pathogenesis of autoimmune diabetes. Immune intervention is effective for the prevention and treatment of T1D by blocking the autoimmune assault to ß cells. The non-structural protein 1(NS1) of influenza A viruses is a non-essential virulence factor encoded on segment 8 that has multiple accessory functions, including suppression of innate immunity and adaptive immunity, inhibition of apoptosis and activation of phosphoinositide 3-kinase (PI3K). This research investigated whether the expression of NS1 can prevent and treat diabetes mellitus induced by Streptozotocin (STZ). The NS1 expressing plasmid pEGFP-C2/NS1 was constructed and injected intramuscularly to both thighs of mice. Its effect on mice was observed. Intramuscular delivery of pEGFP-C2/NS1 resulted in reduction in hyperglycemia and diabetes incidence, with an increase in insulin. pEGFP-C2/NS1 could also increase glycogen and regulated serum cytokine levels. In addition, by comparison to the mice treated with empty vector pEGFP-C2, ameliorative insulitis was observed in the mice treated with recombinant plasmid pEGFP-C2/NS1. This result suggests that the expression of NS1 is effective for the prevention and treatment of diabetes mellitus induced by STZ in a mouse model.
Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Animais , Linhagem Celular , Injeções Intramusculares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Plasmídeos/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
Hepatocellular carcinoma (HCC) is one of the most deadly human cancers. Chronic hepatitis B virus (HBV) infection is one of the predominant risk factors associated with the development of HCC and complicates the treatment of HCC. In this study, we demonstrate that a HBV-positive HCC cell line HepG2.2.15, was more resistant to chemotherapy agents than its parental HBV-negative cell line HepG2. HBV-positive HCC cells exhibited defective Chk1 phosphorylation and increased chromosomal instability. CGK733, a small molecule inhibitor reportedly targeting the kinase activities of ATM and ATR, significantly enhanced taxol-induced cytotoxicity in HBV-positive HepG2.2.15 cells. The mechanism lies in CGK733 triggers the formation of multinucleated cells thus promotes the premature mitotic exit of taxol-induced mitotic-damaged cells through multinucleation and mitotic catastrophe in HBV-positive HepG2.2.15 cells. These results suggest that CGK733 could potentially reverse the taxol resistance in HBV-positive HCC cells and may suggest a novel strategy to treat HBV-infected HCC patients.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Benzenoacetamidas/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Paclitaxel/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Tioureia/análogos & derivados , Proteínas Mutadas de Ataxia Telangiectasia , Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA/antagonistas & inibidores , Células Gigantes/efeitos dos fármacos , Células Hep G2 , Vírus da Hepatite B/isolamento & purificação , Humanos , Neoplasias Hepáticas/virologia , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tioureia/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
Cancer driven by somatic mutations may express neoantigens that can trigger T-cell immune responses. Since T-cell receptor (TCR) repertoires play critical roles in anti-tumor immune responses for oncology, next-generation sequencing (NGS) was used to profile the hypervariable complementarity-determining region 3 (CDR3) of the TCR-beta chain in peripheral blood samples from 68 gastric cancer patients and 49 healthy controls. We found that most hyper-expanded CDR3 are individual-specific, and the gene usage of TRBV3-1 is more frequent in the tumor group regardless of tumor stage than in the healthy control group. We identified 374 hyper-expanded tumor-specific CDR3, which may play a vital role in anti-tumor immune responses. The patients with stage IV gastric cancer have higher EBV-specific CDR3 abundance than the control. In conclusion, analysis of the peripheral blood TCR repertoires may provide the biomarker for gastric cancer prognosis and guide future immunotherapy.
Assuntos
Neoplasias Gástricas , Regiões Determinantes de Complementaridade/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Neoplasias Gástricas/genética , Linfócitos TRESUMO
Double-strand breaks repaired by homologous recombination (HR) are first resected to form single-stranded DNA, which binds replication protein A (RPA). RPA attracts mediators that load the Rad51 filament to promote strand invasion, the defining feature of HR. How the resection machinery navigates nucleosome-packaged DNA is poorly understood. Here we report that in Schizosaccharomyces pombe a conserved DDB1-CUL4-associated factor (DCAF), Wdr70, is recruited to DSBs as part of the Cullin4-DDB1 ubiquitin ligase (CRL4(Wdr70)) and stimulates distal H2B lysine 119 mono-ubiquitination (uH2B). Wdr70 deletion, or uH2B loss, results in increased loading of the checkpoint adaptor and resection inhibitor Crb2(53BP1), decreased Exo1 association and delayed resection. Wdr70 is dispensable for resection upon Crb2(53BP1) loss, or when the Set9 methyltransferase that creates docking sites for Crb2 is deleted. Finally, we establish that this histone regulatory cascade similarly controls DSB resection in human cells.
Assuntos
DNA/genética , Exodesoxirribonucleases/genética , Reparo de DNA por Recombinação , Schizosaccharomyces/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , UbiquitinaçãoRESUMO
Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser392-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser392-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser392-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser392 presents an alternative for HCC chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Imidazóis/farmacologia , Neoplasias Hepáticas/patologia , Fosfosserina/metabolismo , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency.
Assuntos
Técnicas de Transferência de Genes , Compostos Heterocíclicos/química , Lipídeos/química , Animais , Materiais Biocompatíveis/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclamos , DNA/metabolismo , DNA/ultraestrutura , Eletroforese em Gel de Ágar , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Fluorescência , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ácido Linoleico/química , Luciferases/genética , Tamanho da Partícula , Plasmídeos/metabolismo , Coelhos , Eletricidade Estática , TransfecçãoRESUMO
For a clinically effective non-viral gene delivery system, a non-toxic and highly efficient vector is of great importance. A series of linear cationic polymers were synthesized by the ring-opening polymerization between diglycidyl ethers and diamines. Their structure-activity relationships as gene delivery vectors were systematically studied. Besides the amino groups with various densities, these polymers have uniform distribution of hydroxyl groups, which were formed in the polymerization and may benefit their biocompatibility and serum-tolerance. These polymers have good DNA binding ability and could condense DNA into nanoparticles with proper sizes and zeta-potentials. MTT assay revealed that polyplexes formed from title polymers have lower cytotoxicity than that derived from PEI. Most of the polymers have higher transfection efficiency than 25 kDa PEI in the in vitro transfection experiments. Polymers prepared from diglycidyl ethers with less or no N atom (2a and 2b) gave dramatically decreased TE, indicating that secondary amine on the backbone is highly required for efficient gene transfection, and compound 2 may be a good building block in the design of cationic polymers for gene delivery. More importantly, these polymers showed much better serum tolerance. Unlike PEI, the transfection mediated by P5 was seldom affected by the presence of 10% serum. Cellular uptake and intracellular distribution studies also confirmed the good performance of P5 in the transfection process with serum.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Poliaminas/química , Polimerização , Soluções Tampão , Morte Celular , Linhagem Celular Tumoral , DNA/metabolismo , Eletroforese em Gel de Ágar , Endocitose , Compostos de Epóxi/química , Etídio/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Espaço Intracelular/metabolismo , Luciferases/metabolismo , Microscopia de Fluorescência , Peso Molecular , Tamanho da Partícula , Plasmídeos/metabolismo , Polieletrólitos , Polietilenoimina/síntese química , Polietilenoimina/química , Eletricidade Estática , Transfecção , VírusRESUMO
A novel DNA vaccine vector encoding the Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein epitopes, pEGFP/Ag85A-sHA2 (pAg85A-sHA2), was designed to provide protection against influenza. The antigen encoded by the DNA vaccine vector was efficiently expressed in mammalian cells, as determined by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence analyses. Mice were immunized with the vaccine vector by intramuscular injection before challenge with A/Puerto Rico/8/34 virus (PR8 virus). Sera and the splenocyte culture IFN-γ levels were significantly higher in immunized mice compared with the control mice. The novel vaccine group showed a high neutralization antibody titer in vitro. The novel vaccine vector also reduced the viral loads, increased the survival rates in mice after the PR8 virus challenge and reduced the alveolar inflammatory cell numbers. Sera IL-4 concentrations were significantly increased in mice immunized with the novel vaccine vector on Day 12 after challenge with the PR8 virus. These results demonstrated that short HA2 (sHA2) protein epitopes may provide protection against the PR8 virus and that Ag85A could strengthen the immune response to HA2 epitopes, thus, Ag85A may be developed as a new adjuvant for influenza vaccines.