RESUMO
The activation of lymphocytes in patients with lupus and in mouse models of the disease is coupled with an increased cellular metabolism in which glucose plays a major role. The pharmacological inhibition of glycolysis with 2-deoxy-d-glucose (2DG) reversed the expansion of follicular helper CD4+ T cells and germinal center B cells in lupus-prone mice, as well as the production of autoantibodies. The response of foreign Ags was however not affected by 2DG in these mice, suggesting that B and CD4+ T cell activation by autoantigens is uniquely sensitive to glycolysis. In this study, we tested this hypothesis with monoclonal B cells and CD4+ T cells specific for lupus-relevant autoantigens. AM14 Vκ8R (AM14) transgenic B cells are activated by IgG2a/chromatin immune complexes and they can receive cognate help from chromatin-specific 13C2 CD4+ T cells. We showed that activation of AM14 B cells by their cognate Ag PL2-3 induced glycolysis, and that the inhibition of glycolysis reduced their activation and differentiation into Ab-forming cells, in the absence or presence of T cell help. The dependency of autoreactive B cells on glycolysis is in sharp contrast with the previously reported dependency of 4-hydroxy-3-nitrophenyl acetyl-specific B cells on fatty acid oxidation. Contrary to AM14 B cells, the activation and differentiation of 13C2 T cells into follicular helper CD4+ T cells was not altered by 2DG, which differs from polyclonal CD4+ T cells from lupus-prone mice. These results further define the role of glycolysis in the production of lupus autoantibodies and demonstrate the need to evaluate the metabolic requirements of Ag-specific B and T cells.
Assuntos
Linfócitos T CD4-Positivos , Lúpus Eritematoso Sistêmico , Linfoma de Células B , Animais , Camundongos , Autoanticorpos , Autoantígenos/metabolismo , Cromatina/metabolismo , Glucose/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Linfócitos T Auxiliares-IndutoresRESUMO
Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.
Assuntos
Proteínas de Homeodomínio , Lúpus Eritematoso Sistêmico , Camundongos , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Alelos , Isoformas de Proteínas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , DNA , Ubiquitinas/genéticaRESUMO
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which poorly characterized genetic factors lead to the production of proinflammatory or autoreactive T cells. Pre-B cell leukemia homeobox 1 (PBX1) is a transcription factor whose dominant negative isoform (PBX1-D) is overexpressed in the CD4+ T cells of SLE patients and lupus-prone mice. Pbx1-D overexpression favors the expansion of proinflammatory T cells and impairs regulatory T (Treg) cell development. Here we show that Pbx1 deficiency and Pbx1-D overexpression decreased STAT3 expression and activation in T cells. Accordingly, Pbx1 deficiency in T cells and Pbx1-D overexpression reduced STAT3-dependent TH17 cell polarization in vitro, but it had no effect in vivo at steady state. STAT3-dependent follicular helper T (TFH) cell polarization in vitro and splenic TFH cell frequency were not affected by either Pbx1 deficiency or Pbx1-D overexpression. Pbx1 deficiency also increased the expression of cell cycle arrest and pro-apoptotic genes, with an increased apoptosis in T cells. Our results suggest a complex interplay between PBX1 and STAT3, which may contribute to lupus pathogenesis through dysregulation of the cell cycle and apoptosis.
Assuntos
Lúpus Eritematoso Sistêmico , Fator de Transcrição 1 de Leucemia de Células Pré-B , Fator de Transcrição STAT3 , Animais , Humanos , Camundongos , Linfócitos T CD4-Positivos , Regulação da Expressão Gênica , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Linfócitos T Auxiliares-IndutoresRESUMO
The maintenance of regulatory T (Treg) cells critically prevents autoimmunity. Pre-B cell leukemia transcription factor 1 (Pbx1) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4+ T cells. Pbx1-d overexpression impaired Treg cell homeostasis and promoted inflammatory CD4+ T cells. Here, we showed a high expression of Pbx1 in human and murine Treg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of Treg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in Treg cells. Intriguingly, Rtkn2, a Rho-GTPase previously associated with Treg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of Treg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.
Assuntos
Lúpus Eritematoso Sistêmico , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Divisão Celular , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Isoformas de Proteínas/genética , Lúpus Eritematoso Sistêmico/genéticaRESUMO
Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-ß1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-ß1 has an opposite and dominant effect to IL-2. TGF-ß1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.
Assuntos
Membrana Celular/metabolismo , Citocinas/fisiologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores Imunológicos/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacologia , Antagonismo de Drogas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/fisiologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Imunológicos/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The expansion of follicular helper T (Tfh) cells, which is tightly associated with the development of lupus, is reversed by the inhibition of either glycolysis or glutaminolysis in mice. Here we analyzed the gene expression and metabolome of Tfh cells and naive CD4+ T (Tn) cells in the B6.Sle1.Sle2.Sle3 (triple congenic, TC) mouse model of lupus and its congenic B6 control. Lupus genetic susceptibility in TC mice drives a gene expression signature starting in Tn cells and expanding in Tfh cells with enhanced signaling and effector programs. Metabolically, TC Tn and Tfh cells showed multiple defective mitochondrial functions. TC Tfh cells also showed specific anabolic programs including enhanced glutamate metabolism, malate-aspartate shuttle, and ammonia recycling, as well as altered dynamics of amino acid content and their transporters. Thus, our study has revealed specific metabolic programs that can be targeted to specifically limit the expansion of pathogenic Tfh cells in lupus.
RESUMO
Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias da Língua , Antígeno B7-H1/genética , Fusobacterium/genética , Fusobacterium/metabolismo , Humanos , Neoplasias Bucais/genética , RNA Mensageiro , RNA Ribossômico 16S/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Neoplasias da Língua/genética , Microambiente Tumoral/genéticaRESUMO
Estrogen-related receptor γ (Esrrg) is a murine lupus susceptibility gene associated with T cell activation. Here, we report that Esrrg controls Tregs through mitochondria homeostasis. Esrrg deficiency impaired the maintenance and function of Tregs, leading to global T cell activation and autoimmunity in aged mice. Further, Esrrg-deficient Tregs presented an impaired differentiation into follicular Tregs that enhanced follicular helper T cells' responses. Mechanistically, Esrrg-deficient Tregs presented with dysregulated mitochondria with decreased oxygen consumption as well as ATP and NAD+ production. In addition, Esrrg-deficient Tregs exhibited decreased phosphatidylinositol and TGF-ß signaling pathways and increased mTOR complex 1 activation. We found that the expression of human ESRRG, which is high in Tregs, was lower in CD4+ T cells from patients with lupus than in healthy controls. Finally, knocking down ESRRG in Jurkat T cells decreased their metabolism. Together, our results reveal a critical role of Esrrg in the maintenance and metabolism of Tregs, which may provide a genetic link between lupus pathogenesis and mitochondrial dysfunction in T cells.
Assuntos
Lúpus Eritematoso Sistêmico/genética , Mitocôndrias/patologia , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Linfócitos T Reguladores/imunologia , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Mitocôndrias/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
OBJECTIVES: In accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. METHODS: Putative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. RESULTS: Nine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. CONCLUSION: CD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.
Assuntos
Ligante CD27/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Linfócitos T/imunologia , HumanosRESUMO
Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.
Assuntos
Apoptose/fisiologia , Biomarcadores Tumorais/metabolismo , Proteína Ligante Fas/fisiologia , Neoplasias Gástricas/mortalidade , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologiaRESUMO
The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. To elucidate the molecular basis of resistance to cisplatin, we compared cisplatin-induced apoptotic responses of the parental human bladder cancer cell line, T24 and its resistant subclone, T24R2. In T24 cells, cisplatin induce apoptosis and the activation of caspase-8, -9 and -3 and poly(ADP-ribose) polymerase cleavage. The expression levels of Fas, FasL, and FADD were not changed by the treatment with cisplatin. Furthermore, neither Fas-neutralizing antibody nor dominant negative mutant of FADD affected cisplatin-induced apoptosis. Western blot analysis of subcellular fractions showed that cisplatin induced redistribution of Bax and cytochrome c. Thus, cisplatin causes apoptosis in a death receptor-independent and mitochondria-dependent fashion in T24 cells. In contrast, overexpressed Bcl-2 protein inhibited cisplatin-induced Bax translocation and its downstream events in T24R2. Downregulation of Bcl-2 by RNAi potentiated the redistribution of Bax and cytochrome c and reversed cisplatin-resistance. Our results indicate that upregulation of Bcl-2 contributes to the development of cisplatin-resistance and usage of siRNA which targets the Bcl-2 gene may offer a potential tool to reverse the resistance to cisplatin in bladder cancer.
Assuntos
Antineoplásicos/farmacologia , Caspases/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Caspase 3 , Caspase 8 , Caspase 9 , Linhagem Celular Tumoral , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Regulação para Cima , Neoplasias da Bexiga UrináriaRESUMO
The potent anti-cancer agent cis-diamminedichloroplatinum (II) (cisplatin) is currently used for treating bladder cancer. However, clinical use of this drug for long periods is often limited because of the appearance of cisplatin-resistant bladder tumor cells. We employed the method of a differential display reverse transcriptase polymerase chain reaction to identify the differentially expressed genes in the parental human bladder cancer cell line, T24 and three cisplatin-resistant cell lines. We report here that cisplatin-resistant cell lines overexpress Bcl-2 family protein Bcl-2-related gene expressed in fetal liver (Bfl-1)/A1 as compared with their parental cell. Cisplatin and gamma-irradiation induced expression of Bfl-1/A1 in T24R2 cells but not in T24 cells. Among Bcl-2 family members, Bfl-1/A1 showed the most significant alteration of the expression level in resistant cells. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) by cisplatin and gamma-irradiation selectively occurred in T24R2 cells. Mitochondrial depolarization and cell death by cisplatin were also prevented in T24R2 cells. Moreover, Bfl-1/A1 inhibited cisplatin- and TNF-alpha-induced apoptosis in BOSC23 cells. Our findings suggest that the induction of Bfl-1/A1 by NF-kappaB may be important in controlling resistance to cisplatin responses in bladder tumor cells.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , NF-kappa B/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Apoptose , Humanos , Antígenos de Histocompatibilidade Menor , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para CimaRESUMO
BACKGROUND: Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level. METHODS: To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA. RESULTS: EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5'-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (-2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator. CONCLUSIONS: EGF signal is critical for the Mac-2BP expression, and more importantly, STAT3 could work as a negative regulator, while hTERT as a positive regulator in Mac-2BP transcription.
Assuntos
Antígenos de Neoplasias/metabolismo , Glicoproteínas de Membrana/metabolismo , Fator de Transcrição STAT3/metabolismo , Antígenos de Neoplasias/genética , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Fator de Crescimento Epidérmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética , Telomerase/metabolismo , TransfecçãoRESUMO
BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Telomerase/antagonistas & inibidores , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias/terapia , RNA Interferente Pequeno , Telomerase/genética , Telomerase/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Mac-2BP is a ligand of the galectin family that has been suggested to affect tumor proliferation and metastasis formation. We assessed Mac-2BP expression at the transcriptional and translational levels to evaluate nerve growth factor (NGF)-induced Mac-2BP expression. A time kinetic analysis using reverse transcription-polymerase chain reaction showed that NGF-induced Mac-2BP transcript levels were 4-5 times higher than in controls. Mac-2BP enzyme-linked immunosorbent assay and immunofluorescence staining showed a 2-3-fold increase in intracellular and secreted Mac-2BP as a result of NGF stimulation. This increase was regulated by Akt activation and NF-kappaB binding. p65 and p50-NF-kappaB are major transcriptional factors in the Mac-2BP promoter region, and were shown to be regulated in accordance with the Akt activation states. Collectively, these results suggest that NGF induces Mac-2BP expression via the PI3K/Akt/NF-kappaB pathway.
Assuntos
Antígenos de Neoplasias/genética , Glicoproteínas de Membrana/genética , NF-kappa B/fisiologia , Fator de Crescimento Neural/farmacologia , Proteína Oncogênica v-akt/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Regulação para Cima/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Linhagem Celular , DNA/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Mac-2 binding protein (Mac-2BP) is a secreted tumor antigen that is elevated in many cancers and implicated in tumor metastasis, as well as cell adhesion and immune functions. We focused on the human telomerase reverse transcriptase (hTERT) induced Mac-2BP expression and the relationship between Mac-2BP expression and the progression of gastric cancer. A cDNA expression array analysis was performed on the telomerase-negative cell line, SW13, which was engineered to overexpress hTERT when compared with the parental SW13 cell. hTERT-induced Mac-2BP expression was confirmed via RT-PCR and Northern blotting. ELISA and flow cytometric analyses revealed that Mac-2BP protein was increased by 2- to 4-fold in hTERT-overexpressing cells compared with the mock control. Mac-2BP expression was significantly reduced when the overexpressed hTERT was neutralized by the introduction of hTERT-specific siRNA. These results suggest that Mac-2BP expression is modulated by hTERT. Mac-2BP levels in both gastric cancer cells and tumor tissues were determined via Northern blot analysis and immunohistochemistry. Mac-2BP protein was highly expressed in most gastric cancer cell lines, and gastric tumor tissues were stained more densely than normal tissues. The intracellular and secreted Mac-2BP levels were also evaluated via ELISA, indicating that Mac-2BP was expressed and secreted more abundantly in gastric cancer patients than in healthy donors. The elevated serum Mac-2BP level in gastric tumor patients was also significantly associated with distant metastasis (p = 0.05) and higher tumor stage (p = 0.04). Our findings suggest that Mac-2BP is induced by hTERT, and that it may prove to be a useful prognostic marker for the detection of malignant progression of metastatic stomach cancers.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Pequenas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias Gástricas/metabolismo , Telomerase/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/genética , Estudos de Casos e Controles , DNA de Neoplasias/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Mucosa Gástrica/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Neoplasias Gástricas/secundário , Telomerase/antagonistas & inibidores , Telomerase/genética , Células Tumorais Cultivadas , Regulação para CimaRESUMO
BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.