RESUMO
BACKGROUND: Variants of chromodomain helicase DNA binding protein 7 (CHD7) gene are commonly associated with Kallmann syndrome (KS) and account for 5-6% of idiopathic hypogonadotropic hypogonadism (IHH) cases. Here we report a novel mutation of CHD7 gene in a patient with KS, which may contribute to the better understanding of KS. CASE PRESENTATION: A 29-year-old male patient with KS and a chief complaint of delayed puberty for 13 years (Tanner B Stage< 4) was admitted to the Department of Endocrinology of the First Affiliated Hospital of Zhejiang University (Hangzhou, China) in September 2019. Dual-energy X-ray absorptiometry (DEXA) showed low bone density in both lumbar spine (L1 ~ L5 mean Z-score - 3.0) and femoral neck (Z-score - 2.7). Dynamic contrast-enhanced magnetic resonance imaging (MRI) of pituitary and contrast-enhanced computed tomography (CT) showed no abnormal findings. Ophthalmological evaluation showed that his both eyes showed exotropia, and no sight loss was noted. Heterozygous c.1619G > T mutation of TCD7 gene (p.G4856V) was detected, whereas none of his family members had this mutation. Human chorionic gonadotropin (HCG) and human menopausal gonadotropin (HMG) were injected for three times/week to treat idiopathic hypogonadotropic hypogonadism (IHH). After several months of therapy, the patient's health condition improved. His testicles became larger, and his secondary sexual characteristics improved after treatment. CONCLUSION: Exploration of the novel splice-site mutation of CHD7 may further our current understanding of KS.
Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Síndrome de Kallmann/genética , Mutação de Sentido Incorreto , Adulto , China , Análise Mutacional de DNA , Heterozigoto , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/genética , Hipogonadismo/terapia , Síndrome de Kallmann/complicações , Síndrome de Kallmann/diagnóstico , Síndrome de Kallmann/terapia , Imageamento por Ressonância Magnética , Masculino , Polimorfismo de Nucleotídeo Único , Puberdade Tardia/diagnóstico , Puberdade Tardia/etiologia , Puberdade Tardia/genética , Puberdade Tardia/terapia , Tomografia Computadorizada por Raios XRESUMO
Transport and homeostasis of transition metals in chloroplasts, which are accurately regulated to ensure supply and to prevent toxicity induced by these metals, are thus crucial for chloroplast function and photosynthetic performance. However, the mechanisms that maintain the balance of transition metals in chloroplasts remain largely unknown. We have characterized an albino-revertible green 1 (arg1) rice mutant. ARG1 encodes an evolutionarily conserved protein belonging to the ATP-binding cassette (ABC) transporter family. Protoplast transfection and immunogold-labelling assays showed that ARG1 is localized in the envelopes and thylakoid membranes of chloroplasts. Measurements of metal contents, metal transport, physiological and transcriptome changes revealed that ARG1 modulates cobalt (Co) and nickel (Ni) transport and homeostasis in chloroplasts to prevent excessive Co and Ni from competing with essential metal cofactors in chlorophyll and metal-binding proteins acting in photosynthesis. Natural allelic variation in ARG1 between indica and temperate japonica subspecies of rice is coupled with their different capabilities for Co transport and Co content within chloroplasts. This variation underpins the different photosynthetic capabilities in these subspecies. Our findings link the function of the ARG1 transporter to photosynthesis, and potentially facilitate breeding of rice cultivars with improved Co homeostasis and consequently improved photosynthetic performance.
Assuntos
Oryza , Transportadores de Cassetes de Ligação de ATP/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Cobalto/metabolismo , Homeostase , Níquel/metabolismo , Níquel/toxicidade , Oryza/genética , Fotossíntese , Melhoramento VegetalRESUMO
Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women. Extracted from pregnant mare urine, CEEs are composed of nearly a dozen estrogens existing in an inactive sulfated form. To determine whether the hepatic steroid sulfatase (STS) is a key contributor to the efficacy of CEEs in HRT, we performed estrogen-responsive element (ERE) reporter gene assay, real-time PCR, and UPLC-MS/MS to assess the STS-dependent and inflammation-responsive estrogenic activity of CEEs in HepG2 cells and human primary hepatocytes. Using liver-specific STS-expressing transgenic mice, we also evaluated the effect of STS on the estrogenic activity of CEEs in vivo We observed that CEEs induce activity of the ERE reporter gene in an STS-dependent manner and that genetic or pharmacological inhibition of STS attenuates CEE estrogenic activity. In hepatocytes, inflammation enhanced CEE estrogenic activity by inducing STS gene expression. The inflammation-responsive estrogenic activity of CEEs, in turn, attenuated inflammation through the anti-inflammatory activity of the active estrogens. In vivo, transgenic mice with liver-specific STS expression exhibited markedly increased sensitivity to CEE-induced estrogenic activity in the uterus resulting from increased levels of liver-derived and circulating estrogens. Our results reveal a critical role of hepatic STS in mediating the hormone-replacing activity of CEEs. We propose that caution needs to be applied when Premarin is used in patients with chronic inflammatory liver diseases because such patients may have heightened sensitivity to CEEs due to the inflammatory induction of STS activity.
Assuntos
Estrogênios Conjugados (USP)/metabolismo , Esteril-Sulfatase/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios Conjugados (USP)/análise , Estrogênios Conjugados (USP)/farmacologia , Feminino , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Cavalos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Esteril-Sulfatase/antagonistas & inibidores , Esteril-Sulfatase/genética , Espectrometria de Massas em Tandem , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/patologiaRESUMO
The aberrant expression of Pknox1 is associated with hepatic glucose and lipid dysmetabolism status of type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism causing Pknox1 overexpression in this pathological status remains unclear. By using miRNA target prediction programs, we found that the 3'-UTR of the Pknox1 mRNA sequence contains highly conserved target sites of miR-17 family. In a rat model of streptozotocin and high-fat diet-induced T2DM and NAFLD complication, the increased hepatic expression of Pknox1 was consistent with decreased expressions of miR-17 family, especially miR-17 and miR-20a. Furthermore, an inverse correlation was observed between Pknox1 and miR-17 and miR-20a in free fatty acids-induced hepatocyte steatosis. Dual-luciferase reporter assay further showed that Pknox1 was a valid target gene of miR-17 family. The ectopic expression of miR-17 or miR-20a could markedly suppress Pknox1 expression in hepatocytes. MiR-17 or miR-20a overexpression also resulted in significantly enhanced insulin sensitivity and reduced hepatocyte steatosis in HepG2 and L02 cells, which were determined by altered phosphorylation on insulin receptor signaling pathway proteins and decreased intracellular triglyceride and lipid accumulation, respectively. These data implicate the upregulated hepatic expression of Pknox1 in T2DM complicated with NAFLD may be caused by the reduced expression of miR-17 family, indicating that developing miRNA-mediated regulation strategies on Pknox1 may provide new therapeutic options for metabolic disease.
Assuntos
Diabetes Mellitus Tipo 2/genética , Fígado Gorduroso/genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Insulina/genética , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Transdução de Sinais/genéticaRESUMO
To evaluate the role of TCF7L2, a key regulator of glucose homeostasis, in estradiol (E2) and progesterone (P4)-modulated glucose metabolism, mouse insulinoma cells (MIN6) and human liver cancer cells (hepG2 and HUH7) were treated with physiological concentrations of E2 or P4 in the up- and down-regulation of TCF7L2. Insulin/proinsulin secretion was measured in MIN6 cells, while glucose uptake and production were evaluated in liver cancer cells. E2 increased insulin/proinsulin secretion under both basal and stimulated conditions, whereas P4 increased insulin/proinsulin secretion only under glucose-stimulated conditions. An antagonistic effect, possibly concentration-dependent, of E2 and P4 on the regulation of islet glucose metabolism was observed. After E2 or P4 treatment, secretion of insulin/proinsulin was positively correlated with TCF7L2 protein expression. When TCF7L2 was silenced, E2- or P4-promoted insulin/proinsulin secretion was significantly weakened. Under glucotoxicity conditions, overexpression of TCF7L2 increased insulin secretion and processing. In liver cancer cells, E2 or P4 exposure elevated TCF7L2 expression, enhanced the activity of insulin signaling (pAKT/pGSK), reduced PEPCK expression, subsequently increased insulin-stimulated glucose uptake, and decreased glucose production. Silencing TCF7L2 eliminated effects of E2 or P4. In conclusion, TCF7L2 regulates E2- or P4-modulated islet and hepatic glucose metabolism. The results have implications for glucose homeostasis in pregnancy.
Assuntos
Estradiol/metabolismo , Glucose/metabolismo , Homeostase , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Progesterona/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica de Plantas , Código das Histonas , Histonas/genética , Oryza/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Mutação , Oryza/crescimento & desenvolvimento , Oryza/ultraestrutura , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/ultraestrutura , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
BACKGROUND: Hepatogenous diabetes (HD) occurs as a complication of cirrhosis. Whether genetic factors, rather than only liver damage, play roles in the development of HD is unknown. TCF7L2 gene has been reported to be associated with type 2 diabetes and also cancer risks. We aim to evaluate the impact of TCF7L2 gene on the susceptibility of HD and hepatocellular carcinoma (HCC) in a Chinese Han population. PATIENTS AND METHODS: A total of 367 adult liver transplant candidates with liver cirrhosis were included. Fifteen tag single nucleotide polymorphisms (SNPs) were selected from HapMap CHB database with a minor allele frequency of >0.2 and r(2) of >0.8. Another three SNPs were also chosen because of their close association with type 2 diabetes in East Asian. RESULTS: Patients with HD presented significantly poorer liver function, higher incidence of cirrhotic complications and higher insulin resistance compared with non-HD patients. Three SNPs were differentially distributed between HD patients and non-HD patients. In multivariate logistic analysis, TCF7L2 rs290487 and rs6585194 polymorphisms were independently associated with HD after adjustment of clinical factors. The TCF7L2 rs290487 C/C variant homozygote showed much higher insulin resistance and significantly increased HD risk comparing with T/T and T/C genotypes, while the genetic variant of rs6585194 was protectively against HD. Three SNPs (rs290481, rs290487 and rs290489) located near the 3' end of TCF7L2 gene were associated with HCC risk with marginal significance. Patients carrying G-C-A haplotype had a significantly higher HCC risk than those with A-T-G. CONCLUSIONS: TCF7L2 polymorphisms were associated with HD and maybe cancer risk as well. Further studies with large samples are needed to verify these results.
Assuntos
Carcinoma Hepatocelular/genética , Diabetes Mellitus Tipo 2/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Povo Asiático/genética , China , Complicações do Diabetes/genética , Diabetes Mellitus Tipo 2/complicações , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Resistência à Insulina/genética , Cirrose Hepática/etiologia , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
OBJECTIVE: To investigate the effects of overexpression of decorin (DCN), one of the small leucine-rich proteoglycans, on the expression of extracellular matrix molecules in glomerular mesangial and tubular cells. METHODS: Recombinant adenovirus vector containing DCN gene (Ad/DCN) was constructed, and recombinant adenovirus containing LacZ gene (Ad/LacZ) was used as control vector. Rat renal glomerular mesangial cells of the line RMC and tubular cells of the line HK-2 were cultured and divided into following groups: high glucose + Ad/DCN transfection (experimental), high glucose + Ad/LacZ transfection (vector control), high glucose + neutralizing antibody against transforming growth factor (TGF)-beta 1 (positive control), high glucose non-transfection (PBS control), and low glucose culture (normal control) groups. Western blotting was used to detect the protein expression of decorin, TGF-beta 1, collagen type IV, MMP-2, and MMP-9. RESULTS: The DCN protein expression was low only in the low glucose group, and was up-regulated in other groups, especially in the Ad/DCN group, so as the expression of TGF-beta 1 and collagen type IV, while the ratio of TGF-beta 1 to decorin is increased in both RMC and HK-2 cells. The expression levels of MMP-2 and MMP-9 decreased in the high-glucose-cultured RMC cells and increased in the high-glucose-cultured HK-2 cells, however, adenovirus-mediated transfection of decorin gene reversed all of these changes, and had almost the same effects on reducing the protein expression of TGF-beta 1 collagen type IV, MMP-2, and MMP-9 as anti-TGF-beta 1 antibody. CONCLUSION: The agents increasing the decorin expression in mesangial and tubular cells may help prevent the development and progression of diabetic nephropathy. Overexpression of decorin may be a useful tool for developing new therapeutic application for the treatment of diabetic nephropathy.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Túbulos Renais/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células Mesangiais/metabolismo , Proteoglicanas/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Decorina , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Túbulos Renais/citologia , Proteoglicanas/genética , RatosRESUMO
Immunotherapy for cancer is often limited by weak immunogenicity of tumor antigens. However, immune systems are usually strong and effective against foreign invading antigens. To test whether the destructive effect of adaptive immunity against foreign antigens can be redirected to tumors for cancer therapy, we immunized mice with adenovector expressing LacZ (Ad/CMV-LacZ). Subcutaneous syngeneic tumors were then established in the immunized animals or in naïve animals. The immune response against adenovirus or LacZ was redirected to tumors by intratumoral injection of Ad/CMV-LacZ. We found that immunization and treatment with the adenovector dramatically reduced the tumor growth rate compared with intratumoral administration of adenovector in naïve mice. Complete tumor regression was observed in about 50% of the immunized animals but not in the naïve animals. Similar effects were observed when oncolytic vaccinia virus was used to immunize and treat tumors. Lymphocyte infiltration in tumors was dramatically increased in the immunized group when compared with other groups. Moreover, immunity against parental tumor cells was induced in the animals cured with immunization and treatment with Ad/CMV-LacZ, as evidenced by the lack of tumor growth when the mice were challenged with parental tumor cells. Taken together, these results suggest that redirecting adaptive immunity against foreign antigens is a potential approach for anticancer therapy and that pre-existing immunity could enhance virotherapy against cancers.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Experimentais/terapia , Adenoviridae/genética , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Feminino , Fibrossarcoma/terapia , Terapia Genética , Imunização , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Mycobacterium bovis/imunologia , Neoplasias Experimentais/imunologia , Terapia Viral OncolíticaRESUMO
OBJECTIVE: To construct recombinant adenovirus expressing rat proteoglycan II (Ad-DCN) and to study its biological features. METHODS: Rat DCN gene was inserted into an E1 and E3-substituted adenovirus shuttle plasmid. And this shuttle plasmid including DCN was recombined with AdEasy-1 in BJ5183-AD-1 electroporation competent cells to form recombinant adenovirus-DCN plasmid, which was further transfected into Ad293 cells to passage adenovirus. The control recombinant adenovirus, Ad-LacZ, was also constructed in the same way. After plaque forming trail and adjusted with PBS, Ad-DCN and Ad-LacZ were obtained with titer 1x10(9) pfu x ml(-1). PCR, Western blot and MTT analysis were used to detect the expression of DCN or the bioactivity of expressed DCN. RESULT: DCN was detected in Ad-DCN infected CHO cells by PCR and Western blot, but not in Ad-LacZ infected CHO cells. MTT analysis results showed that the supernatant from the culture of Ad-DCN infected CHO cells could abrogate the inhibitive effect of TGFbeta1 on proliferation of CCL-64 cells. The proliferation rate of TGFbeta1 + Ad-DCN treated cells was significantly higher than that of TGFbeta1 + Ad-lacz or TGFbeta1 treated cells [(0.5252 +/-0.04 compared with 0.2826 +/-0.02 or 0.2918 +/-0.02) OD, P <0.05] and lower than that of control cells [(0.9332 +/-0.08) OD, P <0.05]. CONCLUSION: The constructed recombinant adenovirus can express biologically active decorin.
Assuntos
Adenoviridae/metabolismo , Vetores Genéticos , Proteoglicanas/biossíntese , Adenoviridae/genética , Animais , Masculino , Proteoglicanas/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Recombinação GenéticaRESUMO
PURPOSE: The tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector. EXPERIMENTAL DESIGN: We constructed a TRAIL-expressing oncolytic adenovector designated as Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity, and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors. RESULTS: Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing, replication-defective adenovector or an oncolytic adenovector-expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all s.c. xenograft tumors established from a human non-small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term, tumor-free survival. Furthermore, we found no treatment-related toxicity. CONCLUSIONS: Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy.
Assuntos
Neoplasias do Colo/terapia , Neoplasias Pulmonares/terapia , Vírus Oncolíticos/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Vetores Genéticos/genética , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Nus , Terapia Viral Oncolítica , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Relação Estrutura-Atividade , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transplante Heterólogo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Neovascularização Patológica , Neoplasias da Próstata/patologia , Tiazóis/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Masculino , Fosforilação , Tiazolidinas , Células Tumorais CultivadasRESUMO
Small-molecule inhibitors of cyclin-dependent kinases (CDKs) are known to induce cell cycle arrest and apoptosis in certain cancer cells. In order to evaluate the antitumor activity of one such inhibitor, GW8510, against human lung cancers, we analyzed the effects of GW8510 on six nonsmall cell lung cancer (NSCLC) cell lines (A549, H1299, H460, H226, H358 and H322) and normal human fibroblast (NHFB). We treated the cells with GW8510 at concentrations of 0-10 microM, and found that it suppressed cell growth in vitro in all the lung cancer cells but not in NHFB. Subsequent study showed that GW8510 induced apoptosis and cell cycle arrest in the A549, H1299 and H460 cells in a time- and dose-dependent manner. Western blot analysis showed that GW8510 downregulated the expression of X-linked inhibitor of apoptosis (XIAP) but had no detectable effect on the expression of Bax, Bak, or Bcl2. GW8510 also downregulated XIAP mRNA level, suggesting that downregulation of XIAP expression occurs at the transcriptional level. Moreover, ectopic XIAP expression diminished growth inhibition and apoptosis induction by GW8510. Importantly, GW8510 was not capable of inducing apoptosis of NHFB cells. These results suggest that GW8510 might provide a treatment strategy for human NSCLC and XIAP is an important target for GW8510-induced apoptosis of NSCLC cells that occurs through inhibition of XIAP mRNA transcription.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Indóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Transcrição Gênica/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Apoptose , Regulação para Baixo , Humanos , RNA Mensageiro/metabolismoRESUMO
Although gemcitabine is a potent therapeutic agent in the treatment of human non-small cell lung cancer (NSCLC), resistance to gemcitabine is common. In this study, we investigated the molecular mechanisms involved in acquired gemcitabine resistance against NSCLC cells. Gemcitabine-resistant NSCLC H1299 cells (H1299/GR) were selected by long-term exposure of parental H1299 cells to gemcitabine. The median inhibitory concentrations of gemcitabine in H1299 and H1299/GR cells were 19.4 and 233.1 nM, respectively. Gemcitabine induced activation of c-Jun NH2-terminal kinase (JNK) in parental H1299 cells but not in H1299/GR cells after 48 h. Blocking JNK activation by pretreatment with SP600125, a specific JNK inhibitor, or by transfection with dominant-negative JNK vectors abrogated gemcitabine-induced apoptosis in parental H1299 cells as evidenced by interruption of caspase activation. Transient transfection with a JNKK2-JNK1 plasmid expressing constitutive JNK1 partially restored the effect of gemcitabine in H1299/GR cells. Our results indicate that gemcitabine-induced apoptosis in human NSCLC H1299 cells requires activation of the JNK signaling pathway. Attenuated JNK activation may contribute to development of acquired gemcitabine resistance in cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Desoxicitidina/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Resistência a Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , GencitabinaRESUMO
Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias do Colo/tratamento farmacológico , Inibidores de Cisteína Proteinase/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Citocromos c/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leupeptinas/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas Mitocondriais/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Development of new therapeutic agents for colon cancer is highly desirable. To this end, we screened a chemical library for new anticancer agents and identified a synthetic compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), which kills cancer cells more effectively than it kills normal human fibroblasts. The molecular mechanism of the antitumor action of DBPT was further analyzed in three human colorectal cancer cell lines. DBPT effectively inhibited the growth of colorectal cancer cells, independent of p53 and P-glycoprotein status, whereas normal fibroblasts were unaffected at the same IC50. Over time, DLD-1 cancer cells treated with DBPT underwent apoptosis. The general caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone partially blocked DBPT-induced apoptosis in a dose-dependent manner. DBPT-induced apoptosis, including cytochrome c release and caspase activation, was abrogated when c-Jun NH2-terminal kinase (JNK) activation was blocked with either a specific JNK inhibitor or a dominant-negative JNK1 gene. However, constitutive JNK activation alone did not replicate the effects of DBPT in DLD-1 cells, and excessive JNK activation by adenovirus encoding MKK7 had little influence on DBPT-induced apoptosis. Our results suggested that DBPT induces apoptosis in colorectal cancer cell lines through caspase-dependent and caspase-independent pathways and that JNK activation was crucial for DBPT-induced apoptosis. DBPT and its analogues might be useful as anticancer agents.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Tiazóis/farmacologia , Antracenos/farmacologia , Apoptose/fisiologia , Caspases/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Ativação Enzimática/efeitos dos fármacos , Células HCT116 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , TiazolidinasRESUMO
Proteasome inhibitors have emerged as promising anticancer therapeutic agents. Bortezomib (PS-341), a specific proteasome inhibitor, exhibits antitumor activity against a wide range of malignancies and has been approved by the US Food and Drug Administration for the treatment of relapsed or refractory multiple myeloma. However, the molecular mechanisms of bortezomib-mediated apoptosis remain unclear. To characterize the mechanisms of apoptosis induction by proteasome inhibitors, we examined levels of Bcl-2 protein family members (Bik/NBK, Bax, Bak, Bcl-2, and Bcl-XL), release of cytochrome c, and activation of caspase-9 and -3 in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human lung cancer cell line H1299; and human ovarian cancer cell line SKOV3 after they were treated with bortezomib. The result showed that bortezomib induced rapid accumulation of Bik/NBK but not other Bcl-2 family members in all six cell lines. Bortezomib-mediated Bik/NBK accumulation and apoptosis were also observed in human embryonic kidney cells 293 and normal human bronchial epithelial cells. Moreover, dramatic Bik/NBK accumulation and apoptosis induction were observed when cells were treated with proteasome inhibitor MG132 and calpain inhibitor I (ALLN). Furthermore, no detectable changes in IkappaBalpha levels or in NFkappaB functionality were found after treatment with bortezomib. Finally, Bik/NBK accumulation was caused by stabilization of the protein from degradation and was associated with bortezomib cytotoxicity and apoptosis induction. Pretreatment of DLD1 cells with Bik/NBK siRNA reduced bortezomib-mediated Bik/NBK accumulation and cell death. Our results suggested that Bik/NBK is one of the mediators of proteasome inhibitor-induced apoptosis.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Proteínas de Membrana/fisiologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Proteínas Reguladoras de Apoptose , Bortezomib , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo , Feminino , Humanos , Rim , Proteínas Mitocondriais , NF-kappa B/metabolismo , Neoplasias OvarianasRESUMO
AIM: To analyze the factors which influence the serum levels of hyaluronic acid (HA), type III pro-collagen (PCIII), laminin (LN) and type IV collagen (C-IV) in liver fibrosis. METHODS: The serum specimens from 141 chronic hepatitis patients were assayed for fibrosis indexes including HA, PCIII, LN and C-IV with radioimmunoassay (RIA) and liver function indexes by an automatic biochemistry analyzer. The patients were then divided into consistent group and inconsistent group. The patients'clinical manifestations were recorded, routine blood pictures were done by a blood counter and analyzer (AC-900). Liver biopsy specimens were examined path-morphologically. The inner diameters of portal vein, splenic vein and thickness of spleen were all measured by ultrasonography. RESULTS: Sixteen patients (14.16%) had serum fibrosis indexes inconsistent with histological stage of their hepatic fibrosis. Their serum fibrosis indexes did not correlate with the stage of hepatic fibrosis (P>0.05), but were positively correlated with the grade of inflammation (chi2=12.07, P<0.05). At the same time, serum albumin (ALB) and the ratio of albumin and globulin (A/G) were significantly increased (t=3.06, P<0.01), (t=3.70, P<0.01). Serum levels of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), gamma-glutamyl transferase (GGT) and globulin (GLB) were all significantly decreased (t=2.45, P<0.05), (t=2.33, P<0.05), (t=2.08, P<0.05), (t=3.03, P<0.01). Weary degree also decreased more obviously (chi2=7.52, P<0.05), but other clinical manifestations, routine blood indexes, serum levels of alkaline phosphatase (AKP), total bilirubin (TBIL), total protein (TP), width of main portal vein, width of splenic vein and thickness of spleen had no significant change (P>0.05). CONCLUSION: Serum fibrosis indexes can be influenced by the grade of inflammation, some liver function indexes and clinical manifestations. Comprehensive analysis is necessary for its proper interpretation.