RESUMO
Antimicrobial peptides (AMPs) are considered potential alternatives to antibiotics due to their advantages in solving antibiotic resistance. Brevinin-2GUb, which was extracted from the skin secretion of Hylarana guentheri, is a peptide with modest antimicrobial activity. Several analogues were designed to explore the structure-activity relationship and enhance its activity. In general, the Rana box is not an indispensable motif for the bioactivity of Brevinin-2GUb, and the first to the 19th amino acids at the N-terminal end are active fragments, such that shortening the peptide while maintaining its bioactivity is a promising strategy for the optimisation of peptides. Keeping a complete hydrophobic face and increasing the net charges are key factors for antimicrobial activity. With the increase of cationic charges, α-helical proportion, and amphipathicity, the activity of t-Brevinin-2GUb-6K (tB2U-6K), in combatting bacteria, drastically improved, especially against Gram-negative bacteria, and the peptide attained the capacity to kill clinical isolates and fungi as well, which made it possible to address some aspects of antibiotic resistance. Thus, peptide tB2U-6K, with potent antimicrobial activity against antibiotic-resistant bacteria, the capacity to inhibit the growth of biofilm, and low toxicity against normal cells, is of value to be further developed into an antimicrobial agent.
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DMPC-10A (ALWKKLLKK-Cha-NH2) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala1 and Trp3 remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.
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Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro14 substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity.
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BACKGROUND: Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed throughout the central and peripheral nervous system and in cells of the diffuse neuroendocrine system. Aberrant function of UCH-L1 has been associated with neurological disorders such as Parkinson's disease and Alzheimer's disease. Moreover, UCH-L1 exhibits a variable expression pattern in cancer, acting either as a tumour suppressor or promoter, depending on the type of cancer. In non-small cell lung carcinoma primary tumour samples, UCH-L1 is highly expressed and is associated with an advanced tumour stage. This suggests UCH-L1 may be involved in oncogenic transformation and tumour invasion in NSCLC. However, the functional significance of UCH-L1 in the progression of NSCLC is unclear. The aim of this study was to investigate the role of UCH-L1 using NSCLC cell line models and to determine if it is clinically relevant as a prognostic marker for advanced stage disease. METHODS: UCH-L1 expression in NSCLC cell lines H838 and H157 was modulated by siRNA-knockdown, and the phenotypic changes were assessed by flow cytometry, haematoxylin & eosin (H&E) staining and poly (ADP-ribose) polymerase (PARP) cleavage. Metastatic potential was measured by the presence of phosphorylated myosin light chain (MLC2). Tumour microarrays were examined immunohistochemically for UCH-L1 expression. Kaplan-Meier curves were generated using UCH-L1 expression levels and patient survival data extracted from Gene Expression Omnibus data files. RESULTS: Expression of UCH-L1 was decreased by siRNA in both cell lines, resulting in increased cell death in H838 adenocarcinoma cells but not in the H157 squamous cell line. However, metastatic potential was reduced in H157 cells. Immunohistochemical staining of UCH-L1 in patient tumours confirmed it was preferentially expressed in squamous cell carcinoma rather than adenocarcinoma. However the Kaplan-Meier curves generated showed no correlation between UCH-L1 expression levels and patient outcome. CONCLUSIONS: Although UCH-L1 appears to be involved in carcinogenic processes in NSCLC cell lines, the absence of correlation with patient survival indicates that caution is required in the use of UCH-L1 as a potential prognostic marker for advanced stage and metastasis in lung carcinoma.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Proliferação de Células , Ubiquitina Tiolesterase/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Adesão Celular , Movimento Celular , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Taxa de Sobrevida , Análise Serial de Tecidos , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genéticaRESUMO
Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.
Assuntos
Proteínas E1A de Adenovirus/fisiologia , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Núcleo Celular/metabolismo , Progressão da Doença , Métodos Epidemiológicos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Erythropoietin (Epo) regulates erythropoiesis by binding to its receptor (EpoR) and promoting cell proliferation, differentiation and inhibition of apoptosis. Epo is widely used to treat cervical cancer-related anaemia. However, there are data suggesting that administration of Epo is associated with an increment in recurrence rate, and decreased disease-free and overall survival. In the present study, we investigated the expression of Epo and EpoR on cervical cancer cell lines. We observed that both EpoR and extracellular Epo are constitutively expressed in cervical cancer cells. Inhibition of either Epo or EpoR expression with siRNA attenuated cell proliferation, whereas addition of exogenous Epo led to a significant increase in cell growth, both in vitro and in vivo. Epo-induced proliferation was associated with the activation of JAK2, JAK3, STAT3 and STAT5 but not JAK1 and STAT1. Our results are consistent with the existence of a functional, endogenous Epo/EpoR system in cervical cancer with the capacity to activate the transduction of signals resulting in an increased proliferation potential.
Assuntos
Comunicação Autócrina , Proliferação de Células , Eritropoetina/farmacologia , Janus Quinases/metabolismo , Comunicação Parácrina , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Neoplasias do Colo do Útero/metabolismo , Animais , Apoptose , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Janus Quinases/genética , Camundongos , Camundongos Nus , RNA Mensageiro/genética , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Eritropoetina/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Animais , Neoplasias da Mama/genética , Carcinoma/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Inibidores Enzimáticos/farmacologia , Eritropoetina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Janus Quinase 2/efeitos dos fármacos , Janus Quinase 2/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos , Receptores da Eritropoetina/genética , Fator de Transcrição STAT5/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Breast cancer-associated 1 (BRCA1) plays an important role in breast cancer initiation and progression through its functions in the cell cycle and DNA repair processes; however, its role in metastatic development in human breast cancer is still poorly understood. We have previously shown that osteopontin (OPN) expression was suppressed by wild-type BRCA1 (Wt.BRCA1) and that a natural mutant allele of BRCA1 (Mut.BRCA1) diminished the effect of Wt.BRCA1 on OPN in vitro. In this study, we show that while Wt.BRCA1 suppresses OPN-induced metastasis in a rat syngeneic system, Mut.BRCA1 enhances the development of metastasis through OPN, suggesting that OPN and BRCA1 work closely to regulate metastatic development in the rat. To test whether these findings are relevant to human breast cancer, we have investigated the relationship between BRCA1, OPN, and metastatic properties in human breast cancer-related cells. Using western blot analysis, we show that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN protein expression; and in parallel that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN-mediated in vitro properties associated with the metastatic state in both MCF-7 and MDA MB435s cells. Overall, these results suggest that Mut.BRCA1 can elicit some of the changes involved in metastatic progression in human breast cancer via the overexpression of OPN.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Genes BRCA1 , Mutação em Linhagem Germinativa , Osteopontina/fisiologia , Animais , Neoplasias da Mama/genética , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Invasividade Neoplásica , Osteopontina/genética , Ratos , Ratos Wistar , TransfecçãoRESUMO
Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Receptores da Eritropoetina/genética , Sequência de Aminoácidos , Especificidade de Anticorpos , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Receptores da Eritropoetina/imunologiaRESUMO
OBJECTIVE: To investigate the significance of mutation and single nucleotide polymorphism (SNP) of class III receptor tyrosine kinases such as PDGFRbeta and SHIP in acute myeloid leukemia (AML) patients. METHODS: Screening of the mutation and SNP of PDGFRbeta and SHIP by genomic PCR, RT-PCR, directly sequencing and Mass-ARRAY system was carried out in 273 AML patients. RESULTS: The mutations of PDGFRbeta R685C and SHIP Q1153L were detected for the first time in AML patients. The positivity ratio was 0.73% and 0.36% respectively. CONCLUSION: The mutations of PDGFRbeta R685C and SHIP Q1153L may contribute to leukemogenesis of AML.
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Fosfatos de Inositol/genética , Leucemia Mieloide Aguda/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Humanos , Espectrometria de Massas , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigate the role of mutations of receptor tyrosine kinases as well as their downstream scaffold molecules in leukemogenesis of acute myeloid leukemia (AML) in Chinese patients. Genes of interest included FLT3, PDGFRbeta, KDR, CSF2Rbeta, SOCS1, PIAS3 and SHIP. The coding sequence of these genes was analysed by the reverse transcription-polymerase chain reaction to search novel mutations. A novel mutation (A > T, Q1154L) of SHIP (1 of 192, 0.52%) was identified and another novel mutation (C > T, R685C) of PDGFRbeta (2 of 192, 1.04%). In addition, FLT3 mutations were seen in three of five patients with AML following myelodysplastic syndrome (60%) and 39 of 268 (14.6%) de novo AML patients (P < 0.05). No mutations were found in the coding sequence regions of KDR, CSF2Rbeta, SOCS1 or PIAS3.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Polimorfismo de Nucleotídeo Único , Receptores Proteína Tirosina Quinases/genética , Sequência de Bases , China , Primers do DNA/química , Humanos , Dados de Sequência Molecular , Síndromes Mielodisplásicas/genética , Reação em Cadeia da Polimerase , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
OBJECTIVE: To observe the effects of inhibiting stromal cell derived factor-1 (SDF-1) expression by RNA interference (RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells. METHODS: SDF-1 specific short hairpin RNA (shRNA) expressing plasmid was transferred into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A) , SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The un-transfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control. RESULTS: The level of secreted SDF-1 protein (pg/10(5) cells/week) in the supernatants of Group A, B and C were 1920 +/- 205, 12,370 +/- 1355 and 6620 +/- 770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8 +/- 2.6)%, (57.4 +/- 3.8)% and (45.2 +/- 4.0)%, respectively, and the IC50 values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively. CONCLUSION: Down-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.
Assuntos
Células da Medula Óssea/metabolismo , Quimiocina CXCL12/genética , Interferência de RNA , Células Estromais/metabolismo , Adesão Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica , Humanos , Células JurkatRESUMO
OBJECTIVE: To study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells. METHOD: Inhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transferring SDF-1 specific short hairpin RNA (shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles, apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The un-transfected acute leukemic (group B) and normal (group C) bone marrow stromal cells were taken as controls. RESULTS: The content of SDF-1 protein in supernatant of group A\[(384 +/- 41) pg/ml] was significantly lower than that in group B[(2474 +/- 271) pg/ml] or group C[(1324 +/- 154) pg/ml]. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B: 29 h, group C: 33 h), and G(0)/G(1) stage cells increased [group A: (28.47 +/- 2.39)%, vs group B: (19.43 +/- 2.80)%, group C: (27.15 +/- 2.07)%], S stage cells decreased [group A: (25.57 +/- 1.90)%, vs group B: (74.48 +/- 3.23)%, group C: (60.99 +/- 2.33)%], G(2)/M stage cells increased [group A: (45.96 +/- 3.24)%, vs group B: (6.09 +/- 1.96)%, group C: (11.86 +/- 1.98)%], the apoptosis rate increased [group A: (15.2 +/- 0.8)%, vs group B: (5.4 +/- 0.7)%, group C: (9.5 +/- 0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased. CONCLUSION: The inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.
Assuntos
Células da Medula Óssea/metabolismo , Quimiocina CXCL12/genética , Interferência de RNA , Células Estromais/metabolismo , Apoptose/genética , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Expressão Gênica , Humanos , Células Jurkat , TransfecçãoRESUMO
Development of resistance to 1-beta-arabinofuranosylcytosine (AraC) is a major obstacle in the treatment of patients with acute myeloid leukaemia (AML). Deficiency of functional deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We screened 5378 bp sequences of the dCK gene, including all exons and the 5' flanking region, and identified two single nucleotide polymorphisms (SNPs) in the regulatory region (rSNPs) with high allele frequencies. These two rSNPs (-201C>T and -360C>G) formed two major haplotypes. Genotyping with sequencing and MassARRAY system among 122 AML patients showed that those with -360CG/-201CT and -360GG/-201TT compound genotypes (n = 41) displayed a favourable response to chemotherapy whereas those with -360CC/-201CC (n = 81) tended to have a poor response (P = 0.025). Moreover, real-time quantitative reverse transcriptase-polymerase chain reaction showed that patients with -360CG/-201CT and -360GG/-201TT genotypes expressed higher level of dCK mRNA compared to those with the -360CC/-201CC genotype (P = 0.0034). Luciferase-reporter assay showed that dCK 5' regulatory region bearing -360G/-201T genotype alone had an eight-fold greater transcriptional activation activity compared to that with -360C/-201C genotype, whereas co-transfection of both -360G/-201T and -360C/-201C constructs mimicked the heterozygous genotype, which exhibited a four-fold greater activity compared to that with -360C/-201C. These results indicate that rSNP haplotypes of dCK gene may serve as a genetic marker for predicting drug responsiveness, which will be beneficial in establishing more effective AML chemotherapeutic regimens.
Assuntos
Desoxicitidina Quinase/genética , Leucemia Mieloide/genética , Polimorfismo de Nucleotídeo Único , Doença Aguda , Sequência de Bases , Primers do DNA , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/enzimologia , Reação em Cadeia da Polimerase , Resultado do TratamentoRESUMO
Both all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) have proven to be very effective in obtaining high clinical complete remission (CR) rates in acute promyelocytic leukemia (APL), but they had not been used jointly in an integrated treatment protocol for remission induction or maintenance among newly diagnosed APL patients. In this study, 61 newly diagnosed APL subjects were randomized into three treatment groups, namely by ATRA, As(2)O(3), and the combination of the two drugs. CR was determined by hematological analysis, tumor burden was examined with real-time quantitative RT-PCR of the PML-RAR alpha (promyelocytic leukemia-retinoic acid receptor alpha) fusion transcripts, and side effects were evaluated by means of clinical examinations. Mechanisms possibly involved were also investigated with cellular and molecular biology methods. Although CR rates in three groups were all high (> or =90%), the time to achieve CR differed significantly, with that of the combination group being the shortest one. Earlier recovery of platelet count was also found in this group. The disease burden as reflected by fold change of PML-RAR alpha transcripts at CR decreased more significantly in combined therapy as compared with ATRA or As(2)O(3) mono-therapy (P < 0.01). This difference persisted after consolidation (P < 0.05). Importantly, all 20 cases in the combination group remained in CR whereas 7 of 37 cases treated with mono-therapy relapsed (P < 0.05) after a follow-up of 8-30 months (median: 18 months). Synergism of ATRA and As(2)O(3) on apoptosis and degradation of PML-RAR alpha oncoprotein might provide a plausible explanation for superior efficacy of combination therapy in clinic. In conclusion, the ATRA/As(2)O(3) combination for remission/maintenance therapy of APL brings much better results than either of the two drugs used alone in terms of the quality of CR and the status of the disease-free survival.