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Background: Peripheral T-cell lymphoma (PTCL) is a heterogeneous disease with dismal outcomes. We conducted an open-label, phase 2 nonrandomised, externally controlled study to evaluate the efficacy and safety of targeted agents plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) (CHOPX) for PTCL in the front-line setting. Methods: Eligible patients were ≥18 years of age and newly diagnosed PTCL. Patients in the CHOPX group received standard CHOP at Cycle 1. Specific targeted agents were added from Cycle 2, decitabine if TP53 mut, azacytidine if TET2/KMT2D mut, tucidinostat if CREBBP/EP300 mut, and lenalidomide if without mutations above. Patients in the CHOP group received CHOP for 6 cycles. The primary endpoint was the complete response rate (CRR) at the end of treatment (EOT). Secondary endpoints included overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety. The study was registered with ClinicalTrials.gov, NCT04480099. Findings: Between July 29, 2020, and Sep 22, 2022, 96 patients were enrolled and included for efficacy and safety analysis with 48 in each group. The study met its primary endpoint. CRR at EOT in the CHOPX group was superior to the CHOP group (64.6% vs. 33.3%, OR 0.27, 95%CI 0.12-0.64; p = 0.004). At a median follow-up of 24.3 months (IQR 12.0-26.7), improved median PFS was observed in the CHOPX group (25.5 vs. 9.0 months; HR 0.57, 95%CI 0.34-0.98; p = 0.041). The median OS was similar between two groups (not reached vs. 30.9 months; HR 0.55, 95%CI 0.28-1.10; p = 0.088). The most common grade 3-4 hematological and non-hematological adverse events in the CHOPX group were neutropenia (31, 65%) and infection (5, 10%). Interpretation: Targeted agents combined with CHOP demonstrated effective and safe as first-line treatment in PTCL. Biomarker-driven therapeutic strategy is feasible and may lead to promising efficacy specifically toward molecular features in PTCL. Funding: This study was supported by the National Key Research and Development Program (2022YFC2502600) and the General Program of the Shanghai Municipal Health Commission (202040400).
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Background: Orelabrutinib is a novel, small molecule, selective irreversible Bruton tyrosine kinase inhibitor. The purpose of this study was to evaluate the efficacy and safety of orelabrutinib in patients with relapsed or refractory Waldenström's macroglobulinemia (R/R WM). Methods: This is a prospective, multicenter study of orelabrutinib in patients with WM who had at least one prior line of treatment. Orelabrutinib was administered orally at a daily dose of 150 mg until disease progression or unacceptable toxicity. The primary endpoint was major response rate (MRR) assessed by the Independent Review Committee (IRC) according to IWWM-6. This study is registered with ClinicalTrials.gov, NCT04440059. This trial was also registered on Center for Drug Evaluation (www.chinadrugtrials.org.cn) in March 2019, with a number of CTR2019036. Findings: Between August 2019 and December 2020, 66 R/R WM patients were assessed for eligibility. Forty-seven eligible patients were evaluated for efficacy at a median follow-up of 16.4 months (interquartile range: 12.5, 19.5). As assessed by IRC, the MRR was 80.9%, and the overall response rate was 89.4%. The median time to at least a minor response was 1.9 months. The PFS rates was 89.4% at 12 months. For patients with MYD88L265P /CXCR4NEG, MYD88L265P /CXCR4 S338X, and MYD88NEG /CXCR4NEG mutations, the MRRs were 84.6%, 100%, and 25.0%. Most adverse events were Grades 1 or 2 (91.0%). The common grade 3 or higher adverse events occurring were neutropenia (10.6%), thrombocytopenia (6.4%), and pneumonia (4.3%). Serious adverse events (SAE) occurred in 10 patients (21.3%). One treatment-related death was reported (hepatitis B reactivation). Interpretation: Orelabrutinib has shown good efficacy and manageable safety profiles in patients with R/R WM. Funding: InnoCare Pharma.
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CRISPR/Cas system has become one of the most powerful techologies in biomedical research, and has showed great potentials in the gene related diseases. However, efficient delivery systems of CRISPR/Cas to target cells remains challenging. In recent years, nanoparticles have showned great potentials for the delivery of CRISPR/Cas systems. This paper mainly approaches the development and new strategies of CRISPR/Cas delivery systems, as well as their application in the clinical diseases. By summarizing the CRISPR/Cas systems delivery, new strategies are expected for the gene therapy.
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Edição de Genes , Nanopartículas , Sistemas CRISPR-Cas/genética , Terapia GenéticaRESUMO
Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection.
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Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Anticorpos Facilitadores , Vírus da Dengue/imunologia , Precursores de Proteínas/imunologia , Proteínas do Envelope Viral/imunologia , Aedes/citologia , Aedes/virologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Reações Cruzadas , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos/genética , Peptídeos/imunologia , Precursores de Proteínas/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Dengue Grave/imunologia , Dengue Grave/virologia , Proteínas do Envelope Viral/genéticaRESUMO
Growing evidence reported that Golgi phosphoprotein 3 (GOLPH3) was involved in the progression of several human cancers. To determine whether knockout of GOLPH3 enhances the effect of Chemotherapy against cell growth of oral squamous cell carcinoma in vitro. OSCC cells were transfected with Golph3 plasmid, Golph3-RNAi and the relative control plasmids. Transfected Tca-8113 cells treated with cis-Dichlorodiamineplatinum (DDP; 0, 0.05, 0.25, 1.25, 6.25 and 31.25 ug/ml) or Paclitaxe (0, 2, 10, 50, 250 and 1250 nM) or Adriamycin (0, 0.25, 0.5, 1, 2 and 4 ug/ml) for 24 h, respectively, was determined using MTT assay. Apoptosis-related protein expression Cytochrome-C, Caspase3 and Bcl-2 was analyzed by RT-PCR and western blots. Result of MTT showed that Golph3-RNAi transfected Tca-8113 cells enhanced the effect of chemotherapy, and the effect was strengthened with the increasing concentration of drugs, and the Golph3 plasmid transfected Tca-8113 cells showed the opposite effect. RT-PCR and western blots assays revealed that expression of cytochrome-C and caspase3 were up-regulated, while Bcl-2 expression was down-regulated in Golph3-RNAi transfected Tca-8113 cells. Taken together, this study demonstrated that GOLPH3 had potent pro-tumor growth and decreased the effect of Chemotherapy, and its mechanism is primarily via cell anti-apoptosis, down-regulating the expression of cytochrome-C and caspase3, up-regulating Bcl-2 expression.
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AIM: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro. METHODS: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR. RESULTS: Oridonin (2-12 µmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 µmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53. CONCLUSION: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis.
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Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Diterpenos do Tipo Caurano/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Nucleares/genética , Transporte Proteico/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Isodon/química , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas Nucleares/metabolismo , NucleofosminaRESUMO
AIM: To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes. METHODS: The proliferation of human multiple myeloma KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting. RESULTS: Treatment of KM3 cells with BA (5-25 µg/mL) suppressed the cell proliferation in time- and dose-dependent manners. The IC(50) values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 µg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-II and P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis. CONCLUSION: The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells.
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Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mieloma Múltiplo/patologia , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Western Blotting , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Triterpenos Pentacíclicos , Triterpenos/uso terapêutico , Ácido BetulínicoRESUMO
Imatinib mesylate is a molecular targeted agent for treating chronic myeloid leukemia (CML) and gastrointestinal stromal tumor. Although imatinib mesylate is not regarded as an immunosuppressive agent, few studies have also shown that it may impair immune response. In this report, we present a case of transient hepatitis B virus (HBV) reactivation during imatinib mesylate treatment for CML.
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Vírus da Hepatite B/patogenicidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/virologia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Benzamidas , Humanos , Mesilato de Imatinib , Masculino , Replicação Viral/efeitos dos fármacosRESUMO
Multiple myeloma (MM) is the second most commonly diagnosed hematologic malignancy. Although new drugs, including bortezomib and lenalidomide, have improved the treatment landscape for MM patients, MM remains incurable. Therefore, screening for novel anti-myeloma drugs is necessary. Gambogic acid (GA), the main active ingredient of gamboges secreted from the Garcinia hanburryi tree, has been reported to exhibit potent anticancer activity in certain solid tumors and hematological malignancies, while there are few studies that are available concerning its effects on MM cells. In the present study, we investigated the anticancer activity of GA on the MM RPMI-8226 cells and further studied the underlying mechanisms by which GA affected the cells. RPMI-8226 cells were cultured and the effect of GA on cell proliferation was analyzed using MTT assay. Hoechst 33258 staining was used to visualize nuclear fragmentation, and reactive oxygen species (ROS) levels were detected. GA was found to have a significant, dose-dependent effect on growth inhibition and apoptosis induction in RPMI-8226 cells. This activity is associated with the accumulation of ROS, which contributes to the activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP), accompanied with apoptosis in RPMI-8226 cells treated with GA. Mammalian SIRT1, as the closest homolog of the yeast Sir2, was extensively involved in regulating cell processes, including cell senescence, aging and neuronal protection, as well as having anti-apoptotic properties. Moreover, SIRT1 overexpression has been shown to protect cancer cells from chemotherapy and ionizing radiation. In the present study, we demonstrated that GA has the potential to downregulate the expression of SIRT1 via ROS accumulation. In conclusion, our study found that GA is able to induce apoptosis in RPMI-8226 cells via ROS accumulation followed by caspase-3 activation, PARP cleavage and SIRT1 downregulation. These results suggest that GA may have the potential to not only induce apoptosis in MM cells, but also to decrease the relapse rate of MM.
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OBJECTIVE: To probe the cause for triggering HBV reactivation and possible management of the chronic hepatitis B individuals received imatinib. METHODS: This study presented two cases of transient hepatitis B virus (HBV) reactivation and hepatic dysfunction during oral imatinib for chronic myeloid leukemia (CML) and made a literatures review about the pathogenesis, possible prophylactic and therapeutic management of such chronic hepatitis B individuals receiving imatinib. RESULTS: Two CML patients, without prior liver dysfunction but with chronic HBV infection, suffered from transient HBV reactivation occurred during oral imatinib. Both of them finally obtained good outcome following the additional oral nucleotide antiviral therapy. CONCLUSION: It remained unclear whether imatinib induced the reactivation of HBV in patients with a latent HBV infection. From our study, all candidates receiving oral imatinib should be screened for HBsAg and anti-HBc antibodies prior to initiation of imatinib. Prophylactic antiviral therapy should be offered to HBV-infected individuals along with a close monitoring for signs of reactivation.
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Benzamidas/efeitos adversos , Vírus da Hepatite B/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/virologia , Piperazinas/efeitos adversos , Pirimidinas/efeitos adversos , Ativação Viral/efeitos dos fármacos , Adulto , Benzamidas/uso terapêutico , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Piperazinas/uso terapêutico , Pirimidinas/uso terapêuticoRESUMO
AIM: To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro. METHODS: Human multiple myeloma RPMI8266 cells were used. The cell viability was assessed using MTT assay. Morphological changes of apoptosis and autophagy were observed under transmission electron microscope. TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs(605 nm)-Anti-LC3 fluorescent probe. Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe. Protein levels of active caspase 3, Beclin 1 and SIRT1 were determined with Western blot analysis. RESULTS: Exposure to oridonin (1-64 µmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC(50) value of 6.74 µmol/L. Exposure to oridonin (7 µmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells. Both the apoptosis and autophagy were time-dependent, and apoptosis was the main effector pathway of cell death. Exposure to oridonin (7 µmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner. The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis, autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 µmol/L). The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis, which was accompanied by increased intracellular ROS and decreased SIRT1. CONCLUSION: Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein. The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway, whereas the autophagy protects the cells from apoptosis.
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Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diterpenos do Tipo Caurano/farmacologia , Mieloma Múltiplo/patologia , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Diterpenos do Tipo Caurano/uso terapêutico , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Sirtuína 1/genéticaRESUMO
To investigate the anti-cancer effects and molecular mechanism of deguelin on the human leukemia HL-60 cells, to explore the expression and clinical significance of p-AKT, survivin and Bcl-2 in leukemia cell line HL-60 cell. Cell growth rate was assessed by MTT assay. Apoptotic index was evaluated by TUNEL staining. Apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit and transmission electron microscopy (TEM), expression of p-Akt, Bcl-2 and surviving in HL-60 cells was checked by Western blot. Deguelin presented striking proliferation inhibition potency on HL-60 cells in vitro, with the I(C50) value for 48 h being 20.14 nM, and induced apoptosis in HL-60 in a concentration-time-dependent manner. Apoptotic bodies and cell shrinkage and fragmentation were observed by TUNEL and TEM. Deguelin-induced cells morphological changes and degraded several kinase proteins, including Bcl-2 and survivin (members of lap). The degradation of these kinases blocked PI3K/Akt survival signal pathways, inducing apoptosis. Deguelin may induce HL-60 cell apoptosis through depletion of multiple kinase proteins and blockage of survival signal pathways of HL-60 cells.
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Apoptose/efeitos dos fármacos , Rotenona/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases , Rotenona/farmacologiaRESUMO
PI3K-Akt signal pathway is an important intracellular signal transduction pathway. It plays important roles in cell apoptosis and survival by affecting the activity of downstream effector molecules, and it is closely associated with the development and progression of human tumors, therefore, PI3K and Akt might be potential targets for tumor therapy. This article reviewed recent progress on the composition, function, regulation and anti-apoptosis of PI3K-Akt signal pathway, and discussed its potential application to tumor gene therapy.
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Apoptose , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate antitumor activity and molecular mechanism of deguelin to the human U937 leukaemia cells and to explore the mechanisms regulating cell cycle and nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. METHODS: The effects of deguelin on the growth of U937 cells were studied by MTT assay, and the cell cycle of U937 cells by a propidium iodide method. The localization of the nuclear pore complex protein Nup98 and Nup88 was checked by immunofluorescence and immunoelectron microscopy. The expressions of Nup98 and Nup88 in U937 cells were checked by flow cytometry (FCM) and Western blot respectively. RESULTS: The proliferation of U937 cells was significantly inhibited in a time-dose dependent manner in deguelin-treated group with a 24 h IC50 value of 21.61 nmol/L and 36 h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin showed reduction in the percentages of cells in G0/G1, whereas accumulation of cells in S and G2/M phase. The ratio of G1/G0 phase cells were 73.01%, 71.15%, 68.42%, 52.45%, 43.99% and 22.82%, and that of S phase cells were 17.18%, 16.30%, 18.09%, 27.56%, 31.21% and 46.85%, and that of G2/M phase cells were 9.75%, 12.31%, 13.09%, 18.99%, 24.83% and 27.79% at deguelin concentrations of 0, 5, 10, 20, 40, 80 nmol/L respectively. Nup88 and Nup98 were found on both the nuclear and cytoplasmic side of the U937 cells. The expression of Nup98 was up-regulated and Nup88 down-regulated in deguelin treated U937 cells. CONCLUSION: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle. The antitumor activity of deguelin was related to up-regulating the expression of Nup98 and down-regulating Nup88 protein.
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Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Rotenona/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Rotenona/farmacologia , Células U937RESUMO
OBJECTIVE: To investigate the anticancer effects and molecular mechanism of deguelin on human Burkitt's lymphoma Daudi cells in vitro and compare the cytotoxicity of deguelin on Daudi cells and human peripheral blood monocular cells (HPBMC). METHODS: The effects of deguelin on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5 diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was assessed through Hoechst 33258 staining and annexin V/PI double-labeled cytometry. The effect of deguelin on the cell cycle of Daudi cells was studied by propidium iodide method. The expressions of cyclin D1 and pRb were checked by Western blot. RESULTS: The proliferation of Daudi cells was decreased in deguelin-treated group with a 24 h IC50 value of 51. 55 nmol/L. Deguelin induced Daudi cells apoptosis in a time- and dose-dependent manner. Daudi cells treated with deguelin showed an increase of G0/G1 phase and decrease of S phase. The Daudi cells treated with 0, 5, 10, 20, 40 nmol/L deguelin for 24 h, showed an increased Go/G, phase from 37.34% to 56.56% , whereas decreased S phase from 37.72% to 21.36%, respectively. PBMC was less sensitive to the cytotoxic effect of deguelin than Daudi cells. The expression of cyclin D1 and pRb protein were decreased in Daudi cells treated with deguelin. CONCLUSION: Deguelin can inhibit the proliferation of Daudi cells by regulating the cell cycle that arrest cells at G0/G1 phase and inducing apoptosis. Moreover, deguelin demonstrats low toxicity in PBMC but selectively induces apoptosis of Daudi cells. The antitumor effects of deguelin may be related to down-regulation of the expression of cyclin D1 and pRb protein.
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Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Rotenona/análogos & derivados , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Rotenona/administração & dosagem , Rotenona/farmacologiaRESUMO
OBJECTIVE: To study the expression of stromal cell derived factor-1 (SDF-1) and its receptor CXCR4 in non-Hodgkin lymphoma (NHL), and to investigate the role of this biological axis in tumor metastasis of NHL. METHODS: Specimens of bone marrow were collected from 4 patients with NHL with bone marrow infiltration and 4 cases of NHL without bone marrow infiltration, and 4 patients with benign hepatopathy with normal myelogram. Lymphoma specimens were collected from 7 cases of NHL and 3 cases of reactive proliferative lymphadenitis. The level of SDF-1alpha mRNA in the stromal cells of bone marrow and lymph node were determined by RT-PCR. The CXCR4 expression on lymphoma cells freshly isolated from bone marrow and lymph node of the NHL patients were studied by flow cytometry. Transwell assay was performed, the upper chamber being added with suspension of cells newly isolated from lymph nodes or bone marrow undergoing co-incubation with ant-CXCR4 monoclonal antibody or not, and the lower chamber added with culture fluid with or without recombinant human SDF-1alpha (rhSDF-1alpha). The migration rate of the primary lymphoma cells towards rhSDF-1alpha was calculated. RESULTS: The expression levels of CXCR4 on the lymphoma cells isolated from the bone marrow involved or not involved by NHL were both significantly higher than that of the mononuclear cells from normal bone marrow (both P < 0.01), whereas the CXCR4 expression level on the lymphoma cells from the lymph nodes of the NHL patients was also significantly higher than that of the mononuclear cells from the lymph nodes of reactive proliferative lymphadenitis (P < 0.01). The SDF-1alpha mRNA expression level in the stromal cells isolated from the bone marrow involved by NHL was significantly higher than those of the stromal cells of bone marrow not involved by NHL and the normal bone marrow (both P < 0.01). Moreover, the SDF-1alpha expression level in the lymph node stromal cells of all the NHL patients was also significantly higher than that of the patients with reactive proliferative lymphadenitis (P < 0.01). Transwell assay revealed that all three kinds of primary lymphoma cells migrated towards rhSDF-1alpha effectively, and this migration was markedly inhibited by the addition of anti-CXCR4 monoclonal antibody to the lymphoma cells (all P < 0.01). CONCLUSION: SDF-1/CXCR4 axis plays a uniquely important biological role in mediating tumor metastasis of NHL.
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Quimiocina CXCL12/genética , Linfoma não Hodgkin/patologia , Receptores CXCR4/genética , Adolescente , Adulto , Idoso , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/fisiologia , Feminino , Citometria de Fluxo , Humanos , Metástase Linfática , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/biossíntese , Receptores CXCR4/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In order to study the relation of antitumour mechanisms of triptolide with neovascularization, the effect of triptolide and tumour necrosis factor (TNF)-alpha on the expression of vascular endothelial growth factor (VEGF) in Raji cell lines and their effect on angiogenesis in human umbilical vein endothelial cells (HUVECs)-derived cell line ECV304 were investigated. The inhibitory rate of cells treated by triptolide detected by MTT; the ELISA was employed to study the VEGF content secreted by Raji cell lines; angiogenesis was tested by network formation of endothelial cells on Matrigel, and the mRNA level of VEGF was measured by RT-PCR. The results showed that treatment of Raji cells with triptolide resulted in significantly enhanced antiproliferative effects in dose- and time-dependent manner. The content of VEGF secreted by Raji cells was increased by TNF-alpha and was suppressed by triptolide (P < 0.01). The mRNA expressions of VEGF(165) and VEGF(121) (containing 165 and 121 amino acid residues, respectively) could be detected in all fractions. TNF-alpha augmented the expression of VEGF(165) and VEGF(121) mRNA when triptolide reduced the expression (P < 0.01). No network and cord were formed in control and triptolide group. There was tube formation on matrigel in the supernatants of Raji culture group and the supernatants groups treated by VEGF and TNF-alpha in Raji cell. It is concluded that the expressions of VEGF in Raji cells are increased by TNF-alpha and suppressed by triptolide. VEGF and TNF-alpha induce angiogenesis and triptolide inhibits angiogenesis in ECV304 cells.
Assuntos
Inibidores da Angiogênese/farmacologia , Diterpenos/farmacologia , Células Endoteliais/metabolismo , Fenantrenos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Crescimento do Endotélio Vascular/biossíntese , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular , Células Endoteliais/citologia , Compostos de Epóxi/farmacologia , Humanos , Linfoma de Células B/patologia , Neovascularização Fisiológica/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais Cultivadas , Veias Umbilicais/citologia , Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
AIM: To investigate the antiproliferative effect of triptolide on B-NHL cell line Raji cells, to study its effect on lymph node metastasis in patients with non-Hodgkin's lymphoma (NHL) in vitro, and to explore the underlying mechanism regulating SDF-1/CXCR4 axis. METHODS: The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effects of triptolide on SDF-1 mRNA expression in lymph node stromal cells from patients with NHL were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effects of triptolide on CXCR4 expression on lymphoma cells freshly isolated from the lymph nodes of these patients were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of primary lymphoma cells towards recombinant human SDF-1 alpha (rhSDF-1 alpha) or cultured lymph node stromal cells in vitro. RESULTS: Triptolide inhibited the proliferation of B-NHL cell line Raji cells in a dose- and time-dependent manner with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. The expression of SDF-1alpha mRNA in lymph node stromal cells obtained from patients with NHL was decreased after treatment by triptolide at concentrations of 25 and 50 nmol/L for 24 h. Flow cytometry analysis showed that the CXCR4 expression on primary lymphoma cells were downregulated gradually in a dose-dependent manner following triptolide treatment. Chemotaxis assays revealed that the migration of freshly isolated lymphoma cells towards either rhSDF-1 or cultured lymph node stromal cells was markedly inhibited by the addition of triptolide in vitro, and the inhibition was dose-dependent. CONCLUSION: Triptolide can inhibit the proliferation of B-NHL cell line Raji cells. Moreover, triptolide is able to inhibit the migration of lymphoma cells via lymph nodes in vitro. The potential antitumor mechanisms of triptolide are related to the antiproliferative effect and the blockage of SDF-1/CXCR4 axis.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Quimiocina CXCL12/biossíntese , Diterpenos/farmacologia , Linfonodos/patologia , Linfoma não Hodgkin/metabolismo , Fenantrenos/farmacologia , Receptores CXCR4/metabolismo , Antineoplásicos Alquilantes/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/genética , Quimiotaxia/efeitos dos fármacos , Diterpenos/administração & dosagem , Relação Dose-Resposta a Droga , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacologia , Humanos , Metástase Linfática , Linfoma de Células B/patologia , Linfoma não Hodgkin/patologia , Fenantrenos/administração & dosagem , Plantas Medicinais/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Estromais/metabolismo , Tripterygium/químicaRESUMO
AIM: To determine whether deguelin can regulate the expression of nuclear factor kappa B (NF-kappaB) binding protein (IkappaBalpha) in U937 human leukemia cells and Raji human B lymphoma cells. METHODS: The localization of IkappaBalpha protein was investigated by using an immunofluorescence method. The expression of IkappaBalpha and NF-kappaB /p65 proteins in Raji and U937 cells were investigated by using Western blotting. Apoptosis was detected through annexin V/PI double-labeled cytometry. RESULTS: IkappaBalpha localized in the cytoplasm in untreated and deguelin-treated cells. After treatment with tumor necrosis factor alpha (TNF-alpha) or deguelin plus TNF-alpha for 15 min, there was a substantial reduction in the amount of IkappaBalpha protein. The expression of IkappaBalpha was downregulated by deguelin in Raji and U937 cells. Deguelin induced apoptosis in U937 cells. CONCLUSION: Deguelin inhibited the expression of IkappaBalpha protein in U937 and Raji cells. The anti-proliferative activity of deguelin is related to the signal pathway of NF-kappaB.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Linfoma de Células B/patologia , Rotenona/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Linfoma de Células B/metabolismo , Inibidor de NF-kappaB alfa , Rotenona/farmacologia , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
AIM: To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. METHODS: The effects of deguelin on the growth of U937 cells were studied by MTT assay. The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot. RESULTS: The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G(0)/G(1) phase, whereas cells accumulated in the S and G(2)/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin. CONCLUSION: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G(2)/M phases, so that the proportion of cells in the G(0)/G(1) phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.