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1.
Anal Chim Acta ; 1036: 80-88, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30253840

RESUMO

Cronobacter spp. are recognized as a world-wide emerging opportunistic food-borne pathogens that can persist in various food and processing environment for a long time. Therefore, the prevention and detection of them are particularly important. In this study, a micro-spot paper-based analytical device (µPADs) was created by combining PVC pad with filter paper. Detection is achieved by measuring the color change (from colorless to indigo) when a species-specific enzyme associated with the Cronobacter spp. of interest reacts with a chromogenic substrate. When combined with the optimization of specific enrichment process, the method allows for a testing time of 10 h or less and is capable of detecting live bacteria on the inoculated surface of samples in concentrations as low as 101 CFU cm-2. We are surprised to discover that C. dublinensis species and their subspecies had the highest ability to produce α-glucosidase in all genus of Cronobacter spp.. This work demonstrated that the manufacturing method is novel, simple, well reproducible (RSD less than 5%) and low-cost (less than $ 0.15 per micro-spot).


Assuntos
Colorimetria/economia , Cronobacter/isolamento & purificação , Contaminação de Alimentos/análise , Papel , Cronobacter/genética , Microbiologia de Alimentos , Conformação Molecular , Propriedades de Superfície
2.
J Dairy Sci ; 101(5): 3835-3843, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29501338

RESUMO

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 103 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.


Assuntos
Cronobacter sakazakii/isolamento & purificação , Contaminação de Alimentos/análise , Imunoensaio/métodos , Fórmulas Infantis/microbiologia , Cronobacter sakazakii/crescimento & desenvolvimento , Ouro/química , Imunoensaio/instrumentação , Fórmulas Infantis/análise , Nanopartículas Metálicas/química , Pós/análise , Especificidade da Espécie
3.
Blood ; 130(8): 995-1006, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28646116

RESUMO

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.


Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Antígenos/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Técnicas de Inativação de Genes , Centro Germinativo/patologia , Humanos , Linfoma Difuso de Grandes Células B/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
4.
Mol Cancer Ther ; 12(11): 2494-504, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23990113

RESUMO

Mantle cell lymphoma (MCL) remains incurable due to its inevitable pattern of relapse after treatment with current existing therapies. However, the promise of a cure for MCL lies in the burgeoning area of novel agents. In this study, we elucidated the therapeutic effect and mechanism of carfilzomib, a novel long-acting second-generation proteasome inhibitor, in MCL cells. We found that carfilzomib induced growth inhibition and apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells was mediated by the activation of JNK, Bcl-2, and mitochondria-related pathways. In addition, carfilzomib inhibited the growth and survival signaling pathways NF-κB and STAT3. Interestingly, we discovered that expression of immunoproteasome (i-proteasome) subunits is required for the anti-MCL activity of carfilzomib in MCL cells. In MCL-bearing SCID mice/primary MCL-bearing SCID-hu mice, intravenous administration of 5 mg/kg carfilzomib on days 1 and 2 for 5 weeks slowed/abrogated tumor growth and significantly prolonged survival. Our preclinical data show that carfilzomib is a promising, potentially less toxic treatment for MCL. Furthermore, an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.


Assuntos
Antineoplásicos/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Oligopeptídeos/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Linfoma de Célula do Manto/patologia , Masculino , Camundongos , Camundongos SCID , Mitocôndrias/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
5.
Exp Hematol ; 41(1): 67-78.e4, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22986101

RESUMO

Overexpression of the cellular nuclear exportin 1, more commonly called chromosomal region maintenance 1 (CRM1), has been associated with malignant progression and mortality. Therefore, activation of nuclear export can play a significant etiologic role in some forms of human neoplasia and serve as a novel target for the treatment of these cancers. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that remains incurable. The objective of this study was to investigate the functional significance of CRM1 in MCL by evaluating the therapeutic efficacy of CRM1 inhibition in MCL in vitro and in vivo. Our results showed that CRM1 is highly expressed in MCL cells and is involved in regulating growth and survival mechanisms through the critical nuclear factor-κB survival pathway, which is independent of p53 status. Inhibition of CRM1 by two novel selective inhibitors of nuclear export (SINE), KPT-185 and KPT-276, in MCL cells resulted in significant growth inhibition and apoptosis induction. KPT-185 also induced CRM1 accumulation in the nucleus, resulting in CRM1 degradation by the proteasome. Oral administration of KPT-276 significantly suppressed tumor growth in an MCL-bearing severe combined immunodeficient mouse model, without severe toxicity. Our data suggest that SINE CRM1 antagonists are a potential novel therapy for patients with MCL, particular in relapsed/refractory disease.


Assuntos
Acrilatos/farmacologia , Carioferinas/antagonistas & inibidores , Linfoma de Célula do Manto/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/farmacologia , Acrilatos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Linfoma de Célula do Manto/patologia , Camundongos , NF-kappa B/antagonistas & inibidores , RNA Interferente Pequeno/genética , Triazóis/uso terapêutico , Proteína Supressora de Tumor p53/fisiologia , Proteína Exportina 1
6.
Cancer ; 119(4): 782-91, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22915070

RESUMO

BACKGROUND: Mantle cell lymphoma (MCL) is an incurable B-cell malignancy, and patients with this disease have the poorest prognosis among all patients with B-cell lymphomas. The signaling pathways that trigger MCL escape from immune surveillance are unclear. Because Toll-like receptors (TLRs) initiate innate and adaptive immune responses against invading pathogens, the authors investigated the impact of TLR signaling in MCL cells. METHODS: TLR expression was examined in MCL cell lines and in primary tumors. The examination focused on TLR4 and its ligand lipopolysaccharide (LPS) on MCL cells and their function on MCL proliferation and immune evasion. RESULTS: MCL cells expressed multiple TLRs, and TLR4 was among the highest expressed molecules. The activation of TLR4 signaling in MCL cells by LPS induced MCL proliferation and up-regulated the secretion of cytokines like interleukin-6 (IL-6), IL-10, and vascular endothelial growth factor (VEGF). LPS-pretreated MCL cells inhibited the proliferation and cytolytic activity of T cells by secreted IL-10 and VEGF, and neutralizing antibodies against these cytokines restored their functions. Similar results were observed in TLR4-positive/myeloid differentiation 88 (MyD88)-positive primary lymphoma cells but not in TLR4-positive/MyD88-negative primary lymphoma cells from patients with MCL. Knockdown of TLR4 on MCL cells abrogated the effect of LPS on MCL cells in term of cell growth or secretion of the cytokines and evasion of the immune system. CONCLUSIONS: The current results indicated that TLR4 signaling triggers a cascade that leads to MCL growth and evasion from immune surveillance. Thus, TLR4 signaling molecules may be novel therapeutic targets in patients with MCL.


Assuntos
Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/metabolismo , Receptor 4 Toll-Like/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Humanos , Evasão da Resposta Imune , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Linfoma de Célula do Manto/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Carcinogenesis ; 33(12): 2568-77, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22971577

RESUMO

Members of the metallothionein (MT) family are short, cysteine-rich proteins involved in metal metabolism and detoxification, suggesting that MT proteins protect cells from damage caused by electrophilic carcinogens and thereby constitute a critical surveillance system against carcinogenesis. However, the roles of MT proteins in human hepatocellular carcinoma (HCC) are not fully understood. We identified a member of the MT family, termed MT1M. MT1M is expressed in various normal tissues with the highest level in the liver. MT1M expression can be induced by heavy metals and protect Escherichia coli from heavy metal toxicity. However, MT1M expression markedly decreased in human HCC specimens. A methylation profiling analysis indicated that the MT1M promoter is methylated in the majority of HCC tumors examined. Moreover, restored expression of MT1M in the HCC cell line Hep3B, which lacks endogenous MT1M expression, suppressed cell growth in vitro and in vivo and augmented apoptosis induced by tumor necrosis factor α. Furthermore, stable expression of MT1M in Hep3B cells blocked tumor necrosis factor α-induced degradation of IκBα and transactivation of NF-κB. We conclude that MT1M is a novel member of the MT family. Frequent downregulation of MT1M in human HCC may contribute to liver tumorigenesis by increasing cellular NF-κB activity.


Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Metalotioneína/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Metalotioneína/antagonistas & inibidores , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/fisiologia , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/farmacologia
8.
Lancet Oncol ; 13(7): 716-23, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22677155

RESUMO

BACKGROUND: The combination of rituximab and lenalidomide has shown promise for the treatment of mantle-cell lymphoma (MCL) in preclinical studies. We aimed to identify the maximum tolerated dose (MTD) of lenalidomide when combined with rituximab in a phase 1 trial and to assess the efficacy and safety of this combination in a phase 2 trial in patients with relapsed or refractory MCL. METHODS: Patients with relapsed or refractory MCL who had received one to four previous lines of treatment were enrolled in this single-arm, open-label, phase 1/2 trial at MD Anderson Cancer Center. In phase 1, to identify the MTD of lenalidomide, four patient cohorts received escalating doses (10, 15, 20, and 25 mg) of daily oral lenalidomide on days 1-21 of each 28-day cycle. 375 mg/m(2) intravenous rituximab was also administered in four weekly doses during cycle 1 only. In phase 2, patients received rituximab plus the MTD of lenalidomide, following the same cycles as for phase 1. Treatment in both phases continued until disease progression, stem-cell transplantation, or severe toxicity. The primary efficacy endpoint was overall response (complete or partial response). The secondary efficacy endpoint was survival. We used the Kaplan-Meier method to estimate response duration, progression-free survival, and overall survival. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00294632. FINDINGS: 52 patients were enrolled between Feb 10, 2006 and July 30, 2009, 14 in phase 1 and 44 (including six patients who received the MTD of lenalidomide in the phase 1 portion) in phase 2. The MTD was 20 mg lenalidomide. One patient who was treated with 25 mg lenalidomide developed a grade 4 non-neutropenic infection and died. In the phase 2 portion of the study, grade 3-4 haematological toxicities included neutropenia (29 patients), lymphopenia (16 patients), leucopenia (13 patients), and thrombocytopenia (ten patients). There were only two episodes of febrile neutropenia. Among 44 patients in phase 2, 25 (57%) had an overall response: 16 (36%) had a complete response and nine (20%) had a partial response. The median response duration was 18·9 months (95% CI 17·0 months to not reached [NR]). The median progression-free survival was 11·1 months (95% CI 8·3 to 24·9 months), and the median overall survival was 24·3 months (19·8 months to NR). Five of 14 patients who had received bortezomib treatment before enrolment achieved an overall response. INTERPRETATION: Oral lenalidomide plus rituximab is well tolerated and effective for patients with relapsed or refractory MCL. FUNDING: Celgene.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/efeitos adversos , Intervalo Livre de Doença , Feminino , Humanos , Lenalidomida , Linfoma de Célula do Manto/mortalidade , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva , Rituximab , Talidomida/administração & dosagem , Talidomida/efeitos adversos , Talidomida/análogos & derivados
9.
Leuk Res ; 36(3): 363-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22000823

RESUMO

The proteasome inhibitor bortezomib (BTZ) is known to be chemotherapeutic in relapsed or refractory mantle cell lymphoma (MCL). Atiprimod (ATI), a novel cationic amphophilic compound, has been tested in clinical trials in multiple myeloma (MM). We sought to evaluate the effect of an ATI-BTZ combination on MCL and to elucidate its therapeutic mechanisms. The ATI and BTZ combination significantly inhibited growth and induced apoptosis of both cultured MCL cell lines and freshly isolated tumor cells from patients with refractory or relapsed MCL. However, the combination yielded lower cytotoxicity in normal peripheral blood mononuclear cells (PBMC). Furthermore, ATI and BTZ induced apoptosis via two different signaling pathways. More significantly, ATI and BTZ markedly delayed tumor growth and prolonged survival in MCL-bearing NOD-SCID mice. Our results demonstrate that ATI and BTZ confer significant therapeutic effects in MCL in vitro and in vivo and should therefore be investigated in a clinical trial in patients with relapsed or refractory MCL.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ácidos Borônicos/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/patologia , Pirazinas/uso terapêutico , Compostos de Espiro/uso terapêutico , Animais , Fator de Indução de Apoptose/metabolismo , Western Blotting , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma de Célula do Manto/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Taxa de Sobrevida
10.
Leuk Res ; 35(3): 380-6, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21047686

RESUMO

Mantle cell lymphoma (MCL) frequently relapses after therapy and new therapeutic regimens are needed. Lenalidomide (LEN), a thalidomide analogue, displays direct cytotoxicity against MCL cells. This study was undertaken to evaluate the combined therapeutic effect of LEN and dexamethasone (DEX) on MCL. LEN synergized with DEX to induce the growth inhibition and apoptosis of both established MCL cells and freshly isolated MCL cells from refractory or relapsed MCL patients. The synergy was more significant in freshly isolated patients' MCL cells than established MCL cells. Cell cycle analysis showed that LEN enhanced DEX-induced G(0)/G(1) arrest. The effect of the LEN and DEX combination on apoptotic induction was mainly through mitochondrial signaling pathways, as demonstrated by phosphorylation of bcl-2 and up-regulation of proapoptotic proteins Bax, Bad and Bim, and the subsequent activation of caspase-9, caspase-3, and cleavage of PARP. Importantly, the combination of LEN and DEX delayed the tumor growth and improved the survival of MCL-bearing mice. The results support the use of the LEN and DEX combination as a new therapeutic regimen in clinical trials of MCL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/patologia , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dexametasona/administração & dosagem , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Lenalidomida , Masculino , Camundongos , Camundongos SCID , Talidomida/administração & dosagem , Talidomida/análogos & derivados
11.
Cancer Epidemiol Biomarkers Prev ; 19(10): 2445-52, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20805315

RESUMO

BACKGROUND: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription. METHODS: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the "probe level" (a single consensus value) or "bead level" (multiple measurements provided by individual beads). RESULTS: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were <2-fold changed. There was no consistent evidence that TRIzol causes premature reverse transcription termination. Instead, a subset of DEPs were systematically due to increased signals in TRIzol-preserved samples from probes near the 5' end of transcripts, suggesting better mRNA preservation with TRIzol. CONCLUSIONS: TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts. IMPACT: The standard practice of prolonged sample storage in TRIzol is suitable for GEP.


Assuntos
Perfilação da Expressão Gênica/métodos , Guanidinas/química , Fenóis/química , RNA/química , Humanos , Leucemia Mieloide Aguda/genética , Análise em Microsséries , Mieloma Múltiplo/genética , RNA/genética , RNA Neoplásico/química , RNA Neoplásico/genética , Transcrição Gênica
12.
Blood ; 114(18): 3880-9, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19654406

RESUMO

Tumor cell-derived heat shock proteins are used as vaccines for immunotherapy of cancer patients. However, current approaches require the generation of custom-made products and are clinically ineffective. To improve the applicability of heat shock protein-based immunotherapy in cancers and to enhance clinical efficacy, we explored combinational treatments in a myeloma setting using pooled heterogeneous or allogeneic myeloma cell line-derived glycoprotein 96 (gp96) as universal vaccines, and clearly demonstrated that pooled but not single gp96 from heterogeneous or allogeneic myeloma cell lines was as effective as autologous gp96 in protecting mice from tumor challenge and rechallenge and in treating established myeloma. We showed that interferon gamma and CD4+ and CD8+ T cells were required for gp96-induced antimyeloma responses and that pooled gp96 induced broader immune responses that protected mice from developing different myeloma. Furthermore, pooled gp96 plus CpG in combination with anti-B7H1 or anti-interleukin-10 monoclonal antibodies were effective in treating mice with large tumor burdens. Thus, this study strongly suggests that pooled gp96 vaccines from myeloma cell lines can replace gp96 vaccines from autologous tumors for immunotherapy and induce immune responses against broader tumor antigens that may protect against tumor recurrence and development of unrelated tumors in vaccinated myeloma patients.


Assuntos
Vacinas Anticâncer/farmacologia , Proteínas de Choque Térmico/farmacologia , Imunoterapia , Mieloma Múltiplo/terapia , Proteínas de Neoplasias/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/uso terapêutico , Humanos , Ligante Coestimulador de Linfócitos T Induzíveis , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/uso terapêutico , Proteínas/genética , Proteínas/imunologia
13.
Am J Hematol ; 84(9): 553-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19565649

RESUMO

Rituximab (RTX), a chimeric anti-CD20 antibody, is associated with direct induction of apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) with clinical efficacy in mantle cell lymphoma (MCL). Lenalidomide (LEN), a novel immunomodulatory agent, sensitizes tumor cells and enhances ADCC. Our study attempted to elucidate the mechanism of LEN-enhanced RTX-mediated cytotoxicity of MCL cells. We found that LEN and RTX induced growth inhibition of both cultured and fresh primary MCL cells. LEN enhanced RTX-induced apoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases (JNK), Bcl-2, Bad; increasing release of cytochrome-c; enhancing activation of caspase-3, -8, -9 and cleavage of PARP. Meanwhile, LEN activated NK cells and increased CD16 expression on CD56(low)CD16(+) NK cells. Whole PBMCs but not NK cell-depleted PBMCs treated with LEN augmented 30% of RTX-dependent cytotoxicity. Daily treatment with LEN increased NK cells by 10-folds in SCID mice, and combination of LEN and RTX decreased tumor burden and prolonged survival of MCL-bearing SCID mice. Taken together, our study demonstrates that LEN plus RTX provides a synergistically therapeutic effect on MCL cells by enhancing apoptosis and RTX-dependent NK cell-mediated cytotoxicity and may be an optimal combination in the clinical trial of relapsed or refractory MCL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Células Sanguíneas/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Lenalidomida , Leucócitos Mononucleares/efeitos dos fármacos , Linfoma de Célula do Manto/patologia , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Rituximab , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Talidomida/farmacologia
14.
Mol Biol Rep ; 36(8): 2075-85, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19048389

RESUMO

The Gadd45 family of proteins, which includes alpha, beta, and gamma isoforms, has recently been shown to play a role in the G2/M cell cycle checkpoint in response to DNA damage; however, the mechanisms by which Gadd45 proteins inhibit cell cycle control are not fully understood. Using immunohistochemical analysis, we found that protein expression of Gadd45gamma, but not Gadd45alpha, was down-regulated in hepatocellular carcinoma. We thus investigated possible mechanisms by which Gadd45alpha and Gadd45gamma might differentially induce G2/M arrest in the human hepatoma Hep-G2 cell line. Flow cytometric analysis revealed significant G2/M arrest in cells transfected with either Gadd45alpha or Gadd45gamma. Importantly, we found that expression of either Gadd45alpha or Gadd45gamma activated the P38 and JNK kinase pathways to induce G2/M arrest. Taken together, these findings suggest that the induction of G2/M arrest by Gadd45alpha or Gadd45gamma involves activation of two distinct signaling pathways in Hep-G2 hepatoma cell lines.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Nucleares/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Ciclo Celular , Regulação para Baixo , Feminino , Fase G2/fisiologia , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Transfecção
15.
Zhonghua Nan Ke Xue ; 10(12): 932-4, 2004 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-15638028

RESUMO

OBJECTIVE: To investigate the possible roles of MT1M gene on the cell cycle and signaling pathway of Hep-G2. METHODS: Hep-G2 human hepatoma cells made by transfection with expressible MT1M gene, and the cell cycle was detected by flow cytometry, and the signaling pathway was measured by dual luciferase assay in Hep-G2 cells. RESULTS: MT1M gene was able to induce changes of the cell cycle and the activation of NF-kappaB pathway in Hep-G2 cells. CONCLUSION: MT1M gene may affect the cell cycle in Hep-G2 and activate the NF-kappaB-dependent transcription.


Assuntos
Fase G2/fisiologia , Neoplasias Hepáticas/patologia , Metalotioneína/genética , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , NF-kappa B/metabolismo , Plasmídeos/genética , Transfecção
16.
Mol Biol Rep ; 30(4): 249-53, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14672412

RESUMO

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 gamma (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 gamma cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 y caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 gamma in cell growth control was further confirmed in this paper.


Assuntos
Carcinoma Hepatocelular/metabolismo , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Proteínas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , China , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas/genética , Proteínas/genética , Proteínas GADD45
17.
World J Gastroenterol ; 9(4): 692-5, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12679912

RESUMO

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1, 20), and it has been identified as a "hotspot" mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2,3,9, 10,16,24). We evaluated in this paper whether this "hotspot" mutation could be detected in cell-free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker. METHODS: We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products. RESULTS: We found in exon 7 of p53 gene G-T transversion at the third base of codon 249 resulting 249(Arg) - 249(Ser) mutation in 10/25 (40 %) hepatocellular carcinoma cases, 4/20 (20 %) cirrhotics, and 2/30 (7 %) healthy controls. The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2-91.7) for HCC cases compared to controls. CONCLUSION: These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency, in plasma DNA of Qidong cirrhotics and healthy controls; We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249(Ser) p53 mutation should be developed to a new early diagnostic marker for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Códon , DNA/sangue , Éxons , Genes p53 , Neoplasias Hepáticas/genética , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Sequência de Bases , Carcinoma Hepatocelular/epidemiologia , China/epidemiologia , DNA/genética , DNA/isolamento & purificação , Feminino , Antígenos de Superfície da Hepatite B/análise , Humanos , Neoplasias Hepáticas/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Medição de Risco , alfa-Fetoproteínas/análise
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