RESUMO
BACKGROUND AND AIMS: High temperatures may reduce fecal immunochemical test (FIT) positivity and colorectal cancer (CRC) detection sensitivity. We investigated the effect of temperature on hemoglobin concentration [Hb], in the FOB Gold®. Additionally, we examined FIT pick-up, storage, return times and specimen collection. MATERIALS AND METHODS: In vitro experiments with buffer containing FIT devices, inoculated with Hb-spiked stool. For 7 days, 144 samples were stored in groups of 36 at 4 °C, 22 °C, 30 °C, and 50 °C. Additionally, 54 samples were stored in groups of 18 at 34 °C, 42 °C and 50 °C for 20 h. Paired t-tests and repeated measure ANOVA assessed [Hb] change. Sixty-five screening participants completed a FIT-handling questionnaire. RESULTS: After 7 days, mean [Hb] was stable at 30 °C (0.8 µg Hb/g;95 %CI: -1.5 to 3.1;p = 0.50). For 50 °C, mean [Hb] decreased within 2 days (-21.3 µg Hb/g;95 %CI: -30.2 to -12.5;p < 0.001) and after 20 h (-63.0 µg Hb/g;95 %CI: -88.7 to -37.3;p < 0.001), respectively. All other temperature categories showed significant mean [Hb] increase. Same-day FIT return was reported by 80 %. Eighty-seven percent experienced specimen collection as easy and 33 % kept the FIT refrigerated after collection. CONCLUSIONS: The FOB Gold® is suitable for CRC screening in tropical climates. Although most respondents indicated same-day sample return, we recommend avoiding FIT storage above 30 °C for longer than7 days.
Assuntos
Neoplasias Colorretais , Detecção Precoce de Câncer , Fezes , Hemoglobinas , Humanos , Neoplasias Colorretais/diagnóstico , Hemoglobinas/análise , Detecção Precoce de Câncer/métodos , Fezes/química , Região do Caribe , Sangue Oculto , Masculino , Feminino , Pessoa de Meia-Idade , Temperatura Alta , Imunoquímica , IdosoRESUMO
Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of ß5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional ß5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the ß5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.
Assuntos
Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Biblioteca de PeptídeosRESUMO
Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells.
Assuntos
Aminopeptidases/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Regulação para Baixo , Humanos , Gotículas Lipídicas , Fosforilação , Pró-Fármacos/metabolismoAssuntos
Bortezomib/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma Anaplásico de Células Grandes , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores Proteína Tirosina Quinases , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Quinase do Linfoma Anaplásico , Feminino , Humanos , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Masculino , Células Tumorais CultivadasRESUMO
Current treatment strategies for rheumatoid arthritis (RA) consisting of disease-modifying anti-rheumatic drugs or biological agents are not always effective, hence driving the demand for new experimental therapeutics. The antiproliferative capacity of proteasome inhibitors (PIs) has received considerable attention given the success of their first prototypical representative, bortezomib (BTZ), in the treatment of B cell and plasma cell-related hematological malignancies. Therapeutic application of PIs in an autoimmune disease setting is much less explored, despite a clear rationale of (immuno) proteasome involvement in (auto)antigen presentation, and PIs harboring the capacity to inhibit the activation of nuclear factor-κB and suppress the release of pro-inflammatory cytokines such as tumor necrosis factor alpha and interleukin-6. Here, we review the clinical positioning of (immuno) proteasomes in autoimmune diseases, in particular RA, systemic lupus erythematosus, Sjögren's syndrome and sclerodema, and elaborate on (pre)clinical data related to the impact of BTZ and next generation PIs on immune effector cells (T cells, B cells, dendritic cells, macrophages, osteoclasts) implicated in their pathophysiology. Finally, factors influencing long-term efficacy of PIs, their current (pre)clinical status and future perspectives as anti-inflammatory and anti-arthritic agents are discussed.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Humanos , Complexo de Endopeptidases do Proteassoma/efeitos adversosRESUMO
The use of automated blood cell counters (automated hematology analyzers) for diagnostic purposes is inextricably linked to clinical laboratories. However, the need for uniformity among the various methods and parameters is increasing and standardization of the automated analyzers is therefore crucial. Standardization not only involves procedures based on reference methods but it also involves validation, verification, quality assurance, and quality control, and it includes the involvement of several participants. This article discusses the expert guidelines and provides an overview of issues involved in complete blood count parameter reference methods and standardization of reporting units.
Assuntos
Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/normas , Guias como Assunto , Padrões de Referência , Estudos de Validação como AssuntoRESUMO
Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin-proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A4 hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.
Assuntos
Aminopeptidases/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Neoplasias/tratamento farmacológico , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Humanos , Neoplasias/enzimologia , Neoplasias/genéticaRESUMO
BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10-12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive ß5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2-4 fold increase in the mRNA and protein levels of the constitutive ß5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.
RESUMO
Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Artrite Reumatoide/metabolismo , Leucócitos Mononucleares/metabolismo , Mutação/genética , Inibidores de Proteassoma , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células HEK293 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Resultado do TratamentoRESUMO
As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1-2 months of selection with 30-90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34(+) proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34(+) hematopoietic stem and precursor cells.
Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular , Doxorrubicina/farmacologia , Células-Tronco Hematopoéticas/citologia , Células de Langerhans/citologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telomerase/metabolismoRESUMO
BACKGROUND: Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. METHODS: 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. RESULTS: 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). CONCLUSIONS: 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Hidroxiquinolinas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacocinética , Bortezomib , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Humanos , Hidroxiquinolinas/farmacocinética , Immunoblotting , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/metabolismo , Inibidores de Proteases/farmacologia , Pirazinas/farmacocinética , Transplante HeterólogoRESUMO
The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.