RESUMO
INTRODUCTION: Genome-wide association studies link susceptibility to late-onset Alzheimer's disease (LOAD) with EphA1. Sequencing identified a non-synonymous substitution P460L as a LOAD risk variant. Other Ephs regulate vascular permeability and immune cell recruitment. We hypothesized that P460L dysregulates EphA1 receptor activity and impacts neuroinflammation. METHODS: EphA1/P460L receptor activity was assayed in isogenic Human Embryonic Kidney (HEK) cells. Soluble EphA1/P460L (sEphA1/sP460L) reverse signaling in brain endothelial cells was assessed by T-cell recruitment and barrier function assays. RESULTS: EphA1 and P460L were expressed in HEK cells, but membrane and soluble P460L were significantly reduced. Ligand engagement induced Y781 phosphorylation of EphA1 but not P460L. sEphA1 primed brain endothelial cells for increased T-cell recruitment; however, sP460L was less effective. sEphA1 decreased the integrity of the brain endothelial barrier, while sP460L had no effect. DISCUSSION: These findings suggest that P460L alters EphA1-dependent forward and reverse signaling, which may impact blood-brain barrier function in LOAD. HIGHLIGHTS: EphA1-dependent reverse signaling controls recruitment of T cells by brain endothelial cells. EphA1-dependent reverse signaling remodels brain endothelial cell contacts. LOAD-associated P460L variant of EphA1 shows reduced membrane expression and reduced ligand responses. LOAD-associated P460L variant of EphA1 fails to reverse signal to brain endothelial cells.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/genética , Barreira Hematoencefálica , Células Endoteliais , Estudo de Associação Genômica Ampla , Ligantes , Receptor EphA1/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.
Assuntos
Movimento Celular/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B , Proteínas de Neoplasias , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9 , Animais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are incurable hematological malignancies that are pathologically linked with aberrant NF-κB activation. In this study, we identified a group of novel C8-linked benzofused Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and MM cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4h exposure and demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits. In primary CLL cells, sensitivity to DC-1-192 was inversely correlated with RelA subunit expression (r2=0.2) and samples with BIRC3 or NOTCH1 mutations showed increased sensitivity (P=0.001). RNA-sequencing and gene set enrichment analysis confirmed the over-representation of NF-κB regulated genes in the down-regulated gene list. Furthermore, In vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC-1-192 significantly prolonged survival (P=0.017). In addition, DC1-192 showed synergy with bortezomib and ibrutinib; synergy with ibrutinib was enhanced when CLL cells were co-cultured on CD40L-expressing fibroblasts in order to mimic the cytoprotective lymph node microenvironment (P = 0.01). Given that NF-κB plays a role in both bortezomib and ibrutinib resistance mechanisms, these data provide a strong rationale for the use of DC-1-192 in the treatment of NF-κB-driven cancers, particularly in the context of relapsed/refractory disease.
Assuntos
Neoplasias Hematológicas , Leucemia Linfocítica Crônica de Células B , Adenina/análogos & derivados , Animais , Apoptose , Benzodiazepinas/farmacologia , Bortezomib/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B , Piperidinas , Pirróis , Microambiente TumoralRESUMO
Chronic lymphocytic leukaemia (CLL) patients often have abnormal expansions of CD4+ and CD8+ T cells and this can be associated with progressive disease. To characterise the key T-cell populations involved in this phenomenon, we used flow cytometry and 11 phenotypic markers to study 74 CLL patients and 14 controls. T cells of CLL patients were more phenotypically complex than those of healthy controls with significant increases in the frequencies of CD4 and CD8 memory T cells expressing exhaustion-, activation- and senescence-associated markers. Multivariate analysis of 111 different T-cell subsets showed that high frequencies of four subsets (three CD8 and one CD4) were associated with shorter progression-free survival. The most significant association was with CD4+ HLA-DR+ PD-1+ T cells, and patients could be stratified into high- and low-risk groups based on the frequency of these T cells. The expansion of this CD4+ subset could not be accounted for by age, cytomegalovirus infection or increases in Treg cells. Overall, these results highlight two relatively simple biomarkers, percentage CD8+ and percentage CD4+ PD-1+ HLA-DR+ T cells, which can be used to risk-stratify CLL patients, independent of other tumour-associated markers. They also provide further evidence for the pivotal role of T cells in modulating the pathology of CLL.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Antígenos HLA-DR/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Receptor de Morte Celular Programada 1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.
Assuntos
Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Endotélio Vascular/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Migração Transendotelial e Transepitelial/imunologia , Linfócitos T CD4-Positivos/patologia , Endotélio Vascular/patologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , MasculinoRESUMO
Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.
Assuntos
Antineoplásicos/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Ovarianas/enzimologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL), but to date, only static in vitro cultures have been used to study these phenomena. To address this lack of data, we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L and CXCR4 (both P < .0001) and of CD49d and CD5 (both P = .003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the "extravascular" space (mean migration, 1.37% ± 2.14%; n = 21). Migrated CLL cells had significantly higher expression of CD49d (P = .02), matrix metallopeptidase-9 (P = .004), CD38 (P = .009), CD80 (P = .04), and CD69 (P = .04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r(2), 0.47; P = .01), and treatment with the CD49d-blocking antibody natalizumab resulted in significantly decreased migration (P = .01). Taken together, our data provide evidence for a novel, dynamic, and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL.
Assuntos
Técnicas de Cultura de Células , Quimiotaxia de Leucócito , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Linfócitos/patologia , Microfluídica , Pessoa de Meia-Idade , Modelos Teóricos , Resistência ao CisalhamentoRESUMO
Cdk9 is a key elongation factor for RNA transcription and functions by phosphorylating the C-terminal domain of RNA polymerase II. Here we present direct evidence that cdk9 is important for cancer cell survival and describe the characterization of the potent cdk9 inhibitor CDKI-73 in primary human leukemia cells. CDKI-73 induced caspase-dependent apoptosis that was preceded by dephosphorylation of cdk9 and serine 2 of RNA polymerase II. CDKI-73 was more potent than the pan-cdk inhibitor flavopiridol and showed >200-fold selectivity against primary leukemia cells when compared with normal CD34+ cells. Furthermore, CDKI-73 was equipotent in poor prognostic sub-groups of leukemia patients and showed cytotoxic synergy with the nucleoside analog fludarabine. The Mechanism of synergy was associated with CDKI-73-mediated transcriptional inhibition of MCL1 and XIAP that was maintained when used in combination with fludarabine. Our data present a strong rationale for the development of cdk9 inhibitors such as CDKI-73 as anticancer therapeutics.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Vidarabina/análogos & derivados , Animais , Apoptose , Estudos de Casos e Controles , Quinase 9 Dependente de Ciclina/genética , Sinergismo Farmacológico , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Fosforilação , Prognóstico , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagem , Transcrição Gênica/efeitos dos fármacos , Vidarabina/administração & dosagem , Vidarabina/farmacologiaRESUMO
Heat shock protein 90 (HSP90; HSP90AA1) is a molecular chaperone involved in signalling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of HSP90 are being examined as anti-cancer agents, but the critical molecular mechanism(s) of their activity remains unresolved. HSP90 inhibition potentially facilitates the simultaneous targeting of multiple molecules within tumour cells and represents an attractive therapeutic proposition. Here, we investigated HSP90 as a molecular target for acute myeloid leukaemia (AML) using the novel HSP90 inhibitor NVP-AUY922-AG. NVP-AUY922-AG induced dose-dependent killing in myeloid cell lines and primary AML blasts. In primary blasts, cell death in response to NVP-AUY922-AG was seen at concentrations almost 2 logs lower than cytarabine (Ara-C) (50% lethal dose = 0·12 µ mol/l ± 0·28). NVP-AUY922-AG was significantly less toxic to normal bone marrow (P = 0·02). In vitro response to NVP-AUY922-AG did not correlate with response to Ara-C (r(2) = 0·0006). NVP-AUY922-AG was highly synergistic with Ara-C in cell lines and in 20/25 of the primary samples tested. NVP-AUY922-AG induced increases in HSP70 expression and depletion of total AKT, IKKα and IKKß in cell lines and primary blasts. This study shows that the novel HSP90 inhibitor NVP-AUY922-AG has significant single agent activity in AML cells and is synergistic with Ara-C.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Resorcinóis/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anexina A5/metabolismo , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Citarabina/administração & dosagem , Citarabina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Quinase I-kappa B/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Isoxazóis/administração & dosagem , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Resorcinóis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Adulto JovemRESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of multiple over-expressed signaling proteins that promote growth and survival in cancer cells. Chronic lymphocytic leukaemia (CLL) is characterized by increased expression of several Hsp90 client proteins making it a potentially susceptible to Hsp90 inhibition. In this study we showed that the novel Hsp90 inhibitor NVP-AUY922-AG was cytotoxic to primary CLL cells in vitro (LD50=0.18µM±0.20). Importantly, its toxicity was preserved under cytoprotective co-culture conditions that rendered fludarabine ineffective. At the molecular level, NVP-AUY922-AG depleted the expression of multiple Hsp90 client proteins including Akt and activators of NF- κB, IKKα and IKKß. Consistent with this inhibition profile, NVP-AUY922-AG resulted in decreased transcription of the NF-B target genes MCL1, CFLAR, BIRC5. In contrast, fludarabine significantly induced the transcription of MCL1 and BIRC5. Given the anti-apoptotic nature of these genes and the role they play in fludarabine resistance, we considered that the combination of NVP-AUY922-AG with fludarabine might resensitize CLL cells to the effects of fludarabine. In keeping with this hypothesis, the combination of NVP-AUY922-AG and fludarabine was highly synergistic (mean CI=0.110.06) and this synergy was enhanced in co-culture (mean CI=0.06±0.08). Furthermore, the combination maintained the decrease in MCL1, CFLAR and BIRC5 transcription suggesting that the ability of NVP-AUY922-AG to modulate expression of these genes may contribute to the efficacy of this drug under cytoprotective co-culture conditions and for its remarkable synergy with fludarabine. Taken together these findings indicate that Hsp90 inhibition is an attractive therapeutic strategy in CLL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Resorcinóis/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Células Cultivadas , Citoproteção/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Isoxazóis/administração & dosagem , Leucemia Linfocítica Crônica de Células B/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Resorcinóis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Survivina , Microambiente Tumoral , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
BACKGROUND: Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II. RESULTS: The mean lethal dose 50% (LD(50)) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 µM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. CONCLUSIONS: Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Naftiridinas/farmacologia , Tiazóis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Citarabina/farmacologia , Citarabina/uso terapêutico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Deleção de Genes , Genes p53 , Humanos , Dose Letal Mediana , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/metabolismo , Células Mieloides/patologia , Naftiridinas/uso terapêutico , Tiazóis/uso terapêutico , Inibidores da Topoisomerase II/uso terapêuticoRESUMO
Multiple myeloma remains incurable with conventional therapeutics. Thus, new treatments for this condition are clearly required. In this study we evaluated the novel NF-kappaB inhibitor LC-1 in multiple myeloma cell lines and plasma cells derived from multiple myeloma patients. LC-1 was cytotoxic to multiple myeloma cell lines H929, U266, and JJN3, and induced apoptosis in a dose-dependent manner with an overall LD(50) of 3.6 micromol/L (+/-1.8) after 48 hours in culture. Primary multiple myeloma cells, identified by CD38 and CD138 positivity, had a mean LD(50) for LC-1 of 4.9 micromol/L (+/-1.6); normal bone marrow cells were significantly less sensitive to the cytotoxic effects of LC-1 (P = 0.0002). Treatment of multiple myeloma cell lines with LC-1 resulted in decreased nuclear localization of the NF-kappaB subunit Rel A and the inhibition of NF-kappaB target genes. In addition, LC-1 showed synergy with melphalan, bortezomib, and doxorubicin (combination indices of 0.72, 0.61, and 0.78, respectively), and was more effective when cells were cultured on fibronectin. These data show that LC-1 has activity in multiple myeloma cell lines and primary multiple myeloma cells, and its ability to inhibit NF-kappaB seems important for its cytotoxic effects. Furthermore, LC-1-induced transcriptional suppression of survivin and MCL1 provides a potential explanation for its synergy with conventional agents.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Ácidos Borônicos , Bortezomib , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Pirazinas , Sesquiterpenos/toxicidade , Fator de Transcrição RelA/metabolismoRESUMO
2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Arabinofuranosiluracila/síntese química , Arabinofuranosiluracila/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/farmacocinética , Antivirais/farmacocinética , Arabinofuranosiluracila/farmacocinética , Catepsina A/antagonistas & inibidores , Catepsina A/metabolismo , Linhagem Celular Tumoral , Cromatografia em Camada Fina , Desenho de Fármacos , Esterases/antagonistas & inibidores , Humanos , Indicadores e Reagentes , Fígado/efeitos dos fármacos , Fígado/enzimologia , Linfócitos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Pró-Fármacos/farmacocinética , Relação Estrutura-Atividade , Suínos , Vírus/efeitos dos fármacosRESUMO
Activating mutations of the FMS-like tyrosine kinase 3 gene (FLT3) occur in approximately one-third of patients with acute myeloid leukaemia (AML) and predict for a poor outcome. Heat shock protein 90 (Hsp90) is a molecular chaperone that is frequently used by cancer cells to stabilise mutant oncoproteins. Mutant FLT3 is chaperoned by Hsp90 in primary AML blasts whereas unmutated FLT3 is not, making Hsp90 inhibitors potentially useful therapeutically. The present study showed that inhibition of Hsp90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) was cytotoxic to primary AML cells expressing mutant FLT3. Inhibition of Hsp90 results in altered downstream signalling effects in primary AML cells with disruption of Janus kinase-signal transducer and activator of transcription (JAK-STAT), mitogen-activated protein kinase and phosphatidylinositol 3/AKT signalling pathways. Co-treatment of blasts with 17-AAG and cytarabine resulted in a synergistic or additive effect in approximately 50% of AML cases tested. Our results confirm that Hsp90 is a valid molecular target in the therapy of AML. Inhibition of Hsp90 in parallel with conventional AML therapies may have particular benefit in those patients with the poor prognostic FLT3 mutant disease.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Leucemia Mieloide Aguda/patologia , Tirosina Quinase 3 Semelhante a fms/genética , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Morte Celular/efeitos dos fármacos , Citarabina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
BACKGROUND: Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents. DESIGN AND METHODS: Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples. RESULTS: The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies. CONCLUSIONS: AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Organofosfatos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Apoptose , Aurora Quinases , Crise Blástica , Proliferação de Células/efeitos dos fármacos , Histonas/metabolismo , Humanos , Fosforilação , Ploidias , Poliploidia , Células Tumorais CultivadasRESUMO
The FUS gene is overexpressed in acute myeloid leukaemia (AML) patients and has roles in transcription and mRNA processing. We used ectopic expression of FUS and FUS antisense sequences to assess the effect of modulation of FUS expression in all-trans retinoic acid (ATRA)-sensitive (NB4) and insensitive (NB4R2) human acute promyelocytic (APL) cell lines which express the t(15:17) translocation. Growth, viability and differentiation patterns were maintained, but the expression of the FUS antisense construct in both the cell lines altered the response to ATRA: the previously ATRA-sensitive NB4 cells exhibited resistance; whilst the previously resistant NB4R2 cells showed a differentiation response to treatment.