RESUMO
BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.
Assuntos
Citocinas/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Leucemia/genética , Leucemia/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Biossíntese de Proteínas , Estresse Fisiológico/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Acrilamidas/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Células Jurkat , NAD/metabolismo , Fosforilação , Piperidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transcrição GênicaRESUMO
RAS pathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Here, using single-cell, multi-omics technologies, we seek to dissect the biological mechanisms underlying the initiation and progression of RAS pathway-mutated CMML. We identify that RAS pathway mutations induce transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs) and downstream monocytic populations in response to cell-intrinsic and -extrinsic inflammatory signaling that also impair the functions of immune cells. HSPCs expand at disease progression after therapy with HMA or the BCL2 inhibitor venetoclax and rely on the NF-κB pathway effector MCL1 to maintain survival. Our study has implications for the development of therapies to improve the survival of patients with RAS pathway-mutated CMML.
Assuntos
Apoptose , Leucemia Mielomonocítica Crônica , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/patologia , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Humanos , Apoptose/efeitos dos fármacos , Animais , Mutação/genética , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Progressão da Doença , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , NF-kappa B/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Crise Blástica/patologia , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Crise Blástica/metabolismoRESUMO
The molecular mechanisms of venetoclax-based therapy failure in patients with acute myeloid leukemia were recently clarified, but the mechanisms by which patients with myelodysplastic syndromes (MDS) acquire secondary resistance to venetoclax after an initial response remain to be elucidated. Here, we show an expansion of MDS hematopoietic stem cells (HSCs) with a granulo-monocytic-biased transcriptional differentiation state in MDS patients who initially responded to venetoclax but eventually relapsed. While MDS HSCs in an undifferentiated cellular state are sensitive to venetoclax treatment, differentiation towards a granulo-monocytic-biased transcriptional state, through the acquisition or expansion of clones with STAG2 or RUNX1 mutations, affects HSCs' survival dependence from BCL2-mediated anti-apoptotic pathways to TNFα-induced pro-survival NF-κB signaling and drives resistance to venetoclax-mediated cytotoxicity. Our findings reveal how hematopoietic stem and progenitor cell (HSPC) can eventually overcome therapy-induced depletion and underscore the importance of using close molecular monitoring to prevent HSPC hierarchical change in MDS patients enrolled in clinical trials of venetoclax.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Células-Tronco Hematopoéticas/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Sulfonamidas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.
Assuntos
Cromossomos Humanos Par 3 , Inibidores de Histona Desacetilases , Leucemia Mieloide Aguda , Proteína do Locus do Complexo MDS1 e EVI1 , Proteogenômica , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromossomos Humanos Par 3/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteogenômica/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover mechanisms through which MM cells overcome DNA damage, we investigate how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting Interleukin enhancer binding factor 2 (ILF2), a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identify the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage. Our study reveals a mechanism of vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , DNA Helicases/metabolismo , Reprogramação Metabólica , Reparo do DNA , Dano ao DNARESUMO
Estrogen promotes growth in many tissues by activating Wnt/ß-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/ß-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced ß-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of ß-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of ß-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3ß. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/ß-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3ß-dependent activation of ß-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.
Assuntos
Diarileptanoides/farmacologia , Receptor alfa de Estrogênio/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fitoestrógenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Curcuma/química , Diarileptanoides/química , Suplementos Nutricionais , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fitoestrógenos/química , Pós-Menopausa/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.
Assuntos
NAD , Neoplasias de Mama Triplo Negativas , Humanos , NAD/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Fosfotransferases (Aceptor do Grupo Álcool)RESUMO
RAS pathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Using single-cell, multi-omics technologies, we sought to dissect the biological mechanisms underlying the initiation and progression of RAS pathway-mutated CMML. We found that RAS pathway mutations induced the transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs), which underwent proliferation and monocytic differentiation in response to cell-intrinsic and -extrinsic inflammatory signaling that also impaired immune cells' functions. HSPCs expanded at disease progression and relied on the NF- K B pathway effector MCL1 to maintain their survival, which explains why patients with RAS pathway- mutated CMML do not benefit from BCL2 inhibitors such as venetoclax. Our study has implications for developing therapies to improve the survival of patients with RAS pathway- mutated CMML.
RESUMO
Myelodysplastic syndromes (MDS) are a group of incurable hematopoietic stem cell (HSC) neoplasms characterized by peripheral blood cytopenias and a high risk of progression to acute myeloid leukemia. MDS represent the final stage in a continuum of HSCs' genetic and functional alterations and are preceded by a premalignant phase, clonal cytopenia of undetermined significance (CCUS). Dissecting the mechanisms of CCUS maintenance may uncover therapeutic targets to delay or prevent malignant transformation. Here, we demonstrate that DNMT3A and TET2 mutations, the most frequent mutations in CCUS, induce aberrant HSCs' differentiation towards the myeloid lineage at the expense of erythropoiesis by upregulating IL-1ß-mediated inflammatory signaling and that canakinumab rescues red blood cell transfusion dependence in early-stage MDS patients with driver mutations in DNMT3A and TET2 . This study illuminates the biological landscape of CCUS and offers an unprecedented opportunity for MDS intervention during its initial phase, when expected survival is prolonged.
RESUMO
DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting ILF2, a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation. STATEMENT OF SIGNIFICANCE: Metabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.
RESUMO
SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production. SIGNIFICANCE: MDS-RS are characterized by significant anemia. Patients with MDS-RS die from a shortage of red blood cells and the side effects of iron overload due to their constant need for transfusions. Our study has implications for the development of therapies to achieve long-lasting hematologic responses. This article is highlighted in the In This Issue feature, p. 476.
Assuntos
Síndromes Mielodisplásicas , Fosfoproteínas , Humanos , Idoso , Fatores de Processamento de RNA/genética , Fosfoproteínas/genética , Síndromes Mielodisplásicas/genética , Células Precursoras Eritroides , Transdução de Sinais , eIF-2 QuinaseRESUMO
Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Síndromes Mielodisplásicas , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Células-Tronco Hematopoéticas/patologia , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , SulfonamidasRESUMO
OBJECTIVE: Gambogic acid (GA) has been reported to induce apoptosis in cholangiocarcinoma (CCA) cell lines. However, the molecular mechanisms underlying its anti-cancer activity remain poorly understood. This study was aimed to investigate GA's effect on human CCA cell lines, KKU-M213 and HuCCA-1, and its associated mechanisms on Wnt/ß-catenin signaling pathway. METHODS: Cell viability, apoptosis, and cell cycle analysis were conducted by MTT and flow cytometry. The effect of GA mediated Wnt/ß-catenin and ER stress were determined by luciferase-reporter assay, qRT-PCR, and western blot analysis. RESULTS: GA exhibited potent cytotoxicity in CCA cells which was associated with significantly inhibited cell proliferation, promoted G1 arrest, and activated caspase 3 mediated-apoptosis. GA attenuated ß-catenin transcriptional levels, decreased ß-catenin protein, and suppressed the expression of c-Myc, a downstream target gene of Wnt/ß-catenin signaling. GA activated genes involved in ER stress mechanism in KKU-M213 and enhanced CCA's sensitivity to gemcitabine. CONCLUSION: Our findings reveal that the molecular mechanism underpinning anti-cancer effect of GA is partially mediated through the inhibition of Wnt/ß-catenin signaling pathway and induction of ER stress induced-apoptosis. GA may serve as a promising therapeutic modality for amelioration of gemcitabine-induced toxicity in CCA.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Xantonas/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , HumanosRESUMO
The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells' self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells' skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance.
Assuntos
Hematopoese/fisiologia , Encurtamento do Telômero/fisiologia , Animais , Transtornos da Insuficiência da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea/metabolismo , Transtornos da Insuficiência da Medula Óssea/patologia , Autorrenovação Celular , Reprogramação Celular , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interferons/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Análise de Célula Única , Telômero/química , Telômero/fisiologia , Encurtamento do Telômero/genéticaRESUMO
We investigated the effect of a phytoestrogen, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD), from Curcuma comosa Roxb. (Zingiberaceae family) on the adipogenic differentiation of mesenchymal progenitors, human bone marrow-derived mesenchymal stem cells (hBMSCs). DPHD inhibited adipocyte differentiation of hBMSCs by suppressing the expression of genes involved in adipogenesis. DPHD at concentrations of 0.1, 1, and 10 µM significantly decreased triglyceride accumulation in hBMSCs to 7.1 ± 0.2, 6.3 ± 0.4, and 4.9 ± 0.2 mg/dL, respectively, compared to the nontreated control (10.1 ± 0.9 mg/dL) (p < 0.01). Based on gene expression profiling, DPHD increased the expression of several genes involved in the Wnt/ß-catenin signaling pathway, a negative regulator of adipocyte differentiation in hBMSCs. DPHD also increased the levels of essential signaling proteins which are extracellular signal-regulated kinases 1 and 2 (ERK1/2) and glycogen synthase kinase 3 beta (GSK-3ß) that link estrogen receptor (ER) signaling to Wnt/ß-catenin signaling. In conclusion, DPHD exhibited the anti-adipogenic effect in hBMSCs by suppression of adipogenic markers in hBMSCs through the activation of ER and Wnt/ß catenin signaling pathways. This finding suggests the potential role of DPHD in preventing bone marrow adiposity which is one of the major factors that exacerbates osteoporosis in postmenopause.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Curcuma/química , Diarileptanoides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fitoestrógenos/farmacologia , Extratos Vegetais/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diarileptanoides/química , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fitoestrógenos/química , Extratos Vegetais/química , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Triglicerídeos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Sistema de Sinalização das MAP Quinases , Epitélio Pigmentado da Retina/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagia/fisiologia , Linhagem Celular , Humanos , MAP Quinase Quinase 4/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, exhibit anticancer effects in preclinical models. However, continuous exposure to NAMPT inhibitors, such as FK866, can induce acquired resistance. METHODS: We developed FK866-resistant CCRF-CEM (T cell acute lymphoblastic leukemia) and MDA MB231 (breast cancer) models, and by exploiting an integrated approach based on genetic, biochemical, and genome wide analyses, we annotated the drug resistance mechanisms. RESULTS: Acquired resistance to FK866 was independent of NAMPT mutations but rather was based on a shift towards a glycolytic metabolism and on lactate dehydrogenase A (LDHA) activity. In addition, resistant CCRF-CEM cells, which exhibit high quinolinate phosphoribosyltransferase (QPRT) activity, also exploited amino acid catabolism as an alternative source for NAD+ production, becoming addicted to tryptophan and glutamine and sensitive to treatment with the amino acid transport inhibitor JPH203 and with l-asparaginase, which affects glutamine exploitation. Vice versa, in line with their low QPRT expression, FK866-resistant MDA MB231 did not rely on amino acids for their resistance phenotype. CONCLUSIONS: Our study identifies novel mechanisms of resistance to NAMPT inhibition, which may be useful to design more rational strategies for targeting cancer metabolism.
RESUMO
The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ligação Proteica/efeitos dos fármacos , Quinonas/farmacologia , RNA Mensageiro/metabolismo , Abietanos , Linhagem Celular , Desenho de Fármacos , Humanos , Simulação de Dinâmica Molecular , Mimetismo Molecular , Quinonas/síntese química , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 µM) and antagonist at high concentrations (10-50 µM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.