RESUMO
Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Extratos Vegetais/farmacologia , Óleos de Plantas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cromatografia Líquida/métodos , Frutas/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Espectrometria de Massas/métodos , Camundongos , Mitocôndrias/metabolismo , Oxirredução , Persea/química , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
D-cyclins are universally dysregulated in multiple myeloma and frequently overexpressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematologic malignancies, we conducted a high-throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), which inhibited the expression of cyclins D1, D2, and D3 and arrested cells at the G(0)/G(1) phase. After D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of phosphoinositide 3-kinase in intact cells and the activity of the phosphoinositide 3-kinases α, ß, δ, and γ in a cell-free enzymatic assay. In contrast, it did not inhibit the enzymatic activities of other related kinases, including the mammalian target of rapamycin, the DNA-dependent protein kinase catalytic subunit, and phosphoinositide-dependent kinase-1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via the phosphoinositide 3-kinase/protein kinase B signaling pathway. Given its potent antileukemia and antimyeloma activity and minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Ciclina D/antagonistas & inibidores , Ciclina D/genética , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ativação Transcricional/efeitos dos fármacos , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sequência de Bases , Benzopiranos/química , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Primers do DNA/genética , Avaliação Pré-Clínica de Medicamentos , Fase G1/efeitos dos fármacos , Humanos , Células K562 , Leucemia/genética , Leucemia/patologia , Camundongos , Camundongos SCID , Estrutura Molecular , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf-dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate.
Assuntos
Glucocorticoides/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Ciclina D2 , Ciclinas/genética , Ciclinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Glucocorticoides/química , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
We have characterized the rat NK receptors NKR-P1A and -P1B. A cDNA library was constructed from the rat NK cell line, RNK-16. Using the pMX retroviral cloning system, the library was expressed in the human NK cell line, YTSeco, and cells staining with the anti-rat mAb 10/78 identified, FACS sorted and cloned. Two genes, corresponding to rat NK receptors NKR-P1A and -P1B, were identified. YTSeco clones expressing either NKR-P1A or -P1B were functionally tested using (51)Cr-release redirected lysis assays and calcium flux experiments. This demonstrated that NKR-P1A functions as an activation receptor, as previously shown, and that NKR-P1B functions as an inhibitory receptor, as predicted by the presence of an immunoreceptor tyrosine-based inhibition motif. Although annotated as NKR-P1A specific, we found that mAb 10/78 stained YTSeco clones expressing NKR-P1A or -P1B equally well, as did the mAb 3.2.3 used for the original cloning of rat NKR-P1A.