RESUMO
Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure-activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.
Assuntos
Ácidos Aminossalicílicos/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Fator de Transcrição STAT3/antagonistas & inibidores , Sulfonamidas/farmacologia , Ácidos Aminossalicílicos/síntese química , Ácidos Aminossalicílicos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto-oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid-derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon-gamma and demethylating agent 5-azacytidine (5-AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high-risk MDS with a possible role in disease progression and therapy resistance.
Assuntos
Antígenos de Diferenciação/metabolismo , Medula Óssea/metabolismo , Síndromes Mielodisplásicas/metabolismo , Proteínas de Neoplasias/metabolismo , Antígenos de Diferenciação/sangue , Antígenos de Diferenciação/genética , Azacitidina/farmacologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Interferon gama/farmacologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Síndromes Mielodisplásicas/sangue , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Prognóstico , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair.
Assuntos
Dano ao DNA , Reparo do DNA , Corpos de Inclusão Intranuclear/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Técnicas de Inativação de Genes , Humanos , Corpos de Inclusão Intranuclear/efeitos da radiação , Proteína da Leucemia Promielocítica/deficiência , Proteína da Leucemia Promielocítica/genéticaAssuntos
Síndromes Mielodisplásicas , NAD(P)H Desidrogenase (Quinona)/genética , Polimorfismo de Nucleotídeo Único , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Síndromes Mielodisplásicas/enzimologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Taxa de SobrevidaRESUMO
Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis frequently progressing into acute myeloid leukemia (AML), with emerging evidence implicating aberrant bone marrow (BM) microenvironment and inflammation-related changes. 5-azacytidine (5-AC) represents standard MDS treatment. Besides inhibiting DNA/RNA methylation, 5-AC has been shown to induce DNA damage and apoptosis in vitro. To provide insights into in vivo effects, we assessed the proinflammatory cytokines alterations during MDS progression, cytokine changes after 5-AC, and contribution of inflammatory comorbidities to the cytokine changes in MDS patients. We found that IL8, IP10/CXCL10, MCP1/CCL2 and IL27 were significantly elevated and IL12p70 decreased in BM of MDS low-risk, high-risk and AML patients compared to healthy donors. Repeated sampling of the high-risk MDS patients undergoing 5-AC therapy revealed that the levels of IL8, IL27 and MCP1 in BM plasma were progressively increasing in agreement with in vitro experiments using several cancer cell lines. Moreover, the presence of inflammatory diseases correlated with higher levels of IL8 and MCP1 in low-risk but not in high-risk MDS. Overall, all forms of MDS feature a deregulated proinflammatory cytokine landscape in the BM and such alterations are further augmented by therapy of MDS patients with 5-AC.
RESUMO
Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), MTABGNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of MTABGNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, MTABGNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of MTABGNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed MTABGNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.
Assuntos
Exocitose , Ouro/química , Ouro/farmacocinética , Nanotubos/química , Polilisina/química , Polilisina/farmacocinética , Compostos de Amônio Quaternário/química , Compostos de Sulfidrila/química , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Meios de Cultura , Estabilidade de Medicamentos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Citometria de Fluxo , Humanos , Lisossomos/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Nanotubos/análise , Proteoglicanas/química , Proteoglicanas/metabolismo , Compostos de Amônio Quaternário/síntese químicaRESUMO
The Promyelocytic leukemia protein (PML) tumor suppressor is upregulated in several forms of cellular senescence, however the mechanism of its induction is elusive. Here we show that genotoxic drugs that induce senescence, such as 5-bromo-2'deoxyuridine (BrdU), thymidine (TMD), distamycin A (DMA), aphidicolin (APH), etoposide (ET) and camptothecin (CPT) all evoke expansion of PML nuclear compartment and its association with persistent DNA lesions in several human cancer cell lines and normal diploid fibroblasts. This phenomenon was accompanied by elevation of PML transcripts after treatment with BrdU, TMD, DMA and CPT. Chemical inhibition of all JAK kinases and RNAi-mediated knock-down of JAK1 suppressed PML expression, implicating JAK/STAT-mediated signaling in regulation of the PML gene. As PML protein stability remained unchanged after drug treatment, decreased protein turnover was unlikely to explain the senescence-associated increased abundance of PML. Furthermore, binding activity of Interferon Stimulated Response Element (ISRE) within the PML gene promoter, and suppression of reporter gene activity after deletion of ISRE from the PML promoter region suggested that drug-induced PML transcription is controlled via transcription factors interacting with this element. Collectively, our data show that upregulation of the PML tumor suppressor in cellular senescence triggered by diverse drugs including clinically used anti-cancer chemotherapeutics relies on stimulation of PML transcription by JAK/STAT-mediated signaling, possibly evoked by the autocrine/paracrine activities of senescence-associated cytokines.
Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Janus Quinase 1/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
We have recently identified a new class of high affinity ligands for CD69 leukocyte membrane receptor, carboxylated calixarenes. Of the three compounds investigated here, thiacalix[4]arene had the highest affinity for CD69 in direct binding assays, and proved to be the most specific inhibitor of CD69 identified so far in receptor precipitation and cellular activation experiments. Carboxylated calixarenes also proved effective at protection of CD69(high) lymphocytes from apoptosis triggered by a multivalent ligand or antibody. Thus, carboxylated calixarenes set a new paradigm for noncarbohydrate ligands for CD69 making them attractive for protection of killer cells in combined animal tumor therapies.