RESUMO
Fluorizoline is a prohibitin (PHB)-binding compound that induces apoptosis in several cancer cell lines as well as in primary cells from hematologic malignancies. In this study, we show that fluorizoline treatment triggers the activation of the stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 prior to caspase activation in human cell lines. However, the blockage of p38 and JNK activity with chemical inhibitors or siRNA-mediated downregulation of MAPK14 (p38) does not prevent fluorizoline-induced apoptosis, suggesting that the activation of these kinases plays an alternative role in the cell response to fluorizoline treatment. Here, we describe that fluorizoline treatment leads to the secretion of pro-inflammatory cytokines interleukin-8 (IL-8) and interleukin-6 (IL-6). Importantly, we demonstrate that the activation of the stress-activated kinases JNK and p38 mediates the secretion of both IL-8 and IL-6. This study shows novel insights into the pro-inflammatory role exhibited by a compound that binds to PHB, thus supporting the potential of PHBs as anti-inflammatory proteins.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Citocinas , Proibitinas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , ApoptoseRESUMO
Fluorizoline is a synthetic molecule that induces apoptosis, by selectively targeting prohibitins (PHBs), through induction of the BH3-only protein NOXA. This induction is transcriptionally regulated by the integrated stress response (ISR)-related transcription factors ATF3 and ATF4. Here, we evaluate the role of the four eIF2α kinases, to decipher which is responsible for the mechanism of ISR activation triggered by fluorizoline in HeLa and HAP1 cells. First, we demonstrated the involvement of the eIF2α kinases using ISR inhibitor (ISRIB) and by simultaneous downregulation of all four eIF2α kinases, as both approaches were able to increase cell resistance to fluorizoline-induced apoptosis. Furthermore, we confirmed that fluorizoline treatment results in endoplasmic reticulum (ER) stress, as evidenced by PERK activation. Despite PERK activation, this kinase was not directly involved in the ISR activation by fluorizoline. In this regard, we found that the eIF2α kinases are capable of compensating for each other's loss of function. Importantly, we demonstrated that the mitochondrial-stress-related eIF2α kinase HRI mediates ISR activation after fluorizoline treatment.
Assuntos
Proibitinas , eIF-2 Quinase , Humanos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Apoptose , Células HeLa , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos , Fator 4 Ativador da Transcrição/genéticaRESUMO
γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. Novel γδ T-cell-based immunotherapies, primarily focusing on the two major γδ T-cell subtypes that infiltrate tumors (i.e. Vδ1 and Vδ2), are being developed. The Vδ1 T-cell subset is enriched in tissues and contains both effector T-cells as well as regulatory T-cells with tumor-promoting potential. Vδ2 T-cells, in contrast, are enriched in circulation and consist of a large, relatively homogeneous, pro-inflammatory effector T-cell subset. Healthy individuals typically harbor in the order of 50-500 million Vγ9Vδ2 T-cells in the peripheral blood alone (1-10% of the total CD3+ T-cell population), which can rapidly expand upon stimulation. The Vγ9Vδ2 T-cell receptor senses intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of mevalonate pathway dysregulation or upon pharmaceutical intervention. Early clinical studies investigating the therapeutic potential of Vγ9Vδ2 T-cells were based on either ex vivo expansion and adoptive transfer or their systemic activation with aminobisphosphonates or synthetic phosphoantigens, either alone or combined with low dose IL-2. Immune-related adverse events (irAE) were generally \mild, but the clinical efficacy of these approaches provided overall limited benefit. In recent years, critical advances have renewed the excitement for the potential of Vγ9Vδ2 T-cells in cancer immunotherapy. Here, we review γδ T-cell-based therapeutic strategies and discuss the prospects of those currently evaluated in clinical studies in cancer patients as well as future therapies that might arise from current promising pre-clinical results.
Assuntos
Linfócitos Intraepiteliais , Neoplasias , Humanos , Imunoterapia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos TRESUMO
Fluorizoline is a prohibitin-binding compound that triggers apoptosis in several cell lines from murine and human origin, as well as in primary cells from hematologic malignancies by inducing the integrated stress response and ER stress. Recently, it was described that PHB (Prohibitin) 1 and 2 are crucial mitophagy receptors involved in mediating the autophagic degradation of mitochondria. We measured mitophagy in HeLa cells expressing Parkin and in A549, a lung cancer cell line that can undergo mitophagy in a Parkin-independent manner, and we demonstrated that both fluorizoline and rocaglamide A, another PHB-binding molecule, inhibit CCCP- and OA-induced mitophagy. Moreover, we demonstrated that PHBs are mediating Parkin-dependent mitophagy. In conclusion, besides being a potent pro-apoptotic compound, we present fluorizoline as a promising new mitophagy modulator that could be used as anticancer agent.
RESUMO
The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proibitinas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Proteínas Repressoras/metabolismo , Tiazolidinas/farmacologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células HEK293 , Humanos , Proibitinas , Proteínas Repressoras/genética , Tiazolidinas/químicaRESUMO
Fluorizoline is a new synthetic molecule that induces p53-independent apoptosis, in several tumor cell lines and in primary leukemia cells, by selectively targeting prohibitins (PHBs). In this study, we describe how fluorizoline induces BCL-2 homology 3-only protein NOXA, without modulating the protein levels of anti-apoptotic B-cell lymphoma-2 (BCL-2) family members prior to caspase activation, as well as how it synergizes with the BCL-2 and BCL-XL inhibitor ABT-737 to induce apoptosis. Interestingly, fluorizolinetreatment triggers the activation of the integrated stress response (ISR) in HeLa and HAP1 cells, with increased eukaryotic translation initiation factor 2α phosphorylation, and induction of ATF3, ATF4, and CHOP. Moreover, PHB downregulation induces similar ISR activation and apoptosis as with fluorizoline treatment. In addition, we studied the essential role of the pro-apoptotic protein NOXA in fluorizoline-induced apoptosis and we describe its mechanism of induction in HeLa and HAP1 cells. Moreover, we identified ATF3 and ATF4 as the transcription factors that bind to NOXA promoter upon fluorizoline treatment. Furthermore, using ATF3 and ATF4 CRISPR HeLa and HAP1 cells, we confirmed that both factors mediate the induction of NOXA and apoptosis by fluorizoline. In conclusion, fluorizoline treatment triggers the activation of the ISR that results in the induction of ATF3 and ATF4, important regulators of NOXA transcription in fluorizoline-induced apoptosis.
Assuntos
Fator 3 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Hidrocarbonetos Fluorados/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tiazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator 3 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Hidrocarbonetos Fluorados/metabolismo , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proibitinas , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Tiazóis/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In this study, we have assessed the pro-apoptotic effect of fluorizoline in 3 different multiple myeloma cell lines and 12 primary samples obtained from treatment-naïve multiple myeloma patients. Fluorizoline induced apoptosis in both multiple myeloma cell lines and primary samples at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline. Moreover, fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. Finally, NOXA-depletion by CRISPR/Cas9 in cells that do not express BIM conferred resistance to fluorizoline-induced apoptosis in multiple myeloma cells. These results suggest that targeting prohibitins could be a new therapeutic strategy for myeloma multiple.
Assuntos
Antineoplásicos/metabolismo , Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proibitinas , Ligação Proteica/fisiologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismoRESUMO
Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Ribonucleosídeos/farmacologia , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Adenina/análogos & derivados , Aminoimidazol Carboxamida/agonistas , Aminoimidazol Carboxamida/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/agonistas , Sinergismo Farmacológico , Feminino , Humanos , Hidrocarbonetos Fluorados/agonistas , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Piperidinas , Proibitinas , Pirazóis/agonistas , Pirimidinas/agonistas , Ribonucleosídeos/agonistas , Sulfonamidas/agonistas , Tiazolidinas/agonistas , Células Tumorais CultivadasRESUMO
Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins (PHBs). In this study, the pro-apoptotic effect of fluorizoline was assessed in two cell lines and 21 primary samples from patients with debut of acute myeloid leukemia (AML). Fluorizoline induced apoptosis in AML cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespectively of patients' clinical or genetic features. In addition, fluorizoline inhibited the clonogenic capacity and induced differentiation of AML cells. Fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. These results suggest that targeting PHBs could be a new therapeutic strategy for AML.
Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Apoptose , Benzotiazóis/química , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Proibitinas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Tiazóis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HT29 , Células HeLa , Humanos , Células Jurkat , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proibitinas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitinsâ 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.