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
The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-ß) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-ß-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-ß. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-ß, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-ß-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-ß in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-ß1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-ß that may contribute to tumor progression, we found that NOX4 is also required for TGF-ß-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-ß-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-ß-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-ß signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-ß pathway.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fator de Crescimento Transformador beta , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Fator de Crescimento Transformador beta1RESUMO
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
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.