RESUMO
The maturation of RNA from its nascent transcription to ultimate utilization (e.g., translation, miR-mediated RNA silencing, etc.) involves an intricately coordinated series of biochemical reactions regulated by RNA-binding proteins (RBPs). Over the past several decades, there has been extensive effort to elucidate the biological factors that control specificity and selectivity of RNA target binding and downstream function. Polypyrimidine tract binding protein 1 (PTBP1) is an RBP that is involved in all steps of RNA maturation and serves as a key regulator of alternative splicing, and therefore, understanding its regulation is of critical biologic importance. While several mechanisms of RBP specificity have been proposed (e.g., cell-specific expression of RBPs and secondary structure of target RNA), recently, protein-protein interactions with individual domains of RBPs have been suggested to be important determinants of downstream function. Here, we demonstrate a novel binding interaction between the first RNA recognition motif 1 (RRM1) of PTBP1 and the prosurvival protein myeloid cell leukemia-1 (MCL1). Using both in silico and in vitro analyses, we demonstrate that MCL1 binds a novel regulatory sequence on RRM1. NMR spectroscopy reveals that this interaction allosterically perturbs key residues in the RNA-binding interface of RRM1 and negatively impacts RRM1 association with target RNA. Furthermore, pulldown of MCL1 by endogenous PTBP1 verifies that these proteins interact in an endogenous cellular environment, establishing the biological relevance of this binding event. Overall, our findings suggest a novel mechanism of regulation of PTBP1 in which a protein-protein interaction with a single RRM can impact RNA association.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Processamento Alternativo/genética , Sítios de Ligação/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica/genética , RNA/metabolismo , HumanosRESUMO
Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.
RESUMO
Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.
RESUMO
Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin-induced cell death in human oral squamous cell carcinoma (OSCC). The growth-inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit-8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4',6-diamidino-2-phenylindole staining, measurement of the sub-G1 population, and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real-time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase-dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p-STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia-1 (Mcl-1) expression, leading to apoptotic cell death. The genipin-mediated reduction in survivin and Mcl-1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/patologia , Survivina/metabolismo , Survivina/farmacologia , Survivina/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Neoplasias Bucais/patologia , Apoptose , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
PURPOSE: Composite cyclin-dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. METHODS: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. RESULTS: CDK9 inhibitors exhibited potent antiproliferative activities in HCC cells regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1 and survivin. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited significantly slower tumor growth than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving a CDK9 inhibitor also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Knockdown of CDK9 expression reduced the proliferative activities of HCC cells. CONCLUSION: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Apoptose/genéticaRESUMO
The prognostic value of chromosomal 1q21 gain in newly diagnosed multiple myeloma (NDMM) remains controversial. Add-on Myc aberrations may further worsen the outcome. To investigate whether specific genes located at the 1q21 region, such as myeloid cell leukemia 1 (Mcl-1), are involved in NDMM progression, we examined bone marrow cytogenetic abnormalities in 153 patients with NDMM by fluorescence in situ hybridization. Their response to treatment and survival was also analyzed. C-Myc and Mcl-1 expressions in bone marrow samples were analyzed by RT-PCR. The expression of Mcl-1 was evaluated in bone marrow sections by immunohistochemistry. MM cell lines were transfected with Mcl-1 siRNA. 1q21 gain was present in 55/153 (35.9%) patients and strongly associated with Myc rearrangement (31/153, 20.3%, P = 0.004). A positive correlation was observed between Myc and Mcl-1 mRNA levels in bone marrow cells from 47 patients (r = 0.57, P < 0.001). The combination of 1q21 gain and Myc rearrangement was associated with poorer overall survival than Myc rearrangement alone (16.8 vs. 27.9 months, P = 0.077) or 1q21 gain alone (16.8 vs. 60.7 months, P < 0.01). High Mcl-1 protein expression in bone marrow plasma cells was associated with Myc rearrangement. Mcl-1 silencing by siRNA inhibited Myc protein expression in three myeloma cell lines. Treatment with the small-molecule Mcl-1 inhibitor, UMI-77, produced similar results. Overall, the combination of Myc rearrangement and 1q21 gain was associated with particularly poor prognosis in patients with MM. Furthermore, our data are consistent with Mcl-1-dependent Myc protein activation.
Assuntos
Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Aberrações Cromossômicas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Prognóstico , RNA Mensageiro/genéticaRESUMO
Myeloid cell leukemia-1 (Mcl-1) is a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to their resistance to current chemotherapeutics. In this study, more than thirty coumarin derivatives with different substituents were designed and synthesized, and their Mcl-1 inhibitory activities evaluated using a fluorescence polarization-based binding assay. The results showed that the catechol group was a key constituent for Mcl-1 inhibitory activity of the coumarins, and methylation of the catechol group led to decreased inhibitory activity. The introduction of a hydrophobic electron-withdrawing group at the C-4 position of 6,7-dihydroxycoumarin, enhanced Mcl-1 inhibitory capacity, and a hydrophilic group in this position was unbeneficial to the inhibitory potency. In addition, the introduction of a nitrogen-containing group to the C-5 or C-8 position, which allowed an intramolecular hydrogen bond, was also unfavorable for Mcl-1 inhibition. Among all coumarins tested, 4-trifluoromethyl-6,7-dihydroxycoumarin (Cpd 4) displayed the most potent inhibitory activity towards Mcl-1 (Ki = 0.21 ± 0.02 µM, IC50 = 1.21 ± 0.56 µM, respectively), for which the beneficial effect on taxol resistance was also validated in A549 cells. A strong interaction between Cpd 4 and Mcl-1 in docking simulations further supported the observed potent Mcl-1 inhibition ability of Cpd 4. 3D-QSAR analysis of all tested coumarin derivatives further provides new insights into the relationships linking the inhibitory effects on Mcl-1 and the steric-electrostatic properties of coumarins. These findings could be of great value for medicinal chemists for the design and development of more potent Mcl-1 inhibitors for biomedical applications.
Assuntos
Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Bile acid synthesis is restricted to hepatocytes and is rate-limited by CYP7A1 (cholesterol 7α hydroxylase). CYP7A1 expression undergoes tight regulation and is repressed after partial hepatectomy to prevent the accumulation of toxic bile acids. Augmenter of Liver Regeneration (ALR) is a hepatotrophic factor shown to support liver regeneration by augmenting cell proliferation and reducing apoptosis. Nevertheless, less is known about ALR's role in protecting hepatocytes from bile acid accumulation and bile acid-induced apoptosis. Therefore, HepG2 and Huh-7 cells were incubated with recombinant human ALR (rALR) and the expression of CYP7A1, bile acid-induced apoptosis as well as potential molecular mechanisms were analyzed. We found that rALR reduces CYP7A1 expression by increasing nuclear NFκB levels. Moreover, rALR reduced glycochenodeoxycholate (GCDC)-induced-apoptosis by decreased expression of pro-apoptotic Bax and enhanced expression of anti-apoptotic Mcl-1, which is regulated by phosphatidylinositol-3-kinase (PI3K)/Akt activation and glycogen synthase kinase-3ß (GSK3ß) phosphorylation. Inhibitors for PI3K/Akt (GSK690693) and GSK3ß (SB415286) confirmed the specificity of rALR treatment for this pathway. In addition, rALR reduces pro-death signaling by decreasing GCDC-induced JNK phosphorylation. Taken all together, rALR might contribute to protecting hepatocytes from toxic concentrations of bile acids by down-regulating their denovo synthesis, attenuating apoptosis by activation of PI3K/Akt - GSK3ß pathway and inhibition of JNK signaling. Thereby this suggests a new role of ALR in augmenting the process of liver regeneration.
Assuntos
Apoptose , Ácidos e Sais Biliares/biossíntese , Carcinoma Hepatocelular/terapia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hepatócitos/citologia , Neoplasias Hepáticas/terapia , Regeneração Hepática , Ácidos e Sais Biliares/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Colesterol 7-alfa-Hidroxilase , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Animal studies have demonstrated that myeloid cell leukemia-1 (Mcl-1) gene deficiency leads to premature ovarian failure and decreased reproductive ability in mice. This study investigated the relationship between MCL-1 gene variation and idiopathic premature ovarian insufficiency (POI) in Chinese women. METHODS: A total of 200 idiopathic POI patients and 100 healthy controls were recruited for this study, and peripheral blood was collected. First, genomic DNA was extracted from peripheral leukocytes. Then, the entire coding region and splice sites of the MCL-1 gene were amplified by polymerase chain reaction. Chi-squared tests were used to compare the genotype distribution and allele frequency of single nucleotide polymorphisms between the POI and control groups. RESULTS: Three mutations of the MCL-1 gene (c.-36C > T, c.-131C > T and c.78C > T) were identified. After data analysis, c.-36C > T and c.-131C > T in the 5'-untranslated region were both found in the POI group and the control group. No difference was found in the genotype distribution or allelic frequency of either variant between the POI group and the control group (p > 0.05). The synonymous variant (c.78C > T) in exon 1 was discovered in only one of the control subjects and did not result in a change in amino acid sequence (p.Gly26Gly). CONCLUSION: MCL-1 gene mutation may not be associated with idiopathic POI in Chinese women.
Assuntos
Insuficiência Ovariana Primária , Animais , China , Feminino , Humanos , Camundongos , Insuficiência Ovariana Primária/genéticaRESUMO
This study investigated the prognostic effects of genomic biomarkers for predicting chemoradiotherapy (CRT)-based treatment outcomes in patients with adenocarcinoma (AC) of the uterine cervix. In all, 21 patients receiving definitive CRT were included. In accordance with the International Federation of Gynecology and Obstetrics (FIGO) staging system, 5, 8, and 8 patients were classified as having stage IB3, II, and III disease, respectively. Pretreatment biomarkers were analyzed using tissue microarrays from biopsy specimens. Genomic alterations were examined by next-generation sequencing (NGS). The outcome endpoints were disease-free survival (DFS), distant metastasis-free survival (DMFS), and local relapse-free survival (LRFS). A Cox regression model was used to examine the prognostic effects of the biomarkers and clinical parameters. The presence of myeloid cell leukemia-1 (MCL1) gene amplification and a lower immunohistochemical (IHC) marker of tumor necrotic factor alpha (TNF-α) H-score were two prognostic factors for inferior DFS. The four-year DFS was 28% and 68% for patients with or without MCL1 copy number gain, respectively (p = 0.028). In addition, MCL1 amplification predicted poor DMFS. A lower tumor mutation number (TMN) calculated from nonsynonymous mutations was associated with lower LRFS. For patients with adenocarcinoma of the uterine cervix receiving definitive CRT, prognostic information can be supplemented by MCL1 amplification, the TMN, and the TNF-α H score.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/radioterapia , Biomarcadores Tumorais/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/radioterapia , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Quimiorradioterapia , Intervalo Livre de Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Prognóstico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/mortalidadeRESUMO
Mcl-1 is an anti-apoptotic protein overexpressed in hematological malignancies and several human solid tumors. Small molecule inhibition of Mcl-1 would offer an effective therapy to Mcl-1 mediated resistance. Subsequently, it has been the target of extensive research in the pharmaceutical industry. The discovery of a novel class of Mcl-1 small molecule inhibitors is described beginning with a simple biaryl sulfonamide hit derived from a high through put screen. A medicinal chemistry effort aided by SBDD generated compounds capable of disrupting the Mcl-1/Bid protein-protein interaction in vitro. The crystal structure of the Mcl-1 bound ligand represents a unique binding mode to the BH3 binding pocket where binding affinity is achieved, in part, through a sulfonamide oxygen/Arg263 interaction. The work highlights the some of the key challenges in designing effective protein-protein inhibitors for the Bcl-2 class of proteins.
Assuntos
Descoberta de Drogas , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
Norcantharidin (NCTD) is the demethylated form of cantharidin that exhibits anticancer potential in many cancer cell types. Recent reports suggest that NCTD targeting ROS/AMPK and DNA replication signaling pathway could be an effective strategy for the treatment of PCa cells. However, supportive evidence is limited to the effect of NCTD that induction of apoptosis through suppression of the Mcl-1. Here, we show that NCTD induced PCa cell apoptosis and triggered caspase activation, which was associated with mitochondria dysfunction. Mechanistic investigations suggested that NCTD modulated the Akt signaling via increased nuclear translocation and interaction with the myeloid cell leukemia-1 (Mcl-1) promoter by FOXO4, resulting in an apoptotic effect. Moreover, miR-320d, which targets Mcl-1, was significantly upregulated after NCTD treatment. Overexpression of miR-320d by NCTD induced mitochondria dysfunction and apoptosis, which was notably attenuated with a miR-320d inhibitor. In vivo xenograft analysis revealed that NCTD significantly reduced tumor growth in mice with PC3 tumor xenografts. Taken together, our results provide new insights into the critical role of NCTD in suppressing Mcl-1 via epigenetic upregulation of miR-320d, resulting in PCa cell apoptosis.
Assuntos
MicroRNAs/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Fatores de Transcrição Forkhead , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Proteína Oncogênica v-akt/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Osteoarthritis is the most frequent chronic bone and joint diseases in older populations all over the world. Lipopolysaccharide (LPS)-induced murine chondrogenic ATDC5 cell model has been widely used for testing new osteoarthritis therapeutic targets. This study aimed to explore the effects of microRNA-136 (miR-136) on LPS-induced ATDC5 cell injury and inflammatory cytokine expression, as well as underlying potential mechanism. We found that LPS remarkably inhibited ATDC5 cell viability, induced ATDC5 cell apoptosis, and upregulated the expression of inflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, IL-8, and tumor necrosis factor α (TNF-α; P < .01 or < .001). Moreover, LPS obviously upregulated the expression of miR-136 in ATDC5 cells (P < .05). Overexpression of miR-136 markedly exacerbated the LPS-induced ATDC5 cell viability inhibition, cell apoptosis enhancement, and inflammatory cytokine expression (P < .05), and suppression of miR-136 had opposite effects (P < .05). Myeloid cell leukemia 1 (Mcl-1) was a direct target gene of miR-136, which participated in the effect of miR-136 on LPS-induced ATDC5 cell inflammatory injury. Overexpression of Mcl-1 alleviated the LPS-induced inactivation of Wnt/ß-catenin and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways, while suppression of Mcl-1 had opposite effects. To conclude, this study verified that miR-136 promoted LPS-induced ATDC5 cell injury and inflammatory cytokine expression by targeting Mcl-1, and Mcl-1 was involved in the regulatory effects of LPS on Wnt/ß-catenin and JAK/STAT signaling pathways in ATDC5 cells.
Assuntos
Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: We have shown previously that nitidine chloride (NC) induces apoptosis via inhibition of signal transducer and activator of transcription 3 (STAT3). However, its downstream molecules are not fully understood yet. Here, we report that NC as STAT3 inhibitor downregulates myeloid cell leukemia-1 (Mcl-1) protein in HSC-3 and HSC-4 human oral squamous cell carcinoma (OSCC) cells and a nude mouse tumor xenograft model. METHODS: This study investigated the effects of NC on Mcl-1 expression in HSC-3 and HSC-4 cells using Western blotting, RT-PCR, and dual-luciferase assay. Immunohistochemistry was employed to evaluate Mcl-1 expression levels in mouse tumor tissues. Construction of Mcl-1 overexpression vector and transient transfection was done to test the apoptosis of HSC-3 cells. RESULTS: Nitidine chloride did not affect either mRNA level or promoter activity of Mcl-1, and the decrease in Mcl-1 protein by NC was caused by lysosome-dependent degradation, but not proteasome-dependent degradation. The overexpression of Mcl-1 protein in OSCC cell lines was sufficient to block the induction of apoptosis. In addition, NC strongly reduced the expression level of Mcl-1 protein compared with other STAT3 inhibitors such as cryptotanshione and S3I-201 in OSCCs. CONCLUSIONS: Our findings suggest that NC triggers apoptosis via lysosome-dependent Mcl-1 protein degradation and could be chosen as a promising chemotherapeutic candidate against human OSCCs.
Assuntos
Antineoplásicos , Benzofenantridinas/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Expressão Gênica/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteólise/efeitos dos fármacos , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Humanos , Lisossomos/patologia , Camundongos , Camundongos Nus , Neoplasias Bucais/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/fisiologiaRESUMO
Angiotensin II (Ang II) plays a central role in cardiovascular diseases by causing endothelial apoptosis and dysfunction. Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of apoptosis-regulating proteins. It has been reported that Mcl-1 plays a pivotal role in protecting cells against apoptosis. Presently, the effects of Ang II on the expression of Mcl-1 remain unknown. In this study, we report, for the first time, that the antiapoptotic protein Mcl-1 is degraded by the proteasome during Ang II-induced apoptosis in HUVECs. Notably, our results demonstrate that prior phosphorylation by GSK-3ß is required for proteasomal degradation of Mcl-1. Notably, the reduced level of Mcl-1 was abolished by a specific GSK-3ß inhibitor, suggesting that the phosphorylation of Mcl-1 by GSK-3ß is required for proteasomal degradation of Mcl-1. Overexpression of Mcl-1 rescued apoptosis induced by Ang II, however, knockdown of Mcl-1 exacerbated Ang II-induced apoptosis, thereby indicating that the protein level of Mcl-1 determines the response of endothelial cells to this drug. © 2017 IUBMB Life, 69(5):321-327, 2017.
Assuntos
Angiotensina II/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Angiotensina II/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fosforilação/efeitos dos fármacos , Tiazóis/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Oncostatin M (OSM), a cytokine in the interleukin-6 (IL-6) family, has been proposed to play a protective role in the central nervous system, such as attenuation of excitotoxicity induced by N-methyl-D-aspartate (NMDA) and glutamate. However, the potential neuroprotective effects of OSM against mitochondrial dysfunction have never been reported. In the present study, we tested the hypothesis that OSM may confer neuronal resistance against 3-nitropropionic acid (3-NP), a plant toxin that irreversibly inhibits the complex II of the mitochondrial electron transport chain, and characterized the underlying molecular mechanisms. We found that OSM preconditioning dose- and time-dependently protected cortical neurons against 3-NP toxicity. OSM stimulated expression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 family member expressed in differentiating myeloid cells, that required prior phosphorylation of Janus kinase-1 (JAK1), JAK2, extracellular signal-regulated kinase-1/2 (ERK1/2), signal transducer and activator of transcription-3 (STAT3), STAT1, and cAMP-response element-binding protein (CREB). Pharmacological inhibitors of JAK1, JAK2, ERK1/2, STAT3, STAT1, and CREB as well as the siRNA targeting at STAT3 and Mcl-1 all abolished OSM-dependent 3-NP resistance. Finally, OSM-dependent Mcl-1 induction contributed to the enhancements of mitochondrial bioenergetics including increases in spare respiratory capacity and ATP production. In conclusion, our findings indicated that OSM induces Mcl-1 expression via activation of ERK1/2, JAK1/2, STAT1/3, and CREB; furthermore, OSM-mediated Mcl-1 induction contributes to bioenergetic improvements and neuroprotective effects against 3-NP toxicity in cortical neurons. OSM may thus serve as a novel neuroprotective agent against mitochondrial dysfunction commonly associated with pathogenic mechanisms underlying neurodegeneration.
Assuntos
Córtex Cerebral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metabolismo Energético/fisiologia , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neurônios/metabolismo , Oncostatina M/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Anti-Hipertensivos/efeitos adversos , Anti-Hipertensivos/farmacologia , Córtex Cerebral/citologia , Metabolismo Energético/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neurônios/citologia , Nitrocompostos/efeitos adversos , Nitrocompostos/farmacologia , Propionatos/efeitos adversos , Propionatos/farmacologia , RatosRESUMO
BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sec6 and Sec8, which are components of the exocyst complex, has been concerned with various roles independent of its role in secretion, such as cell migration, invadopodia formation, cytokinesis, glucose uptake, and neural development. Given the vital roles of the exocyst complex in cellular and developmental processes, the disruption of its function may be closely related to various diseases such as cancer, diabetes, and neuronal disorders. Malignant peripheral nerve sheath tumors (MPNSTs) have high malignant potential and poor prognosis because of aggressive progression and metastasis. To date, no chemotherapeutic agents have been validated for MPNSTs treatment because how MPNSTs are resistant to chemotherapeutic agents remains unknown. This study demonstrates that combination of doxorubicin and sorafenib induces apoptosis in MPNST cells through downregulation of B cell lymphoma protein 2 (Bcl-2), Bcl-2-related protein long form of Bcl-x (Bcl-xl), and myeloid cell leukemia 1 (Mcl-1). Moreover, both Sec6 and Sec8 levels decreased after treatment with doxorubicin and sorafenib and were found to be associated with Bcl-2 and Mcl-1 expressions, but not Bcl-xl. Although Sec8 was found to be involved in the regulation of both Bcl-2 and Mcl-1 at the mRNA level, Sec6 regulated Bcl-2 at the mRNA level and the binding affinity of F-box and WD repeat domain containing 7 and Mcl-1, thereby controlling Mcl-1 at the protein level. Bcl-2 or Mcl-1 mRNA suppression by Sec6 or Sec8 depletion resulted in significant changes in nuclear factor-kappa B, cAMP response element, and p53 transcriptional activity. These results suggest that Sec6 and Sec8 are therapeutic target molecules in MPNST.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias de Bainha Neural/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas de Transporte Vesicular/metabolismo , Apoptose , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/fisiopatologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sorafenibe , Transcrição Gênica , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Helicobacter pylori induces the antiapoptotic protein myeloid cell leukemia 1 (Mcl1) in human gastric epithelial cells (GECs). Apoptosis of oncogenic protein Mcl1-expressing cells is mainly regulated by Noxa-mediated degradation of Mcl1. We wanted to elucidate the status of Noxa in H. pylori-infected GECs. For this, various GECs such as AGS, MKN45, and KATO III were either infected with H. pylori or left uninfected. The effect of infection was examined by immunoblotting, immunoprecipitation, chromatin immunoprecipitation assay, in vitro binding assay, flow cytometry, and confocal microscopy. Infected GECs, surgical samples collected from patients with gastric adenocarcinoma as well as biopsy samples from patients infected with H. pylori showed significant up-regulation of both Mcl1 and Noxa compared with noninfected samples. Coexistence of Mcl1 and Noxa was indicative of an impaired Mcl-Noxa interaction. We proved that Noxa was phosphorylated at Ser(13) residue by JNK in infected GECs, which caused cytoplasmic retention of Noxa. JNK inhibition enhanced Mcl1-Noxa interaction in the mitochondrial fraction of infected cells, whereas overexpression of nonphosphorylatable Noxa resulted in enhanced mitochondria-mediated apoptosis in the infected epithelium. Because phosphorylation-dephosphorylation can regulate the apoptotic function of Noxa, this could be a potential target molecule for future treatment approaches for H. pylori-induced gastric cancer.
Assuntos
Apoptose , Células Epiteliais/patologia , Infecções por Helicobacter/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Gástricas/patologia , Estômago/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/virologia , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Citometria de Fluxo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/patologia , Infecções por Helicobacter/virologia , Helicobacter pylori/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas Imunoenzimáticas , Imunoprecipitação , MAP Quinase Quinase 4 , Mitocôndrias , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Estômago/virologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologiaRESUMO
Heat shock protein 90 (Hsp90) is an attractive therapeutic target. Geldanamycin (GA), the first identified Hsp90 inhibitor, exhibited potent antitumor activity, but possessed significant hepatotoxicity. To overcome the hepatotoxicity derived from the quinone structure of GA, a non-quinone GA derivative 17-demethoxy-reblastatin (17-DR) was obtained from a genetically modified strain of Streptomyces hygroscopicus. In the present study, we examined the anticancer effects of 17-DR on human hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, and its underlying mechanisms. The results indicated that 17-DR could concentration-dependently inhibit the proliferation, and decrease the colony formation in HCC cells. It also induced significant apoptosis in HCC cells, which was mediated by mitochondria via a caspase-dependent pathway. The mechanisms involved in 17-DR-induced apoptosis included the downregulation of myeloid cell leukemia-1 (Mcl-1), and upregulation of Bcl-2 antagonist killer 1 (Bak). And the upregulated Bak expression resulted from downregulation of Mcl-1 played an essential role in this process. Taken together, these results indicated that 17-DR possessed potent anticancer effects on HCC cells by inhibiting cell proliferation and inducing apoptosis.