RESUMEN
In a previous study, a novel anthraquinone analog BW-AQ-101 was identified as a potent inducer of MDM2 degradation, leading to upregulation of p53 and apoptosis in cell culture studies. In animal models of acute lymphocytic leukemia, treatment with BW-AQ-101 led to complete disease remission. In this study, we systematically investigated the effect of substitution patterns of the core anthraquinone scaffold. Through cytotoxicity evaluation in two leukemia cell lines, the structure-activity relationship of thirty-two analogs has been examined. Several analogs with comparable or improved potency over BW-AQ-101 have been identified. Western-blot assays verified the effect of the potent compounds on the MDM2-p53 axis. The study also suggests new chemical space for further optimization work.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Antraquinonas/síntesis química , Antraquinonas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Células K562 , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: It is known that the MDM2 protein is stabilized when it forms a heterodimer with its partner MDM4, but MDM2 protein stability in its homodimer form is not known. The MDM2 protein contains a C-terminal RING domain that not only functions as an E3 ligase to regulate ubiquitination of p53 and MDM2 itself, but also is characterized to be able to bind several specific cellular mRNAs to regulate gene expression. In this study, we evaluate whether the MDM2 protein stability is regulated by the binding of a specific small RNA (XIAP IRES mRNA). METHODS: We performed chemical cross-linking and bimolecular fluorescence complementation (BiFC) assay to measure the human MDM2 protein stability in its homodimer form and the effect of XIAP IRES on MDM2 homodimerization and protein stabilization. Ubiquitination and pulse-chase assays were used to detect MDM2 self-ubiquitination and protein turn-over. Fluorescent titration and ITC were used to examine the binding between MDM2 RING protein and XIAP IRES. Western blot assay was used for determining protein expression. Clonogenic assay, WST and flow cytometry were used to test the effects of XIAP IRES, siXIAP and IR on cancer cell growth and apoptosis. RESULTS: We found that self-association (homodimerization) of MDM2 occurs through the C-terminal RING domain of MDM2 and that the MDM2 protein becomes unstable when it is homodimerized. MDM2 homodimerization resulted in an increased function of the RING domain for MDM2 self-ubiquitination. Binding of XIAP IRES to the RING domain inhibited MDM2 homodimerization and self-ubiquitination, which resulted in stabilization of MDM2, as well as increased XIAP expression. Upregulation of XIAP and MDM2 that led to inhibition of p53 by the XIAP IRES resulted in cell growth and survival in both p53-normal and -deficient cancer cells. CONCLUSIONS: Our study identified a new IRES RNA that interacts with MDM2 protein and regulates its stabilization, which suggested that targeting of MDM2 through disruption of MDM2 protein-RNA interaction might be a useful strategy for developing novel anti-cancer therapeutics.
Asunto(s)
Supervivencia Celular/genética , Multimerización de Proteína/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Unión Proteica/genética , Estabilidad Proteica , Proteína p53 Supresora de Tumor/genética , Ubiquitinación/genética , Regulación hacia Arriba/genéticaRESUMEN
Inactivation of the tumor suppressor Ras-association domain family 1 isoform A (RASSF1A) due to epigenetic silencing occurs in a variety of human cancers, and still largely unknown are the regulators and mechanisms underlying RASSF1A gene promoter methylation. Herein, we report that this methylation is regulated by p53 and death-associated protein 6 (DAXX) in acute lymphoblastic leukemia (ALL). We found that p53 bound to the RASSF1A promoter, recruiting DAXX as well as DNA methyltransferase 1 (DNMT1) for DNA methylation, which subsequently resulted in inactivation of RASSF1A in wild-type p53 ALL cells. Although the presence of p53 was required for the recruitment of DAXX and DNMT1 to the RASSF1A promoter, fluctuation in p53 protein levels did not affect the rates of RASSF1A methylation. Conversely, methylation of RASSF1A promoter was critically controlled by DAXX, as the enforced overexpression of DAXX led to enhanced RASSF1A promoter methylation, whereas inhibition of DAXX reduced RASSF1A methylation. Interestingly, we found that the p53/DAXX-mediated RASSF1A methylation regulated murine double minute 2 (MDM2) protein stability in ALL. Our results reveal a novel function for p53 in the methylation of RASSF1A promoter by its interaction with DAXX. Discovery of this mechanism provides new insight into the interactions among the tumor-related factors p53, RASSF1A, DAXX, and MDM2 in cancer pathogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Metilación de ADN , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Línea Celular Tumoral , Proteínas Co-Represoras , ADN , Cartilla de ADN , Humanos , Chaperonas Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The MDM2 oncogene is amplified and/or overexpressed in various human cancers and elevated expression of MDM2 protein acts as a survival factor promoting cancer progression through both p53-dependent and -independent pathways. Here, we report a novel small-molecule chemical compound (MX69-102) that we identified to induce MDM2 protein degradation, resulting in reactivation of p53, inhibition of XIAP, and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL) in vitro and in vivo. We have previously identified a compound (MX69) that binds to the MDM2 C-terminal RING domain and induces MDM2 protein degradation. In the present study, we performed structural modifications of MX69 and selected analog MX69-102, showing increased MDM2-targeting activity. MX69-102 exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of about 0.2 µM, representing an approximately 38-fold increase in activity compared to MX69. MX69-102 also showed effective inhibition on xenografted human MDM2-overexpressing ALL in SCID mice. Importantly, MX69-102 had minimal or no inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in vivo in animal models. Based on the strong inhibitory and apoptotic activity against MDM2-overexpressing ALL, along with minimal or no toxicity to normal cells/tissues, MX69-102 is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory/MDM2-overexpressing ALL.
Asunto(s)
Apoptosis , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Humanos , Animales , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Apoptosis/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Línea Celular Tumoral , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Relación Dosis-Respuesta a Droga , Ratones , Femenino , Relación Estructura-ActividadRESUMEN
Death-domain-associated protein (DAXX) is a multifunctional protein that regulates a wide range of cellular signaling pathways for both cell survival and apoptosis. Regulation of DAXX gene expression remains largely obscure. We recently reported that berberine (BBR), a natural product derived from a plant used in Chinese herbal medicine, downregulates DAXX expression at the transcriptional level. Here, we further investigate the mechanisms underlying the transcriptional suppression of DAXX by BBR. By analyzing and mapping the putative DAXX gene promoter, we identified the core promoter region (from -161 to -1), which contains consensus sequences for the transcriptional factors Sp1 and Ets1. We confirmed that Sp1 and Ets1 bound to the core promoter region of DAXX and stimulated DAXX transcriptional activity. In contrast, BBR bound to the DAXX core promoter region and suppressed its transcriptional activity. Following studies demonstrated a possible mechanism that BBR inhibited the DAXX promoter activity through blocking or disrupting the association of Sp1 or Ets1 and their consensus sequences in the promoter. Downregulation of DAXX by BBR resulted in inhibition of MDM2 and subsequently, activation of p53, leading to cancer cell death. Our results reveal a novel possible mechanism: by competitively binding to the Sp1 and Ets1 consensus sequences, BBR inhibits the transcription of DAXX, thus inducing cancer cell apoptosis through a p53-dependent pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Berberina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas Co-Represoras , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Fluorescencia , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Overexpression of both human murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) is detected in tumor cells from several cancer types, including childhood acute leukemia lymphoma (ALL), neuroblastoma (NB), and prostate cancer, and is associated with disease progression and treatment resistance. In this report, we described the design and syntheses of a series of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold from our previously reported lead compound JW-2-107 and tested their cytotoxicity in a panel of human cancer cell lines. The best compound identified in this study is compound 3e. Western blot analyses demonstrated that treatments with 3e decreased MDM2 and XIAP protein levels and increased expression of p53, resulting in cancer cell growth inhibition and cell death. Furthermore, compound 3e effectively inhibited tumor growth in vivo when tested using a human 22Rv1 prostate cancer xenograft model. Collectively, results in this study strongly suggest that the tetrahydroquinoline scaffold, represented by 3e and our earlier lead compound JW-2-107, has abilities to dual target MDM2 and XIAP and is promising for further preclinical development.
Asunto(s)
Leucemia Mieloide Aguda , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Niño , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/farmacología , Apoptosis , Línea Celular Tumoral , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
TRAF1 is a member of the TRAF family, which plays important roles in signal transduction that mediate cell life and death in the immune response, inflammatory and malignant diseases. It is known that TRAF1 transcription is inducible by various cytokines, but little is known about the regulation of its mRNA translation. In the present study, we demonstrated that the human TRAF1 mRNA has an unusually long 5'-UTR that contains internal ribosome entry segment (IRES) regulating its translation. By performing gene transfection and reporter assays, we revealed that this IRES sequence is located within the 572 nt upstream from the AUG start codon. An element between nt -392 and -322 was essential for the IRES activity. Interestingly, we found that the TRAF1 expression is induced in cancer cells by chemotherapeutic drug vincristine that regulates cytoplasmic localization of polypyrimidine tract binding protein, which may contribute to the IRES-dependent translation of TRAF1 during vincristine treatment. These results indicate that TRAF1 translation is initiated via the IRES and regulated by vincristine, and suggest that regulation of the IRES-dependent translation of TRAF1 may be involved in effecting the cancer cell response to vincristine treatment.
Asunto(s)
Regiones no Traducidas 5' , Antineoplásicos Fitogénicos/farmacología , Regulación Neoplásica de la Expresión Génica , Biosíntesis de Proteínas , Factor 1 Asociado a Receptor de TNF/genética , Vincristina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Proteína de Unión al Tracto de Polipirimidina/análisis , Biosíntesis de Proteínas/efectos de los fármacos , Eliminación de Secuencia , Factor 1 Asociado a Receptor de TNF/biosíntesis , Células Tumorales CultivadasRESUMEN
Amplification of the MYCN gene leads to its overexpression at both the mRNA and protein levels. Overexpression of MYCN mRNA may also have an important role in promoting neuroblastoma (NB) beyond the translation of MYCN protein. In the present study, we report a small molecule compound (MX25-1) that was able to bind to the 3'UTR of MYCN mRNA and induce MYCN mRNA degradation; this resulted in potent cell-growth inhibition and cell death specifically in MYCN-amplified or MYCN 3'UTR overexpressing NB cells. To evaluate the role of MYCN 3'UTR-mediated signals in contributing to the anticancer activity of MX25-1, we examined the status and activation of the tumor suppressor microRNA (miRNA) let-7, which is a target of MYCN 3'UTR in MYCN-amplified NB. We first observed that overexpression of MYCN mRNA was associated with high-level expression of the let-7 oncogenic targets DICER1, ARID3B and HMGA2. Following MYCN mRNA degradation, the expression of DICER1, ARID3B and HMGA2 was downregulated in MX25-1-treated cells. Inhibition of let-7 reversed the downregulation of these oncogenic mRNAs and significantly increased resistance of NB cells to MX25-1. Our results from this study supported the notion that overexpression of MYCN mRNA due to gene amplification has an independent function in NB cell growth and disease progression and suggest that targeting MYCN mRNA may represent an attractive strategy for therapy of MYCN amplified NB, both by inhibiting MYCN's cell-survival effects and activating the tumor-suppressor effect of let-7.
RESUMEN
Murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) are important cell survival proteins in tumor cells. As a dual MDM2/XIAP inhibitor reported previously, compound MX69 has low potency with an IC50 value of 7.5 µM against an acute lymphoblastic leukemia cell line EU-1. Herein, we report the structural optimization based on the MX69 scaffold, leading to the discovery of a 25-fold more potent analogue 14 (IC50 = 0.3 µM against EU-1). We demonstrate that 14 maintains its mode of action by dual targeting of MDM2 and XIAP through inducing MDM2 protein degradation and inhibiting XIAP mRNA translation, respectively, which resulted in cancer cell growth inhibition and cell death. The results strongly suggest that the scaffold based on 14 is promising for further optimization to develop a new therapeutic agent for leukemia and possibly other cancers where MDM2 and XIAP are dysregulated.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Quinolinas/farmacología , Sulfonamidas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Quinolinas/síntesis química , Quinolinas/metabolismo , Ratas Sprague-Dawley , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lymphoblastic leukemia (ALL). We used a set of ALL cell lines with wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survivin expression in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3-resistant cells were either pretransfected with the PTEN gene or simultaneously treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfering RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3-resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapoptotic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Imidazoles/farmacología , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Enfermedad Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Muerte Celular , Cromonas/farmacología , Sinergismo Farmacológico , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Morfolinas/farmacología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfohidrolasa PTEN/metabolismo , ARN Interferente Pequeño , Survivin , Transfección , Células Tumorales CultivadasRESUMEN
MDM4 and topoisomerase IIα (TOP2A) are overexpressed in various human cancers. MDM4 acts as an oncoprotein which promotes cancer progression by inhibiting tumor suppressor p53. As a DNA replication- and cell division-regulating enzyme, TOP2A is the main target of many anticancer therapy regimens; however, the exact role of TOP2A in cancer remains elusive. Herein, we report that MDM4 and TOP2A bind to each other and are mutually upregulated at the post-translational level, leading to TOP2A protein stabilization, inhibition of p53, and increased tumor-cell proliferation. We demonstrate that the C-terminal region (CTR) of TOP2A binds to a unique sequence (residues: 188-238) of MDM4, which contains an auto-inhibitory segment regulating the MDM4-p53 interaction. TOP2A binding in turn activates MDM4 for p53 binding, resulting in enhanced inhibition of p53 and cancer cell proliferation. Conversely, binding of the MDM4 sequence to the CTR of TOP2A stabilizes TOP2A protein, leading to increased TOP2A protein expression. These results reveal novel functions of MDM4 and TOP2A as well as their interactions in oncogenesis, suggesting that inhibition of the MDM4-TOP2A interaction may represent a novel strategy in specifically and simultaneously targeting TOP2A and MDM4 for cancer treatment.
Asunto(s)
Proteínas de Ciclo Celular , Proliferación Celular , ADN-Topoisomerasas de Tipo II , Regulación Neoplásica de la Expresión Génica , Neoplasias , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Proto-Oncogénicas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Dominios Proteicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines. METHODS: Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively. RESULTS: Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2. CONCLUSION: This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.
Asunto(s)
Apoptosis/fisiología , Doxorrubicina/farmacología , Factor VIIa/metabolismo , Genes bcl-2/fisiología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Tromboplastina/metabolismo , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation of CD34(+) hematopoietic progenitor cells. Although signal transducers and activators of transcription 3 (STAT3) is believed essential for transduction of GM-CSF-induced cell proliferation, the signaling mediated by STAT3 is not completely understood. Because survivin regulates cell proliferation and survival via its antiapoptotic function, we studied the link between STAT3 signaling and survivin expression in CD34(+) cells. METHODS: GM-CSF-induced STAT3 and survivin expression in CD34(+) cells was examined by Western blot assay. GM-CSF-activated survivin promoter activity was analyzed by gene transfection and reporter assays. The binding of STAT3 to the survivin promoter was evaluated by chromatin immunoprecipitation and electrophoretic mobility shift assay. Western blotting and flow cytometry were utilized to test the effect of Janus family of tyrosine kinases (JAK) inhibitor and STAT3 small interfering RNA (siRNA) on cell apoptosis. RESULTS: We found that GM-CSF stimulates survivin promoter activity in CD34(+) KG-1 cells, and STAT3 binds to the core survivin promoter containing a STAT response element TT(N)(5)AA at sites -264 to -256. Mutation or deletion of this STAT response element completely abolished the effects of GM-CSF on survivin promoter activity. Furthermore, addition of either JAK inhibitor or STAT3 siRNA was able to inhibit GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent, CD34(+) line TF-1 decreased cell growth and increased apoptosis. CONCLUSION: Altogether, our results suggest that survivin is a transcriptional target of STAT3, and that GM-CSF-stimulated CD34(+) cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.
Asunto(s)
Antígenos CD34 , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/citología , Humanos , Proteínas Inhibidoras de la Apoptosis , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/fisiología , Inhibidores de Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , SurvivinRESUMEN
A novel small-molecule anthraquinone (AQ) analogue, AQ-101, which was synthesized through chemical modification of the core structures of rhein, exhibited potent anticancer activity. In the present study, we evaluated the cancer-inhibiting mechanism of AQ-101 and tested the therapeutic potential of this compound for treating cancer in mice. We found that AQ-101 was able to induce MDM2 protein degradation through a self-ubiquitination and proteasome-mediated mechanism. This AQ-101-induced MDM2 downregulation led to activation of p53, which contributed to apoptosis of acute lymphoblastic leukemia (ALL), especially those with a wild-type p53 phenotype and MDM2 expression in vitro and in vivo When given for a period of 2 weeks (20 mg/kg/day, 3×/week), AQ-101 inhibited development of ALL in nude or SCID mice with a human ALL xenograft and achieved cure by the end of the 5-month experiment. Importantly, AQ-101 showed minimal or no inhibitory effect on normal human hematopoiesis in vitro and was well tolerated in vivo in animal models. Given that MDM2-overexpressing cancers are commonly refractory to current treatment options, our study results suggest that further development of AQ-101 is warranted, as it represents a potentially new, safe anticancer drug with a novel strategy for targeting MDM2. Mol Cancer Ther; 17(2); 497-507. ©2017 AACR.
Asunto(s)
Antraquinonas/uso terapéutico , Neoplasias/genética , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Animales , Antraquinonas/farmacología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológicoRESUMEN
The tumor suppressor PTEN has been associated with the cellular localization of MDM2 in regulation of apoptosis through inhibiting PI3k/Akt signaling. To investigate whether expression of PTEN is involved in MDM2-mediated chemoresistance, we examined a set of acute lymphoblastic leukemia (ALL) cell lines for the expression of PTEN and sensitivity to doxorubicin. Testing 9 ALL cell lines selected for wild-type p53 phenotype and uniformly high levels of MDM2 expression, we initially demonstrated that cell lines with high levels of PTEN expression were sensitive to doxorubicin, whereas lines lacking PTEN expression were generally resistant. Forced expression of PTEN in a PTEN-negative and doxorubicin-resistant ALL line (EU-1) resulted in decreased cell growth and enhanced sensitivity to doxorubicin. Examining the cellular localization of MDM2, we confirmed that the majority of MDM2 is localized in the nucleus in PTEN-negative doxorubicin-sensitive ALL cells, whereas MDM2 is expressed predominantly in the cytoplasm in either PTEN-positive or PTEN-transfected cells. Furthermore, by coimmunoprecipitaton and cotransfection assays, we found that PTEN physically binds p53 in vitro as well as in vivo. Binding of PTEN to p53 attenuated MDM2-mediated p53 inhibition. These results suggest that PTEN inhibits MDM2 and protects p53 through both p13k/Akt-dependent and -independent pathways. Furthermore, loss of PTEN can result in resistance to apoptosis by activating MDM2-mediated antiapoptotic mechanism.
Asunto(s)
Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/metabolismo , Proteínas Nucleares , Monoéster Fosfórico Hidrolasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antibióticos Antineoplásicos/farmacología , Linfoma de Burkitt/enzimología , Linfoma de Burkitt/genética , División Celular/fisiología , Línea Celular Tumoral , Niño , Daño del ADN , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Humanos , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-mdm2 , Transfección , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
MDM2 and XIAP are mutually regulated. Binding of MDM2 RING protein to the IRES region on XIAP mRNA results in MDM2 protein stabilization and enhanced XIAP translation. In this study, we developed a protein-RNA fluorescence polarization (FP) assay for high-throughput screening (HTS) of chemical libraries. Our FP-HTS identified eight inhibitors that blocked the MDM2 protein-XIAP RNA interaction, leading to MDM2 degradation. The compound-induced MDM2 downregulation resulted not only in inhibition of XIAP expression, but also in activation of p53, which contributed to cancer cell apoptosis in vitro and inhibition of cancer cell proliferation in vivo. Importantly, one of the MDM2/XIAP inhibitors, MX69, showed minimal inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in animal models.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To investigate the possible role of inhibiting NF-kB activation in sensitizing tumor cells to chemotherapy-induced apoptosis, we transfected the dominant-negative mutant inhibitor of NF-kB (IkBm) into the EU-1 cell line, an acute lymphoblastic leukemia (ALL) line with constitutive NF-kB activation. Overexpression of IkBm significantly reduced constitutive NF-kB activity in EU-1 cells, resulting in decreased cell growth. In response to apoptosis induced by chemotherapeutic drugs, IkBm-transfected cells (EU-1/IkBm) exhibited increased sensitivity to vincristine (VCR), whereas sensitivity to doxorubicin (Dox) was not changed as compared to neo-transfected control (EU-1/neo) cells. To further evaluate the link between IkBm and sensitivity to Dox and VCR, we demonstrated that both endogenous IkBalpha and ectopic IkBm bind to p53. In response to Dox, the cytosolic p53.IkBalpha complex rapidly dissociated due to downregulation of IkBalpha. However, the p53.IkBm complex did not dissociate under these conditions. Although treatment of EU-1/IkBm cells with Dox increased the expression of p53, the nondissociating p53.IkBm complex resulted in decreased p53 function, as demonstrated by absence of cell-cycle arrest and induction of p53 target genes. Contrastingly, VCR-induced cell death neither downregulated IkBalpha nor induced p53, as shown by the lack of NF-kB activation and p53-mediated gene expression in VCR-treated cells. Our data suggest that IkBm simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas I-kappa B/genética , FN-kappa B/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Humanos , Proteínas I-kappa B/metabolismoRESUMEN
Survivin is a unique member of the inhibitor of apoptosis protein family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DeltaEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild-type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53-143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53-143. When EU-4/p53-143 cell culture was shifted from 37.5 degrees C to the wt-p53-permissive temperature (32.5 degrees C), the expression of survivin and survivin-DeltaEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a proapoptotic factor and its expression is upregulated by p53.
Asunto(s)
Empalme Alternativo , Antineoplásicos/uso terapéutico , Leucemia/genética , Proteínas Asociadas a Microtúbulos/genética , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/efectos de los fármacos , Secuencia de Bases , División Celular , Línea Celular , Cartilla de ADN , Doxorrubicina/farmacología , Humanos , Proteínas Inhibidoras de la Apoptosis , Leucemia/tratamiento farmacológico , Leucemia/patología , Proteínas de Neoplasias , Survivin , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia ArribaRESUMEN
The MYCN oncogene is amplified in 20% of neuroblastomas, leading to its overexpression at both the mRNA and protein levels. MYCN overexpression is strongly associated with advanced disease stage, rapid tumor progression and a worse prognosis. In the present study, we identified microRNA-375 (miR-375) as a negative regulator of MYCN: enforced expression of miR-375 inhibited MYCN-amplified neuroblastoma in vitro and in vivo. Upon searching the website miRbase for possible miR-375 binding sites within the whole MYCN mRNA, we found that the MYCN 5'-UTR had significant sequence complementarity to miR-375, yet no complementary sequences existed within the MYCN 3'-UTR and coding regions. Enforced overexpression of miR-375 efficiently inhibited MYCN mRNA translation and protein synthesis, via an IRES-dependent mechanism. In athymic nude mouse model with human MYCN-amplified neuroblastoma, MYCN downregulation by miR-375 led to inhibition of tumor cell growth and tumorigenicity. In particular, miR-375-regulated inhibition of MYCN translation was enhanced when MYCN-amplified neuroblastoma cells were exposed to stress stimulation, such as ionizing irradiation (IR), resulting in a remarkable increase in the neuroblastoma's sensitivity to IR-induced cell death. Our results identified a novel mechanism by which IRES-dependent translation of MYCN is repressed by miR-375, particularly during cellular stress, highlighting a potential anticancer strategy: the development of miR-375 as a novel therapeutic agent to treat MYCN-amplified neuroblastoma.
Asunto(s)
Proliferación Celular/genética , Genes myc , Neuroblastoma/genética , Tolerancia a Radiación/genética , Ribosomas/metabolismo , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular Tumoral , Regulación hacia Abajo , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neuroblastoma/patologíaRESUMEN
Nilotinib is a selective BCR-ABL tyrosine kinase inhibitor related to imatinib that is more potent than imatinib. Nilotinib is widely used to treat chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The present study identifies Mouse double minute 2 homolog (MDM2) as a target of nilotinib. In studying ALL cell lines, we found that the expression of MDM2 in both Philadelphia positive (Ph+) and Philadelphia negative (Ph-) ALL cells was remarkably inhibited by nilotinib, in a dose- and time-dependent manner. Further studies demonstrated that nilotinib inhibited MDM2 at the post-translational level by inducing MDM2 self-ubiquitination and degradation. Nilotinib-mediated MDM2 downregulation did not result in accumulation and activation of p53. Inhibition of MDM2 in nilotinib-treated ALL cells led to downregulation of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP), a translational target of MDM2, resulting in activation of caspases. Inhibition of XIAP following nilotinib-mediated downregulation of MDM2 resulted in apoptosis of MDM2-expressing ALL; however, similar nilotinib treatment induced stronger apoptosis in Ph+/MDM2+ ALL than in Ph-/MDM2+ or Ph+/MDM2- ALL. The ALL cells that were Ph-/MDM2- were totally resistant to nilotinib. These results suggested that nilotinib can inhibit MDM2 and induce a p53-independent apoptosis pathway by downregulating XIAP; thus, nilotinib can treat not only Ph+, but also Ph- ALL patients whose cancer cells overexpress MDM2.